Taxonomy and Phylogeny of Prokaryotes

Abstract

Classification of prokaryotes is hierarchically organized into seven levels: kingdoms, phyla, classes, orders, families, genera, and species. In prokaryotes, because they reproduce by clonal fission, the species, considered as the basic unit of the biological diversity, faces several problems such as the definition of an individual. A bacterial strain can be recognized as an individual belonging to a species. However, many inconsistencies exist between phenotypic similarity levels and evolutionary relationships deduced from molecular phylogenies. Most taxonomic groups have been reconsidered through phylogenetic analysis in the 1980s, and a consensus has been reached on the need for coherence between taxonomy and phylogeny. Thus, the multiple revisions of species, genera, or higher taxonomic levels pose many complex problems that are solved gradually. Prokaryotic microorganisms correspond to two of the three domains of life: Archaea and Bacteria. Their systematics is described in the “Bergey’s Manual for Systematic Bacteriology, second edition” published in five volumes.

In the text, the Latin terms used are those accepted by the Nomenclature Committee, and the organization of the bacterial and archaeal domains is presented as they appear in the “Bergey’s Manual for Systematic Bacteriology.” They are discussed according to the recent data of the hierarchical classification of Prokaryotes.

Chapter Coordinator

1 The Concept of Species in Prokaryotes and Its Evolution

Classification of prokaryotes has long been based on the same hierarchical systems on which Linnaeus (Linnaeus 1753) based his nomenclatural system applied initially to plants and animals. These classifications are hierarchically organized into seven levels: kingdoms*, phyla*, classes*, orders*, families*, genera*, and species*. In the kingdoms corresponding to the animal and the plant worlds, a robust classification has been established. Its success depends in part on the definition of a species that has been proposed for eukaryotes: a species is a group of individuals who have the ability to reproduce and yield fecund offspring and share many functional and morphological characteristics. This definition of species based on the “interfertility” criterion, which aims to be universal, is not applicable to prokaryotes because they reproduce by clonal reproduction*. However, the species is considered as the basic unit of biological diversity, and its definition should be unambiguous and strong. In relation to this, the systematic of prokaryotes faces several problems such as the definition of an individual* and the definition of a species without being able to rely on the criterion of interfertility.

For years, the species concept in prokaryotes was based on morphological and physiological criteria (morphologic and physiologic are very seldom used) that have proven not to be really effective. Today molecular biology provides new tools to strengthen the concept of species replacing interfertility criteria and proposing quantitative criteria (Box 6.1). Thus, strains of prokaryotes for which hybridization of genomic DNA considered pairwise reaches 70 % are considered as belonging to the same species (Wayne et al. 1987). Other taxonomic levels are left to the discretion of the researchers and the consideration of a set of rules (priority, uniqueness, consistency, etc.). However, it is important to remember that the goal of this approach is to provide a solid framework in which the defined taxa*, must be, inasmuch as possible, “natural” and “consistent” (i.e., reflecting relationships between microorganisms), which is not always possible. Also, Stackebrandt and Goebel (1994) have suggested an equivalence between the classical definition of a species based on the labor-intensive and delicate genomic hybridization* technique and an identification technique most commonly used in recent years. This approach is based on the comparison of genes encoding RNA of the small subunit of the ribosome (16S rRNA). By this method, which is being reevaluated (Stackebrandt and Ebers 2006), two strains are considered as not belonging to the same species if their 16S rRNA sequences have a similarity below a threshold currently set at 97 %.

Box 6.1: The Species Concept Paradox

Xavier Nesme

Laboratoire d’écologie microbienne, UMR 5557 Université Claude Bernard Lyon 1, Villeurbanne Cedex, France

Species and speciation are constant topics of discussion in biology. In prokaryotes, this issue is particularly crucial at the time of metagenomics, which generates large volumes of nucleotide sequences from a variety of strains, species, genera, etc., that should be classified and ranked automatically with maximum biological sense. However, the bacterial species is paradoxically both well definable and lacking a consensual concept that would highlight the causes and biological consequences that this definition covers.

Bacterial Species Versus Eukaryotic Species

The bacterial species is defined simply on a technical basis by measuring the reassociation rate (“relative binding ratio” or RBR) of the genomic DNA of pairs of bacterial strains. For its part, the species in eukaryotes has benefited from profound reflections that led to the biological species concept (“biological species concept” or BSC), which highlights the role of the sexual isolation of species with, as a result, the containment of gene flow and therefore of genetic innovations to a given species.

The genomic definition of bacterial species (Wayne et al. 1987) is as follows:

“The bacterial species includes strains with both a relative DNA reassociation % superior to 70 % and 5 °C or less ΔTm; the two must be considered.”

The biological species concept (BSC) in eukaryotes (Mayr 1942) is as follows:

“Species are groups of actually or potentially interbreeding populations, which are reproductively isolated from other such groups.”

A simply operational definition of the species in bacteria corresponds to a biological species concept in eukaryotes: Are they the same thing? Can a biological concept be associated to the definition of bacterial species?

The genomic basis of the definition of a bacterial species was confirmed in 2002 by the International Committee of the definition of bacterial species, because since its publication in 1987, this definition proved operational in most lineages of prokaryotes (Stackebrandt et al. 2002). This definition, based on a rigorous physical measurement, has greatly reduced taxonomists’ strifes about what should or not be grouped in the same species. This is unfortunately not the case for other taxonomic levels, especially the genus that do not currently have a definition as consensual, with as consequences technical and regulatory debates where purely scientific arguments are sometimes rare to find.

However, the same committee requested that alternative methods to RBR be proposed to define the “genomic” species. In addition, the committee strongly encourages research to find a biological basis for the bacterial species.

An Empirical but Not Arbitrary Definition

The canonical aspect of the 70 % RBR value is disturbing. In today’s world of biology where Darwinian-derived concepts constitute a paradigm, the genomics definition of bacterial species has undeniably creationist’s undertones. It seems that the RBR allows to capture the “essence” of the species and therefore suggests that such an “essence” exists. This Aristotelian view is reinforced by the declared justification of the polyphasic approach, in which different methodologies are used in conjunction with RBR to associate to it various phenotypic markers. This approach would give more highlights to this “essence” and yield more “shadows” on the wall of “Plato’s cave.”

In addition, setting a threshold at 70 % on RBR may seem arbitrary. In fact, this threshold was empirically obtained by comparing a large amount of RBR values. What Grimont (Grimont 1988) and others have shown is that there was no continuum in the distribution of RBR values between 100 and 0 %, but that there was a discontinuity around the value of 70 %. In addition, pairs of strains with RBR values above that threshold (Box Fig. 6.1) had all been previously classified in the same “species” on the basis of their biological similarities, using methods of numerical taxonomy applied to a large number of morpho-biochemical characters.

Box Fig. 6.1

Frequency distribution of reassociations measures between bacterial DNAs considered pairwise (RBR). There is a clear break in the distribution of values around 70% (arrow). This break may be observed with other methods for measuring the similarity or dissimilarity, with genetic or genomic sequences at 5°C for ΔTm, at 4–5 % of nucleotide divergence in MLSA, at 11 % of average genomic divergence (CGM) in AFLP, and at 93–94 nucleotide identity (ANI) between genomes. The left part of the curve corresponds to measurements involving pairs of strains belonging to the same species as they can be identified by morpho-biochemical criteria (Modified and redrawn from Grimont 1988)

Finally, such a gap in the distribution of RBR obtained initially with Enterobacteriaceae has also been found with similar values in many if not in all bacterial taxa. This explains the operational success of this definition. One must recall, however, that the 70 % value is not an absolute one, but that it can and should be adjusted according to taxa. By virtue of its robustness, it is likely that this definition will persist because it guarantees the stability of nomenclature. This is however a regulatory argument that may not be totally “scientific.” Therefore, the methodology for determining genomic species should be simplified to facilitate its implementation and understand the realities covered by such a definition.

Alternative Methodologies to RBR

Determining the RBR is quite tedious and requires equipment that is not available in all laboratories. Moreover, the method requires the availability of genomic DNA in large amounts for each strain analyzed and yields values for only pairs of strains. The method is not easily “portable” (that is to say used easily and without adaptation) from one laboratory to another and hardly lends itself to the analysis of many isolates as it is necessary in population genetics. Validated alternative methodologies are based on the molecular analysis of individual genomes. We can mention in that respect the amplified fragment length polymorphism or AFLP methodology, which proceeds by random sampling of different portions of the genome. AFLP allows to estimate the average mismatch rate or current genome mispairing (CGM). As it is perfectly correlated with RBR, it helps delineate genomic species with correspondence between the 11 % CGM threshold and the 70 % RBR threshold (Mougel et al. 2002). This method allows the phylogenomic analysis of numerous isolates and thus lends itself to the study of populations. It is now challenged by phylogenetic analysis of several housekeeping genes known as the multi-locus sequence analysis or MLSA. MLSA, which is completely portable, appears as the method of choice to determine the bacterial species. A value of 70 % RBR roughly corresponds to nucleotide mismatch rates on the order of 5 % in housekeeping genes. This is exactly what the ΔTm measures, with a drop of 1 °C per % genomics mismatch.

However, it is the significance of phylogenetic groupings measured, for instance, by the iterative “bootstrap” resampling method – and not an absolute threshold value – that must be used to delimit species in MLSA (Gevers et al. 2005). Other criteria, such as the average nucleotide identity or ANI (Goris et al. 2007) or other methods also based on the alignments of sequenced genomes, reveal not only discontinuities between species but also between genera. Because of easy access to complete genome sequencing, it can be expected that in the future, depositing the genomic reference sequence will be a prerequisite to the definition of new bacterial species.

As shown in Box Fig. 6.1, all these methods show that there is a discontinuity in the distribution of divergence measures (AFLP, MLSA, ΔTm) – or symmetrically of the similarity (RBR, ANI) – between genomes. This determination is empirical. What may be arbitrary is the choice of the degree of genomic difference to define entities called “species” about which one may wonder how they compare or not to eukaryotic species.

Speciation of Genomic Species Is “Fixed” in the Past

It must be understood that this definition implies that genomic species are bacterial lineages that have differentiated and have been isolated long enough that their genomic differences have led to the thresholds described above. An immediate consequence of this is that the events that led to this divergence have occurred long ago: speciation of genomic species was fixed in the past. In addition, since the infraspecific divergence is of the same order of magnitude for all species, speciation would have occurred roughly at the same time in various lineages! However, the phenomenon of speciation occurred and still occurs now, as is the case for many bacterial pathogens such as Yersinia pestis, Mycobacterium tuberculosis, or Bacillus anthracis that belong to wider “genomic” species. Since the genomic bacterial species definition does not integrate contemporary speciation, it is therefore irreconcilable with the BSC of eukaryotes.

Homologous Recombination and Sexual Isolation of Genomic Species

In eukaryotes, BSC is based on the sexual isolation of species. In prokaryotes, “sexuality” is both more promiscuous because it can involve very distantly related taxa (e.g., Firmicutes vs. Proteobacteria) and, very partial, because it concerns only a fraction of the genome. However, it has been suggested that the genetic divergence between species is such that it could result in a significant decrease in the frequency of homologous recombination (i.e., the mechanism by which foreign DNA is integrated into the bacterial genome) and thus lead to relative sexual isolation of species. Attempts have been made to explore this idea because it was attractive to be able to reconcile the two concepts. Briefly, it appears that in a model organism (the Agrobacterium tumefaciens species complex) and for a given marker gene, the decrease in the rate of homologous recombination was from 8 times, between very distant strains belonging to the same species, to 9 times, between strains belonging to different but closely related species (Costechareyre et al. 2009). This difference does not appear very significant, and homologous recombination is probably not sufficient to explain the genetic isolation of bacterial species. A sexual isolation, however, can have other causes. Studies of population genetics, for example, the work of Bailly et al. (2006), showed that sympatric species of Sinorhizobium are sufficiently isolated that gene exchanges occur significantly more between members of the same species than between species. The nature of the barrier, physiological, geographical, or otherwise, which leads to the sexual isolation of these species, is not known.

The Cause of Discontinuities Between Genomic Species

The existence of genomic groups is the result of forces that have swept or “purged” the diversity among these groups. Two types of models are available for these forces. One is based on selection. The other involves genetic drift with a predominant role of the founder effect.

Genomic Species Versus Ecological Species

For Cohan (2001), in the world of prokaryotes, each genomic group could correspond to an “ecotype” defined as a population of cells occupying the same ecological niche that are in intense competition with any adaptive mutant from this population. Well defined, the ecotypes share many properties attributed to eukaryotic species: the genetic diversity within ecotypes is limited by a cohesive force (here, the periodic selection), and the ecotypes are ecologically distinct. Therefore, ecotypes can be discovered and classified as genomic groups even when one remains ignorant of their ecology. This model is attractive because it is now possible to find the specific genome of a bacterial species and to then infer its specific ecological functions. This was done by Lassalle and collaborators (2011) who combined comparative genomics and reverse ecology to unmask the species-specific genes and then the species-specific ecological traits that differentiate Agrobacterium fabrum from its sister species within the A. tumefaciens complex. It is a great challenge for comparative genomic programs to try to find these features in the variable part of the genome. Conversely, the conserved genome part (the “core genome”) has likely little effect on these specificities even if it bears the phylogenetic signatures used to characterize the species by MLSA. However, again for reasons of stability of the taxonomy, it may seem dangerous to raise each prokaryotic group, even a single clone, with proven ecological specialization and thus an “ecotype,” to the status of a good and valid species. In connection with this model, the idea circulates that there may be a nesting of ecological specialization levels corresponding to nested taxonomic levels. Thus, genomic species could be ecological species even if ecotypes can also differentiate in these species (Box Fig. 6.2).

Box Fig. 6.2

Stable ecotype concept. One bacterial strain differentiates into two sublines which differ in certain aspects of their ecology. Selection periodically sweeps virtually all diversity occurred since the last episode of selection. As the two populations are ecologically distinct – that is, ecotypes – the periodic selection in a line does not influence the diversity of the other line. Ecotypes can then diverge to form separate species. In this model, the transfer of genes carrying genetic innovations plays an important role in adaptation to new ecological niches (Modified and redrawn from Cohan 2001)

Metapopulations, Founder Effect, and Genetic Drift

The force that holds together the genomic cohesion of bacterial populations does not necessarily result from episodes of intense selection (Fraser et al. 2009). In models where populations go through bottlenecks that significantly reduce their effective size, the diversity is affected through genetic drift. In the island metapopulation model (“islands” in the sense of exploitable resources), islands of various sizes can be colonized by single founder genotypes coming at random from other islands. Strains may differentiate in an island, and one of these new genotypes can colonize at random another island. If some islands become unable to support colonization, they lose their inhabitants and there follows a purge of diversity. This leads to a population differentiation between islands without selection of genotypes but just resulting from successive colonizations. Such a model could very well apply to microorganisms in the soil that undergo intense explosion of populations – for example, in contact with roots – followed by drastic reduction in disconnected soil microhabitats. It remains to be seen whether the model applies to all taxonomic levels such as from strains to species.

Conclusion

The “genomic” definition of Eubacteria or Archaea species is efficient and confers stability to the taxonomic nomenclature. This is, however, a fixed version of the species concept that does not fit with the contemporary evolutionary concepts of speciation. Nevertheless, it may well be that it is within the “genomic species” that the majority of genetic exchanges would occur and thus that ecological innovations would be shared. Genomic species could well be “ecological species” adapted to specific ecological niches, at least at the time of speciation. However, it is also possible that genetic drift has played a major role in the individualization of genomic species with the result that their differentiations are purely contingent to the vagaries of the history of each species. The advantage of prokaryotes is that these alternatives are testable via comparative analysis of their genomes. In practice, it is possible to find the genes and functions that determine the specific niche of each species.

In addition to its use for the definition of species in prokaryotes, the 16S rDNA has also established itself as a reference via molecular phylogeny for the delimitation of higher taxonomic levels. However, the gene* for 16S ribosomal RNA is not the “yardstick” of bacterial taxonomy. It is sometimes too conserved to be able to distinguish between some close species such as in the case of the Bacillus cereus species complex*. In addition, the genomes* of prokaryotes can contain multiple copies of this gene, reaching 10 (Bacillus subtilis) or even 15 copies (Rainey et al. 1996).

While in most cases, within a genome, the multiple copies of genes coding for 16S rRNA are identical or very similar, due to gene conversion (i.e., homologous recombination events leading to a homogenization of copies of a same gene in a genome), there are also taxa which are found to have markedly different copies. For example, in Escherichia coli, one of the seven copies present in the genome has up to 1 % difference (15 different bases/1,500 nt; Cilia et al. 1996), or in Thermomonospora, one of the six copies present in the genome has 10 % difference (Yap et al. 1999), showing thus the difficulty for phylogenetic studies depending on the selected 16S rRNA gene copy.

Finally, the use of a single marker*, for the identification of new microorganisms or to study their position in a phylogeny, can lead to errors when there are gene transfers (cf. Sect. 12.2; Daubin et al. 2001) or when mutations in different lineages are convergent. This is why other genes are frequently used in addition to 16S rRNA. For example, for the identification of bacterial strains, additional markers are considered as the 23S ribosomal RNA gene, which is longer and more variable than 16S, allowing comparisons at finer scale. But a number of genes also known as housekeeping genes* like gyrB, rpoB, etc., are more and more used through approaches called multi-locus sequence typing or MLST*.

Despite the problems mentioned above, the use of the 16S rRNA gene has many advantages, the first of these being related to its abundance in public sequence databases. For example, the Ribosomal Database Project II entirely dedicated to rRNA contains 2,765,278 16S rRNA sequences aligned and annotated (release 10, update 32, May 14, 2013, Fig. 6.1). This abundance of 16S rRNA sequences is due to the fact that this molecule was used early as a reference marker. Indeed, the 16S rRNA genes have a number of advantages such as (1) ubiquity, that is, the presence in all living beings without exception; (2) the stability of the function of the gene product; (3) a low rate of mutation (allowing comparison across the living world) and making possible to design primers called universal, which allow amplification of almost all-known rRNA genes¹; (4) a sufficient length; and (5) a low frequency of horizontal transfer.

Fig. 6.1

Increase in the number of available 16S rRNA sequences. Graph showing the increasing number of 16S rRNA sequences in the databases, over the past 20 years (data extracted from the Ribosomal Database Project II http://rdp.cme.msu.edu/index.jsp)

2 Obtaining a Prokaryotic Strain: Strains Collection

Classical microbiology is based on obtaining prokaryotes strains* or isolates, that is to say on prokaryote cultures that may be kept for years, exchanged between laboratories, and compared with other isolates. However, isolates evolve over time so that subcultures can sometimes undergo major changes such as loss of plasmids, genomic recombination events, invasion by insertion elements (Polzin and McKay 1991), or have had point mutations with major phenotypic consequences, etc. It is therefore important to define the approach that will permit conservation of isolates as stable as possible. After being characterized, an isolate can become a reference strain which must be deposited in a reference collection*, for example, ATCC in the USA (www.atcc.org), the DSMZ in Germany (www.dsmz.de), the NCIMB in England (http://www.ncimb.com/), the JCM in Japan (http://www.jcm.riken.go.jp/), or the collection of the Institute Pasteur in France (www.crbip.pasteur.fr). The mission of these reference collections is to maintain the different isolates and make them accessible to the entire international community of researchers in microbiology. Microorganisms are maintained as pure strains coded and referenced, maintained under freezing conditions (at −80 ° C or in liquid nitrogen), or freeze-dried.

If strain cultures are well preserved, microorganisms can be “revived” and subcultured when requested to be studied or used. Sometimes, however, strains with unusual physiological requirements may get lost, which is why it is so important that international collections of microorganisms should share their strains, and reference strains should be deposited in several international collections.

3 Characterization of a Prokaryotic Strain (Minimum Standards)

Strains of prokaryotes are characterized using two types of criteria: phenotypic and genotypic criteria. The “minimum standards” are the minimum phenotypic and genotypic characters requested by the International Committee on Systematics of Prokaryotes (ICSP) which guarantees the description of species, their nomenclature, and their taxonomic position. The International Committee on Systematics of Prokaryotes brings together researchers in microbiology from different countries chosen for their expertise in systematics of defined bacterial groups. The committee is assisted by subcommittees corresponding to different bacterial groups, for example, the subcommittee on photosynthetic bacteria, the subcommittee for sulfate-reducing bacteria, the subcommittee for Gram-positive bacteria, the subcommittee for thermophilic archaea, etc.

3.1 Phenotypic Criteria

These correspond to phenotypic traits expressed by microorganisms maintained in pure cultures. They must be carefully chosen to distinguish clearly between microorganisms (Table 6.1). They must be easy to determine and provide reproducible and reliable results. For a long time, these phenotypic criteria were limited to morphological characters: shape and size of the cells which constitute the pure strains, types of cell aggregates, response to Gram staining, presence of flagella, spore formation, the presence of capsules, cell particularities, etc. If morphological criteria have been used extensively (and successfully) for the classification of eukaryotes, the low morphological diversity of prokaryotes (cocci, rods, spirilla, cf. Sect. 3.1.1) did not allow developing a system for precise classification. However, this low morphological diversity is compensated by the high prokaryotic metabolic diversity especially in energy metabolism (cf. Sect. 3.3).

Table 6.1 Phenotypic criteria commonly used in taxonomy: classic minimum standards required by the International Committee for Bacterial Systematics

In prokaryotic microorganisms, the metabolic diversity, whether relating to catabolism (energy metabolism: donors and electron acceptors, respiratory, fermentative or photosynthetic types) or anabolism (sources of carbon, nitrogen, sulfur, etc.), has been used to characterize strains of prokaryotes relatively accurately. Many additional criteria were used in some cases to refine the characterization and allow a better classification. These are structural criteria (membrane lipids, coenzymes of the respiratory chains, etc.), ecophysiological criteria (adaptations to environmental conditions: pH optima, temperature, salinity, etc.), and antigenic criteria and resistance (presence of certain antigens, pathogenesis, antibiotic resistance, etc.) (Imhoff and Caumette 2004).

It is obvious that phenotypic properties have special meaning: not only do they allow classifying prokaryotes (called artificial classification), but they also tell something about the capabilities of microorganisms. They are therefore essential to understand the role of microorganisms in the environment where they live. For example, with respect to pathogenic bacteria, it is essential to know their metabolic and pathogenic features in order to develop sanitary applications and treatment of infectious diseases.

To identify a prokaryote, a taxonomist typically performs a series of phenotypic assays, some general, others specific to the type of microorganism studied. Identification keys can then be used. These dichotomous keys are constructed from the most general characteristics (cell shape, Gram stain, motility, etc.) to more specific characters (using specific substrates, the presence of antigens, etc.). The three major weaknesses of these methods are as follows:

• 1. The criteria tested may vary according to culture conditions.

• 2. A greater weight is given to taxonomic criteria used in the first part of the dichotomous key.

• 3. They allow the identification of organisms, but not the development of an evolutionary classification.

An attempt to resolve the first bias lies in the development of standardized methods for the determination provided by a single manufacturer and therefore still using the same components and the same culture conditions. This standardization of methods has to overcome the possible variations in the criteria used from one laboratory to another. Two methods are commonly used today:

• 1. The “API system” method using plastic plates comprising wells in which the substrates are in a lyophilized form to be assayed, the result is very often based on the change in pH during use of the substrate.

• 2. The “BIOLOG” method using microplates with wells in which the substrates tested can also be found in lyophilized form with a developer of redox potential, thereby indicating the respiratory activity of the microorganism following the use of the substrate.

These micromethods (API and BIOLOG) are mainly used in medical laboratories or hygiene control for rapid identification of pathogenic bacteria. However, additional tests (search for antigens, antibiotic resistance, etc.) are often needed to better characterize and confirm the taxon tested. These methods have been developed for specific groups of prokaryotes, including bacterial pathogens present in hospital environments (enterobacteria, clostridia, staphylococci, streptococci, etc.). They are thus not appropriate for all groups, including the prokaryotes grown from environments (sulfur-oxidizing or sulfate-reducing bacteria, methanogenic archaea, photosynthetic bacteria, etc.) which often have metabolic characteristics not covered by these tests.

3.2 Genetic Criteria

Generally for isolates, it is possible to match phenotypic criteria with genotypic criteria such as the percentage of bases G and C in the genomic DNA (% G + C), the gene sequence of the 16S ribosomal RNA (16S rDNA), MLST data, and DNA/DNA hybridization of genomic DNA between strains. These criteria are minimum standards required in the International Committee on Systematics of Prokaryotes. In the absence of phenotypic criteria distinguishing a group of isolates, it is possible to speak of genomic species*, that is to say defined solely from molecular criteria. The genomic species will be unnamed, but listed.

3.2.1 The Percentage of G and C Bases in Genomes

The G + C content can be characterized by genomic DNA hydrolysis and chromatography or by measuring the melting temperature for denaturing DNA. Despite this criterion being not discriminating enough, it is still requested by scientific journals. This technique is based on the fact that the ATGC bases of DNA allow pairing of the two DNA strands with two hydrogen bonds between the A and T bases (T = A) and three hydrogen bonds between the G and C bases (G ≡ C). The calculation of G + C% is

$$ \mathrm{G}+\mathrm{C}\%=\frac{\mathrm{G}+\mathrm{C}\kern0.5em \mathrm{mol}\times 100}{\mathrm{Mol}\left(\left(\mathrm{G}+\mathrm{C}\right)+\left(\mathrm{A}+\mathrm{T}\right)\right)}. $$

Due to the wide range of G + C percentages in genomic DNA in prokaryotes (13–75 %), this criterion has been proposed to differentiate groups of prokaryotes, assuming that bacterial strains or archaea belonging to the same species should have G + C percentages very similar if not identical (Fig. 6.2). Similarly, the variability within a genus was supposed to be very low. However, it appeared that the use of this criterion, although always required, allows differentiating neither species, genera, nor phyla (Fig. 6.3). Indeed, some important variations exist between organisms of the same genus. For example, within the genus Mycobacterium, the G + C% ranges from 57.8 % (Mycobacterium leprae TN) to 69.3 % (Mycobacterium avium K-10), or within the genus Mycoplasma rate varies from G + C 23.8 % (Mycoplasma capricolum ATCC 27343) to 40 % (Mycoplasma pneumoniae M129). The same applies to taxonomic levels of higher rank, such as phyla*, for which G + C variations are too large to be used (Fig. 6.3). Other genetic criteria have therefore been subsequently proposed (DNA/DNA hybridization, 16S rRNA sequencing, etc.), whereas the G + C% is now mainly used to identify gene regions exchanged laterally in sequenced genomes (cf. Sect. 12.2).

Fig. 6.2

Composition range in DNA bases (G + C%)

Fig. 6.3

Range of composition of major taxonomic groups in prokaryotic DNA base (G + C%)

3.2.2 The DNA/DNA Hybridization of Genomes

The gold standard for the identification of species is the DNA/DNA hybridization (Fig. 6.4). It is required by taxonomic flagship journals (e.g., International Journal of Systematic and Evolutionary Microbiology). This approach provides a 70 % threshold of DNA hybridized to define the membership of two strains of the same species. The two main problems of this approach are the need for culturing the strain in order to extract its DNA (thus excluding non-cultivated strains) and non-archivability, which therefore requires the cultivation of all the type strains of species with which a new strain should be compared.

Fig. 6.4

Method of DNA/DNA hybridization; schematic and principle of the method. The DNA/DNA hybridization is done between the DNA of two organisms, 1 and 2. The DNA of one of the microorganisms is labeled with radioactive P³² phosphate. The DNA is then denatured, separated into single-stranded fragments. The denatured DNA of both organisms are then mixed by adding large excess in the unmarked DNA of organism 2, so as to limit the autohybridation between the labeled DNA of an organism. After hybridization (reconstitution of DNA double-stranded hybrids between the two microorganisms’ DNA) and removal of remaining single strands by enzymatic digestion, the radioactivity of the double-stranded DNA hybrid is measured and compared to a control where 100 % of the labeled denatured DNA a microorganism are hybridized with each other. The radioactivity value obtained in the hybridization sample compared to that of the control 100 %, and thus gives the percentage hybridization between the two microorganisms

3.2.3 The 16S rRNA Gene

The sequence of the gene coding for 16S ribosomal RNA is now provided by almost all the authors describing a new species. To be informative, it must be of good quality (less than 1 % of undetermined bases) and have a length of at least 1,000 nucleotides out of the 1,500 that make up the gene (cf. Sect. 17.7.4). The phylogenetic analysis of 16S rRNA sequences allows specifying the relationship of the studied microorganism compared to other microorganisms whose 16S rRNA sequences are known (see below). An advantage of this approach is that the sequencing of the gene coding for 16S rRNA can be done not only for isolates but also for complex communities (Stackebrandt et al. 1993) or in organs infected by a microorganism. Thus, the microorganisms are characterized by their 16S rRNA sequences and compared to other microorganisms. If the studied microorganism is not isolated, the name Candidatus* must be used (Murray and Stackebrandt 1995).

The sequences of other genes are sometimes used in order not to depend on the identification of a single marker. This approach called MLST was developed following the observation that the phylogenies of different genes are not always consistent with that of 16S rRNA. Indeed, the amount of information present in the 16S rRNA is sometimes not sufficient to reliably position strains. The use of alternative phylogenetic markers is therefore important to refine the phylogenetic position of studied microorganisms based on 16S rRNA gene alone. In addition, because of frequent genetic exchanges among prokaryotes, the use of several alternative markers is recommended. With the increase in sequencing capacity, it is quite possible that in a few years, the complete genome sequence will be required for the identification of an isolate.

4 Dendrograms and Phylogenetic Trees

4.1 Phenotypic Dendrograms, Numerical Taxonomy

Numerical taxonomy is associated with artificial classifications, as opposed to phylogenies that are associated with natural classifications (see below). Historically, numerical taxonomy appeared before molecular phylogenies, and today it can be used in addition to phylogenetic analyses for characterization at the species level. These dendrograms should not be confused with phylogenetic trees. The former represent phenotypic similarities, whereas the latter represent relations of kinship.

The principle of numerical taxonomy relies on comparisons of numerous phenotypic features for a set of strains in order to group them according to their degree of phenotypic similarity. Usually, more than 50 independent characters are used, but in some cases, a higher number may be required (e.g., up to 150 characters for the study of aerobic heterotrophic bacteria). Phenotypic characteristics tested must be independent, namely, overlapping characters must be avoided. The features of each strain are translated into “positive” or “negative” in a binary manner and gathered into a character matrix (Fig. 6.5a). The similarity between strains is subsequently quantified by a similarity coefficient S_(AB), such as that of Jaccard, through pairwise comparisons of the strains. The S_(AB) coefficient between two strains (Jaccard coefficient) is defined as follows:

$$ {\mathrm{S}}_{\mathrm{j}\left(\mathrm{AB}\right)}=\mathrm{a}/\left(\mathrm{a}+\mathrm{b}+\mathrm{c}\right) $$

In this formula, a represents the number of “positive” characters shared by the two trains, b corresponds to the number of “positive” features for strain A that are “negative” in strain B, whereas c is the number of “negative” characters in strain A that are “positive” in strain B. The resulting coefficient is expressed as a percentage of similarity between two strains. It is important to note that the Jaccard coefficient considers only characters with a positive result in one or other of the two strains compared. There are other indices such as the Sokal and Michener index that take into account the sharing of negative characters. A S_(AB) of 70 % is expected at the species level. The calculation of similarity coefficients among all pairs of strains provides a similarity matrix (Fig. 6.5b) that can be plotted as a dendrogram where the most similar strains are placed close together (Fig. 6.5c).

Fig. 6.5

(a) Matrix of characters used to classify 25 strains of sulfur-oxidizing photosynthetic bacteria isolated from three different lagoon environments. 1 positive test, 0 negative test. (b) Distance matrix derived from the matrix of S_(AB) (1-S_(AB)). The S_(AB) was calculated using the Jaccard coefficient. (c) Construction of the dendrogram from the distance matrix (b). (Chr) Chromatium, (Tca) Thiocapsa, (Tcs) Thiocystis, (Rbi/Rps) Rhodobium/Rhodopseudomonas, (Rdv/Rba) Rhodovulum/Rhodobacter, (Rps) Rhodopseudomonas, (Prs) Prosthecochloris. In most cases, strains belonging to the same genus are grouped together. The scale represents 10 % difference between strains. The difference is calculated as the sum of all horizontal distances between two strains. This dendrogram is constructed iteratively creating couples with the shortest distance and then recalculating a new matrix with this couple. This approach is called UPGMA

4.2 Phylogenetic Trees

In addition to phenotypic characterization, the identification and the classification of new isolates relies more and more frequently on a phylogenetic analysis. Besides deciphering of relationships among taxa, this approach allows studying the biodiversity and classifying prokaryotes via molecular techniques that do not require the direct cultivation of the corresponding microorganisms (cf. Sect. 8.4.2). A phylogenetic analysis is used to determine kinship relationships between taxa (strains, species, genera, etc.) and therefore allows identifying the closest known relatives of the strain of interest. The reference marker used for this analysis is the gene coding for 16S rRNA. Identification based on this gene opens the question of the similarity threshold above in which it is assumed that two closely related isolates belong to the same species which is classically defined by DNA/DNA hybridization (Wayne et al. 1987). Stackebrandt and Goebel (1994) suggested that a threshold of 70 % of genomic DNA hybridization between strains of the same species corresponded to a percent identity of at least 97 % of their genes encoding 16S rRNA.

One has to understand the 97 % level as a threshold below which there is no need to make a DNA/DNA hybridization because the two strains belong to different species, but this does not mean that two strains harboring more than 97 % identity at the 16S rDNA level always belong to the same species. Therefore, in this latter case, DNA/DNA hybridization between the genomes of two strains is required (Fig. 6.6). The lack of equivalence agreed between the two techniques lies in the fact that 16S rDNA corresponds to one locus among the thousands of loci present in a genome. Therefore, it is recommended to use a multi-locus approach such as AFLP (amplified fragment length polymorphism) and correlate the results with data from DNA/DNA hybridization (Stackebrandt et al. 2002) (Fig. 6.7).

Fig. 6.6

Comparison of the DNA/DNA hybridization and the percentage of homology between 16S sequences. This meta-analysis yields a threshold of 97 % below in which it is considered that two bacterial strains are not likely to have a DNA/DNA hybridization greater than 70 % and thus belong to the same bacterial species. The converse is not true, because greater than 97 % similarity at the 16S rDNA sequences may correspond to a rate of DNA/DNA hybridization less than 70 % and thus to different species (redrawn from Stackebrandt and Goebel’s work (1994) with permission of the authors)

Fig. 6.7

AFLP method compared to DNA/DNA hybridization. According to work done on the genus Vibrio, valid also for many bacterial genera including Xanthomonas, Burkholderia, Rhizobium, and Bacillus (Modified from Thompson et al. (2004) with permission of the author and ASM)

Phylogenetic analysis of molecular markers* (e.g., 16S rRNA) is a multistep process (Fig. 6.8), the first of which is to compare the sequence of interest to investigate whether homologous sequences are present in sequence public databases (e.g., general databases such as the nr database at the NCBI or more specialized databases such as the RDP II or silva in the case of 16S rRNA). This initial research is based on sequence similarity comparisons among the sequences of the database and the studied sequence. Indeed, the closer related the two strains are, the closer the sequences of their genes (including 16S rRNA) will be. One of the most frequently used software to perform these searches is the BLAST (Basic Local Alignment Search Tool). It helps to identify similar regions between two protein or nucleic acid sequences (Fig. 6.8a). The identification of most similar sequences provides only a crude taxonomic indication (genus level or higher). To get a finer taxonomic affiliation, it is thus important to perform a phylogenetic analysis. For the phylogenetic analysis, all homologous sequences from organisms close to the one analyzed are extracted from the database and aligned using multiple alignment software such as Clustal omega or Muscle (Fig. 6.8b, c). The resulting alignments are trimmed in order to remove regions where homology between sites is ambiguous (i.e., poorly conserved regions containing insertions/deletions that cannot be unambiguously positioned). The remaining sites are used to infer the phylogenetic trees (Fig. 6.8d). One of the tree reconstruction methods commonly used in taxonomy is the neighbor-joining (Saitou and Nei 1987) because of its simplicity and speed. The neighbor-joining method belongs to the family of distance methods because the first step of neighbor-joining requires the quantification of evolutionary distances among all pairs of sequences studied. This estimate of the evolutionary distances is based upon the use of an evolutionary model. The calculation of evolutionary distances between each pair of sequences allows the establishment of a pairwise distance matrix (Fig. 6.8e). These distances are then represented as phylogenetic trees where branch lengths are proportional to evolutionary distances among sequences (Fig. 6.8f). One of the advantages of the neighbor-joining is the ability to process a large number of sequences in a very short time (few seconds to analyze hundreds of sequences). Its use is preferable to the UPGMA* that assumes that all sequences are evolving at the same rate (molecular clock hypothesis, Box 5.3; Ochman et al. 1999), but less satisfactory than the Maximum Likelihood* that is time-consuming.

Fig. 6.8

Key steps in phylogenetic analysis. (a) Similarity search for sequences homologous to the sequence of interest in the sequence databases, (b) extraction of counterparts from closely related species, (c) multiple alignment of sequences, (d) filtering the alignment so that only regions where homology between sites is reliable remain, (e) construction of a distance matrix, (f) reconstruction of the phylogenetic tree and identification of the closest known relatives (see explanations in the text), the distance scale for the phylogenetic tree represents 1 % of difference

5 Classification of Prokaryotic Microorganisms: Phylogeny Versus Phenotype

Classifications based on phenotypic criteria are more ancient than those based on molecular phylogeny. With regard to the prokaryotic microorganisms, many inconsistencies exist between phenotypic similarity levels and evolutionary relationships deduced from molecular phylogenies. Most taxonomic groups have been reconsidered through phylogenetic analysis in the 1980s, and a consensus was reached on the need for coherence between taxonomy and phylogeny. Reconciliation between the two schools of thought, similar in their basic goals but historically distinct, if it makes sense, is not easy. Thus, the multiple revisions of species, genera, or higher taxonomic levels pose many complex problems that are solved gradually. The difficulties are still present in some taxa where a tangle exists among species, leading to three possibilities all unsatisfactory: (1) accepting the existence of polymorphic taxa, with significant differences at the phenotypic level; (2) grouping all bodies taxon in a large catch-all using the rule of precedence; (3) subdividing heterogeneous taxa into smaller homogeneous subtaxa with the risk of having to cut into smaller and smaller units and thus increase beyond the reasonable the number of subgroups.

There is no solution that can be applied arbitrarily to all situations. For example, Shigella flexneri and Escherichia coli, two Gammaproteobacteria (family Enterobacteriaceae), exhibit a level of DNA/DNA hybridization above 70 %, which means that these two genera represent in fact the same species. However, there is a consensus to keep the two names because of the risks for human health due to their different pathogenicity characteristics. In this case, the phenotype has precedence over the genotype to maintain both species (and genera). However, the existence of pathogenic Escherichia coli strains may reduce the need to distinguish between the two genera and bring forward a name change in Escherichia and Shigella. Conversely, some broad and highly diversified genera such as Bradyrhizobium (Alphaproteobacteria), Bacillus (Firmicutes), or Clostridium (Firmicutes) gather groups of species which likely represent distinct genera. Finally, it should be remembered that any revision of the nomenclature associated with the reconsideration of some taxa raises the issue of names priority rules.

6 Systematic of Prokaryotic Microorganisms: Hierarchical Organization and Phylogenetics

Prokaryotic microorganisms correspond to two of the three domains* of life: Archaea and Bacteria (cf. Sect. 5.1.4, Fig. 5.9). Their systematics is described in the “Bergey’s Manual for Systematic Bacteriology, second edition” published in five volumes (Boone and Castenholz 2001; Brenner et al. 2005; de Vos et al. 2008; Krieg et al. 2011; Goodfellow et al. 2012). For more information, the reader may consult the website http://www.bacterio.cict.fr/.

In the text of this book, the Latin terms used are those accepted by the Nomenclature Committee as they appear in the second edition of the “Bergey’s Manual for Systematic Bacteriology” (Table 6.2).

Table 6.2 High taxonomic ranks of prokaryotic microorganisms proposed in Bergey’s Manual of Systematic Bacteriology

The use of phylogenetic criteria for establishing a natural classification of prokaryotes (work of Carl Woese and George Fox in the late 1970s) (Woese and Fox 1977; Fox et al. 1980) revealed that no or very few phenotypic features can be used to define the highest taxonomic levels. For example, initial analysis of the 16S rDNA showed that there were not one but two groups of prokaryotes not distinguishable on the basis of morphological criteria, but as distant from each other in terms of their genetic distance as they are from eukaryotes. Similarly, these studies showed that no phenotypic feature can be specifically associated to a given phylum. For example, the phylum Proteobacteria was initially called purple bacteria because of their purple pigmentation resulting from carotenoids associated with bacteriochlorophylls. In fact this phylum gathers a large number of lineages related to these photosynthetic pigments (hence, the purple pigmentation characteristic). Similarly, the so-called Gram-positive bacteria (testing positive for Gram stain) form two distinct bacterial phyla (i.e., the Firmicutes and Actinobacteria); this indicates that they are not specifically related to each other. In fact, only two phyla, the Spirochetes and the Cyanobacteria, were found to have been properly defined on the basis of phenotypic traits, meaning a spiral shape and the ability to achieve an oxygenic photosynthesis, respectively. This was a disappointment for those who believed that the use of molecular markers would allow discerning evolutionary traits associated with each phylum, which is not the case. An analogy can be made between the major phylogenetic groups and neighborhoods within large cities; knowing the large group to which a given microorganism belongs does not reveal much of its physiology and its characteristics, rather it may give some indications. An analogy can be made with the fact of belonging to a city neighborhood: it tells little about the personal characteristics of individuals who live there, and at most, it indicates membership in the area. This underlies the great plasticity of phenotypic and physiological features within high taxonomic groups.

In 1990, Woese and collaborators (1990) proposed a definition of each of the three domains of life:

• 1. The Eucarya from the Greek adjective “eu” meaning true and the Greek name “Karyon” signifying kernel include organisms with cells carrying genetic information in linear chromosomes enveloped by a nuclear membrane (core), containing eukaryotic-type ribosomes, and surrounded by a phospholipid bilayer (cf. Sect. 3.1.2) that consists of a glycerol core attached to two fatty acid molecules by ester bonds.

• 2. The Bacteria (from the Greek name “bakterios” meaning stick) denote cells with an prokaryotic-type organization (pro “Karyon” that does not have a true nucleus) surrounded by a phospholipids bilayer (cf. Sect. 3.1.1), consisting of a glycerol molecule attached to two molecules of fatty acid by ester bonds and which contain bacterial-type ribosomes.

• 3. The Archaea from the Greek adjective “arkeos” meaning ancient, primitive, group cells with archaeal-type ribosomes, having a prokaryotic-type organization, and surrounded by either a phospholipid bilayer mainly made of phospholipids consisting of a glycerol molecule attached to two molecules of isoprenoid by ether bonds or a phospholipid monolayer consisting of two glycerol molecules connected together by two isoprenoid chains (cf. Sect. 4.1.10, Fig. 4.5).

6.1 Domain Archaea

6.1.1 The Discovery of Archaea

The domain Archaea has been revealed by Carl R. Woese and George E. Fox who analyzed the oligonucleotide profiles derived from the digestion of the RNA component of small ribosomal subunits (16S for prokaryotes/18S for eukaryotes) (Woese 2007). Briefly, the 16S (or 18S) rRNAs of each organism are extracted and digested with different restriction enzymes (including the RNase T1). Digestion products are then subjected to bidirectional electrophoresis to build migration profiles (also called oligonucleotide catalogs) specific to each organism. The pairwise comparison of these catalogs (via Sokal and Michener index calculation) allows quantifying the similarities between rRNA profiles and thus studying and classifying the corresponding organisms. In their seminal study, Woese and Fox analyzed the rRNAs from the cytoplasm of various eukaryotes and prokaryotes (including methanogens), mitochondria and chloroplast. The results were surprising. As expected, the eukaryotes and prokaryotes rRNA sequences appeared very different from each other. However, a similar difference was observed between the prokaryotic 16S rRNA sequences, which form two distinct and distantly related groups. This means that at the genetic level, 16S rRNA sequences from two prokaryotes displaying strong phenotypic similarities can be more different than the 18S rRNA sequences from Homo sapiens and a plant or even than the 18S rRNA from Homo sapiens and the 16S rRNA from E. coli. These results have profoundly changed our view of the living world, by shifting from a eukaryote/prokaryote dichotomy to a tripartite divide. This divide was rapidly confirmed by subsequent phylogenetic analyses. At that time, the first group of prokaryotes gathered a wide variety of bacteria, while the second corresponded to methanogenic bacteria (i.e., carrying out the biosynthesis of methane). The former was named Eubacteria (eu = true) and the latter Archaebacteria (archaea = old) by Carl Woese. The name Archaebacteria reflects the widespread belief at the time that methanogenesis could have been one of the earliest metabolisms on Earth. Accordingly, present-day methanogens (and thus Archaebacteria) would have conserved this ancestral metabolism. Contradicting this hypothesis, Archaebacteria were rapidly enriched with new members, most of them being non-methanogens: the thermoacidophilic Thermoplasma that were formerly classified with Mycoplasma because like the latter they are devoid of cell wall; the Halobacteria which are extreme halophiles; the Sulfolobales, another group of thermoacidophiles; and various lineages of hyperthermophiles with optimal growth temperatures greater than 80 °C such as the Thermococcales.

A few years later, Carl Woese proposed to rename Eubacteria and Archaebacteria as Bacteria and Archaea, respectively, to remove the “bacteria” suffix which implies that Archaea are somehow bacteria and that both prokaryotic domains share a closer evolutionary link compared to Eucarya. In fact, the tree of life based on 16S/18S rRNA being unrooted (cf. Sect. 4.1.10), it is impossible to determine the relationships between the three domains.

6.1.2 Diversity of Archaea

While more than 10 bacterial phyla were described in the seminal works of Carl Woese, Archaea were divided into two phyla, Crenarchaeota and Euryarchaeota, corresponding to only three and nine cultivated orders (Woese 1987). Euryarchaeota (also referred to as euryotes or euryarchaeotes, from Greek “Euryos” meaning varied/diverse) bring together various lineages presenting very different lifestyle, such as hyperthermophilic or mesophilic methanogens (Methanococcales, Methanobacteriales, Methanocellales, Methanomicrobiales, Methanosarcinales, Methanopyrales, and the recently proposed “Methanoplasmatales”), extreme halophiles (Halobacteriales and the recently discovered “Nanohaloarchaea”), sulfate reducers (Archaeoglobales), thermoacidophiles (Thermoplasmatales), and some hyperthermophiles (Thermococcales). In contrast, the second phylum gathers exclusively thermophiles or hyperthermophiles. It was named Crenarchaeota (also referred to as crenotes or crenarchaeotes) from the Greek “Krenos” meaning source/origin because Carl Woese thought that the ancestor of Archaea was hyperthermophile, a feature retained by Crenarchaeota. For a long time, only two archaeal phyla were recognized, but new phyla (i.e., Korarchaeota, “Nanoarchaeota”, Thaumarchaeota, and more recently the Aigarchaeota) have been proposed in the last few years.

Most archaea and bacteria look alike. They are similar in size, shape, and cellular organization. However a few atypical cell morphologies are found in archaea as polygonal, triangular, or ultrathin square cells or very irregular cells. As bacteria, archaea exhibit a variety of phenotypes and physiologies. In fact, except for methanogenesis, all metabolisms described in archaea exist also in bacteria. Conversely no photosynthesis involving chlorophyll, spore production*, or pathogens have been reported in archaea so far. Archaea can be heterotrophic or autotrophic, can use various electron acceptors and electron donors, be aerobic, or anaerobic, etc. The main feature distinguishing bacteria from archaea is the nature of their cell envelope. In contrast to bacteria, the archaeal membranes contain lipids made of isoprene (and not fatty acid) chains, ether (and not ester) linkages, and l-glycerol (and not d-glycerol) moiety, in addition to the phosphate group. Moreover, archaeal envelope does not contain peptidoglycan or murein, resulting in insensitivity to the most common antibiotics targeting the bacterial cell wall (for a recent and complete review on the archaeal envelope, see Albers and Meyer 2011). Worth noting, the archaeal cell envelope is highly variable even among closely related lineages (Table 6.3), highlighting an unexpectedly very dynamic structure.

Table 6.3 Main features of the archaeal cell envelope for a subset of representative archaea

To the exception of Ignicoccus hospitalis (the host of Nanoarchaeum equitans), which harbors two membranes, the archaeal cell envelope is composed of a single membrane. In nearly all archaea characterized to date, this membrane is surrounded by a proteinaceous protein layer called the S-layer, which form a crystalline array (Table 6.3). S-layers are also found in bacteria. S-layer contributes to the shape, osmoprotection, and permeability of the cell. In most cases, the S-layer is composed of a single protein (or glycoprotein) aligned in lattices with oblique (p1 or p2), tetragonal (p4), or hexagonal (p3 or p6) symmetry. S-layer proteins have sizes ranging from 40 to 200 kDa and are also found in many bacteria. The composition and structure of the S-layer varies among archaea. While the hexagonal symmetry is predominant in archaeal S-layers, oblique or tetragonal lattices exist (Albers and Meyer 2011). In addition to S-layer, very atypical and stable proteinaceous structures have been reported in two unrelated species, namely, Methanospirillum hungatei (Methanomicrobiales) and Methanosaeta concilii (Methanosarcinales) (Table 6.3). It consists in tubular sheaths enclosing linear chains of cells, the individual cells within the chains being themselves surrounded either by an S-layer (M. hungatei) or by an amorphous granular protein layer (M. concilli) (Albers and Meyer 2011).

In many archaea, the S-layer is the only component of the cell wall, whereas in others various additional components can be found in the cell envelope. For instance, some methanogens (e.g., Methanobacteriales or Methanopyrales) harbor a cell wall composed of pseudopeptidoglycan (also called pseudomurein), which superficially resemble bacterial peptidoglycan. The comparison of the proteins involved in the biosynthesis of archaeal pseudomurein and bacterial murein showed no homology, suggesting that the two pathways emerged twice independently during evolution. A fibrillar polymer, called methanochondroitin, is found in the cell wall of members of the Methanosarcina genus when they form aggregates, but not in single cells. Strikingly a very similar polymer, the chondroitin, is part of the connective tissue matrix of vertebrates (Albers and Meyer 2011). Other polymers can be encountered in the cell envelope of some extreme halophiles belonging to Halobacteriales such as glutaminylglycan (Natronococcus occultus), highly sulfated heteropolysaccharides (Halococcus morrhuae), or halomucin (Haloquadratum genus) (Table 6.3). Worth noting, halomucin is the largest archaeal protein (9,159 amino acids) known to date. Its amino acid composition and domain organization are similar to mammalian mucin, which acts as a shield against dehydration of various tissues, such as the bronchial epithelium and the eyes (Albers and Meyer 2011). Finally, a few archaea belonging to Thermoplasmatales harbor highly pleomorphic shapes, due to the lack of any cell wall (Table 6.3).

At the genetic level, Archaea are a mosaic. Their informational systems (i.e., systems involved in the transmission and expression of genetic information, namely, the machineries of transcription, translation, replication, and repair) are similar to eukaryotes (cf. Sect. 4.1.10), whereas their housekeeping and metabolic genes and their general cell organization are similar to bacteria. Since their discovery, Archaea have led to some of the most exciting discoveries in the field of biochemistry and biotechnologies, but archaeal genetics has been slow to get off the ground, until recently. In fact, the last past years have witnessed spectacular progress, and genetic tools and models are now available for the two major archaeal phyla, the Euryarchaeota (i.e., Halobacteriales, Methanococcales, Methanosarcinales, and Thermococcales) and the Crenarchaeota (i.e., Sulfolobales) (for more details on archaeal genetics, see the excellent review of Leigh et al. 2011). This will accelerate our understanding of the biology of Archaea.

Because the first described archaea inhabited some of the more inhospitable places on earth (from an anthropomorphic point of view), members of this domain have been considered for a long time as “exotic microbes” or “curiosities” by most microbiologists. Because archaea are the only living organisms able to grow optimally at temperatures above 100 °C, the dominance of archaea over bacteria in extremely hot environments was early recognized. In contrast, bacteria were considered as dominant over archaea in all other ecosystems. As a consequence, and to the exception of methanogens, the relevance of archaea in microbial ecosystems and in global biogeochemical cycles has been underestimated for years (Forterre et al. 2002; Gribaldo and Brochier-Armanet 2006). The situation has changed with the birth of molecular ecology at the end of the 1980s. The investigation of microbial ecosystems with molecular tools has uncovered the incredible genetic, physiological, and phenotypic diversity of Archaea (Schleper et al. 2005; Lopez-Garcia and Moreira 2008). A large number of new lineages, such as groups called I, II, III, IV, SA1, SA2, ARMAN, ANME-1, ANME-2, “Nanohaloarchaea,” Miscellaneous Crenarchaeotal Group (MCG), etc., many of them representing likely high-level taxonomic groups were discovered. Importantly, these uncultured archaeal lineages could represent an important fraction of the biomass of some ecosystems (Narasingarao et al. 2011). Despite great advance in cultivation techniques, most of these lineages have resisted all cultivation attempts and remain poorly characterized. This underlines that our knowledge of Archaea based on cultured lineages is far from being representative of the real diversity of this domain. However, thanks to rapid progresses in DNA sequencing and metagenomics, complete genomes of representatives of some of these groups have been sequenced. Such culture-independent investigations will accelerate our understanding of these uncultured lineages and of Archaea in general.

6.1.3 Classification of Archaea

6.1.3.1 The Crenarchaeota

Based on 16S rRNA phylogenies, Crenarchaeota are divided into three orders (5 families and 22 genera) with cultured representatives: The Sulfolobales, the Desulfurococcales, and Thermoproteales, the first two being more closely related to each other (Fig. 6.9). Recently two additional orders have been proposed, namely, the Acidilobales and the Fervidicoccales, living in acidic hot springs. However, subsequent phylogenetic and genomic analyses suggested that they are rather Desulfurococcales (Brochier-Armanet et al. 2011). Crenarchaeota can be anaerobic, facultative anaerobic, or aerobic extreme thermophiles or hyperthermophiles. Their energetic metabolism is mainly based on sulfur, even if some of them are also able to use organic and other inorganic compounds or have lost the ability to use sulfur. Some of them are also acidophilic, being able to grow in pH ranging from 2 to 5.

Fig. 6.9

Phylogeny of Archaea. Phylogeny of Archaea based on a concatenation of ribosomal proteins present in archaea for which complete genome sequences are available. These proteins are a good alternative to 16S rRNA because they allow inferring better resolved (and less biased) phylogenies. They are now commonly used to infer reference phylogenies of microorganisms

6.1.3.1.1 The Sulfolobales

This order was proposed by Karl Stetter in 1989. Sulfolobales are extreme thermophilic or hyperthermophilic acidophiles thriving at temperatures ranging from 65 to 90 °C and at pH ranging from 1 to 5. Cells are regular to irregular cocci of about 1.0 up to 5 μm in diameter, occurring usually singly or in pairs. Most members of the Sulfolobales have been isolated from continental solfataric fields, from acidic hot soils, acidic hot springs, and smoldering slag heaps. In contrast, a few strains only have been isolated from submarine hydrothermal systems. This order contains a single family, the Sulfolobaceae divided into six genera: Sulfolobus, Acidianus, Metallosphaera, Stygiolobus, Sulfurisphaera, and Sulfurococcus. They can be aerobic, facultative anaerobic, or anaerobic. When growing autotrophically, they gain energy by oxidizing S⁰, S₂O₃ ²⁻, sulfidic ores, or H₂ and use CO₂ as a carbon source. In contrast, organotrophic growth occurs by aerobic respiration or anaerobic sulfur respiration or by fermentation of organic substrates. More precisely, Sulfolobus are obligate aerobes. Some of them can grow mixotrophically or heterotrophically by using complex organic compounds, sugar or amino acids, and oxidizing H₂ or S⁰; lithoautotrophically by oxidizing sulfidic ores (pyrite, sphalerite, and chalcopyrite) or S⁰; or chemolithotrophically to chemoheterotrophically by using complex organic compounds and amino acids and by oxidizing S⁰. Finally some of them are also able to oxidize Fe²⁺ to Fe³⁺. They are mainly found in terrestrial volcanic hot springs. Sulfolobus are very popular models for studies among others on informational processes (i.e., translation, transcription, replication, and repair), cell division, RNA processing, and metabolism and have been developed for genetic experiments (Leigh et al. 2011). A neighbor genus, Acidianus, harbors facultative aerobes that can be either obligate lithoautotrophs or facultative chemolithoautotrophs. Under aerobic conditions, they oxidize sulfur compounds as S⁰, sulfidic ores, H₂, and Fe²⁺, while under anaerobic conditions, they use H₂ as electron donor to reduce S⁰, leading to the formation of H₂S as metabolic end product (S⁰-H₂ autotrophy). Metallosphaera are aerobic facultative chemolithoautotrophs that oxidize sulfur compounds as S⁰, sulfidic ores, metal sulfides (FeS), H₂S, S₄O₆ ²⁻, H₂ and can also grow on complex organic substrates. In contrast, Stygiolobus are obligate chemolithoautotrophs growing under anaerobic conditions via the S⁰-H₂ autotrophy. Sulfurisphaera are facultative anaerobes that grow mixotrophically or heterotrophically through S⁰-H₂ autotrophy or the oxidation of complex organic compounds. Sulfurococcus are aerobic facultative chemolithoautotrophs which oxidize sulfidic ores or S⁰ and use complex organic compounds, various sugars, and amino acids. Some of them are also able to oxidize Fe²⁺ to Fe³⁺. For additional information on Sulfolobales, see Dworkin and collaborators (2006).

6.1.3.1.2 The Desulfurococcales

This order was defined by Harald Huber and Karl Stetter in 2001. Desulfurococcales cells are regular to irregular cocci (of about 0.5–15 μm), discs or dishes, which occur singly, in pairs, short chains, or aggregates. Some of them are flagellated. Most are neutrophilic or weakly acidophilic hyperthermophiles, living at temperatures ranging from 85 to 106 °C. Desulfurococcales occur mainly in hot marine environments, such as shallow marine sediments, springs, and venting waters (Aeropyrum, Ignicoccus, Staphylothermus, Stetteria, Thermodiscus, Pyrodictium, and Hyperthermus) or deep-sea hydrothermal systems and black smokers (Ignicoccus, Staphylothermus, Pyrodictium, and Pyrolobus). However, some representatives are also found in hot volcanic terrestrial ecosystems with low salinity and acidity to slightly alkaline pH values, such as hot springs, mud holes, and soils of continental solfataric fields (Desulfurococcus, Sulfophobococcus, Thermosphaera, and Acidilobus). Desulfurococcales gathers anaerobic, facultative anaerobic, or aerobic organisms. Under autotrophic conditions, they oxidize H₂ using S⁰, S₂O₃ ²⁻, NO₃ ⁻, or NO₂ ⁻ as electron acceptor and CO₂ as a carbon source. Alternatively, they are also able to grow organotrophically through aerobic respiration, anaerobic sulfur respiration, or fermentation of organic substrates. The most hyperthermophilic organisms known to date belong to Desulfurococcales, among which Pyrolobus fumarii has an optimal growth temperature of 106 °C. It is able to grow at 113 °C but not below 90 °C. In contrast, even if some bacterial spores (e.g., Morella, Firmicutes) are able to resist 121 °C, no bacterial cells are able to survive above 100 °C. Desulfurococcales are divided into two main families: the Pyrodictiaceae and the Desulfurococcaceae. Pyrodictiaceae form a rather coherent cluster containing the genera Geogemma (which contain the famous strain 121 which was claimed to grow at 121 °C), Hyperthermus, Pyrodictium, and Pyrolobus, all having optimal growth temperature ranging from 95 to 106 °C. Pyrodictium forms networks of hollow cannulae, in which the disk- or dish-shapes cells are embedded. They are marine obligate anaerobes that can be chemolithoautotrophs to mixotrophs able to use S⁰ or S₂O₃ ²⁻ and H₂ under autotrophy with grow on complex organic compounds, or obligate heterotrophs fermenting complex organic compounds or peptides. Hyperthermus are obligate marine anaerobic heterotrophic cocci that ferment peptides products leading to the formation of organic acids, butanol, and CO₂. Finally Pyrolobus are marine anaerobic or microaerobic, obligately chemolithoautotrophic irregular cocci. They reduce NO₃ ⁻, S₂O₃ ²⁻, or O₂ with H₂. In contrast, Desulfurococcaceae grow optimally at 85–95 °C and are much more diverse than Pyrodictiaceae. They encompass the genera Aeropyrum, Desulfurococcus, Ignicoccus (the host of Nanoarchaeum equitans), Ignisphaera, Staphylothermus, Stetteria, Sulfophobococcus, Thermodiscus, Thermogladius, and Thermosphaera. Desulfurococcus are anaerobic cocci isolated from continental environments. They grow mixotrophically or heterotrophically by respiring S⁰ or fermenting complex organic compounds, peptides, amino acids, starch, or glycogen. Aeropyrum are the only aerobic members of Desulfurococcaceae. These cocci are marine heterotrophs that respire complex organic compounds with O₂. Ignicoccus are marine anaerobic cocci which grow lithoautotrophically via S⁰-H₂ autotrophy and produce exclusively H₂S as the metabolic end product. Staphylothermus are anaerobic marine coccoid organisms that grow in aggregates and gain energy by fermentation of complex organic substrates in the presence of S⁰. Stetteria are coccoid marine anaerobic mixotrophs that respire S⁰ or S₂O₃ ²⁻ on complex organic compounds in presence of H₂. Sulfophobococcus are continental anaerobic obligate heterotrophic cocci in which growth is inhibited by S⁰. Thermodiscus are dish- or disk-shaped anaerobic marine obligate heterotrophs, which carry out S⁰ respiration and fermentation of complex organic compounds. Thermosphaera forms short chains or aggregates of cocci. They are anaerobic continental obligate heterotrophs which ferment complex organic compounds. Their growth is inhibited by elemental sulfur and also by H₂. Finally, Desulfurococcales include additional genera, namely, the Acidilobus, Caldisphaera, and Fervidicoccus. The former and the latter were recently proposed to represent new crenarchaeotal orders (Acidilobales and Fervidicoccales), whereas more recent analyses have confirmed that their membership of Desulfurococcales (see Brochier-Armanet et al. 2011), Acidilobus are acidophiles which grow optimally at pH close to 4 in terrestrial acidic hot springs. They are obligate heterotrophs growing via the fermentation of complex organic compounds. For additional information on Desulfurococcales, see Dworkin and collaborators (2006).

6.1.3.1.3 The Thermoproteales

This order was defined by Wolfram Zillig and collaborators in 1981. Thermoproteales are extreme thermophiles or hyperthermophiles with optimal growth temperatures ranging from 75 to 100 °C and from neutral to slightly acidic pH (from 3.7 to 7). Currently, the order is represented by two families: the Thermoproteaceae and the Thermofilaceae, which contain a single genus (Thermofilum) and five genera (Caldivirga, Pyrobaculum, Thermocladium, Thermoproteus, and Vulcanisaeta), respectively. The former are rods of at least 0.4 μm in diameter, whereas the latter are ultrathin filaments of only 0.15–0.35 μm in diameter and 1–100 μm in length. Some Thermoproteales bear spherical bodies (“golf clubs”) at their terminals (e.g., Vulcanisaeta) and are flagellated (Pyrobaculum) or not (Thermofilum). Thermofilaceae and some Thermoproteus are obligate heterotrophic anaerobes that use peptides and S⁰ as donor and accepter of electrons, respectively. Other Thermoproteus are facultative lithoautotrophic anaerobes that use H₂, simple or complex compounds as electron donors, and S⁰ or malate as electron acceptors. In contrast, most Pyrobaculum are anaerobes, but some are also aerobes. They can be either obligate heterotrophs or facultative lithoautotrophs. They can use a wide range of substrates as electron donors (e.g., H2, S₂O₃ ²⁻, and complex organic compounds) and electron acceptors (e.g., S⁰, S₂O₃ ²⁻, SO₃ ⁻, O₂, NO₃ ⁻, NO₂ ⁻, Fe³⁺, selenate, selenite, arsenate, l-cysteine, and oxidized glutathione). Caldivirga grows heterotrophically under anaerobic or microaerobic conditions at weakly acidic pH (3.7–4.2) by using complex organic compounds as electron donors and S⁰, S₂O₃ ²⁻, and SO₄ ²⁻ as electron acceptors. Thermocladium are obligate heterotrophs that use S⁰, S₂O₃ ², SO₄ ²⁻, and l-cystine as electron acceptor under anaerobic or microaerobic conditions. Finally, Vulcanisaeta grows optimally at pH 4.0–4.5 on proteinaceous substrates as carbon sources and use S⁰or S₂O₃ ²⁻as electron acceptor. To the exception of Pyrobaculum aerophilum, which was isolated from a marine hydrothermal system, all Thermoproteales have been obtained from terrestrial volcanic habitats (e.g., springs, water holes, mud holes, and soils of continental solfataric fields, etc.) with low salinity and acidity to neutral pH. In their ecosystem, Thermoproteales are important component of food webs. They can function as primary producers and/or as consumers of organic material. For additional information on Thermoproteales, see Dworkin and collaborators (2006).

6.1.3.2 The Euryarchaeota

Based on phylogenies of 16S rRNA, the Euryarchaeota have been divided initially into nine orders: Methanobacteriales, Methanococcales, Methanopyrales, Methanomicrobiales, Methanosarcinales, Halobacteriales, Thermoplasmatales, Thermococcales, and Archaeoglobales. Recently additional orders have been proposed: Methanocellales, Methanoplasmatales, etc. Euryarchaeota are much more diverse than Crenarchaeota. They present very different physiologies, live all types of environments: from temperate environments to the coldest or the hottest, from most alkaline to most acidic, and to most hypersaline ones. Large-scale phylogenetic analyses indicate that Thermococcales represent the earliest diverging lineage within Euryarchaeota; methanogens class I (Methanobacteriales, Methanococcales, Methanopyrales) and the large cluster containing Thermoplasmatales, “Methanoplasmatales,” DHEV2, and group II, occupy an intermediate position, whereas Archaeoglobales and the large group gathering Halobacteriales and methanogens class II (i.e., Methanosarcinales, Methanomicrobiales, and Methanocellales) branch apically in the Euryarchaeota tree (Fig. 6.9).

6.1.3.2.1 The Methanogens (Fig. 6.10a–d)

Fig. 6.10

Photomicrographs of methanogenic archaea and/or halophilic (a–d) bacteria. (a) Methanosarcina mazei, (b) Methanocalculus halophilus, (c) Methanobacterium bryantii, (d) Methanobacterium oryzae, (e) Thermotoga hypogea, (f) Spirochaeta smaragdinae. (a and c) Photographs: courtesy of Prof. Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany; (b, d, e) Photographs: courtesy of Dr Jean-Luc Cayol, University of the Méditerranée, Marseille; (f) Photography: courtesy of Marie-Laure Fardeau, University of the Méditerranée, Marseille. The bars represent 10 μm

Methanogens represent a large and highly diversified group of unrelated strictly anaerobic archaea, which produce large amount of methane (CH₄) as the major end product of their energy metabolism. Methanogens are key components of most anaerobic ecosystems due to their capacity to achieve the final step in the decomposition of organic matter. However, many methanogens are also autotrophs, being able to use CO₂ as sole source of carbon. Different types of reactions can lead to CH₄ production. The former ones are based on CO₂ reduction to CH₄. In this case, CO₂ is reduced by electrons provided by H₂ (hydrogenotrophic methanogens), formate, CO, or certain alcohols (e.g., 2-propanol, ethanol, etc.). Nearly all methanogens can use H₂ as electron donor, but many are also able to utilize formate, whereas very few use alcohols. The second type of reaction is based on methyl compounds. Indeed, some methanogens are able to use methyl-containing C-1 compounds (e.g., methanol, methylamine, dimethylamine, trimethylamine, dimethylsulfide, formate, etc.) as substrate for methanogenesis. The methyl groups of these molecules are used as electron acceptors and are reduced directly to CH₄. A small amount of these molecules are used as electron donor through oxidation processes leading to the formation of CO₂. Finally, some Methanosarcinales species can catabolize acetate molecules by reducing their methyl carbon to CH₄ and by oxidizing their carboxyl carbon to CO₂. CH4 production has been reported in non-methanogen organisms (e.g., aerobic bacteria, plants, mitochondria, etc.). However, this production results from side reactions of their normal metabolism and leads to very small amount of CH₄. Methanogens are currently divided into six orders, forming two distinct lineages. Methanogen class I gathers Methanobacteriales, Methanococcales, and Methanopyrales, whereas Methanomicrobia (i.e., methanogen class II) encompasses Methanocellales, Methanomicrobiales, and Methanosarcinales. However, the relationships among orders within each class are not resolved (Fig. 6.9). Most methanogens are mesophilic, although extremophiles living in hot (e.g., Methanocaldococcus and Methanopyrus), cold (e.g., Methanogenium frigidum), or hypersaline (e.g., Methanothermobacter, Methanohalobium) environments are known. Methanogens vary in shape (e.g., long or short rods, regular or irregular cocci, filaments, loops, etc.) and cell wall composition (see above). Methanogens have been developed as models to study archaeal informational processes, regulation, osmoregulation, protein structures, and microbial syntrophic associations (i.e., associations between organisms that facilitate the transfer of nutrients) (Leigh et al. 2011). Well-developed genetic tools are now available for Methanococcus and Methanosarcina (Leigh et al. 2011). It has been suggested that methanogenesis was ancestral in Archaea, meaning that the last common ancestor to all present-day archaea was a methanogen. However, recent analyzes suggest rather that this metabolism appeared secondarily during the diversification of Euryarchaeota, namely, in the last common ancestor of all present-day methanogens (Bapteste et al. 2005). This implies that methanogenesis would have been secondarily lost in their non-methanogen relatives (e.g., Archaeoglobales, Halobacteriales, “Nanohaloarchaea,” Thermoplasmatales and relatives, Fig. 6.9). Supporting this hypothesis, all the genes involved in methanogenesis are present in Archaeaoglobales, excepted those involved in the last step of this pathway, preventing the biosynthesis of CH₄ in these archaea (see below). A seventh order tentatively called “Methanoplasmatales,” which likely correspond to the RC-III (Rice cluster III), has been recently proposed (Paul et al. 2012). These archaea have been detected in various habitats including marine environments, and soils, but also in the intestinal tracts of termites and mammals. This lineage is closely related to Thermoplasmatales, marine group II, and DHVE groups.

The order Methanomicrobiales has been proposed by William E. Balch and Ralph S. Wolfe in 1981. Members of this order are strictly anaerobic. They present very diverse morphologies: short rods, curved rods, plates, irregular cocci, filaments, etc., ranging from 0.4 to 2.6 μm in diameter and from 0.1 to 1 in width × 1.5–10 μm in length. Some of them are flagellated. All Methanomicrobiales are able to use H₂ and CO₂ as a substrate for methanogenesis, many of them can utilize formate, and some can also use alcohols. In contrast to their close relatives Methanosarcinales, they can use neither acetate nor methylated C-1 compounds (such as methanol, methylamines, or methyl sulfides) for methanogenesis, even if acetate can be used as a carbon source by some species. In contrast to Methanobacteriales and Methanopyrales, Methanomicrobiales cell wall does not contain pseudomurein. Some of them (Methanospirillum hungatei) are surrounded by proteinaceous sheaths (Table 6.3). Most Methanobacteriales are mesophilic, but psychrophilic (e.g., Methanogenium frigidum) and thermophilic (e.g., Methanoculleus thermophilus) are known. They have been reported in various anaerobic habitats (e.g., marine and freshwater sediments, swamps, anaerobic digesters, rumens of various animals, oil fields, underground waters, etc.). Methanomicrobiales are generally free-living, but some species (e.g., Methanoplanus endosymbiosus) are endosymbionts of anaerobic H₂ producer protists, such as ciliates living in freshwater sediments (e.g., Metopus contortus). Methanomicrobiales has been divided into four families. Methanocorpusculaceae and Methanospirillaceae each contain a single genus Methanocorpusculum and Methanospirillum. Both genera use H₂ with CO₂ and formate for methanogenesis, whereas some representatives can also use 2-propanol (or 2-butanol) with CO₂. Methanoregulaceae group three genera (Methanolinea, Methanoregula, and Methanosphaerula), whereas Methanomicrobiaceae gather six genera (Methanoculleus, Methanofollis, Methanogenium, Methanolacinia, Methanomicrobium, and Methanoplanus). Members of both families use H₂ with CO₂, but some are also able to use formate for CH₄ production. In addition Methanomicrobiaceae can produce CH₄ from 2-propanol and CO₂, from 2-butanol and CO₂, or from cyclopentanol and CO₂. From an evolutionary point of view, Methanomicrobiales belong to class II methanogens and are closely related to Methanocellales and Methanosarcinales and to Halobacteriales (Fig. 6.9). For additional information on Methanomicrobiales, see Dworkin and collaborators (2006).

The order Methanosarcinales has been proposed by David R. Boone and collaborators in 2002. Members of this order are strictly anaerobic. They present very diverse morphologies: coccoid, flat, polygonal, irregular cocci, spheroids, rods, pseudosarcinae, or sheathed rods, etc., ranging in size from 0.5 to 100 μm in diameter and from 0.8 wide × 7 μm long. They can form filaments and aggregates (that can be massive). Some of them are flagellated. Like Methanomicrobiales, Methanosarcinales cell wall does not contain pseudomurein, and some are surrounded by a proteinaceous sheath (Methanosaeta concilii) or methanochondroitin (Methanosarcina mazei and Methanosarcina acetivorans, Table 6.3). Most of them are mesophiles, even if some species are thermophilic (e.g., Methanosaeta thermophila). They live at pH neutral to weakly alkaline. Some Methanosarcinales are also halotolerant and halophilic (e.g., Methanohalobium evestigatum). They are able to use numerous substrates for methanogenesis. Contrarily to Methanomicrobiales, Methanosarcinales can grow by splitting acetate to CH₄ and CO₂. They can also dismutate methyl compounds (methanol, methyl amines, methyl sulfides, etc.) producing CO₂ and CH₄, or use H₂ to reduce methyl compounds. They are important components of ecosystems due to their capacity to achieve the terminal steps of the degradation of organic matter in anoxic environments where light and terminal electron acceptors other than CO₂ are limiting. Representatives of the Methanosarcinales are widespread and are found in very diverse anaerobic environments (e.g., freshwater, ocean, muds, sediments (even in extremely halophilic ones), gas industry pipelines, underground waters, sludge from anaerobic sewages, rumen and gastrointestinal tracts of metazoa, deep terrestrial subsurface, etc.). This order comprises three families: Methanosaetaceae, Methanosarcinaceae, and Methermicoccaceae. Methanosaetaceae are represented by a single genus (Methanosaeta). They are able to use acetate as sole energy source leading to production of CH₄ and CO₂, and acetate is the sole energy substrate. In contrast, Methanosarcinaceae encompass nine genera (Halomethanococcus, Methanimicrococcus, Methanococcoides, Methanohalobium, Methanohalophilus, Methanolobus, Methanomethylovorans, Methanosalsum, and Methanosarcina (Fig. 6.10a)) of coccoidal or pseudosarcinal bacteria. All representatives of this family can dismutate methyl compounds. Some are able to reduce acetate or CO₂ with H₂ but none catabolize formate. Methermicoccaceae contain a single genus Methermicoccus of small, thermophilic cocci, able to use methanol, methylamine, and trimethylamine as substrates for methanogenesis. From an evolutionary point of view, Methanosarcinales belong to class II methanogens and are closely related to Methanomicrobiales and Methanocellales and to Halobacteriales (Fig. 6.9). For additional information on Methanosarcinales, see Dworkin and collaborators (2006).

The Methanocellales order has been proposed by Sanae Sakai and colleagues in 2008. It corresponds to the taxon formerly designated RC-I (Rice Cluster I). This order contains a single family Methanocellaceae and a single genus Methanocella. Most cells are rod-shaped and occur singly; however, coccoid shaped appear in late-exponential culture. Described strains to date are nonmotile. They produce CH₄ from H₂ and formate. Optimal growth occurs at 35–37 °C, at neutral pH. Based on 16S rRNA environmental survey, Methanocellales appear to be widely distributed, especially in rice paddies that are one of the major sources of CH₄ on Earth, contributing about 10–25 % of global CH₄ emission. From an evolutionary point of view, Methanocellales belong to class II methanogens and are closely related to Methanomicrobiales and Methanosarcinales and to Halobacteriales (Fig. 6.9).

The order Methanobacteriales has been proposed by William E. Balch and Ralph S. Wolfe in 1981. Methanobacteriales are strict anaerobic mesophilic, thermophilic, or hyperthermophilic microorganisms. They grow at temperature ranging from 15 to 97 °C. Optimal growth of most members of this order occurs at nearly neutral pH even if alkaliphilic (e.g., Methanobacterium alcaliphilum) or moderately acidophilic (e.g., Methanobacterium espanolense) strains have been characterized. They are found in anoxic habitats (e.g., freshwater and marine sediments, hot springs, groundwater, oil fields, peat bogs, rice paddies, terrestrial subsurface environments, anaerobic sewages, sludge, and gastrointestinal tracts of animals including humans, etc.). Methanobacteriales are generally free-living, but some species are endosymbionts of anaerobic H₂ producer protists, such as ciliates (e.g., Nyctotherus ovalis) thriving in the intestinal tracts of cockroaches, millipedes, and frogs. Cells are either rod-shaped or coccoid, often forming chains or aggregates. They can also form long filaments (up to 120 μm in length). Similarly to Methanopyrales but in contrast to other methanogens, the cell wall of Methanobacteriales contains pseudomurein. Most Methanobacteriales use H₂ as electron donor to reduce CO₂ leading to CH₄ formation. Some representatives of this order can also use formate, CO, or secondary alcohols as electron donors for CO₂ reduction, whereas members of a single genus (e.g., Methanosphaera) can form CH₄ by using H₂ to reduce methanol. Methanobacteriales are divided into two families: the Methanobacteriaceae and the Methanothermaceae. Methanobacteriaceae encompass rod-shaped mesophiles and thermophiles. Most of them are nonflagellated. This family is divided into four genera: Methanobacterium, Methanobrevibacter, Methanosphaera, and Methanothermobacter. They are H₂ oxidizers, although some species can also oxidize formate, CO, and/or secondary alcohols. Methanosphaera reduce methanol, whereas other Methanobacteriaceae use CO₂ as an electron acceptor. Methanobrevibacter smithii and Methanosphaera stadtmanae are two main methanogens found in human gut. The Methanothermaceae is represented by a single genus, Methanothermus, which grows by reducing CO₂ with H₂. Members of this family are hyperthermophilic, living at optimal temperature ranging from 80 to 85 °C, at pH 6.5. In contrast to Methanobacteriaceae, Methanothermaceae are flagellates. From an evolutionary point of view, Methanobacteriales belong to class I methanogens and are closely related to Methanococcales and Methanopyrales (Fig. 6.9). For additional information on Methanobacteriales, see Dworkin and collaborators (2006).

The order Methanopyrales has been proposed by Harald Huber and Karl Setter in 2001. It is represented by a single family Methanopyraceae and a single genus Methanopyrus, a single species Methanopyrus kandleri that was isolated from hydrothermally heated deep-sea sediment. Methanopyrus are the only methanogens known to date growing optimally at temperatures greater than 100 °C. Methanopyrus cells are rod-shaped and flagellated. Similarly to Methanobacteriales but in contrast to other methanogens, the cell wall of Methanopyrales contains pseudomurein. Methanopyrus kandleri uses H₂ as electron donor to reduce CO₂ leading to CH₄ formation. It is an obligate chemolithoautotroph that uses CO₂ as sole carbon source. From an evolutionary point of view, Methanopyrales belong to class I methanogens and are closely related to Methanococcales and Methanobacteriales (Fig. 6.9).

The order Methanococcales has been proposed by William E. Balch and Ralph S. Wolfe in 1981. Representatives of this order have been isolated from various anaerobic habitats (e.g., shores, estuary sediments, salt-marsh, coastal geothermally heated marine sediments, reservoir water, deep-sea hydrothermal vents, high-temperature oil reservoirs, etc.). Methanococcales have been divided into two families, each being represented by two genera. Methanocaldococcaceae gather Methanocaldococcus and Methanotorris, whereas Methanococcaceae encompass Methanococcus and Methanothermococcus. All Methanococcales produce CH₄ by using H₂ to reduce CO₂, but Methanococcaceae are also able to use formate are electron donors. Cells occur as irregular flagellated cocci ranging from 0.9 to 3 μm in diameter occurring singly or in pairs. This order gathers mesophiles (e.g., Methanococcus vannielii), thermophiles (e.g., Methanothermococcus thermolithotrophicus), and hyperthermophiles (e.g., Methanotorris igneus, Methanocaldococcus infernus) at weakly acidic (e.g., Methanotorris igneus), neutral (e.g., Methanothermococcus okinawensis), or weakly alkaliphilic pH. Most members of this order are fast growing and require salt for growth. From an evolutionary point of view, Methanococcales belong to class I methanogens and are closely related to Methanopyrales and Methanobacteriales (Fig. 6.9). For additional information on Methanococcales, see Dworkin and collaborators (2006).

6.1.3.2.2 The Halobacteriales

This order was proposed by William D. Grant and Helge Larsen in 1989. Halobacteriales live in environments containing high salt levels exceeding 150–200 g/l. Most Halobacteriales cannot grow at salt concentrations below 2.5–3 M and are irreversibly damaged (or even lyse) when suspended in solutions containing less than 1–2 M salt. It comprises a single family the Halobacteriaceae, which gather 42 genera: Haladaptatus, Halalkalicoccus, Halarchaeum, Haloarchaeobius, Haloarcula, Halobacterium, Halobaculum, Halobellus, Halobiforma, Halococcus, Haloferax, Halogeometricum, Halogranum, Halomarina, Halomicrobium, Halonotius, Halopelagius, Halopenitus, Halopiger, Haloplanus, Haloquadratum, Halorhabdus, Halorientalis, Halorubellus, Halorubrum, Halorussus, Halosarcina, Halosimplex, Halostagnicola, Haloterrigena, Halovivax, Natrialba, Natrinema, Natronoarchaeum, Natronobacterium, Natronococcus, Natronolimnobius, Natronomonas, Natronorubrum, Salarchaeum, and Salinarchaeum. Most representatives of this order live in hypersaline marine biotopes or freshwaters, such as salt lakes (e.g., the Great Salt Lake, the Dead Sea, etc.) or saltern crystallizer ponds. These environments can be thalassohaline, meaning that they are dominated by Na⁺ and Cl⁻ ions. In contrast, athalassohaline environments present greatly different ionic compositions. Among them, the Dead Sea is dominated by Mg²⁺ and Ca²⁺, in addition to Na⁺ and K⁺. Halobacteriales are also present in saline soils, mines, and arid areas (e.g., coasts, plains, mountains, deserts, etc.). Some Halobacteriales develop on products preserved by salt (e.g., food, hides, etc.) but contaminated through the use of crude solar salt. Indeed, during crystallization of halite, Halobacteriales cells can be trapped inside the growing crystals, and these may remain viable for a long time. The economic damages caused by these halophilic Archaea have triggered many of the early researches on Halobacteriales. In many hypersaline environments, Halobacteriales coexist with eukaryotes (such as green algae Dunaliella) and diverse bacteria such as Salinibacter ruber (Cytophagales). However, in the most extreme ones, Halobacteriales dominate microbial communities over bacteria. Some Halobacteriales (e.g., Natronobacterium) are haloalkaliphilic, living in alkaline hypersaline lakes characterized by salinity at (or close) to saturation and very high pH (9–11) due to high concentrations of carbonates. Many Halobacteriales are mesophilic to moderate thermophilic, having optimal growth temperatures ranging from 35 to 50 °C. This is not surprising given that many hypersaline environments inhabited by Halobacteriales are formed by evaporation processes occurring in warm areas. However, psychrotolerant members of Halobacteriales exist, such as those living in the very cold (but ice-free) hypersaline Deep Lake (Antarctica), which water temperature varies seasonally between below 0 and +11.5 °C. Some Halobacteriales cells are flagellated. Halobacteriales present many different morphotypes: rods, cocci, flat pleomorphic types, perfectly square flat cells, or even triangular and trapezoid cells. Halobacteriales are also known to carry the largest plasmids known to date, some of them being referred as to minichromosomes due to the presence of important or essential genes. For instance, 547 of the 2,674 (20 %) of the genes of Halobacterium sp. NRC1 are located on two megaplasmids, whereas 27 % of the Haloferax volcanii genes are carried on two megaplasmids and two small plasmids. In addition, many Halobacteriales are polyploid, for instance, there are 15–30 genome copies in Haloferax volcanii and Halobacterium salinarum.

The Halobacteriales are chemoorganotrophic. They oxidize various organic compounds under aerobic conditions and use O₂ as terminal electron acceptor. However, the availability of O₂ is often limited due to high microbial densities and the limited solubility of O₂ at high salt concentrations. Without surprise many Halobacteriales are able to use alternative pathways to produce their energy under microaerophilic or anaerobic conditions, including denitrification or fermentation of l-arginine. They are also able to use various compounds as electron acceptors, such as DMSO, TMAO, and fumarate. In addition, some Halobacteriales (e.g., Halobacterium) are phototrophic, meaning that they can use light to produce ATP. This process is carried out by a membrane protein called bacteriorhodopsin (cf. Sect. 3.3.4, Fig. 3.30) under anaerobic conditions. This protein is a light-driven H⁺ pump that expulses H⁺ from the cell, generating a H⁺ gradient which in turn is converted into ATP by ATP synthases. Bacteriorhodopsin may be very abundant in cell membranes. It is responsible for the pink/red coloration of the cells. This phototrophic process is different from chlorophyll-based photosynthesis because it requires neither chlorophyll nor electron transport chains. Furthermore and contrarily to bacteriorhodopsin-based phototrophy, photosynthesis requires additional pigments known as “antennas” and is coupled to carbon fixation. In 2001, homologues of bacteriorhodopsins were discovered in uncultured planktonic marine SAR86 gammaproteobacteria (Beja et al. 2000). Accordingly, these were referred as proteorhodopsins. Since then, homologues of proteorhodopsin were shown to be widespread in the oceans and in many prokaryotic lineages including the ubiquitous and abundant SAR11 alphaproteobacteria, archaea from group II, etc. This suggests that proteorhodopsin-based phototrophy is a significant oceanic microbial process, and therefore could represent a significant source of energy for microbial communities living in the ocean photic zone.

To thrive in environments rich in salts, Halobacteriales must cope with high osmotic pressures. Most halophilic or halotolerant microorganisms use a “salt-out” strategy consisting in the expulsion of salt from the cytoplasm to maintain low intracellular ionic concentrations and the use of organic solutes (e.g., glycerol or glycine betaine) to maintain an osmotic balance with the extracellular medium. In contrast, Halobacteriales, a few anaerobic halophilic bacteria (Haloanaerobiales), and Salinibacter ruber (Bacteroidetes) have adopted a radically different strategy referred as to “salt-in,” which consists in the molar accumulation of ions, especially K⁺ and Cl⁻ ions (and often Na⁺) in their cytoplasm. The import of K⁺ allows balancing the high osmotic pressure generated by high environmental Na⁺ levels. This equilibrium between intracellular K⁺ and extracellular Na⁺ is essential to prevent cells from dehydration. Some haloalkaliphilic archaea use organic osmotic solutes (e.g., 2-sulfotrehalose) in addition to high intracellular salt levels. The salt-out strategy is energetically costly due to the importance of organic-compatible solutes and less suitable at saturating salt levels, which probably explain why organisms using the salt-in strategy predominate under extreme hypersaline conditions. Due to the salt-in strategy, halobacteriales proteins must be adapted to function at molar salt levels. The side effect is that these proteins commonly denature in low-salt solutions. In addition, the cell wall of many Halobacteriales is composed of glycoproteins enriched in acidic amino acids (i.e., aspartate and glutamate). The negative charges provided by the carboxyl groups of these amino acids are surrounded by the Na⁺ ions which stabilize these glycoproteins, ensuring the integrity of the wall. If Na⁺ concentration becomes too low, the negatively charged glycoproteins repel each other, leading to destabilization of the cell wall and cell lysis. It has been early noticed that cytoplasmic proteins are also enriched in acidic amino acids. In addition they are depleted in hydrophobic amino acids, compared with their homologues found in nonhalophilic species. The replacement of large hydrophobic residues by small hydrophilic residues on cytoplasmic protein surface increases their overall polarity, preventing their aggregation and allowing them to remain functional. In parallel their increase in acidic residues creates a high density of negative charges coordinating a network of hydrated cations, which help to maintain the proteins in solution. Halobacteriales have been developed as genetic model because they are efficiently transformable. Moreover, they are simple to manipulate, in particular they are easy to culture, fast growing, and resistant to contamination by nonhalophilic microorganisms. They have been exploited to uncover genes involved in osmotic stress. Furthermore, they are also good models for structural biology because their proteins function under conditions of low water availability and biotechnology (Leigh et al. 2011). From an evolutionary point of view, Halobacteriales represent a relatively late diverging order within Euryarchaeota. They are related to “Nanohaloarchaea,” a lineage of uncultured nanometric archaea (0.6 μm in diameter), which are prevalent in worldwide distributed hypersaline environments (Narasingarao et al. 2011). Halobacteriales are grouping with methanogens class II (i.e., Methanocellales, Methanomicrobiales, and Methanosarcinales) (Fig. 6.9). For additional information on Halobacteriales, see Dworkin and collaborators (2006).

6.1.3.2.3 The Thermococcales

This order has been proposed by Wolfram Zillig and collaborators in 1987. It gathers anaerobic heterotrophic hyperthermophiles which grow optimally at neutral pH (6.0–7.0), even if a few alkaliphilic strains able to grow at pH 9 have been reported. Thermococcales cells are spherical and some of them are flagellated. Their metabolism is based on fermentation. They use polymeric organic substrates like peptides and carbohydrates as energy and carbon sources. The fermentation process produces H₂ which in turn can be used to reduce S⁰ to H₂S. Depending of the considered strains, S⁰ can either be required for growth or to stimulate growth. In some strains (e.g., Palaeococcus), S⁰ can be replaced by Fe²⁺. From an evolutionary point of view, Thermococcales have diverged early within Euryarchaeota (Fig. 6.9). Members of this group are very abundant and commonly found within marine hot water environments. They represent therefore a major constituent of the biomass in these ecosystems. Thermococcales contain a single family Thermococcaceae, represented by three genera: Pyrococcus, Thermococcus, and Paleococcus, the two first being more closely related to each other. Pyrococcus live in marine hydrothermal vents, whereas Thermococcus are also found in terrestrial freshwater, marine solfataric ecosystems, deep-sea hydrothermal vents, and offshore oil wells. Thermococcales have been developed as genetic models for studying DNA replication and repair, transcription and its regulation, carbon and energy metabolism, CRISPR systems, and cellular responses to stress, such as oxidative, osmotic, temperature, and pressure (Leigh et al. 2011). Thermococcales are also used as models in various fields of biotechnology due to their capacity to efficiently utilize polymeric substrates and their capacity to produce a vast array of stable, polymer-degrading hydrolases. For additional information on Thermococcales, see Dworkin and collaborators (2006).

6.1.3.2.4 The Thermoplasmatales

This order was proposed by Reysenbach in 2001. It gathers aerobic and facultative anaerobic acidophiles which can be autotrophic or heterotrophic. The most acidophilic organisms known to date belong to this order. Most representatives of this order maintain their intracellular pH near neutrality, thanks to sophisticated mechanisms such as very effective H⁺ pumps that expel H⁺ outside of the cell and membranes that are highly impermeable to H⁺. Some Thermoplasmatales are flagellated. The name is a reminder that the first described representatives of this order were thermophilic acidophiles, although currently mesophilic representatives are known. Thermoplasmatales are divided into three families: Ferroplasmaceae, Picrophilaceae, and Thermoplasmataceae, each containing a single genus, namely, Ferroplasma, Picrophilus, and Thermoplasma. Thermoplasma cells are of 0.2–5 μm in size. As they are devoid of cell wall or envelope (see above), the cells are pleomorphic, a feature shared with bacterial Mycoplasma. Because of this lack of cell wall and because the colonies of Thermoplasma resemble those of bacterial Mycoplasma, both were initially classified in the same taxonomic group. Molecular phylogeny studies have proven later that these features resulted from independent evolutionary processes (i.e., convergence) and that Mycoplasma and Thermoplasma are in fact unrelated. Thermoplasma are facultative aerobes growing at temperatures ranging from 33 to 67 °C (optimal near 60 °C) at pH 1–2. They are not able to survive at neutral pH. They are chemoorganotrophs. They can grow anaerobically through S⁰ respiration which leads to the release of large amounts of H₂S. They are also able to grow anaerobically without S⁰, suggesting that additional unidentified molecules can be used as electron acceptors. Thermoplasma can also grow aerobically. Hot springs, solfataric fields, warm acidic tropical swamps, marine hydrothermal systems, as well as aerobic and anaerobic zones of continental volcanic areas constitute natural habitats of Thermoplasma. They are also found in anthropized environments as coal refuse piles and associated water samples. Ferroplasma (as Thermoplasma) are pleomorphic. They are mesophilic chemolithoautotrophs. They are able to use CO₂ as a carbon source and Fe²⁺, pyrite, or Mn²⁺ as energy source. Some of them are also able to grow heterotrophically. They occur in many sulfidic ore-containing mines and heaps on earth, as well as in acidic geothermal pool. Picrophilus are hyperacidophilic obligate aerobic heterotrophic irregular cocci of 1–1.5 μm. They grow optimally at temperatures near 60 °C and pH 0.7, but can still divide at pH 0. No growth occurs below 47 °C and above 65 °C, or at pH higher than 3.5. Unlike other Thermoplasmatales, intracellar pH of Picrophilus is low (approximately 4.6), suggesting specific adaptations allowing enzymatic activity in acidic medium. However, the nature of these modifications remains to be determined. They thrive in geothermal solfataric soils and springs, and terrestrial geothermal environments. Thermoplasmatales group with DHEV2, the recently proposed “Methanoplasmatales,” and the uncultured group II (Fig. 6.9). For additional information on Thermoplasmatales, see Dworkin and collaborators (2006).

6.1.3.2.5 The Archaeoglobales

This order was proposed by Karl Stetter in 1989. It gathers regular to irregular lobe-shaped cocci of 0.3–1.3 μm in diameter occurring singly or in pairs that may be flagellated. Archaeoglobales grow anaerobically at temperatures ranging from 65 to 95 °C, at neutral or weakly acidic pH. This order contains a single family Archaeoglobaceae, which is divided into three genera Archaeoglobus, Ferroglobus, and Geoglobus. Most of them live in anoxic shallow and abyssal submarine hydrothermal vents, hot oil field waters but some have been identified in terrestrial environments including hot springs and terrestrial oil wells where SO₄ ²⁻ are abundant. Archaeoglobus are chemoorganotrophic sulfate reducers that oxidize H₂ or organic compounds (e.g., lactate, glucose, pyruvate, acetate, or formate) and reduce SO₄ ²⁻, SO₃ ²⁻, or S₂O₃ ²⁻ (but not S⁰) leading to H₂S formation. Some of them are also chemolithoautotrophic, being also able to use CO₂ as carbon source. In contrast, Ferroglobus are chemolithotrophic which grow anaerobically by using aromatic compounds such as benzoate as electron donors and Fe³⁺ as electron acceptor. They are also able to use H₂ and H₂S as energy sources and NO₃ ⁻ as a terminal electron acceptor leading to the formation of NO₂ ⁻. S₂O₃ ²⁻ can also be used as a terminal electron acceptor. Geoglobus are anaerobic chemoorganotrophs that grow by oxidizing acetate, pyruvate, palmitate, and stearate coupled to reduction of Fe³⁺. They can also grow autotrophically with H₂ as electron donor and poorly crystalline Fe³⁺ oxide as electron acceptor. From an evolutionary point of view, Archaeoglobales branch after the methanogens class I (Methanopyrales, Methanococcales, Methanobacteriales) and Thermoplasmatales, but before the diversification of the large group containing Halobacteriales, Methanomicrobiales, Methanosarcinales, and Methanocellales (Fig. 6.9). Evolutionary and genomic studies have shown that Archaeoglobales are descendants of methanogens that have secondarily lost the capacity to produce methane. This hypothesis was strongly reinforced by the discovery in the genome of Archaeoglobus fulgidus of almost all genes encoding enzymes and cofactors involved in methanogenesis. The only missing genes are those encoding methyl CoM reductase (the enzyme involved in the final step of methanogenesis), which prevents the biosynthesis of methane by this organism. Instead these genes are used in the reverse direction during the oxidation of lactate. For additional information on Archaeoglobales, see Dworkin and collaborators (2006).

6.1.3.3 The Nanoarchaeota

This lineage was first described as a new phylum tentatively named “Nanoarchaeota” by Harald Huber and colleagues in 2002. The only representative of this group is “Nanoarchaeum equitans,” a nanometric hyperthermophilic archaeon that grows attached to the surface of the surface of Ignicoccus hospitalis (Desulfurococcales). The nature of the association (i.e., symbiotic or parasitic) between these two archaea remains debated, even if most recent analyses favor the latter hypothesis. Environmental surveys suggest that members of the “Nanoarchaeota” are broadly distributed in hot biotopes, such as the deep-sea hydrothermal vents, shallow marine areas, and terrestrial solfataric fields. Nanoarchaeota are anaerobic cocci of only 400 nm in diameter. “Nanoarchaeum equitans” cells are not flagellated. They grow under strictly anaerobic conditions at temperatures ranging from 75 to 98 °C. Early phylogenetic analyses suggested that “Nanoarchaeota” branch deeply in archaeal tree, meaning before to the speciation of Crenarchaeota and Euryarchaeota. However, later analyses suggested that this position results from a tree reconstruction artifact called long branch attraction and that “Nanoarchaeota” represent in fact a fast-evolving lineage of Euryarchaeota, likely related to Thermococcales (Brochier et al. 2005) (Fig. 6.9). The relationship between “Nanoarchaeota” and Euryarchaeota has been strengthened by comparative genomic analyses. The 16S rRNA of “Nanoarchaeota” are highly divergent even in previously “universally” conserved regions, meaning that they cannot be easily amplified by PCR using universal 16S rRNA primers. The genome of “Nanoarchaeum equitans” is the smallest genome of archaea known to date (490 kb, corresponding to ~552 genes). It is G + C poor (31.6 %), has high gene density (95 %), and presents a number of unique genomic rearrangements (e.g., split genes such as tRNA, etc.) that were sometime interpreted as ancestral archaeal features in agreement with the deeply branching position of “Nanoarchaeota” observed in initial studies. Many biosynthetic pathways (e.g., lipids, cofactors, amino acids, nucleotides, etc.) and metabolic pathways (e.g., glycolysis, pentose phosphate pathway, carbon assimilation, etc.) are lacking. This suggests that “Nanoarchaeum equitans” is strictly dependent of its host and why all isolation attempts were unsuccessful so far. The genome sequence of a second representative of “Nanoarchaeota” (distantly related to “Nanoarchaeum equitans”) has been obtained from an enrichment culture obtained from a terrestrial hot spring (Podar et al. 2013). In contrast to “Nanoarchaeum equitans,” this new strain could be a symbiont of an uncultured Sulfolobales, but this remains to be formerly demonstrated. The genome of this new nanoarchaeon is larger than the genome of “Nanoarchaeum equitans,” which contains less split genes, suggesting that the former has experienced less severe genome reduction than the latter. It encodes a complete gluconeogenesis pathway as well as a full set of archaeal flagellum proteins, suggesting that this new nanoarchaeon is motile. Altogether these findings indicate that the features of “Nanoarchaeum equitans” that were interpreted as ancestral are in fact derived characters linked to their particular lifestyle (Podar et al. 2013).

6.1.3.4 The Korarchaeota

Korarchaeota (from the Greek “koros” meaning young) was proposed by Susan M. Barns and colleagues in 1996 based on 16S rRNA environmental surveys of a hot spring in the Yellowstone National Park in the USA (Barns et al. 1996). This phylum is represented by a handful of 16S rRNA gene sequences from uncultured representatives. For a long time, these archaea remained elusive. This situation has changed following the report of the genome sequencing of the “Candidatus Korarchaeum cryptofilum” from an enrichment culture (Elkins et al. 2008). This ultrathin filamentous korarchaeaon is 0.16–0.18 μm wide × 26 μm long. The analysis of the genome suggests that “Candidatus Korarchaeum cryptofilum” ferments peptides to obtain carbon and energy. It lacks the ability to synthesize de novo many compounds and cofactors such as purines and CoA. Phylogenetic analyses based on 16S rRNA genes and on various conserved proteins support a closer relationship of Korarchaeota with Crenarchaeota and Thaumarchaeota (including “Aigarchaeota”) than with Euryarchaeota (Fig. 6.9).

6.1.3.5 The Thaumarchaeota

Thaumarchaeota (from the Greek “thaumas” meaning wonder) was proposed in 2008 by Céline Brochier-Armanet and collaborators (Brochier-Armanet et al. 2008). Thaumarchaeota (formerly referred as group I or mesophilic crenarchaeota) have been discovered independently by the teams of Jed Fuhrman and Ed DeLong in 1992 through 16S rRNA surveys of environmental marine samples. This phylum constitutes one of the most abundant and diversified archaeal taxonomic groups (Brochier-Armanet et al. 2012). It gathers numerous sublineages (e.g., group I.1a, I.1b, I.1c, 1A/pSL12, ThAOA/HWCG III, SAGMCG-I, SCG, FSCG, etc.). Most Thaumarchaeota are uncultured free-living archaea occurring in very diverse environments (e.g., freshwaters, lakes, soils, sediments, oceans, hot springs, etc.), but some of them live in association with animals (e.g., Cenarchaeum symbiosum, a sponge symbiont): Worth noticing, Takuro Nunoura and colleagues have recently assembled a composite genome (“Candidatus Caldiarchaeum subterraneum”) from a metagenomic library prepared from a geothermal water stream collected in a subsurface gold mine, which was proposed to represent an additional phylum tentatively called “Aigarchaeota” (corresponding to the formerly uncultured HWCG I group). However, subsequent phylogenetic and comparative genomics analyses suggest that “Aigarchaeota” rather represent an early branching lineage within Thaumarchaeota (Brochier-Armanet et al. 2011). Thaumarchaeota are the subject of much attention since environmental metagenomic surveys have shown that some members of this phylum carry ammonia monooxygenase genes, suggesting that they are nitrifiers (Treusch et al. 2005). This was confirmed after the isolation of Nitrosopumilus maritimus, a marine thaumarchaeon that grows chemolithoautotrophically by aerobically oxidizing ammonia to nitrite (Könneke et al. 2005). This key and limiting step of the nitrogen cycle was previously thought to be restricted to a few sublineages of autotrophic betaproteobacteria and gammaproteobacteria and a few heterotrophic nitrifiers. The discovery of nitrifying archaea extends the taxonomic range of microorganisms capable of nitrification and therefore opens up new perspectives on the origin and evolution of this key metabolism on Earth. Moreover, the widespread distribution of putative archaeal nitrifiers and their numerical dominance over their bacterial counterparts in most marine and terrestrial environments suggested that ammonia oxidizer thaumarchaeota play a major role in global nitrification (Pester et al. 2011). However, recent studies suggest that thaumarchaeotal nitrifiers dominate over bacteria only in environments containing low ammonium concentrations. Three orders of Thaumarchaeota have been proposed to date: Cenarchaeales, Nitrosopumilales, and Nitrososphaerales. The first two correspond to the former group I.1a, whereas the latter was previously referred as group I.1b. Cenarchaeales and Nitrososphaerales contain a single family and a single genus (Cenarchaeaceae/Cenarchaeum and Nitrososphaeraceae/Nitrososphaera, respectively), whereas Nitrosopumilales contains a single family (Nitrosopumilaceae) and two genera (Candidatus Nitrosoarchaeum and Nitrosopumilus). However, these lineages represent a small part of the real diversity of Thaumarchaeota, meaning that additional thaumarchaeota taxonomic groups should be proposed in the near future. Currently, it is not clear whether ammonia-oxidizing thaumarchaeota are strict autotrophs or also able to use other substrates (such as amino acids, oligopeptides, and glycerol) for their growth, indicating they could also be mixotrophs or even heterotrophs. Phylogenetic analyses of various conserved proteins support a closer relationship of Thaumarchaeota (including “Aigarchaeota”) with Crenarchaeota and Korarchaeota than with Euryarchaeota (Fig. 6.9).

6.2 Domain Bacteria

In the second edition of “Bergey’s Manual of Systematic Bacteriology,” the domain is described with 30 bacterial phyla. Relationships between these phyla are not yet resolved and represent a major challenge in microbial evolution.

The 30 bacterial phyla are presented in Table 6.2. They bring together some 7,500 species characterized phenotypically from cultures of bacterial strains, and therefore they are defined from these bacterial isolates fully characterized.

6.2.1 Phyla B1 to B9

Phyla B1 to B9 correspond to bacteria generally positioned in the lower branches of the phylogenetic tree based on 16S rRNA bacteria gene sequences (Fig. 6.11) or bacteria having extremophilic characters and sometimes isolated from extreme environments. However, this position is not found in most phylogenetic analyses based on protein markers (Lopez-Garcia and Moreira 2008). Many species belonging to the phyla B1 to B9 are represented by bacteria capable of living in environments considered extreme, either hyper-hot or highly contaminated by metals or radiation. The phyla Aquificae and Thermotogae, and genus Thermus contain the most known thermophilic members among domain Bacteria.

Fig. 6.11

Phylogeny of Bacteria. Phylogeny of Bacteria based on 8493 16S rRNA gene sequences of type strains (>1,200 bp) available at the Ribosomal Database Project II (release 10, http://rdp.cme.msu.edu/) in 2013. The tree was inferred with the neighbor-joining method. Bacterial phyla are shown in color. The width of areas is proportional to the number of sequences available for each phylum

6.2.1.1 The Phylum Aquificae

These Gram-negative aerobic autotrophic and hydrogenophilic bacteria can grow up to temperatures between 80 and 95 ° C. This phylum includes a single class (Aquificae) containing a single order, Aquificales, in which there is a single family Aquificaceae. The main genera are Aquifex, Calderobacterium, Hydrogenobacter, and Thermocrinis. Representatives of these genera are hyperthermophilic bacteria isolated from marine and terrestrial hot springs, and developing either aerobic or microaerophilic or anaerobic and are chemoorganotrophs and some chemolithotrophs with hydrogen or thiosulfate as electron donors. These microorganisms occupy a basal position in the phylogenetic tree of Bacteria based on 16S rRNA. It was therefore suggested that these were very old lines having inherited from LUCA (cf. Sect. 4.1.1) the ability to live at high temperatures. However, this phylogenetic position is controversial, and these microorganisms may actually be descendants of mesophilic microorganisms which are secondarily adapted to life at high temperature (cf. Sect. 4.1.6).

6.2.1.2 The Phylum Thermotogae (Fig. 6.10e)

Represented by a single class and a single order, Thermotogales includes hyperthermophilic bacteria, Gram-negative, anaerobic, and that has an outer membrane larger than the bacterial body and wrapping it as a “toga.” One family (Thermotogaceae) includes several genera including types Thermotoga, Geotoga, Petrotoga, Marinitoga, and Kosmotoga, isolated for most of them from deep environments (aquifers, oil reservoirs) or volcanic sources in land or underwater. These bacteria are fermentative; some may use thiosulfate or sulfur as electron acceptors, and reduce H₂S.

6.2.1.3 Phyla Thermodesulfobacteria, Thermomicrobia, and Deferribacteres

They also include Gram-negative thermophiles or moderately thermophilic bacteria. They are also phyla represented by one class, one order, and one family. In Thermodesulfobacteriaceae, the genus Thermodesulfobacterium is strictly an anaerobic chemoorganotroph capable of sulfate-reducing and fermenting. In Thermomicrobiaceae, the genus Thermomicrobium is formed of irregular short rods that are aerobic and chemoorganotrophic. The Deferribacteraceae family includes several genera (among them Deferribacter and Geovibrio) which are constituted of straight or curved rods, anaerobes isolated from marine sediments or oil tanks, and are chemoorganotrophic using nitrate, iron, manganese, or cobalt as electron acceptors for anaerobic respirations.

6.2.1.4 The Phylum Chrysiogenetes

With one class, one order (Chrysiogenales), and one family (Chrysiogenaceae), this phylum is represented by a single genus Chrysiogenes formed of Gram-negative, curved, mesophilic, chemoorganotrophic performing anaerobic respiration with arsenate as electron donor.

6.2.1.5 The Phylum Deinococcus/Thermus

This phylum is a dream for biotechnologists. The only class includes two orders, Deinococcales and Thermales. The Deinococcales with a single family (Deinococcaceae) include Gram-positive chemoorganotrophs, aerobic mesophilic or marginally thermophilic, of the genus Deinococcus from which some representatives (Deinococcus radiodurans) are resistant to gamma irradiation of about 5,000 Gy, doses sufficient to eliminate most other microorganisms. The radiation resistance of Deinococcus could be linked to the presence of a mechanism specific (and very effective) for DNA repair (Cox and Battista 2005). These bacteria have a special envelope with a thick Gram-positive wall and in addition, an outer membrane, and an outer layer of S protein.

The Thermales, with one family, Thermaceae, combine several genera including genus Thermus isolated from hot springs (70–90 ° C) which gave the thermostable DNA polymerase used for PCR, an enzyme that represents an annual market of 300 million dollars. This genus comprises bacteria in short filaments or rods, hyperthermophilic, chemoorganotrophic, and aerobic, with certain strains that are capable of using nitrate as electron acceptor under anaerobic conditions. The members of this phylum contain ornithine in their cell wall.

6.2.1.6 The Phylum Chloroflexi

This phylum contains Gram-negative photosynthetic anoxygenic bacteria (also called phototrophic green nonsulfur bacteria) formed of multicellular filaments living in microbial mats or in the water in hot environments anoxic and exposed to light, often in hot springs. Class Chloroflexi with order Chloroflexales contains Chloroflexaceae families (genera Chloroflexus, Chloronema) and Oscillochloridaceae (genus Oscillochloris). This class includes bacteria which contain bacteriochlorophyll c and carotenoids present in structures attached to the cytoplasmic membrane, the chlorosomes (cf. Sect. 3.3.4, Fig. 3.27), and the genus Heliothrix that contains bacteriochlorophyll a with no apparent membrane structures. These bacteria perform anoxygenic photosynthesis and use organic compounds in photoorganotrophic metabolism, and they can fix CO₂ by the pathway called hydroxypropionate cycle (cf. Sect. 3.4.1, Fig. 3.34). In this class, there are also many non-photosynthetic species grouped in order Herpetosiphonales. Classes of Anaerolineae (Anaerolinea) and Caldilineae (Caldilinea) include many genera and species of filamentous bacteria isolated from anaerobic fermenters. Other non-photosynthetic bacteria are also present in this group and have been detected so far by genetic analysis. These bacteria are in the majority inhabitants of environments contaminated by organic pollutants and metal, whether in soils or aquatic environments.

6.2.1.7 The Phylum Nitrospirae

With a single order (Nitrospirales) and one family (Nitrospiraceae), this phylum includes bacteria belonging to genera quite different, consisting of spiral or curved rods, Gram-negative. These microorganisms are aerobic chemolithotrophic nitrifiers (Nitrospira), iron-oxidizers (Leptospirillum), anaerobic chemoorganotrophic sulfate reducers, and thermophilic microbes (Thermodesulfovibrio) or microbes with magnetic iron inclusions (“Magnetobacterium” Fig. 14.46), mostly isolated from terrestrial and marine aquatic environments.

6.2.2 The Phylum B 10 Synergistetes

This phylum is Gram-negative, anaerobic, chemo- and fermentative organotrophic isolated from animals (rumen, genus Synergists) or man (genus Jonquetella). These bacteria are involved in anaerobic digestion operation in the rumen of animals and the human intestinal flora.

6.2.3 The Phylum Cyanobacteria B11 (Figs. 6.12 and 6.13)

This phylum consists of Gram-negative bacteria, performing oxygenic photosynthesis with photosystems I and II inserted in membrane systems (thylakoids) and producing oxygen (cf. Sect. 3.3.4, Fig. 3.28). This bacterial group would be considered as the ancestors of chloroplasts (cf. Sects. 3.3.4, 4.3.1, and 5.4.3). They form a group of bacteria of varied forms ranging from unicellular to multicellular filamentous and colonial types (Figs. 6.12 and 6.13). They contain chlorophyll, carotenoids, and phycobilins or biliproteins, giving them a dark blue-green pigmentation. Some cyanobacteria have the divinyl chlorophyll. They are present in surface waters as well as all kinds of wet surfaces exposed to light where their photosynthetic capacity gives them a competitive advantage. Many kinds of cyanobacteria fixing dinitrogen, including filamentous genera Anabaena and Nostoc and a proportion of them lives in symbiosis with Fungi (lichens) or with Viridiplantae (Cycas, Azolla). Some species of cyanobacteria in algal blooms can produce enterotoxic, hepatotoxic, or neurotoxic cyanotoxins that cause poisoning, which are sometimes fatal like microcystin in Microcystis spp. (Box 15.2). Cyanobacteria use the Calvin cycle for CO₂ fixation. For many years, cyanobacteria have been considered algae (hence the name blue-green algae) due to their ability to perform oxygenic photosynthesis. Their phylogenetic positioning has allowed classifying them as members of the domain Bacteria where they form a major phylum organized into five subsections. Classification within cyanobacteria is problematic because of their membership to the bacterial and botanical systematic codes. Numerous discussions between botanists and bacteriologists are still underway to harmonize the classification of cyanobacteria in both systematic codes.

Fig. 6.12

Photomicrographs of Cyanobacteria. (a) Cyanothece sp., (b) Planktothrix rubescens, (c) Oscillatoria sancta, (d) Gloeothece membranacea, (e) Spirulina subsala, (f) Anabaena cylindrical, (g) Gloeobacter violaceus, (h) Spirulina sp., (i) Oscillatoria sp. (a–g) Photographs, courtesy of Thierry Laurent (cyanobacteria Unit, Institut Pasteur Paris) and Jean-François Humbert (Institute Pasteur, INRA); and (h, i) Photographs, Rémy Guyoneaud. The bars represent 10 μm

Fig. 6.13

Confocal photomicrographs of cyanobacteria observed in a microbial mat from the Ebro Delta in Spain. (a) Microcoleus chtonoplastes, (b) Morphology of Phormidium, (c) Morphology of Lyngbia, (d) Morphology of Oscillatoria, (e) Morphology of Halomicronema, (f) Representatives of the group “Gloeocapsa.” (a–d) Photographs: courtesy of Antonio Solé (Autonomous University of Barcelona); (e, f) Photographs, courtesy of Elia Diestra (Autonomous University of Barcelona). The bars represent 10 μm

• Subsection 1 (Chroococcales): it relates unicellular cyanobacteria that can form clusters of cells such as Microcystis, Synechococcus, Cyanobium, Cyanothece, and Gloeothece.

• Subsection 2 (Pleurocapsales): it includes cyanobacteria forming colonial structures and small spherical structures called beocytes for multiplication (such as genera Pleurocapsa, Dermocarpa).

• Subsection 3 (Oscillatoriales): it is formed of filamentous cyanobacteria that divide by binary fission in a single plane constituting long gliding filaments of identical non-individualized cells as Oscillatoria, Spirulina, and Microcoleus.

• Subsection 4 (Nostocales): it corresponds to the filamentous cyanobacteria with heterocysts – the filaments are formed of photosynthetic cells sometimes interspersed with differentiated cells (heterocysts). These do not make photosynthesis and do not produce oxygen but possess an enzyme (nitrogenase) sensitive to oxygen which enables them to fix atmospheric dinitrogen (N₂) by reducing it to ammonium used by other cells of the filament (Stacey et al. 1979). The best known are the genera Nostoc, Anabaena, and Scytonema.

• Subsection 5 (Stigonematales): it includes branched filamentous cyanobacteria as genera Fischerella and Stigonema.

Many cyanobacteria even non-heterocystous ones are able to fix nitrogen from atmospheric dinitrogen. In this case, nitrogenase must be protected from oxygen produced during photosynthesis, either by being active especially in dark phase or by a spatial cell organization in clusters, allowing the cells at the center of the cluster to find an anoxic environment (cf. Sect. 3.4.4).

In the phylum, Cyanobacteria are also prochlorophytes with Prochlorococcus, Prochloron, and Prochlorothrix genera, which are unicellular or filamentous, abundant in marine phytoplankton. They contain chlorophyll a (as other cyanobacteria) and b (as Viridiplantae chloroplasts), but do not have phycobilins. These microorganisms thrive in the euphotic zone of oligotrophic oceanic waters in the wide open ocean. They are grouped within Chroococcales (Subsection 1).

6.2.4 Other Gram-Negative Bacteria

This set consists of phyla B12 to B30 except phyla B14, B15, and B16 (which include most of the Gram-positive bacteria), that is to say 16 phyla in total. It includes the majority of described species in the domain Bacteria. A phylum is especially important; it is the phylum Proteobacteria originally called the phylum of purple bacteria.

6.2.4.1 The Phylum Proteobacteria

This phylum was originally called “purple bacteria” because it includes pigmented photosynthetic bacteria. Anoxygenic photosynthesis is carried out by these microorganisms by using as an electron donor either an organic compound (phototrophic purple nonsulfur bacteria) or inorganic compounds such as sulfides, hydrogen, or iron (phototrophic purple sulfur bacteria) via bacteriochlorophylls a or b (cf. Sect. 3.3.4). Originally C. Woese had proposed the term “purple bacteria phylum” to emphasize the photosynthetic origin of this group; however, these photosynthetic bacteria are found only in three classes among the six classes that make up this phylum. In fact, in addition to the purple bacteria, this phylum contains a large number of non-photosynthetic bacteria corresponding to the majority of Gram-negative bacterial genera currently known; it is a very large and very varied phylum with different types of bacterial metabolisms: phototrophy, chemoorganotrophy, and chemolithotrophy. Analyses suggest that the absence of photosynthesis observed in a majority of representatives of this phylum is the result of a loss of this metabolism initially present in their ancestor. Indeed, some non-photosynthetic proteobacteria still have a portion of bacteriochlorophyll gene in their genome.

6.2.4.1.1 The Class Alphaproteobacteria

It includes many kinds of phototrophic purple nonsulfur bacteria such as Rhodospirillum (Fig. 6.14g), Rhodobacter, Rhodovulum, Rhodobium, and Rhodopseudomonas (Fig. 6.14d). These genera are grouped into three levels (Rhodospirillales, Rhodobacterales, and Rhizobiales). Many chemoorganotrophic bacteria with varied ecological niches (soil saprophytes, pathogens of plants or animals, symbionts, etc.) are also present in this class with a large number of taxa in close interaction with host animals or plants. Among the best-known representatives, there are Sinorhizobium meliloti, the dinitrogen fixing root symbiont in alfalfa; Agrobacterium tumefaciens, the pathogen that transforms its host plant by injecting it with its own DNA; Rickettsia conorii, the agent of Q fever; and Nitrobacter hamburgensis, nitrifying soil bacteria.

Fig. 6.14

Photomicrographs of Gram-negative bacteria of the phylum Proteobacteria. (a) Beggiatoa sp. (Photograph: Pierre Caumette); (b) Halothiocapsa halophila (Photograph: Pierre Caumette); (c) Chromatium sp., electron microscopy (Photograph: courtesy of Michael Madigan, University of Illinois, Michigan, USA); (d) Rhodopseudomonas palustris (Photograph: Pierre Caumette); (e) Allochromatium vinosum (Photograph: Pierre Caumette); (f) Allochromatium sp. containing polyhydroxybutyrate globules, electron microscopy (Photograph: courtesy of Michael Madigan, University of Illinois, Michigan, USA); (g) Rhodospirillum sp. (Photograph: Pierre Caumette); (h) Desulfovibrio halophilus (Photograph: Pierre Caumette); (i) Desulfobacterium vacuolatum (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany); (j) Desulfonema ishimotoi (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany); (k) Ectothiorhodospira sp. (Photograph: courtesy of Remy Guyoneaud, IPREM, University of Pau, France); (l) Desulfobulbus propionicus (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany); (m) Desulfovibrio sp. (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany); (n) Chromatium sp. interference contrast (Photograph: courtesy of Heribert Cypionka, University of Oldenburg, Germany); (o) Desulfovibrio desulfuricans (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany; (p) Desulfobacterium autotrophicum (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany); (q) Thiodictyon sp., iron-oxidizing bacteria purple (Photograph: courtesy of Armin Ehrenreich, MP Institute of Microbiology, Bremen, Germany); (r) Desulfobacter latus (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany). The bars represent 10 μm except for figures c and f where they are 1 μm. With the exception of figures c, f, and n, all photographs correspond to phase contrast microscopy

In Rhodospirillales, an order consisting of two families (Rhodospirillaceae and Acetobacteraceae), the purple bacterium Rhodospirillum and other relatives (Rhodovibrio, Rhodospira, etc.) coexists with Acetobacter, Gluconobacter (bacteria oxidizing ethanol to acetic acid), and various other bacteria isolated from soil and aquatic continental or marine environments (Azospirillum, Magnetospirillum).

The Rhodobacterales with a single family (Rhodobacteraceae) contain many purple nonsulfur bacteria (Rhodobacter, Rhodovulum, etc.) and marine aerobic or denitrifying bacteria (Amaricoccus, Antarctobacter, Paracoccus, etc.) or containing bacteriochlorophyll a (Roseobacter, Roseovivax, etc.).

The Rhizobiales with 10 families contain some purple phototrophic bacteria in Rhodobiaceae (Rhodobium) or in Bradyrhizobiaceae (Rhodopseudomonas). Other families of this order include many soil bacteria, fixing dinitrogen. It is the case of Rhizobiaceae (Rhizobium), of Phyllobacteriaceae (Phyllobacterium, Mesorhizobium), of Beijerinckiaceae (Beijerinckia), and of Bradyrhizobiaceae (Bradyrhizobium) which also contain nitrifying bacteria (Nitrobacter). Other families concern methylotrophic bacteria as Methylocystaceae, Methylobacteriaceae (Methylobacterium). The Hyphomicrobiaceae (Hyphomicrobium, Aquabacter, and Blastochloris) isolated from soil or aquatic environments, Brucellaceae (Brucella) responsible for brucellosis, and Bartonellaceae (Bartonella) are also part of Rhizobiales.

The three other orders of this class are the Rickettsiales with three bacteria family symbionts or parasites, Rickettsiaceae (Rickettsia, Wolbachia) which are intracellular parasites or symbionts, the Ehrlichiaceae (Ehrlichia), and the Holosporaceae. The Sphingomonadales with family Sphingomonadaceae (Sphingomonas) and Caulobacterales with family Caulobacteraceae (Caulobacter) are the last two orders of this class which includes as a whole many bacteria which are diverse in their metabolism (phototrophic, chemolithotrophic, chemoorganotrophic) and multiply by budding or form stalks, enabling them to live on fixed supports (Hyphomicrobium, Rhodopseudomonas, Rhodomicrobium, Blastochloris, Caulobacter, Ancalomicrobium, and Prosthecomicrobium).

6.2.4.1.2 The Class Betaproteobacteria

It includes some phototrophic purple nonsulfur bacteria but also chemolithotrophic or chemoorganotrophic bacteria that live in varied ecological niches with a predominance of contaminated sites. It consists of six orders with two that contain some purple bacteria. These are Burkholderiales with family Comamonadaceae (Rubrivivax, Rhodoferax) which also contains many non-photosynthetic bacteria and Rhodocyclales with family Rhodocyclaceae (Rhodocyclus) which also includes the chemoorganotrophic genera Propionibacter or Zooglea.

The order Burkholderiales includes five families especially chemoorganotrophic using metals (Ralstoniaceae with Ralstonia metallidurans) or isolated from polluted soils (Burkholderiaceae with Burkholderia, Alcaligenaceae with Alcaligenes) capable of biodegrading hydrocarbons or xenobiotics (pesticides). The family Comamonadaceae includes many chemoorganotrophic bacteria (Comamonas, Aquabacterium) isolated from aquatic environments, or polluted environments including wastewaters (filamentous bacteria: Leptothrix, Sphaerotilus) and chemolithotrophic bacteria using hydrogen (Hydrogenophaga) or sulfur (Thiomonas) as electron donors. Family Oxalobacteraceae (Oxalobacter) is part of this order. The order Hydrogenophilales with a single family (Hydrogenophilaceae) corresponds to aerobic chemolithotrophic bacteria using hydrogen (Hydrogenophilus) or sulfur (Thiobacillus) as electron donors.

The order Methylophilales concerns methylotrophic bacteria (cf. Sect. 3.4.2) grouped in a family (Methylophilaceae) with genera Methylophilus, Methylobacillus, etc. The order Neisseriales with a family (Neisseriaceae) contains very pathogenic Gram-negative cocci (Neisseria gonorrhoeae, Neisseria pneumoniae, Neisseria meningitidis) but also bacteria isolated from soil or water (Aquaspirillum, Chromobacterium).

The order Nitrosomonadales concerns chemolithotrophic bacteria with ammonium oxidizing family Nitrosomonadaceae (Nitrosospira, Nitrosomonas), ferro-oxidant family Gallionellaceae (Gallionella), and family Spirillaceae with chemoorganotrophic bacteria (Spirillum). Many filamentous bacteria are also present in this class with filamentous bacteria in sewage sludge (Sphaerotilus, Zooglea) or iron-oxidizing bacteria (Leptothrix).

6.2.4.1.3 The Class Gammaproteobacteria

It includes the largest number of Proteobacteria grouped in 13 orders. One (the Chromatiales) contains all the phototrophic purple sulfur bacteria oxidizing sulfide during anoxygenic photosynthesis. These bacteria accumulate intracellular sulfur globules (family Chromatiaceae with Chromatium as the type genus (Fig. 6.14c, e, f) and related genera, and Thiocapsa, Halothiocapsa (Fig. 6.14b), Lamprobacter, Thiodictyon (Fig. 6.14q) Thiococcus, Thiocystis, and Thiopedia) or extracellular (Ectothiorhodospiraceae family with the halophilic genus Ectothiorhodospira (Fig. 6.14k) or extreme halophilic Halorhodospira) and non-photosynthetic bacteria (Nitrococcus).

The Xanthomonadales with a family (Xanthomonadaceae) contain chemoorganotrophic aerobic bacteria often isolated from soil and plant pathogens (Xanthomonas, Xylella, etc.).

The Pseudomonadales include two families, Pseudomonadaceae with the genera Pseudomonas, common bacteria of soil and water, some plant pathogens, or nosocomial disease-causing (Pseudomonas aeruginosa), Azomonas, Rhizobacter, and Moraxellaceae with Moraxella, Acinetobacter, which are encountered in soils.

In the class Gammaproteobacteria, there is in particular the order Enterobacteriales for all Enterobacteriaceae, bacteria capable of chemoorganotrophic aerobic or anaerobic respiration (respiring nitrate) and fermentation (cf. Sect. 3.3.3), with coliforms (Klebsiella, Hafnia, Citrobacter, Enterobacter) and Escherichia coli, commensal or pathogenic bacteria being the microorganism most studied in terms of physiology and genetics, Salmonella, agents of typhoid and paratyphoid fever (Salmonella typhi, Salmonella paratyphi), Proteus, and Shigella (Shigella sonnei). In this order are also present Yersinia pestis, the agent of plague, and symbionts of insects or nematodes as Photorhabdus.

The order Vibrionales with a family Vibrionaceae, contains chemoorganotrophic bacteria isolated from waters, particularly coastal waters or estuaries as the genus Vibrio with Vibrio cholerae (cholera agent) and many marine Vibrio causing infections or poisoning of animal (fish) or human, and the genera Salinivibrio or Photobacterium, the latter containing luminescent bacteria with an enzyme, luciferase, also present in prokaryote light organs of certain deep marine organisms.

The Aeromonadales as Vibrionales and Enterobacteriales are also aerobic bacteria, facultative anaerobes (capable of respiring and fermentation) or strict anaerobes include two families: Aeromonadaceae with the genus Aeromonas, freshwater bacteria, some of which are pathogenic of fish (Aeromonas salmonicida), and Succinivibrionaceae which contain genera represented by strict fermentative bacteria (Succinivibrio, Ruminobacter, Anaerobiospirillum) isolated from fermenters or rumen.

The order Pasteurellales with a family (Pasteurellaceae) includes the genus Pasteurella chemoheterotrophic bacteria responsible for pulmonary infections in animals and Haemophilus influenzae pathogen of respiratory track, the first bacterium completely sequenced in 1995 (Fleischmann et al. 1995).

The order Legionellales with a single family (Legionellaceae) contains a single genus (Legionella) corresponding to Legionella pneumophila, the agent of Legionnaires’ disease, surprise guest at the American Legionnaires Convention Philadelphia 1976, often isolated from ventilation systems, water circulation or refrigeration industries and communities, and responsible for serious lung infections.

The order Oceanospirillales includes aerobic chemoorganotrophic marine bacteria also capable of respiring nitrate, grouped into two families, Oceanospirillaceae with genera Oceanospirillum, Marinospirillum, and Halomonadaceae with genera Halomonas or Alcanivorax, bacteria capable of totally degrading hydrocarbons.

The Alteromonadales with the family Alteromonadaceae also comprise chemoorganotrophs marine halophilic or halotolerant bacteria (Alteromonas, Marinobacter, Marinobacterium) or bacteria living at very cold temperatures (Psychromonas).

The order Thiotricales is represented by chemolithotrophic sulfur-oxidizing bacteria filamentous and often gliding (Thiothrix, Thiomargarita, Thiobacterium, Thioploca, Beggiatoa Fig. 6.14a, Thiospira) grouped in the family Thiotricaceae; two other families (Piscirickettsiaceae and Francisellaceae) are also part of this order.

The methylotrophic bacteria order (Methylococcales) is represented by family Methylococcaceae (Methylococcus, Methylobacter), and the Cardiobacteriales order with family Cardiobacteriaceae (Cardiobacterium) are also present in this Gammaproteobacteria class.

6.2.4.1.4 The Class Deltaproteobacteria

With seven orders, it includes among others the majority of sulfate-reducing bacteria which are chemoorganotrophic anaerobic bacteria for most, some may be chemolithotrophic, using sulfate as electron acceptor (Desulfovibrio, Fig. 6.14h, m, o; Desulfobacter, Fig. 6.14r; Desulfobulbus, Fig. 6.14l; Desulfobacterium, Fig. 6.14i, p) and anaerobic bacteria using ferric iron as electron acceptor (Geobacter, Pelobacter). These bacteria are grouped into four orders of this class.

The order Desulfurellales with family Desulfurellaceae contains the genus Desulfurella.

The order Desulfovibrionales includes three families, Desulfovibrionaceae (Desulfovibrio), especially encountered in coastal environments and capable of degrading partially simple organic compounds, Desulfomicrobiaceae (Desulfomicrobium), and Desulfohalobiaceae (Desulfohalobium, Desulfonatronovibrio, etc.), these latter including the halophilic sulfate-reducing bacteria isolated from hypersaline environments.

The Desulfobacterales with families Desulfobacteraceae (Desulfobacter, Fig. 6.14r; Desulfobacterium, Fig. 6.14i, p), Desulfobulbaceae (Desulfobulbus, Fig. 6.14l), and Desulfoarculaceae (Desulfoarculus, Desulfomonile) correspond to sulfate-reducing bacteria isolated from diverse environments, such as oceans, soils, deep earth, oil deposits, etc. They are able to degrade some complex organic compounds and of biotransformation of metals.

The order Desulfomonadales contains sulfate-reducing bacteria in the family Desulfomonadaceae (Desulfomonas) and anaerobic chemoorganotrophic iron-reducing bacteria in families Geobacteraceae (Geobacter) and Pelobacteraceae (Pelobacter).

In this class, there is also the order Syntrophobacterales with two families, Syntrophobacteraceae (Syntrophobacter) and Syntrophaceae (Syntrophus), which includes anaerobic bacteria living in syntrophy requiring interspecies hydrogen transfer but also some sulfate-reducing bacteria (Desulfacinum).

The order Bdellovibrionales (Bdellovibrionaceae) contains bacteriological predatory bacteria (Bdellovibrio or Vampirovibrio).

Finally, the order Myxococcales with the two families Myxococcaceae (Myxococcus, etc.) and Polyangiaceae (Polyangium, Chondromyces, etc.) is composed of bacteria with fruiting structures (myxobacteria).

6.2.4.1.5 The Class Epsilonproteobacteria

The fifth class of this great phylum contains some specific bacteria such as Campylobacter, sulfur-oxidizing bacteria (Thiovulum) grouped in the family Campylobacteraceae, and the pyloric gastric pathogen (Helicobacter pylori) in the family Helicobacteraceae.

6.2.4.1.6 The Class Zetaproteobacteria

This sixth and last class of Proteobacteria includes an order, the Mariprofundales, containing a single family, the Mariprofundaceae with a single genus Mariprofundus corresponding to microaerophilic chemolithotrophic curved rods, using iron as electron donor, with filamentous structures containing iron oxyhydroxide. One species Mariprofundus ferrooxydans was isolated from iron-rich microbial mats near deep hydrothermal vents of the Pacific Ocean.

6.2.4.2 The Phylum Bacteroidetes

It is also an important phylum of Gram-negative bacteria that includes aerobic and anaerobic heterotrophic bacteria living in soil or in water bodies and aquatic sediments. It is often called CFB group (Cytophaga-Flavobacterium-Bacteroides). It is divided into three classes.

Bacteroidia class with order Bacteroidales which contains four families corresponding to anaerobic bacteria encountered in environments rich in organic matter often from urban waste or fermenting materials, feces, or composted material. Families Bacteroidaceae (Bacteroides, etc.), Rickenellaceae (Rickenella, etc.), Porphyromonadaceae (Porphyromonas), and Prevotellaceae (Prevotella) constitute this order.

Flavobacteria class with Flavobacteriales order includes the families of Flavobacteriaceae (Flavobacterium, Cellulophaga, Polaribacter) of Myroidaceae (Myroides) and Blattabacteriaceae (Blattabacterium) including chemoorganotrophic aerobic or denitrifying bacteria which play an important role in the degradation of organic matter in soils and biodegradation of xenobiotics.

Sphingobacteria class in which the order Sphingobacteriales includes five families of chemoorganotrophic aerobic or denitrifying flexuous filamentous moving or gliding bacteria involved in the degradation of organic pollutants and xenobiotics: the Sphingobacteriaceae (Sphingobacterium), the Saprospiraceae (Saprospira), the Flexibacteaceae (Flexibacter, Cytophaga), the Flammeovirgaceae (Flammeovirga, Flexithrix, etc.), and Crenotrichaceae (Crenothrix, Toxothrix). From an evolutionary point of view, it would be close to the Chlorobi phylum.

6.2.4.3 The Phylum Chlorobi

Class Chlorobia contains one order (Chlorobiales) and a single family (Chlorobiaceae) with several genera (Chlorobium, Chloroherpeton, Pelodyction, etc.). This phylum corresponds to anoxygenic phototrophic green sulfur-oxidizing bacteria, containing bacteriochlorophyll c, d, or e in chlorosomes, structures attached to the inner layer of the cytoplasmic membrane. These bacteria use sulfur compounds as electron donors during anoxygenic photosynthesis (cf. Sect. 3.3.4, Fig. 3.29). From an evolutionary point of view, it would be close to the phylum Bacteroidetes.

6.2.5 The Major Phyla of Gram-Positive Bacteria

6.2.5.1 The Phylum Acidobacteria

It is composed of chemoorganotrophic bacteria commonly found in soils and sediments but of which very few representatives have been grown. Besides order Acidobacteriales represented by a family (Acidobacteriaceae), it contains, in addition to the genus Acidobacterium which corresponds to one of many taxa revealed as a result of direct in situ sequencing programs from soils, homoacetogenic bacteria (Holophaga), and iron-reducing bacteria (Geothrix).

6.2.5.2 The Phylum Chlamydiae

It contains chemoorganotrophic bacteria, obligatory intracellular parasites, and pathogens; it is constituted by one order (the Chlamydiales) containing four families: Chlamydiaceae (Chlamydia) responsible for lungs or urogenital infections, Parachlamydiaceae (Parachlamydia), Simkaniaceae (Simkania), and Waddliaceae (Waddlia). It is close to the phyla Planctomycetes and Verrucomicrobia [super phylum PVC (Planctomycetes/Verrucomicrobia/Chlamydiae)].

6.2.5.3 The Phylum Planctomycetes

This phylum, close to Chlamydiae and Verrucomicrobia (super phylum PVC), is formed by budding bacteria, unicellular or filamentous devoid of peptidoglycan, which was initially classified with Fungi. With the order Planctomycetales and one family Planctomycetaceae (Planctomyces, Gemmata, Pirellula, etc.), it consists of specific bacteria with internal membranes that keep DNA isolated from the cytoplasm. From the representatives of this family, the anaerobic oxidation of ammonia reaction (Anammox) was discovered, a reaction that takes place in a special membrane compartment (anammoxosome, Figs. 14.32 and 14.33).

6.2.5.4 The Phylum Verrucomicrobia

It includes two classes, Verrumicrobiae and Opitutae. The first class, with order Verrucomicrobiales and family Verrucomicrobiaceae (Verrucomicrobium), consists of unusual bacteria abundant in soil microbial communities but of which very few representatives have been isolated. The second class, with two orders, Opitutales and Puniceicoccales, includes Gram-negative chemoorganotrophic cocci, aerobic or fermentative, isolated from marine environments, hot springs, or paddy fields. This phylum would be close to phyla Planctomycetes and Chlamydiae (constituting together the PVC super phylum).

6.2.5.5 The Phylum Spirochaetes

It contains bacteria with a particular cell morphology: where cells are long, flexuous, and much spiraled. They are all motile by means of an axial filament. These are aerobic or anaerobic inhabitants of soils and aquatic environments. The spirochaetes are grouped in a single order, Spirochaetales, divided into three families. The family Spirochaetaceae contains pathogenic bacteria such as the well-known Treponema pallidum (agent of syphilis), Borrelia (relapsing agents transmitted by vectors such as ticks or lice and responsible for Lyme disease, relapsing fever or hemorrhagic fevers in animals and men), and many spirochetes (Spirochaeta, Cristispira) living under aerobic or anaerobic conditions in soils or aquatic sediments (Fig. 6.10f). Serpulinaceae family with genus Serpulina and the family Leptospiraceae with Leptospira consist of strict aerobic bacteria, the two families containing parasitic agents, pathogens of the oral cavity or causing kidney disease, and intestinal or liver infections in animals and man.

6.2.5.6 The Phylum Fibrobacteres

This phylum consists of one order, Fibrobacterales with the family Fibrobacteraceae containing one genus (Fibrobacter) corresponding to anaerobic chemoorganotrophic fermentative bacteria, some of which live in the digestive tract of animals and can degrade cellulose.

6.2.5.7 The Phylum Fusobacteria

Composed of one order, Fusobacteriales, with one family, Fusobacteriaceae, the phylum Fusobacteria contains obligate anaerobic bacteria commonly found in the oral cavity and in the intestines of animals, such as the genera Fusobacterium, Leptotrichia, and Streptobacillus, some of which are exclusively responsible for ENT infections and angina.

6.2.5.8 The Phylum Gemmatimonadetes

It includes a single order, Gemmatimonadales, with one family and one genus (Gemmatimonadaceae: Gemmatimonas) in which one species was isolated from a laboratory reactor, Gemmatimonas aurantiaca. It is a Gram-negative, motile, aerobic, and chemoorganotrophic bacterium that can divide by binary fission or budding.

6.2.5.9 The Phylum Lentisphaerae

In this phylum, the class Lentisphaeria includes two orders, order Lentisphaerales (Lentisphaeraceae family, genus Lentisphaera) with a single marine species isolated in Oregon (Lentisphaera araneosa) composed of Gram-negative spherical cells, chemoorganotrophs, and aerobic cocci, producing a transparent exopolymer, and order Victivallales with a single species corresponding to Victivallis vadensis, Gram-negative chemoorganotrophic anaerobic cocci isolated from feces.

6.2.5.10 The Phylum Dictyoglomi

This phylum (Dictyoglomales order, Dictyoglomaceae family) includes thermophilic bacteria and fermentative obligate anaerobes (genus Dictyoglomus).

6.2.5.11 The Phylum Caldiserica

With the order Caldisericales and the family Caldisericaceae, it comprises a single species, Caldisericum exile, isolated from a hot spring in Japan, consisting of multicellular filaments with a common envelope, motile by a polar flagellum, anaerobic and chemoheterotrophic, realizing anaerobic respirations with sulfur compounds such as thiosulfate, sulfite, and elemental sulfur as electron acceptors.

6.2.5.12 The Phylum Elusimicrobia

This phylum (order of Elusimicrobiales, family of Elusimicrobiaceae) comprises a single species in a single genus Elusimicrobium corresponding to pleomorphic rods, nonmotile, Gram-negative, anaerobic, fermentative, isolated from the intestinal tract of the larva Pachnoda ephippiata which feeds on humus.

6.2.5.13 The Phylum Armatimonadetes

This phylum includes two classes. Class Armatimonadia (order Armatimonadales, family Armatimonadaceae) consists of one genus, Armatimonas, formed of sticks to ovoid cells, nonmotile, Gram-negative, aerobic, chemoorganotrophic, isolated from the rhizoplane of an aquatic plant, Phragmites australis, in a freshwater lake in Japan. Class Chthonomonadetes (order Chthonomonadales, family Chthonomonadaceae) includes a genus, Chthonomona, corresponding to Gram-negative, aerobic, chemoorganotrophs, moderately thermophilic and acidophilic isolated from geothermal soils in New Zealand.

6.2.6 The Major Phyla of Gram-Positive Bacteria

6.2.6.1 The Phylum Firmicutes

It corresponds to the Gram-positive bacteria with low G + C% (GC% DNA in <50 %), but there are some exceptions with high G + C percentages (e.g., Symbiobacterium with a GC% as high as 69 %) (Fig. 6.3). The characteristic of having a thick peptidoglycan wall which gives its name to the taxon is shared with the Actinobacteria, which had resulted in many authors grouping into a category called “Gram-positive,” a position which was reexamined on sequence analysis of many genes.

This group of microorganisms has a striking character, which is its ability to produce heat-resistant spores. Consisting of aerobic or anaerobic bacteria, it includes well-known taxa such as Bacillus subtilis, the model soil organism, or Bacillus anthracis, responsible of anthrax which has earned it a great reputation since biological weapons programs were announced in the 1990s. This group also contains bacteria that produce toxins such as the highly virulent Clostridium botulinum, the botulism agent that also contains microorganisms used in several biological weapons programs, or Clostridium tetani, the tetanus agent. In this group is also found Desulfotomaculum, a spore-forming sulfate-reducing bacterium. These spore-forming bacteria are found in two classes that constitute the phylum.

The class Clostridia contains three orders of chemoorganotrophic anaerobic fermentative bacteria. The order Clostridiales is composed of eight families. The family Clostridiaceae contains the genera Clostridium and Acetonoma (Fig. 6.15b, c), Sporobacter (Fig. 6.15i), Anaerobacter, etc. These are bacteria that live in soil and anoxic sediments, manures, and composts where they carry out fermentations. Among Clostridium, spore-forming bacteria, some are pathogens, producing toxins (botulism, tetanus). The families Lachnospiraceae (Anaerophilum, Butyrivibrio, Coprococcus), Peptostreptococcaceae (Peptostreptococcus), Eubacteriaceae (Eubacterium), and Peptococcaceae (Peptococcus, Desulfotomaculum (Fig. 6.15f), etc.) involve bacteria, cocci, or rods, obligately fermentative or sulfate-reducing bacteria, living in sediments, manure, anoxic sludges of organic-rich wastewater treatment plants, and feces. The family Heliobacteriaceae (Heliobacterium, Heliobacillus) corresponds to the phototrophic bacteria with bacteriochlorophyll g performing anoxygenic photosynthesis in photoorganotrophy (cf. Sect. 3.3.4). The families Acidaminococcaceae (Acidaminococcus, Sporomusa) and Syntrophomonadaceae (Syntrophomonas, Anaerobaculum) contain anaerobic cocci or rods developed by fermentation or syntrophy in the rumen or intestinal flora as well as in fermenters or anoxic organic sediments. The order Thermoanaerobacteriales with the family Thermoanaerobacteriaceae (Thermoanaerobacterium; Thermoanaerobacter, Fig. 6.15d) and the order Haloanaerobiales with the families Haloanaerobiaceae (Haloanaerobium, Fig. 6.15g; Halothermothrix, Fig. 6.15l) and Halobacteroidaceae (Halobacteroides, Haloanaerobacter) group fermentative bacteria living in sediments, or isolated from thermophilic or halophilic composts or fermenters, some of the bacteria being moderate halophiles or hyperhalophiles (Haloanaerobiales).

Fig. 6.15

Photomicrographs of Gram-positive bacteria. – Phylum Firmicutes (a) Bacillus sp. (Photograph: Pierre Caumette); (b) Clostridium mayombei (Photograph: courtesy of Alain Brauman); (c) Acetonema longum (Photograph: Jean-Luc Cayol); (f) Desulfotomaculum acetoxidans containing spores and gas vesicles (Photograph: courtesy of Friedrich Widdel, MP Institute of Microbiology, Bremen, Germany); (g) Haloanaerobium sp. (Photograph: Jean-Luc Cayol); (h) Fusibacter paucivorans (Photograph: Jean-Luc Cayol); (i) Sporobacter termitidis (Photograph: Jean-Luc Cayol); (j) Thermoanaerobacter brockii (Photograph: Jean-Luc Cayol); (k) Thermohalobacter berrensis (Photograph: Jean-Luc Cayol); (l) Halothermothrix orenii (Photograph: Jean-Luc Cayol). – Phylum Actinobacteria (d, e): Frankia sp. (Photograph: Philippe Normand). The bars represent 10 μm except for figures g, h, and i where they are 1 μm

The Bacilli class includes two orders.

The Bacillales order which is composed of nine families of chemoorganotrophic aerobic or facultative anaerobic cocci or rods with a fermentative and respiratory metabolism. Family Bacillaceae (genera Bacillus, Fig. 6.15, or Saccharococcus) comprises spore-forming bacilli abundant in soils and waters, ubiquitous due to the dissemination of their spores, and some being pathogens such as Bacillus anthracis (anthrax). Families Planococcaceae (Planococcus) and Staphylococcaceae (Staphylococcus, Macrococcus) involve aerobic or facultative anaerobic cocci isolated from soil, air, or water, some of which may be pathogenic of mucosa, skin, or responsible for poisoning (staphylococcal enteropathogenic Staphylococcus aureus). Families Caryophanaceae (Caryophanon), Paenibacillaceae (Paenibacillus), Alicyclobacillaceae (Alicyclobacillus, Pasteuria), Thermoactinomycetaceae (Thermoactinomyces), and Sporolactobacillaceae (Sporolactobacillus) group aerobic or fermentative bacteria isolated from soil and water sometimes thermophiles. The family Listeriaceae (Listeria) contains bacteria common in soil and water with a species (Listeria monocytogenes) which can be pathogenic and is responsible for food poisoning especially in dairy products.

The order Lactobacillales consisting of six family includes lactic acid bacteria and many cocci. The family Lactobacillaceae (Lactobacillus, Pediococcus) corresponds to chemoorganotrophic aerotolerant and fermentative lactic acid bacteria such as Lactobacillus acidophilus and Lactobacillus bulgaricus used in the manufacture of yogurt. The families Aerococcaceae (Aerococcus), Carnobacteriaceae (Carnobacterium), and Enterococcaceae (Enterococcus) include a majority of aerobic or anaerobic fermentative cocci, isolated from soil and water; some may reduce nitrates, iron, or manganese. The family Leuconostocaceae concerns aerotolerant fermentative or aerobic cocci used in the food industry of lactic fermentation such as Leuconostoc and Oenococcus, this latter being used in malolactic fermentation of grapes juice (winemaking) or cabbage (sauerkraut). The family Streptococcaceae corresponds to the genera Streptococcus with certain pathogens such as hemolytic streptococci (Streptococcus pyogenes) and Lactococcus, lactic acid fermentation bacteria.

6.2.6.2 The Phylum Tenericutes

This phylum, formerly grouped with Firmicutes was elevated to the rank of phylum in the second edition of Bergey’s Manual of Systematic Bacteriology. It contains mycoplasma bacteria that are devoid of peptidoglycan but derive from Gram-positive bacteria while reacting negatively to this test. They are grouped into a single class, the class Mollicutes, including Mycoplasma genitalium, pathogen of animals and man, and Spiroplasma citri, citrus trees pathogen. This class Mollicutes is structured in five orders. The order Mycoplasmatales with the family Mycoplasmataceae (Mycoplasma), the order Entomoplasmatales with the families Entomoplasmataceae (Entomoplasma) and Spiroplasmataceae (Spiroplasma), the order Acholeplasmatales with the family Acholeplasmataceae (Acholeplasma), the order Anaeroplasmatales with the family Anaeroplasmataceae (Anaeroplasma), this family concerning anaerobic mycoplasma, and the order Haloplasmatales with the genus Haloplasma (halophilic mycoplasma). Mycoplasmas are obligate parasites of plants or animals and thus often pathogens in many infections (pulmonary or urogenital animals and man, leaf necrosis and degeneration in plants). A genus Incertae sedis* in the family Erysipelotrichaceae the Erysipelothrix genus is also present in this class Mollicutes, whereas it has a wall normal for Gram-positive bacteria. It is a bacterial pathogen of humans and animals (agent of swine erysipelas, zoonosis transmitted to man). This group has, in addition to unusual morphology (no wall), a high mutation rate (Dybvig and Voelker 1996).

6.2.6.3 The Phylum Actinobacteria

It concerns Gram-positive bacteria with high G + C% (% by GC in the DNA > usually 50 %) (Fig. 6.3). This group of microorganisms has as most striking character varied morphologies ranging from unicellular organisms to irregular shapes consisting of mycelial branched hyphae. This character, also found in Fungi, has been a source of confusion for years when taxonomy was not based on molecular sequences. It is now recognized unequivocally that this is an instance of convergent evolution with Fungi. Actinobacteria, especially the most famous of them, Streptomyces coelicolor, and related genera (Actinomyces, Nocardia) producing structures called conidiophores useful for dissemination. Actinobacteria are dominant in the soil where their dissemination structures (conidiophores and hyphal cells) give them a competitive advantage. They also have the ability to synthesize antibiotic compounds, some of which are used as important drugs in therapy of infectious diseases, and they also confer a competitive advantage vis-à-vis other soil bacteria (cf. Sect. 9.5.1). This phylum contains a class (Actinobacteria) which is composed of five subclasses (the Acidimicrobidae, the Rubrobacteridae, the Coriobacteridae, the Sphaerobacteridae, and the Actinobacteridae).

The first four subclasses contain very few species, often with only one representative and only one family. The Acidimicrobidae with the order Acidimicrobiales and the family Acidimicrobiaceae (Acidimicrobium), the Rubrobacteridae with the order Rubrobacterales and the family Rubrobacteraceae (Rubrobacter), the Coriobacteridae with the order Coriobacteriales which contains a single family and several Coriobacteriaceae genera (Coriobacterium, Atopobium, Cryptobacterium), and with the Sphaerobacteridae with the order Sphaerobacterales containing a family Sphaerobacteraceae (Sphaerobacter) correspond to aerobic Gram-positive facultative anaerobic or aerobic, with stick-shaped, more or less regular, isolated from soils or aquatic sediments, and which perform aerobic or nitrate respiration or fermentation.

The fifth subclass (Actinobacteridae) contains most of the bacteria of the phylum and is divided into two orders (Actinomycetales and Bifidobacteriales) containing numerous suborders and many families.

The order Actinomycetales comprises 10 suborders under which are grouped most of the bacteria of the phylum Actinobacteria. The suborder Actinomycinae with family Actinomycetaceae (Actinomyces, Actinobaculum) contains filamentous, branched bacteria, which are aerobic, chemoorganotrophic, producing antibiotics, colonizing the surface soil, sediment, or decaying leaves in the litter of forests. The suborder Micrococcinae includes several families with a Gram-positive cocci or irregularly shaped cells or sticks depending on the age of the culture: the family Micrococcaceae (Micrococcus, Leucobacter, Arthrobacter, etc.) consists of aerobic bacteria isolated from soil and freshwater or coastal or in the oral cavity (Stomatococcus), the Brevibacteriaceae (Brevibacterium) involve irregularly shaped bacteria, isolated from soils rich in organic matter, and the Cellulomonadaceae (Cellulomonas) include aerobic and fermentative bacteria capable of degrading cellulose in soils; families Dermabacteraceae (Dermabacter), Dermatophilaceae (Dermatophilus), Intrasporangiaceae (Intrasporangium), Jonesiaceae (Jonesia), Microbacteriaceae (Microbacterium, Agromyces, etc.), Beutenbergiaceae (Beutenbergia), and Promicromonosporaceae (Promicromonospora) correspond to different soil isolated bacteria, irregularly shaped or filamentous, important in the degradation of organic matter including macromolecules and polymers. The suborder Corynebacterineae includes several soil bacteria, many of which are pathogens: Corynebacteriaceae family (Corynebacterium) with Corynebacterium diphtheriae (diphtheria agent) and Corynebacterium glutamicum, a microorganism used in biotechnology for the synthesis of many molecules; it also contains other corynebacteria, organisms commonly found in soil and water, capable of degrading macromolecules of some xenobiotics; the family Mycobacteriaceae with the genus Mycobacterium consists of microorganisms of soil and water, some of which are pathogens such as Mycobacterium tuberculosis, the agent of tuberculosis, the bacterial disease that causes the greatest number of deaths worldwide, and Mycobacterium leprae, the agent of leprosy; these bacteria are characterized by their resistance to environmental factors that gives them their envelopes made of waxy mycolic acids. Family Nocardiaceae with Nocardia and Rhodococcus consists of chemoorganotrophic bacteria of soil and aquatic sediments, active in the biodegradation of organic pollutants in soils. Some Nocardia (N. asteroides, N. cyriacigeorgica) are pathogens of animals, and finally, families Dietziaceae, Gordoniaceae, Tsukamurellaceae, and Williamsiaceae contain heterotrophic and saprophytic irregularly shaped soil bacteria forming branches and buds. The suborder Micromonosporineae with a single family (Micromonosporaceae) includes nine types of bacteria in soil and aquatic environments, chemoorganotrophic, some of which are capable of producing spores (Micromonospora). They are irregularly shaped bacteria with buds that develop into filamentous mycelium (Actinoplanes) and produce spores inside a sporangium (Dactylosporangium). The suborder Propionibacterineae includes two families: Propionibacteriaceae (Propionibacterium, Luteococcus, Propioniferax) gathering aerotolerant or anaerobic bacteria, producing lactic acid fermentation, producing propionic acid (flavor of some cheeses), used in the dairy industry. Some species may be pathogenic, while others are inhabitants of the digestive tract and contribute to the intestinal flora. Nocardioidaceae (Nocardioides) bacteria are chemoorganotrophic, filamentous soil saprophytes, and some are able to degrade hydrocarbons. The suborder Pseudonocardineae with two families Pseudonocardiaceae (Pseudonocardia) and Actinosynnemataceae (Actinosynnema) group heterotrophic saprophytic filamentous soil bacteria. The suborder Streptomycineae with the family Streptomycetaceae including the genus Streptomyces contains chemoorganotrophic filamentous soil saprophytic bacteria. These bacteria form branched filaments, producing hyphae, and develop aerial mycelia with sporophores (bodies containing spores that are later released), which led to a confusion with the microscopic Fungi for a long time. These bacteria produce antibiotics and are the source of the majority of known antibiotics. There are also pathogenic species of plants, especially potato (S. scabies). The suborder Streptosporangineae with three families (Streptosporangiaceae, Nocardiopsaceae, Thermomonosporaceae) groups filamentous bacteria producing sporangia and containing spores which are close to the Streptomycetaceae. The suborder Frankineae is divided into six families. The family Frankiaceae with the genus Frankia (Fig. 6.15d, e) is a filamentous bacteria of the soil, often in symbiotic association with plants such as Frankia alni (symbiont of alder roots; Normand and Lalonde 1982). Families Acidothermaceae, Cryptosporangiaceae, Geodermatophilaceae, Nakamurellaceae, and Sporichaetaceae also contain heterotrophic saprophytic soil bacteria, forming mycelia. Some of these bacteria have special habitats such as Acidothermus found in hot springs in Yellowstone (the USA) or Geodermatophilus and Modestobacter found in irradiated soils. The suborder Glycomycineae with the genus Glycomyces also corresponds to mycelial saprophytic soil bacteria.

The order Bifidobacteriales with a family (Bifidobacteriaceae) includes Bifidobacterium bifidus, a bacterium of the digestive tract used as an additive in some yogurts.

7 Conclusion

Microorganisms have been on earth for billions of years (cf. Chap. 4) and have colonized almost all ecological niches, even those created by humans in recent decades. They have a very important adaptive potential. If a science fiction apocalyptic scenario such as that initiated by a meteorite impact or a massive volcanic episode of the kind that produced the Deccan Traps were to occur and eliminate a large proportion of taxa living on earth today, it is likely that life would not completely disappear because many microorganisms would survive and reinitiate evolutionary branches.

The emergence of new pathogens always surprises, either as a new genus Legionella in 1976 or as a particularly virulent new strain of a well-known species such as E. coli strain O157: H7 in 1985. Many environments are still poorly known, and probably only 1–10 % of prokaryotes have been described as of 2012. We must therefore expect many discoveries of new species in partly known environments including manmade and natural extreme environments, in particular, the deep subsurface environment and possibly in extraterrestrial environments that are being explored by metagenomic approaches (cf. Chap. 18). Moreover, it is clear that with many emerging pathogens and the accumulation of xenobiotic compounds in various environmental pollutants that cause the rapid evolution of new pathways of biodegradation, the world of prokaryotic microorganisms is in constant evolution. Consequently, their taxonomy cannot be frozen, and microbial taxonomists must describe a fluid world, with rapid modifications.