Oligoflexia, the Newest Class of the Phylum Proteobacteria, Consisting of only One Cultured Species and Uncultured Bacterial Phylotypes from Diverse Habitats

The phylum Proteobacteria has recently gained a new taxon Oligoflexia that represents the seventh or eighth (if yet-to-be-validated “Zetaproteobacteria” is included) class, described by the only cultured species (as of December 2014), Oligoflexus tunisiensis, as the type species. This bacterium exhibits cellular polymorphism and presence of the uncommon fatty acid C16: 1ω5c as high as >65% of total fatty acids, besides its unique 16S rRNA gene sequence. The class Oligoflexia is characterized by the distinct phylogenetic cluster within the greater proteobacterial cluster, and certain environmentally-derived 16S rRNA gene sequences, a.k.a. environmental clones or phylotypes, of uncultured bacteria are now grouped into the Oligoflexia cluster; however, the content and extent of the cluster has not been clearly depicted. This mini-review illustrates that the Oligoflexia cluster hosts a variety of environmental clones from diverse sources. Currently 20 phylotypes (or clones) are affiliated with the Oligoflexia cluster, and the sources were ranging from soils to cyanobacterial mat, bio-filter, human skin, ant colony, desert, glacial ice, earthworm intestine, and seawater. However, their frequencies in respective clone libraries were generally as low as <1%, which indicates their corresponding species are only minor in respective microbial communities. Moreover, 61 environmental metagenome libraries yielded only 1198 partial sequences having >85% similarities (class-level affiliation) to the 16S rRNA gene sequence of O. tunisiensis, which accounts for merely 0.04% of those registered in Meta-Metagenomic Data Base (MetaMeta DB). On the other hand, >97%-similarities sequences were found in rhizosphere, and <95%-similar sequences were found from hydrocarbon-rich habitats such as petroleum reservoir. Thus, it is suggested that members of Oligoflexia may display cosmopolite distribution in general as well as endemism in certain geochemical settings. *Corresponding author: Takeshi Naganuma, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, Hiroshima 739-8528, Japan, Tel: 81-82-424-7986; Fax: 81-82-424-7916; E-mail: takn@ hiroshima-u.ac.jp Received November 17, 2014; Accepted December 17, 2014; Published December 20, 2014 Citation: Nakai R, Naganuma T (2015) Oligoflexia, the Newest Class of the Phylum Proteobacteria, Consisting of only One Cultured Species and Uncultured Bacterial Phylotypes from Diverse Habitats. J Phylogen Evolution Biol 3: 141. doi:10.4172/2329-9002.1000141 Copyright: © 2015 Nakai R, Naganuma T. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
The largest eubacterial phylum Proteobacteria accommodates about 1600 bacterial species exhibiting extremely diversified morphologies (as expressed in its name as "Proteus"), phylogenies, and metabolisms relevant to geochemical cycling of carbon, nitrogen, sulphur, etc. [1]. The phylum Proteobacteria hosts the greatest number of "culturable" described and deposited species among the prokaryotic phyla [1,2], while the phylum harbors numerous environmentally-derived 16S rRNA gene sequences, a.k.a. environmental clones or phylotypes, of "uncultured" bacteria whose physiological properties are largely unknown [3,4].
The proteobacterial taxa were formerly grouped as "purple bacteria" [5], then re-organized phylogenetically based on 16S rRNA gene sequences into five classes of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria , Deltaproteobacteria and Epsilonproteobacteria [6] and, two more classes were proposed thereafter; they are yetto-be-validated 'Zetaproteobacteria' [7] and already-validated Acidithiobacillia [8] which is a revision of part of the species formerly affiliated with Gammaproteobacteria [8].
We have proposed the seventh or eighth class, if 'Zetaproteobacteria' is excluded or included, respectively, in the phylum Proteobacteria, with the description of the type species Oligoflexus tunisiensis [9] that is the only "cultured" species of the class currently. In proposing the new class, we have constructed a phylogenetic tree of the Oligoflexia cluster consisting of a number of environmental clones of uncultured bacteria from various sources. Thus, this mini-review discusses their environmental and geographical features as well as some unique properties of O. tunisiensis.

Unique morphological and phenotypic traits of O. tunisiensis
Sterile filtration has been a common technique to remove microorganisms from fluids in pharmaceutical and food industries as well as in many areas of biology. For sterilization purposes, membrane filters having a pore size of 0.2 µm are generally used. Nevertheless, not all bacteria are trapped with the filters; certain novel taxa have been found in the 0.2 µm-filtrates [10,11]. Likewise, O. tunisiensis was first obtained from the 0.2 µm-filtrate of liquid suspended with Saharan sand and pebbles [9]. While the cells of O. tunisiensis indeed pass the 0.2 µm-filters, they also show polymorphism with filamentous, spiral, spherical, or curved rod shapes ( Figure 1). Though factors controlling the cell shapes have been unclear, their morphological flexibility and versatility would be associated with the nature to pass through 0.2 µmfilters. Therefore, it is considered that our 0.2 µm-filtration was effective to remove fast-growers and preferably selective for slow-growing species. In addition, O. tunisiensis resumes growth when transferred to fresh agar plates from senile colonies as old as half a year (M. Nishijima, personal communication). Physiological and biochemical properties of O. tunisiensis are listed in Table 1. This bacterium shows mesophilic and neutrophilic growth characteristics with optimum temperature and pH of 25-30°C and 7.0-8.0, respectively, as seen in many other bacterial species. In contrast, this bacterium has unique cellular fatty acid contents. More than 90% of total fatty acids is composed only of two fatty acids, C16: 1ω5c (65.7 %) and C16: 0 (27.5 %), the former being rarely seen in other species [12,13]. It is unclear whether high content of C16: 1ω5c would be general feature of the whole class Oligoflexia; this rare fatty acid could be taken as a biomarker at least of the species O. tunisiensis.
The C16: 1ω5c fatty acid has also been detected in myxobacteria belonging to Deltaproteobacteria but to a lesser content of 15-39% (of total fatty acids) at most [13]. It was proposed to be a possible biomarker of microbial consortia responsible for anaerobic methane oxidation, or ANME, in methane-seeping marine sediments [14]. The ANME consortia are composed of archaea capable of reverse methanogenesis as anaerobic methane oxidizers and sulfate-reducing bacteria belonging to Deltaproteobacteria as potential hydrogendonors in the sediments [15]. About half of the sediment fatty acids is dominated by various C16: 1 acids, most of which is due to C16: 1ω5c probably from bacterial part of the consortia [14]. In O. tunisiensis, C16: 1ω5c solely accounts for 65.7% of total fatty acids, and thus should serve as a useful biomarker.

Not-yet-cultured bacteria within the class Oligoflexia
In the phylogenetic tree of the phylum Proteobacteria depicted in Nakai et al. [9], the class Oligoflexia was shown to harbour environmental clones or phylotypes of uncultured or "not-yet-cultured" bacteria from various sources. Table 2 shows a set of sequence information of 20 phylotypes, which are selected from clone libraries consisting of PCR-amplified near-full-length 16S rRNA gene sequences registered in DDBJ/EMBL/GenBank databases (partly published). Their isolation sources are ranging from soils to cyanobacterial mat, bio-filter, human skin, ant colony, desert, glacial ice, earthworm intestine and seawater ( Table 2). This implicates that the class Oligoflexia hosts a variety of not-yet-cultured bacterial species occurring in wide-ranging habitats.
So far, the closest phylotype to the O. tunisiensis 16S rRNA gene sequence has been TSBAR001_G23 acession no. AB486128; (Table  2) with a similarity of 98.3%. The phylotype was found in a clone library constructed from a Japanese paddy field microflora, and its occurrence frequency was as low as 1/1036, i.e., only one out of total 1036 clones [16]. The paddy field soil was added experimentally with nitrate (in reference to nitrate reduction) and yielded the emerging clone TSNIR001_J18 (AB487112; 16) that is only distantly related to O. tunisiensis at 84.6% similarity but within the Oligoflexia cluster. The emerging clone may have been associated with nitrate reduction. On the other hand, O. tunisiensis shows no nitrate reduction activity with a phenotypic test using API 20NE kit (bioMérieux) [9]. Nonetheless, involvement of the Oligoflexia members in nitrate cycling should be surveyed.
In contrast to their wide-ranging sources, Oligoflexia-affiliated clones are rarely found in many clone libraries. For example, no Oligoflexia clones are seen in a soil clone library (consisting of 1700 clones of PCR-amplified near-full-length 16S rRNA gene sequences), 32% of which are affiliated with the phylum Proteobacteria [17]. Another soil clone library (consisting of 13,001 clones) focusing on "rare members of the soil biosphere" yielded no Oligoflexia clones, either [18]. Thus, Oligoflexia-affiliated bacteria are likely very minor in soils, and their biogeochemical importance may be low accordingly. Yet, quantitative techniques such as real-time PCR and cell counting with fluorescence in situ hybridization are needed to quantify the occurrences of Oligoflexia-affiliated bacteria in environments.
There have been six phylotypes so far that show >97% similarities with the O. tunisiensis 16S rRNA gene sequence (Table 2). They are second to seventh closest to and presumably regarded as the same species as O. tunisiensis. The second to fifth closest phylotypes are from a lacustrine cyanobacterial mat in China and a microalgal photobioreactor in Germany; the latter yielded an occurrence frequency of 3/171 [19], which suggests co-occurrence relationship between O. tunisiensis and phototrophs. The sixth and seventh closest phylotypes were obtained from a bio-filter in China and from American children's knee-pit skin, respectively. Combining with the phylotypes to the tenth closest, occurrence of the O. tunisiensis kins extend to wider habitats ( Table 2).
Previous affiliations of certain clones were revised to be members of the class Oligoflexia. The clones' glb351c (EU978839) and glb342c (EU978830) were obtained from glacial ice if the Northern Schneeferner in Germany, and originally affiliated with the classes Deltaproteobacteria and Betaproteobacteria, respectively [20]: the glacial phylotypes are now re-affiliated with the class Oligoflexia [9]. Although their similarities are as only 89.8% and 87.1%, respectively, to the O. tunisiensis 16S rRNA gene sequence ( Table 2), but they are still placed within the Oligoflexia cluster.
The original clone library of the glacial clones consisted of 338 near-full-length clones, dominated by Proteobacteria phylotypes at a frequency of 190/338, most of which were affiliated with the class Betaproteobacteria [20]. The Betaproteobacteria dominance has commonly been evident in other microflorae of cold habitats such as glacial ice [21], alpine snow [22] and sub-glacial sediment [23]. In contrast, no phylotypes were affiliated to Oligoflexia from these cold habitats (Table 2), and only the few clones were seen at a frequency as low as 4/338 of the German glacier clones [20].Additionally, the glacier phylotype glb351c and the earthworm intestine clone A02-05D (FJ542822) [24] show a 97.0% similarity and form a small but monophyletic cluster [9]. This close relationship leads to an idea that the phylotypes may not be specific to cold habitats.
The MetaMetaDB also provides a "habitability index" (Figure 2) to infer possible habitats of a queried sequence or a "query" and its related sequences [28] based on the BLASTN search [29]. The result suggested that the >97%-similar sequences (to the O. tunisiensis 16S rRNA gene sequence) were likely from rhizosphere, while <95%-similar sequences were mostly from underground habitats such as groundwater and hydrocarbon (e.g. petroleum and gas) reservoir. Figure 2 illustrates "habitability indices" as percentages for source habitats of the matched sequences. Certain geographical tendencies of the source habitats are shown, despite phylogenetic limitation due to short lengths (less than 300-500 bp) of the sequences generated by 454-pyrosequencing.
In summary, general features of the phylotypes (clones) and partial sequences affiliated with the class Oligoflexia are: 1) that they are derived from a variety of source habitats, suggestive of cosmopolite distribution; and 2) that they are minor in occurrence frequencies, possibly associated with the slow-growing nature of the type species, O. tunisiensis. An emerging character is "cosmopolite but minor" or "minor cosmopolitan", which should be tested with other Oligoflexia species to be cultured in future studies. Figure 2: Inferred source habitats for partial 16S rRNA gene sequences of notyet-cultured bacteria potentially affiliated with the class Oligoflexia The microbial habitability indices (y-axis) calculated using MetaMetaDB were based on a set of normalized ratios of environmental categories among the significant hits exhibiting greater than 97%, 95%, 90%, and 85% identity from the BLASTN search with the 16S rRNA gene sequence of Oligoflexus tunisiensis (accession no. AB540021) against curated metagenomic sequences in MetaMetaDB.