Review of the taxonomy of the genus Arthrobacter , emendation of the genus Arthrobacter sensu lato , proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen . nov . , Paeniglutamicibacter gen . nov . , Pseudoglutamicibacter gen . nov . , Paenarthroba

IP: 54.70.40.11 On: Wed, 12 Jun 2019 21:00:27 Review Review of the taxonomy of the genus Arthrobacter, emendation of the genus Arthrobacter sensu lato, proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., Pseudoglutamicibacter gen. nov., Paenarthrobacter gen. nov. and Pseudarthrobacter gen. nov., and emended description of Arthrobacter roseus

The genus Arthrobacter was proposed by Conn & Dimmick (1947) to encompass three species, including the type species of the genus, 'Arthrobacter globiforme'.The type species was later renamed as Arthrobacter globiformis (Skerman et al., 1980); the second species, Arthrobacter tumescens, was reclassified in another genus (Collins et al., 1989;Manaia et al., 2004), and the third species, 'Arthrobacter helvolum', was never mentioned again in a taxonomic paper.It has to be emphasized here that it was not reclassified as 'Pseudoclavibacter helvolum', as stated by Busse et al. (2012).Originally, the genus was described as follows: Arthrobacter Fischer, emend.
Morphology.Varied, with a tendency to go through a more or less definite life cycle, the most characteristic features of which are Gram-negative rods in young cultures and Gram-positive coccoid forms (arthrospores?) in old cultures, with intermediate stages that may be clubs, branched forms, or short unbranched filaments.Large (1 to 2 m) spherical bodies are sometimes observed which have been termed 'cystites'.
Cultural characteristics.Growth on surface of solid media soft and smooth, not dry and wrinkled or hard and leathery, as ordinarily in Mycobacterium and the Actinomycetoceae.Colonies on poured plates ordinarily small (punctiform).Growth in broth usually slow and never profuse.glucose and sometimes other sugars as sources of carbon and energy, but ordinarily without producing sufficient quantities of acid to have appreciable effect on the pH of highly buffered media (e.g. containing peptone).Gelatin usually slowly liquefied.Ordinarily cause blackening of Mueller's tellurite agar.
Concerning the description of the genus Arthrobacter, the Approved Lists of Bacterial Names (Skerman et al., 1980) refer to Keddie (1974), which concentrates on cultural, morphological and physiological characteristics.This description has been the basis for the classification of numerous novel species of the genus Arthrobacter: Cells which in complex medium undergo a marked change in form during the growth cycle.Older cultures (generally 2-7 days) are composed entirely or largely of coccoid cells.In some strains the coccoid cells are uniform in size and spherical, and resemble micrococci; in others they are spherical to ovoid or slightly elongate.In some cultures larger coccoid cells some 2-4 times the size of the remainder may occur; these may predominate under some cultural conditions.On transfer to fresh complex medium growth occurs by enlargement (swelling) of the coccoid cells followed by elongation from one or occasionally two parts of the cell to give rods which usually have a diameter less than that of the enlarged coccoid cell (referred to a figure).In the larger coccoid cells outgrowth may occur at two, three or rarely four parts of the cell (referred to a figure).In both cases subsequent growth and division give rise to irregular rods which vary considerably in size and shape and include straight, bent and curved, wedge-shaped and clubshaped forms (referred to a figure).A proportion of the rods are arranged at an angle to each other giving V formations but more complex angular arrangements often occur.Post-fission outgrowths, usually from the proximal ends of one or both cells of a pair of rods (referred to a figure), and bud-like outgrowths from segments of septate rods (referred to a figure), especially in richer media, may give the appearance of rudimentary branching but true mycelia are not formed.As the exponential phase proceeds the rods become shorter and are eventually replaced by the coccoid cells characteristic of stationary phase cultures (referred to a figure).The coccoid cells are formed either by a gradual shortening of the rods at each successive division or, especially in richer media, by multiple fragmentation of larger rods (referred to a figure).The rods are nonmotile or motile by one subpolar or a few lateral fla-International Journal of Systematic and Evolutionary Microbiology 66 Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Mon, 04 Feb 2019 19:10:17 2012), Arthrobacter cupressi (Zhang et al., 2012), Arthrobacter siccitolerans (SantaCruz-Calvo et al., 2013) and Arthrobacter gyeryongensis (Hoang et al., 2014).However, none of these descriptions considered the phylogenetic and chemotaxonomic heterogeneities found among species of the genus Arthrobacter that have been known for some decades (Fiedler, 1971;Schleifer & Kandler, 1972;Stackebrandt et al., 1983Stackebrandt et al., , 1988;;Collins et al., 1982b;Collins & Kroppenstedt, 1983;Koch et al., 1994).More often than not, novel species were described as members of the genus only when an established species of the genus Arthrobacter was identified as the nearest relative, the peptidoglycan was shown to contain the diagnostic diamino acid lysine and a rod-coccus life cycle was observed.
At the time of writing, the genus Arthrobacter contains 70 species, including Arthrobacter viscosus, which is obviously misnamed, because it shares the highest 16S rRNA gene sequence similarity with members of the genus Rhizobium such as Rhizobium gallicum (98.1 % 16S rRNA gene sequence similarity to the type strain), Rhizobium mongolense (98.0 %) and Rhizobium leguminosarum (97.6 %) (Heyrman et al., 2005).This affiliation is in agreement with chemotaxonomic traits of this species, including its quinone system (ubiquinone Q-10 predominant), fatty acid profile (hydroxy fatty acids present, C 18 : 1 and C 19 : 0 cyclo predominant) and a polar lipid profile that suggests the presence of phosphatidylethanolamine (PE), monomethylethanolamine, phosphatidylglycerol (PG), diphosphatidylglycerol (DPG) and phosphatidylcholine (Collins, 1986).Phylogenetic analyses employing the neighbour-joining algorithm and embracing the vast majority of species of the genus Arthrobacter (Ding et al., 2009;Ganzert et al., 2011;Yassin et al., 2011b;Margesin et al., 2012) do not indicate a stable internal structure of the genus.This is indicated by the fact that many branching nodes are not supported by high bootstrap values (.70 %) and numerous nodes were not found with the maximumlikelihood or maximum-parsimony algorithm.Furthermore, certain species occupy varying positions in different trees, including A. crystallopoietes, Arthrobacter nasiphocae, Arthrobacter roseus, Arthrobacter russicus, Arthrobacter sanguinis and Arthrobacter woluwensis.However, it is worth mentioning that the latter species always occupies a rather deeply branching subline in all trees, often loosely associated with the 'Arthrobacter sulfureus group' (Fig. 1).When representatives of additional members of the family Micrococcaceae are included in tree reconstructions in addition to species of the genus Arthrobacter (Busse et al., 2012), the phylogenetic relationships become more unclear.In this maximum-likelihood tree (Busse et al., 2012), several representatives of other genera branch within the radiation of established species of the genus Arthrobacter, indicating that the genus is not monophyletic.
In order to examine the phylogenetic arrangements within the genus Arthrobacter, including the most recently described species, 16S rRNA gene sequences were extracted from gene banks, multiply aligned using CLUSTAL_X (Thompson et al., 1997) and edited manually to remove gaps and ambiguous nucleotides using BioEdit (Hall, 1999).Phylogenetic calculations were carried out applying the maximum-likelihood, maximum-parsimony and neighbour-joining algorithms implemented in the PHYLIP package (Felsenstein, 2009).Confidence levels of branchings were determined by bootstrap analysis implemented in the PHYLIP package.The results from phylogenetic calculations carried out in the present study, including all established species of the genus Arthrobacter and the type species of other members of the family Micrococcaceae, applying the maximum-likelihood algorithm, are shown in Fig. 1.In this tree, only the branching of a few major lines is supported by high bootstrap values or by the same branching order in the maximum-parsimony and neighbour-joining algorithms.One of these sublines is designated the 'Arthrobacter protophormiae group', comprising the species Arthrobacter creatinolyticus, Arthrobacter soli, A. protophormiae, A. uratoxydans, A. nicotianae, Arthrobacter arilaitensis, A. mysorens, Arthrobacter bergerei and Arthrobacter ardleyensis.Within this subline, close relatedness is suggested from a relatively high bootstrap value between A. ardleyensis and A. bergerei.The second subline, designated the 'Arthrobacter psychrolactophilus group', embraces the species Arthrobacter alpinus, A. cryoconiti, Arthrobacter psychrochitiniphilus, Arthrobacter livingstonensis, Arthrobacter stackebrandtii and Arthrobacter psychrolactophilus, and several internal nodes are supported by high bootstrap values.The third subline is designated the 'Arthrobacter agilis group' and contains the species Arthrobacter flavus, A. agilis, Arthrobacter subterraneus, Arthrobacter tecti, Arthrobacter tumbae and Arthrobacter parietis.High bootstrap support suggests that the latter four species form the stable core of the group.A fourth subline comprises A. citreus, Arthrobacter luteolus, Arthrobacter koreensis and Arthrobacter gandavensis.A deeply branching subline designated the 'Arthrobacter albus/cumminsii group' comprises the species Arthrobacter albus and Arthrobacter cumminsii.A rather stable branching position within the tree suggests a close relatedness between the pairs of species Arthrobacter niigatensis/Arthrobacter defluvii, Arthrobacter gangotriensis/Arthrobacter antarcticus, Arthrobacter castelli/Arthrobacter pigmenti and Arthrobacter oryzae/Arthrobacter humicola.It has to be mentioned here that the latter two species can be considered to be the nearest relatives of A. pascens and A. globiformis (the type species of the genus), with which they also share .98 % 16S rRNA gene sequence similarity, and the common branching node is also found with a second treeing algorithm (Fig. 1).The representatives of the 'Arthrobacter pigmenti group', the 'Arthrobacter citreus group' and the 'A.albus/cumminsii group' and A. russicus, A. nasiphocae and A. crystallopoietes are most distantly related to the type species of the genus and, apparently, more closely related to other members of the family Micrococcaceae.Rather separate positions within the tree are occupied by A. woluwensis and A. sanguinis.The existence of these major sublines is also shown in other phylogenetic analyses, even though the complete set of species of the genus Arthrobacter was not always included in these calculations (Ganzert et al., 2011;Busse et al., 2012;Zhang et al., 2012).
In a study on the diversity of isolates of the genus Arthrobacter from terrestrial deep-subsurface sediments, van Waasbergen et al. (2000) also investigated the recA phylogeny of their isolates in relation to 12 established species of the genus.On the basis of a fragment of the recA gene (360 bases), they showed that the reference species were separated into at least six groups.These authors could also show that the 16S rRNA gene and recA phylogenies were well in accordance with each other.Reanalysis of the recA-based phylogeny including sequences of type strains of species of the genus Arthrobacter from van Waasbergen et al. (2000) supplemented with additional sequences since available from gene banks supported the earlier study (Fig. 2), and is also in agreement with the 16S rRNA gene-based Chemotaxonomically, members of the genus Arthrobacter are rather diverse with respect to peptidoglycan structure, and differences in the quinone system and polar lipid profiles are also observed.
All species of Arthrobacter analysed contain the diagnostic diamino acid lysine, and the peptidoglycan type of arthrobacters is either A3a (only monocarboxylic amino acids present in the interpeptide chain) or A4a (dicarboxylic amino acids present in the interpeptide chain), as defined by Schleifer & Kandler (1972).However, additional differences occur in the presence of amino acids in the crosslinking interpeptide chain of both peptidoglycan types A3a and A4a (Table 1).To some extent, these amino acid compositions reflect relatedness as indicated by 16S rRNA gene sequence analyses.Where analysed, the amino acids of the interpeptide chain usually occur in the L-form.Among species that show the A3a peptidoglycan type, the interpeptide chain is composed of either Lys-Ala 1-4 , Lys-Ala-Thr-Ala, Lys-Ser-Thr-Ala, Lys-Thr-Ala 1-3 , Lys-Ala-Ser-Ala 3 (only found in species of Arthrobacter reclassified in the genus Sinomonas and in Arthrobacter

Overview of groupings within the genus Arthrobacter
In the descriptions of the quinone system, MK-8, MK-9 and MK-10 indicate a menaquinone with eight, nine and ten isoprenoic units in the side chain and (H 2 ) indicates that one isoprenoic unit is dihydrogenated.(Kallimanis et al., 2009).Combinations of completely unsaturated and monosaturated quinones are well known among the class Actinobacteria, but they usually share the same length of isoprenoic side chain.Hence, there are some reservations about the reproducibility of the quinone systems of these two species.
A fatty acid profile that contains predominantly iso-and anteiso-branched fatty acids with the major compound anteiso-C 15 : 0 is common to all species of Arthrobacter.Usually, relatively large amounts of iso-C 15 : 0 , anteiso-C 17 : 0 and/or iso-C 16 : 0 are also present.Some species may also contain significant amounts of the straightchain fatty acid C 16 : 0 .Unfortunately, based on the available data, the importance of fatty acid profiles for identification or differentiation of species of Arthrobacter cannot be evaluated.Fatty acid profiles of species of Arthrobacter have been reported that were analysed from biomass that was grown at different temperatures, with different medium compositions and without indication of the physiological age at which the cells were harvested.Dependence of the fatty acid composition in bacteria on cultural conditions and physiological age has been known for a long time, as demonstrated for representatives of different lines of descent, including cyanobacteria, aerobic endospore-formers, enterobacteria and the genera Listeria and Shewanella (Marr & Ingraham, 1962;Olson & Ingram, 1975;Gill & Suisted, 1978;Bezbaruah et al., 1988;Suzuki & Komagata, 1983;Abu Hatab & Gaugler, 1997;Nichols et al., 2000Nichols et al., , 2002)), and the importance of standardization of growth conditions and physiological age is also emphasized for fatty acid-based identification of bacteria (Sasser, 2009).Keddie & Jones (1981) discussed the heterogeneity among species of Arthrobacter and, based mainly on the diamino acid in the peptidoglycan and differences in the DNA G+C content, they subdivided arthrobacters into three groups ( Though the first studies on polar lipids in arthrobacters were published more than 40 years ago, and interesting differences in the polar lipid compositions of arthrobacters were reported approximately 30 years ago, this approach has only rarely been considered in the description of novel species of Arthrobacter and their differentiation.In an early study, Shaw & Stead (1971) examined the polar lipid profiles of the type strains of A. crystallopoietes and A. pascens and A. globiformis strain 616 and reported no significant qualitative differences.Major lipids in the three species were reported to be the phospholipids DPG, PG and phosphatidylinositol (PI) and the glycolipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacyglycerol (DGDG) and dimannosyldiacylglycerol (DMDG) and probably also small amounts of trimannosyldiacylglycerol (TMDG) and tetramannosyldiacylglycerol (TeMDG).The three major glycolipids showed properties identical to those of three glycolipids of A. globiformis strain 616 identified earlier by Walker & Bastl (1967) Kostiw et al. (1972) also detected the glycolipids MGDG and DGDG in A. crystallopoietes, but no indication of the presence of DMDG, TMDG or TeMDG was reported.
A. ilicis was the first species of the genus Arthrobacter to be described with the inclusion of the polar lipid profile (Collins et al., 1981).This species was described to contain DPG, PG, PI and two glycolipids designated G A and G B and supposed to represent the diglycosyldiacylglycerols DGDG or DMDG and the monoglycosyldiacylglycerol MGDG, respectively.Very similar polar lipid profiles were reported for the type strain of A. aurescens, a close relative of A. ilicis, and the more distantly related species A. polychromogenes (Collins et al., 1982b).The latter authors also detected two major glycolipids in A. crystallopoietes that show chromatographic motilities similar to the monoglycosyldiacylglycerol and diglycosyldiacylglycerol of A. ilicis.In the same study, Collins et al. (1982b) reported on the polar lipid profiles of the type strain of A. globiformis and two strains of A. citreus, including the type strain.The type strain of A. globiformis showed a polar lipid profile that is qualitatively in agreement with that reported by Shaw & Stead (1971) Shaw & Stead (1971) and Collins et al. (1982b).In A. globiformis and A. oxydans, four glycolipids were detected, G 1 , G 3 , G 4 and G 5 ; in A. crystallopoietes and A. ramosus, the glycolipids G 1 and G 3 were detected; and, in A. polychromogenes, the glycolipids G 1 , G 3 and G 5 were detected.The polar lipid profile of A. ramosus is well in agreement with its close relatedness to A. ilicis and A. aurescens (Fig. 1).
The polar lipid profiles of A. globiformis and A. polychromogenes are most similar to those reported for these two species by Collins et al. (1982b).Though A. oxydans and A. polychromogenes are close phylogenetic relatives (Fig. 1), G 4 was not reported for the latter species.Like in the report of Kostiw et al. (1972), A. crystallopoietes was shown to contain only two glycolipids but, instead of DGDG, the presence of DMDG was reported.Collins & Kroppenstedt (1983) confirmed only the presence of the glycolipid of medium hydrophobicity in A. citreus.These authors also showed that the type strains of A. nicotianae, A. protophormiae (formerly Brevibacterium protophormiae) and A. sulfureus (formerly 'Brevibacterium sulfureum') lack PI and contain a single glycolipid of medium hydrophobicity.However, the glycolipid of A. nicotianae and A. protophormiae shows a chromatographic motility different from that of the glycolipid of A. sulfureus.
Considering the information given by Keddie et al. (1986) and the report of Shaw & Stead (1971), the identity of the different glycolipids shown by Collins et al. (1982b) and Collins & Kroppenstedt (1983) can be determined.The glycolipid with the highest hydrophobicity corresponds to MGDG.
The glycolipid with the lowest hydrophobicity corresponds to TMDG, because, in the first chromatographic dimension [corresponding to the solvent system applied by Shaw & Stead (1971)], it shows the same motility as PG (Shaw & Stead, 1971).Since the glycolipid of medium hydrophobicity of A. sulfureus was suggested to correspond to DGDG, whereas all other species of Arthrobacter studied contain DMDG (Keddie et al. 1986), the medium-hydrophobic glycolipid with the slightly higher R f in both chromatographic  (2010).These authors confirmed the identity of one major glycolipid in A. globiformis to be monogalactosyldiacylglycerol but, in contrast to earlier reports, the other major glycolipid was identified as a monoacyldimannosylmonoacylglycerol (MDMMG).The same two major glycolipids were also found in A. scleromae.Interestingly, MDMMG was also reported to be a major lipid in other members of the family Micrococcaceae, including the type strains of S. atrocyanea (Niepel et al., 1997) and Rothia dentocariosa, and it was demonstrated that Rothia mucilaginosa, Rothia amarae and M. luteus contain a major glycolipid (Pas ´ciak et al., 2002(Pas ´ciak et al., , 2010) that shows the same chromatographic motility as MDMMG detected in A. globiformis, A. scleromae and R. dentocariosa.For A. globiformis and M. luteus, this result is surprising, because Walker & Bastl (1967), Shaw & Stead (1971) and Pakkiri et al. (2004) reported that the major mannolipid of A. globiformis strain 616 and the type strain of M. luteus is DMDG.It appears unlikely that these species switch between DMDG and MDMMG depending on environmental conditions and/or physiological age, because it can be supposed that different enzymes would be required to transfer a fatty acid to the mannose and the glycerol.Hence, it has to be supposed that the result from one structural analysis of this mannolipid is due to an error.
Arthrobacter psychrophenolicus, a close phylogenetic relative of A. sulfureus, was reported to contain PG, DPG, PI and an unidentified glycolipid (Margesin et al., 2004).The presence of PI in A. psychrophenolicus is surprising; this lipid would be expected to be absent, as in its close relative A. sulfureus.Since no image of the total lipid profile of this species was provided, it is not possible to evaluate whether the unidentified glycolipid corresponds to DGDG, found in A. sulfureus.
The most recently described species, A. gyeryongensis (Hoang et al., 2014), was described to contain DPG, two unidentified glycolipids and two unidentified phospholipids.The two glycolipids show chromatographic motilities similar to a monoglycosyldiacylglycerol and diglycosyldiacylglycerol and the two phospholipids might represent PG and PI.This polar lipid profile is similar to that of A. ramosus, with which A. gyeryongensis shares the highest 16S rRNA gene sequence similarity (98.2 % between the type strains).
The polar lipid profiles of Arthrobacter castelli, Arthrobacter monumenti, A. pigmenti and A. parietis (Heyrman et al., 2005) share the presence of the lipids PG, DPG and PI and one or two unidentified glycolipids with many species of Arthrobacter.Unidentified phospholipids were also reported in some of these species.In contrast, A. tecti and A. tumbae possess, in addition to PG, DPG and PI, one unidentified phospholipid but no glycolipids (Heyrman et al., 2005).A. russicus was reported to contain exclusively DPG, PG and PI (Li et al., 2004b).The absence of any glycolipid in A. russicus, A. tecti and A. tumbae is most surprising, because the presence of a diglycosyldiacylglycerol is apparently a characteristic of members of the family Micrococcaceae.Hence, reanalyses of the polar lipid profiles of these three species would be desirable in order to confirm these unexpected traits.A. livingstonensis and Arthrobacter cryotolerans were reported to contain PG only (Ganzert et al., 2011), but a polar lipid profile with only a single lipid appears unlikely, because bacterial cytoplasmic membranes are generally composed of several different lipid components.However, in the corresponding paper, the polar lipid profiles of the latter two species were incomplete.In addition to PG, HPLC-MS-MS analyses of the polar lipid profiles demonstrated that A. livingstonensis also contains PI, DPG and two types of unidentified glycolipid, and that A. cryotolerans contains DPG and an unidentified glycolipid (Kai Mangelsdorf, personal communication).
The detection of PE, as reported for A. phenanthrenivorans, A. antarcticus, A. cupressi, A. flavus and A. roseus (Kallimanis et al., 2009;Pindi et al., 2010;Reddy et al., 2000Reddy et al., , 2002;;Zhang et al., 2012), like the absence of any glycolipid, is surprising for representatives of the family Micrococcaceae because, like the presence of at least one glycolipid, the absence of PE appears to be a common trait in polar lipid profiles of members of the family.Hence, reanalyses of the polar lipids of these five species would also be desirable.However, exceptions concerning the presence of PE may occur in selected strains of Arthrobacter, as indicated from the observation that, in the genome of Arthrobacter sp.MA-N2, a sequence was annotated to encode a putative phosphatidylserine decarboxylase (Ru ¨diger Pukall, personal communication), which catalyses the formation of PE from phosphatidylserine.However, when compared to sequences of almost the same length in a FASTA search (Pearson & Lipman, 1988), the amino acid sequence showed the highest similarity to the phosphatidylserine decarboxylase of the epsilonproteobacterium Sulfurimonas gotlandica (50.6 %).Among hits sharing 40-50 % sequence identity, numerous betaproteobacterial strains, a few alpha-, gamma-and deltaproteobacterial strains and several fungal strains were found, but only two actinobacterial strains, assigned to the genus Frankia.Two additional actinobacterial strains assigned to the genus Streptomyces show only 36.2 and 37.7 % identity.This observation suggests that this type of phosphatidylserine decarboxylase is not common among the actinobacteria.Hence, annotation as a phosphatidylserine decarboxylase in Arthrobacter sp.MA-N2 can be considered to be questionable.
In a pioneering study dealing with the dissection of the genus Micrococcus (Stackebrandt et al., 1995), the authors also provided polar lipid data of groups I and II of Arthrobacter, corresponding to the 'A.globiformis/A.citreus group' and the 'A.nicotianae group', respectively.With reference to Jones & Collins (1986), the two groups were listed to contain DMDG and PI.Since no information  (1995) only has been cited in eight papers in the IJSEM for the presence of DMDG and PI in both groups of Arthrobacter (Stackebrandt & Schumann, 2000;Altenburger et al., 2002;Li et al., 2004aLi et al., , 2005c;;Zhang et al., 2007;Schumann et al., 2009;Zhou et al., 2009;Yassin et al., 2011a).
Phylogenetic calculations have clearly demonstrated that the genus is not monophyletic but is mixed with other representatives of the family Micrococcaceae, including the genera Acaricomes, Auritidibacter, Citricoccus, Micrococcus, Nesterenkonia, Renibacterium, Sinomonas, Yaniella and Zhihengliuella (Munoz et al., 2011;Busse et al., 2012).Hence, careful evaluation of the taxonomy of the genus Arthrobacter is desirable.In this context, it is interesting that certain groupings within the radiation of the genus Arthrobacter, as indicated from comparative 16S rRNA gene sequence analyses, are also reflected by identical or at least highly similar chemotaxonomic traits.
The first move was made by Busse et al. (2012), by dissecting the genus Arthrobacter into 11 groups, mainly on the basis of robust phylogenetic clusters or high 16S rRNA gene sequence similarities and group-specific chemotaxonomic traits.A phylogenetic tree showing these groups and including most recently described novel species of Arthrobacter is shown in Fig. 1.
The 'A. globiformis group' contains candidates for the genus Arthrobacter sensu stricto.It is composed of the type species of the genus, A. globiformis, and A. pascens, which are closely related in all phylogenetic trees.A. humicola and A. oryzae were assigned to this group on the basis of high 16S rRNA gene sequence similarity to A. globiformis (.98 %), similar peptidoglycan composition (Lys-Ala 2-3 ) corresponding to peptidoglycan structures A11.5/A11.6 (Schumann, 2011; http://www.dsmz.de/?id5449) and the quinone system menaquinone MK-9(H 2 ). A. crystallopoietes was tentatively assigned to the group on the basis of high 16S rRNA gene sequence similarity (97.6 % between the type strains) and similar chemotaxonomic traits.However, in phylogenetic trees, A. crystallopoietes is usually the sole species on a separate branch (Fig. 1; Ding et al., 2009;Ganzert et al., 2011;Yassin et al., 2011b;Margesin et al., 2012;Busse et al., 2012).The peptidoglycan structure also does not assign this species clearly to the 'A.globiformis group'.Hence, A. crystallopoietes might be considered as the sole species of an as-yet undefined group.was later reported for the type strain of A. rhombi (Chen et al., 2009).Furthermore, the glutamic acid in the interpeptide chain occurs in the D-configuration (Osorio et al., 1999) and not in the L-configuration, like other members of the group.These two chemotaxonomic traits clearly distinguish A. rhombi from members of the 'A.protophormiae group'.Hence, A. rhombi should not be considered to be a member of the 'A.protophormiae group'.Since the combination of menaquinone MK-9(H 2 ) and a peptidoglycan type A4a (variation Lys-Ala-D-Glu) is unique among arthrobacters, A. rhombi can be considered to be a representative of another group of the genus Arthrobacter that was not defined by Busse et al. (2012).Arthrobacter halodurans, which was described by Chen et al. (2009) to share its quinone system with A. rhombi and to belong to the same line of descent but with peptidoglycan type A4a (variation Lys-Ala-L-Glu), should hence not be considered a second species of this unnamed group of the genus Arthrobacter.
The 'A. sulfureus group' contains the species A. antarcticus, A. gangotriensis, Arthrobacter kerguelensis, Arthrobacter psychrophenolicus and A. sulfureus.In many trees, these species form a clade though, in Fig. 1, this branch is not supported by a significant bootstrap value (37 %).However, the same degree of branching is also shown in the neighbour-joining and maximum-parsimony tree (results not shown).16S rRNA gene sequence similarity among the species is .97.0 %.All species share a peptidoglycan A4a (Lys-Glu) corresponding to peptidoglycan structure A11.54.According to Stackebrandt et al. (1983) and Margesin et al. (2004), the glutamic acid in the interpeptide bridge of A. sulfureus and A. psychrophenolicus occurs in the L-configuration.Quinone systems of representatives of this group exclusively contain completely unsaturated menaquinones (MK-8, MK-9 and/or MK-10).
Recently, the species Arthrobacter cryotolerans was described, which was placed phylogenetically at the root of the branch comprising the members of the 'A.sulfureus group' (Ganzert et al., 2011).This species contains a quinone system with MK-9 predominant and lesser amounts of MK-10, MK-8 and MK-7 and peptidoglycan type Lys-Glu.Since these traits are in accordance with the characteristics of the 'A.sulfureus group', the assignment of A. cryotolerans to this group appears to be justified.The 'A. psychrolactophilus group' was defined based on 16S rRNA gene sequence similarities of .97.0 % among its members, a peptidoglycan with either Lys-Thr-Ala 1-3 or Lys-Ala 2 corresponding to peptidoglycan structures A11.25/A11.27/A11.28 and A11.5, respectively, and MK-9(H 2 ) as the major respiratory quinone.Also, the ability of all members of this group to grow at low temperatures (0-4 8C) was also pointed out as a common characteristic.This group comprises the species A. psychrolactophilus, A. stackebrandtii, A. psychrochitiniphilus and A. alpinus.
Recently, two species of Arthrobacter, A. livingstonensis and A. cryoconiti, have been described and placed phylogenetically among the species representing the 'A.psychrolactophilus group' (Ganzert et al., 2011;Margesin et al., 2012).The two species share 97.0-97.9 and 97.7-98.3% 16S rRNA gene sequence similarity, respectively, with the members of this group and exhibit the same quinone system and grow at low temperatures ((6 and 1 8C, respectively).Data from analysis of the peptidoglycan of A. livingstonensis suggest that it is Lys-Thr-Ala (A11.25).This peptidoglycan type is similar to those found in A. psychrolactophilus, A. psychrochitiniphilus and A. alpinus.The peptidoglycan of A. cryoconiti is Lys-Ala 4 (A11.7),resembling that of A. stackebrandtii, with Lys-Ala 2 (A11.5).
Because of their almost identical quinone systems and peptidoglycan variations, the 'A.agilis group', the 'A.citreus group' and several species of the 'A.psychrolactophilus group' are distinguishable from each other only on the basis of 16S rRNA gene sequence similarity (94.2-96.8% similarity between representatives of the three groups) and phylogenetic clustering.Each of these three groups forms a stable clade (Fig. 1).Hence, they can be considered to represent taxonomic entities separate from the core of the genus, Arthrobacter sensu stricto.
The 'A. pigmenti group' comprises the two core species A. castelli and A. pigmenti, the type strains of which share 97.9 % 16S rRNA gene sequence similarity.these two species and slightly higher similarity values with species of other groups, A. monumenti is placed at the root of this group in many phylogenetic trees.Common to the three species is a quinone system with the major menaquinone MK-9(H 2 ), the presence of the cell-wall sugars galactose and rhamnose and a peptidoglycan with four alanines in the interpeptide bridge.However, A. castelli shows a peptidoglycan type Lys-Ala-Ser-Ala 3 (not listed by Schumann, 2011; http://www.dsmz.de/?id5449), whereas A. pigmenti and A. monumenti have peptidoglycan type Lys-Ala 4 corresponding to peptidoglycan structure A11.7.
The 'A. albus/cumminsii group' contains only the two species A. albus and A. cumminsii.The type strains of the two species share 99.1 % 16S rRNA gene sequence similarity and form a rather deeply branching subline within the genus Arthrobacter in phylogenetic trees.The two species possess peptidoglycan type A4a (Lys-Ala-Glu or Lys-Ser-Glu) corresponding to peptidoglycan structures A11.35 and A11.58, respectively.Analysis of the quinone system revealed the presence of the major menaquinone MK-8(H 2 ) in the two species.This quinone system distinguishes this group unambiguously from the vast majority of species of the genus Arthrobacter.Only A. scleromae was reported to exhibit a similar quinone system, but this species is assigned to the 'A.oxydans group', with which it shares the peptidoglycan type (Huang et al., 2005) and high 16S rRNA gene sequence similarity.
The 'Sinomonas group', as defined recently (Busse et al., 2012), harbours species of the genera Sinomonas and Arthrobacter.After reclassification of Arthrobacter albidus and Arthrobacter echigonensis as Sinomonas albida and Sinomonas echigonensis (Zhou et al., 2012), the 'Sinomonas group' no longer contains species of Arthrobacter, and hence it is not be dealt with further in this discussion.
All other established species of the genus Arthrobacter not mentioned above were only tentatively assigned to certain groups or remained unassigned.
During this study on the taxonomy and chemotaxonomy of the genus Arthrobacter, reanalyses of the quinones and polar lipids of several species of Arthrobacter were carried out due to availability, including A. globiformis, A. pascens, A. histidinolovorans, A. roseus, A. agilis, A. nicotinovorans, A. psychrophenolicus, A. polychromogenes, A. alpinus and A. cryoconiti.Additionally, the polar lipids and quinones of A. cumminsii and A. albus, which had been described without including these traits, were also analysed.Extraction and analyses of quinones and polar lipids were carried out as described by Tindall (1990a, b).For HPLC analyses of quinones, the equipment described by Altenburger et al. (1996) or Stolz et al. (2007) was used.
A globiformis DSM 20124 T showed a polar lipid profile that was composed of the major lipids DPG, PG and glycolipid GL3 and moderate amounts of PI and glycolipid GL1 (Fig. 3a).Furthermore, minor to trace amounts of glycolipids GL2, GL4 and GL5 and two polar lipids (L1, L2) were detected.A. pascens WS 1766 T showed a rather similar profile, differing mainly from A. globiformis DSM 20124 T with respect to the amounts of certain lipids including DPG, PG and PI, glycolipids GL1, GL3, GL4 and GL5 and the polar lipids L2 and L3.The major differing characteristic in the lipid profile of A. pascens was the presence of the second highly hydrophobic glycolipid GL6 and absence of a second diglycosyldiacylglycerol (GL2; Fig. 3b) that was previously reported to be present in this species (Shaw & Stead, 1971).The qualitative polar lipid composition of A. globiformis DSM 20124 T was in good agreement with the data reported by Shaw & Stead (1971).In contrast, equal amounts of the two diglycosyldiacylglycerols (GL2 and GL3) were not found, which might be related to the fact that, in the present study, the type strain of A. globiformis was subjected to analysis and not A. globiformis strain 616, which had been examined by the latter authors.The quinone system of A. globiformis DSM 20124 T was composed of 85 % MK-9(H 2 ), 6 % MK-8(H 2 ), 6 % MK-10(H 2 ), 3 % MK-9 and traces (,1 %) of MK-7(H 2 ) and the quinone system of A. pascens WS 1766 T was composed of 69 % MK-9(H 2 ), 28 % MK-10(H 2 ), 2 % MK-8(H 2 ) and 2 % MK-7(H 2 ).
A. agilis DSM 20550 T showed a rather simple polar lipid profile that consisted of the major lipids DPG, PG, PI and GL3 and minor amounts of GL5, L1 and L2.Furthermore, a reddish pigment spot could be detected (Fig. 3e).
The fact that PE was not detected, which is in accordance with the majority of species of the genus Arthrobacter, but contrasts with its reported presence in the next phylogenetic relative, A. flavus (Reddy et al., 2000), supports concerns about the reliable identification of PE in the latter species.The detection of PE and the absence of a diglycosyldiacylglycerol and other glycolipids in species of the family Micrococcaceae including species of the genus Arthrobacter is most surprising.The type strain of A. roseus, reported to contain PE and accessible in the course of this study, was subjected to reanalysis of its polar lipid profile.The results did not provide any indication of the presence of PE (Fig. 3f).Like other arthrobacters, it had a polar lipid profile containing predominantly DPG and PG, moderate amounts of PI, GL3 and three polar lipids (L1, L2, L4) and minor amounts of GL5 and two unidentified highly polar glycolipids GL8 and GL9, which so far are unique among polar lipid profiles of members of the genus Arthrobacter.These results suggest the need to emend the description of the species A. roseus.
The polar lipid profile of A. nicotianae WS1765 T (Fig. 3h) consisted of the major lipids DPG, PG and GL3, moderate amounts of GL1 and polar lipids L1 and L2 and minor amounts of GL5.In agreement with the observation of Collins & Kroppenstedt (1983), PI was not detected, whereas the presence of GL1, GL5, L1 and L2 was not reported by these authors.The quinone system of A. nicotianae WS1765 T contained 65 % MK-8, 31 % MK-9, 3 % MK-7 and 1 % MK-10, confirming earlier results published for this species (Yamada et al., 1976;Collins & Kroppenstedt, 1983).
In A. psychrophenolicus AG31 T , the next phylogenetic relative of A. sulfureus, and the close relative A. cryotolerans LI3 T , less complex polar lipid profiles were detected (Fig. 3i, j), showing only three major lipids, namely DPG, PG and GL2.Additionally, in A. cryotolerans LI3 T , minor amounts of L1  and L2 were detectable.As suggested from the very close phylogenetic relationship to A. sulfureus, which was reported to lack PI (Collins & Kroppenstedt, 1983), this lipid could also not be detected in A. psychrophenolicus AG31 T and A. cryotolerans LI3 T .For A. psychrophenolicus AG31 T , this observation is in contrast to the data reported by Margesin et al. (2004).The quinone system of A. psychrophenolicus AG31 T was composed of 81 % MK-10, 13 % MK-9, 5 % MK-7, 1 % MK-8 and traces (,1 %) of MK-11, which is similar to that reported by Margesin et al. (2004).
Simple polar lipid profiles were detected in A. alpinus S6-3 T and A. cryoconiti Cr6-08 T (results not shown).Both strains exhibited a polar lipid profile consisting of DPG, PG, PI and GL3.Minor amounts of PL5 were detected in A. alpinus S6-3 T .The similarity in the polar lipid profiles is in agreement with their close phylogenetic relationship (Margesin et al., 2012).
In order to identify the motility of DMDG in our chromatographic system, the type strain of Micrococcus luteus, which was reported to contain DMDG (Lennarz & Talamo, 1966;Pakkiri et al., 2004), was subjected to polar lipid analysis.The chromatographic motility of DMDG of M. luteus was compared by co-chromatography in a mixture of extracts of A. globiformis DSM 20124 T and A. polychromogenes WS 1989 T .After staining with anaphthol, only a single positive spot was detected (results not shown), demonstrating that the diglycosylglycerides of M. luteus, A. globiformis and A. polychromogenes show the same chromatographic motility.Applying sophisticated methods, A. globiformis was described to contain MDMMG (Pas ´ciak et al., 2010) and M. luteus to contain DMDG (Pakkiri et al., 2004), but the results from the present study indicate either that MDMMG and DMDG show identical chromatographic motility in the system applied here or that, in one of the studies on the structure of the diglycosylglycerides of A. globiformis and M. luteus, the data from MS analysis were misinterpreted.Since the detailed chemical structure of the mannolipid cannot be clarified here, nor can the question be answered whether MDMMG and DMDG show the same chromatographic motility, the mannolipid will be designated dimannosylglyceride (DMG).
Except A. psychrophenolicus and A. cryotolerans, all representatives of Arthrobacter examined in this study showed the presence of a major glycolipid (GL3) with chromatographic motility corresponding to DMG in A. globiformis and A. polychromogenes.Hence, it is justified to conclude that they all contain DMG. A. psychrophenolicus showed the presence of a glycolipid with the chromatographic motility of a diglycosyldiacylglycerol corresponding to glycolipid Gb reported to be present in its close phylogenetic relative A. sulfureus (Collins & Kroppenstedt, 1983).Glycolipid Gb was supposed to represent DGDG (Keddie et al., 1986).Hence, glycolipid GL2 of A. psychrophenolicus and A. cryotolerans can also be identified as DGDG.This finding suggests extending the statement of Keddie et al. (1986) that, among arthrobacters, only A. sulfureus contains DGDG, to species included in the 'A.sulfureus group' as defined by Busse et al. (2012).
Using information concerning the chromatographic motility of glycolipids reported in the literature (Shaw & Stead, 1971;Collins et al., 1982b;Collins & Kroppenstedt, 1983;Keddie et al., 1986) and the results presented in this study, another two glycolipids (GL1 and GL5; Fig. 3) can be preliminarily identified.Glycolipid GL1 corresponds to MGDG, indicated by the highly hydrophobic chromatographic motility reported for this lipid (Shaw & Stead, 1971) and its presence in A. globiformis, A. pascens and A. crystallopoietes; GL5 corresponds to TMDG, as it exhibits the same R f as PG in the first chromatographic dimension (Shaw & Stead, 1971).
The presence of DGDG in arthrobacters that also contain DMG deserves closer consideration.Shaw & Stead (1971) reported almost equal amounts of these two diglycosyldiacylglycerols in A. globiformis strain 616 and the type strains of A. pascens and A. crystallopoietes (Shaw & Stead, 1971).In contrast, Collins et al. (1982b) showed a polar lipid profile for the type strain of A. globiformis that contained a major lipid with the chromatographic motility of DGDG and significantly smaller amounts of a lipid with the chromatographic motility of DMG whereas, in the present study, DMG was the predominant glycolipid in A. globiformis and DGDG was detected only in minor amounts (Fig. 3a).The presence of almost equal amounts of DGDG and DMG in A. pascens (Shaw & Stead, 1971) could not be reproduced in the present study, in which A. pascens showed DMG as the major glycolipid (Fig. 3b).For the type strain of A. crystallopoietes, Collins et al. (1982b) could not reproduce the relatively large amounts of DGDG reported by Shaw & Stead (1971).This variability of the amounts of DGDG in polar lipid profiles of A. globiformis, A. pascens and A. crystallopoietes may suggest that the expression of this glycolipid depends on growth conditions and/or the physiological age of biomass that was subjected to extraction of polar lipids.Hence, the presence of DGDG in arthrobacters that also contain significant amounts of DMG should not be given too much importance in drawing taxonomic conclusions.
A. phenanthrenivorans, A. antarcticus, A. flavus and A. roseus were reported to contain PE and to lack any glycolipid.The close relatives of A. phenanthrenivorans (A.oxydans and A. polychromogenes), A. antarcticus (A.sulfureus) and A. flavus (A.agilis, A. parietis, A. tecti and A. tumbae) were shown to lack PE, and most of them have been shown to contain at least one glycolipid (Amadi & Alderson, 1982;Collins & Kroppenstedt, 1983;Heyrman et al., 2005;Fig.3c, f, g, i, j).Hence, there is some probability that the named species contain at least one glycolipid and lack PE.The absence of PE and any other aminophospholipid and the presence of four glycolipids in the profile of A. roseus was shown in the present study (Fig. 3f), which is in line with the core polar lipid profile supposed to characterize members of the family Micrococcaceae.For A. cupressi (Zhang et al., 2012), a polar lipid profile was shown containing PE and a glycolipid exhibiting chromatographic motility that can be supposed to represent TMDG.The PE shows a chromatographic motility that would be expected for a diglycosyldiacylglycerol and, hence, it is supposed here that the corresponding spot had been misidentified.However, the presence of PE and the absence of a diglycosyldiacylglycerol has not been shown in any of the nearest related species to A. cupressi (.96.5 % 16S rRNA gene sequence similarity) analysed for polar lipids, including A. globiformis, A. pascens, A. histidinolovorans, A. equi and A. aurescens (Shaw & Stead, 1971;Collins et al., 1982b;Yassin et al., 2011b;Fig. 3a, b, d). A. tecti, A. tumbae and A. russicus differ from the core polar lipid profile in the absence of any glycolipid.However, the close relatives of A. tumbae and A. tecti (A.agilis and A. parietis) were shown to contain two glycolipids (Fig. 3e; Heyrman et al., 2005).Also, Renibacterium salmoninarum, the nearest phylogenetic relative of A. russicus (Pukall et al., 2006;Munoz et al., 2011;Busse et al., 2012;Fig. 1), was reported to contain glycolipids (Collins, 1982).Hence, the presence of a glycolipid in the polar lipid profile would be also expected in A. russicus.A. psychrophenolicus was reported to contain PI, distinguishing it from its close relative A. sulfureus.Reanalysis of the polar lipid profile did not show the presence of PI in A. psychrophenolicus (Fig. 3i), confirming the reservations expressed above.Because of these reservations, supported in two cases by data gained in this study, the reported polar lipid profiles of the other species of the genus Arthrobacter mentioned above are not considered in the following taxonomic conclusions.
Chemotaxonomic data that can be considered to be sufficiently conserved to characterize genera are summarized in Table 2.These data suggest that five groups of the genus Arthrobacter, as recently defined (Busse et al., 2012), are characterized by traits that distinguish them from the core of the genus Arthrobacter (Arthrobacter sensu stricto), which is composed of the type species of the genus, A. globiformis, and the additional species A. pascens, A. oryzae and A. humicola, and from other groups of the genus Arthrobacter, including the 'A.aurescens group', the 'A.oxydans group', the 'A.protophormiae group', the 'A.sulfureus group' and the 'A.albus/cumminsii group'.Except the 'A.oxydans group', these groups form clades in the phylogenetic tree, but only the branching nodes of the 'A.aurescens group' and the 'A.albus/cumminsii group' were supported by another tree-calculating algorithm.High bootstrap support was only found for the branching node of the 'A.albus/cumminsii group'; the branching node of the 'A.protophormiae group' was supported by a relatively high bootstrap value (Fig. 1).Members of the 'A.oxydans group' are split into several branches, with the core consisting of A. oxydans, A. scleromae and A. polychromogenes and the neighbouring species A. defluvii, A. sulfonivorans and A. niigatensis.The species pair A. chlorophenolicus/A.phenanthrenivorans and the single species A. equi and A. siccitolerans each occupy separate positions in the vicinity of the core species of the group.However, each of the species A. chlorophenolicus, A. phenanthrenivorans, A. equi and A. siccitolerans shares high 16S rRNA gene sequence similarity (98.4-98.9, 97.9-99.2, 97.6-99.2 and 98.4-99.3 %, respectively) with the core species and their closest neighbours.These data demonstrate that the high 16S rRNA gene sequence similarities between the species of the group are not well reflected in the phylogenetic tree (Fig. 1) and, hence, the splitting of the members of the 'A.oxydans group' is of little or no significance for taxonomic reorganization of the genus.
DNot listed at http://www.dsmz.de/?id5449.dGlycolipid(s) was detected but chromatographic motility was not shown, which would be required in order to assign them to those identified as present in other species.§Chromatographic motilities of two glycolipids detected in Nesterenkonia suensis suggest that they correspond to MGDG and DMG (Govender et al., 2013) The peptidoglycan type is A3a (variation Lys-Ala-Thr-Ala; A11.17).The quinone system contains menaquinone MK-9(H 2 ) predominantly.The polar lipid profile is composed of the major compounds diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, dimannosylglyceride and monogalactosyldiacylglycerol and minor amounts of trimannosyldiacylglycerol (Collins et al., 1981(Collins et al., , 1982b; this study).The major fatty acid is anteiso-C 15 : 0 .Large amounts of iso-C 15 : 0 , iso-C 16 : 0 , anteiso-C 17 : 0 and iso-C 14 : 0 may also be present.The G+C content of the genomic DNA is in the range 61.3-62.5 mol%.The type species is Paenarthrobacter aurescens.
The description is as provided by Phillips (1953) for Arthrobacter aurescens with the following additional properties.Shares the characteristics listed in the genus description.According to Kodama et al. (1992), the predominant fatty acid of the type strain (IAM 12340 T ) is anteiso-C 15 : 0 , followed by anteiso-C 17 : 0 .Minor fatty acids are iso-C 15 : 0 , iso-C 16 : 0 , iso-C 17 : 0 and C 16 : 0 .Starch is hydrolysed.
L-Arginine, L-asparagine, L-histidine, L-arabinose, D-galactose, D-glucose, D-ribose, D-xylose, 4-aminobutyrate and p-hydroxybenzoate are utilized as carbon sources, but not L-leucine, L-rhamnose, inositol or malonate.Citric acid, formic acid and uric acid are assimilated, but not benzoic acid, glutaric acid, malonic acid, pimelic acid or propionic acid.Utilization of butanediol and histidinol and assimilation of adipic acid are weak.Urea is formed from uric acid.Nitrate is not reduced to nitrite, and no growth occurs in the presence of 10 % (w/v) NaCl.Does not produce nicotine blue.
The description is as provided by Adams (1954) for Arthrobacter histidinolovorans with the following additional properties.In addition, it shares the characteristics listed in the genus description.According to Kodama et al. (1992), the predominant fatty acids of the type strain (JCM 2520 T ) are anteiso-C 15 : 0 , followed by anteiso-C 17 : 0 .Minor amounts of iso-C 14 : 0 , iso-C 15 : 0 , iso-C 16 : 0 , iso-C 17 : 0 , C 14 : 0 and C 16 : 0 and traces of C 18 : 0 may also be present.L-Arginine, Lasparagine, L-histidine, L-arabinose, D-galactose, D-glucose, D-ribose, D-xylose, histidinol, inositol, 4-aminobutyrate and p-hydroxybenzoate are utilized as carbon sources, but not L-leucine, butanediol or malonate.Citric acid, formic acid, propionic acid and uric acid are assimilated, but not adipic acid, benzoic acid, malonic acid or pimelic acid.Utilization of L-rhamnose and assimilation of glutaric acid are weak.Urea is formed from creatinine and uric acid.Nitrate is not reduced to nitrite, and no growth occurs in the presence of 10 % (w/v) NaCl.Starch is not hydrolysed.Does not produce nicotine blue.
The description is as provided by Collins et al. (1981) for Arthrobacter ilicis with the following additional properties.Shares the characteristics listed in the genus description.Predominant fatty acid of the type strain is anteiso-C 15 : 0 , followed by anteiso-C 17 : 0 .Minor fatty acids are iso-C 15 : 0 , iso-C 16 : 0 , C 14 : 0 and C 16 : 0 and traces of iso-C 14 : 0 may also be present (Kodama et al., 1992).According to Kodama et al. (1992), the type strain is positive for utilization of the carbon sources L-arginine, L-asparagine, L-his- tidine, L-arabinose, D-galactose, D-glucose, D-ribose, D- xylose, inositol, 4-aminobutyrate and p-hydroxybenzoate, but negative for L-leucine, butanediol and malonate.Citric acid, propionic acid and uric acid are assimilated, but not adipic acid, benzoic acid, glutaric acid, malonic acid or pimelic acid.Utilization of L-rhamnose and histidinol and assimilation of formic acid are weak.Urea is formed from uric acid but not from creatinine.Does not reduce nitrate to nitrite.Does not hydrolyse starch.Does not grow in the presence of 10 % (w/v) NaCl.Does not produce nicotine blue.According to Kotouc ˇkova ´et al.
(2004), the type strain uses D-xylose, uridine and sucrose for respiration and uses propionic acid, melibiose, 3methyl glucose, raffinose and salicin weakly.Gluconate is not utilized and arbutin and a-cyclodextrin are not utilized for respiration.Positive for pyrrolidonyl arylamidase and negative for elastase and oxidase.Aesculin and starch are not hydrolysed.
The type strain is ATCC 14264 T 5DSM 20138 T 5NCPPB 1228 T .The description is as provided by Kodama et al. (1992) for Arthrobacter nicotinovorans with the following additional properties.Shares the characteristics listed in the genus description, but the polar lipid profile is unknown.

2004.
The description is as provided by Kotouc ˇkova ´et al. (2004) for Arthrobacter nitroguajacolicus with the following additional properties.Shares the characteristics listed in the genus description, but the polar lipid profile is unknown.
The description is as provided by Clark (1955) for Arthrobacter ureafaciens with the following additional properties.Shares the characteristics listed in the genus description, but the polar lipid profile is unknown.Predominant fatty acid of the type strain is anteiso-C 15 : 0 , followed by anteiso-C 17 : 0 .Minor fatty acids are iso-C 14 : 0 , iso-C 15 : 0 , iso-C 16 : 0 , iso-C 17 : 0 , C 16 : 0 and C 18 : 0 (Kodama et al., 1992).According to Kodama et al. (1992), the type strain does not reduce nitrate to nitrite, does not hydrolyse starch and does not grow in the presence of 10 % (w/v) NaCl.Utilizes L-arginine, L-asparagine, L-arabinose, D-galactose, D-glucose, D-xylose, inositol, 4-aminobutyrate and p-hydroxybenzoate, but not L-histidine, L-leucine, L-rhamnose, butanediol or malonate.Citric acid, formic acid, propionic acid and uric acid are hydrolysed, but not adipic acid, benzoic acid, malonic acid or pimelic acid.Utilization of D-ribose and histidinol and assimilation of glutaric acid are weak.Does not reduce nitrate to nitrite.Does not produce nicotine blue.
Description of Pseudarthrobacter gen.nov.
The type strain is ATCC 15216 T 5BCRC 12114 T 5CCUG 23891 T 5CIP 106989 T 5CGMCC 1.1927 T 5DSM 20136 T 5 IFO (now NBRC) 15512 T 5IAM 14590 T 5JCM 2523 T 5 KCTC 3384 T 5LMG 3821 T 5NCIB 10267 T 5VKM Ac-1955 T .The G+C content of the genomic DNA of the type strain is 62.9 mol% (HPLC; Kodama et al., 1992).The description is as provided by Westerberg et al. (2000) for Arthrobacter chlorophenolicus with the following additional properties.The peptidoglycan type, quinone system and fatty acid profile are in agreement with the genus description, but the polar lipid profile is unknown.
Basonym: Arthrobacter defluvii Kim et al. 2008.The description is as provided by Kim et al. (2008) for Arthrobacter defluvii with the following additional properties.The peptidoglycan type, quinone system and fatty acid profile are in agreement with the genus description, but the polar lipid profile is unknown.
The type strain is 4C1-a T (5KCTC 19209 T 5DSM 18782 T ).The description is as provided by Yassin et al. (2011b) for Arthrobacter equi with the following additional properties.

Description of
The peptidoglycan type, quinone system, polar lipid profile and fatty acid profile are in agreement with the genus description, but a glycolipid corresponding to trimannosyldiacylglycerol is not detected.
The type strain is IMMIB L-1606 T (5CCUG 59597 T 5DSM 23395 T ).The description is as provided by Ding et al. (2009) for Arthrobacter niigatensis with the following additional properties.The peptidoglycan type, quinone system and fatty acid profile are in agreement with the genus description, but the polar lipid profile is unknown.

Description of
The type strain is LC4 T (5CCTCC AB 206012 T 5JCM 30147 T ).The description is as provided by Sguros (1954) for Arthrobacter oxydans with the following additional properties.The quinone system, fatty acid profile, polar lipid profile and peptidoglycan type are in accordance with the genus description but, in addition, the polar lipid profile is also reported to contain digalactosyldiacylglycerol (Amadi & Alderson, 1982).The predominant fatty acid is anteiso-C 15 : 0 , followed by anteiso-C 17 : 0 , C 16 : 0 , iso-C 16 : 0 and iso-C 15 : 0 .Minor fatty acids are iso-C 14 : 0 , iso-C 17 : 0 , C 14 : 0 and C 15 : 0 (Kodama et al., 1992).According to Kodama et al. (1992), the type strain is positive for utilization of the carbon sources L-arginine, L-asparagine, L-his- tidine, L-arabinose, D-galactose, D-glucose, L-rhamnose, D- ribose, D-xylose and malonate but does not utilize inositol.Utilization of L-leucine and butanediol is weak.Citric acid, formic acid, propionic acid and uric acid are assimilated, but not adipic acid, benzoic acid, glutaric acid, malonic acid or pimelic acid.Reduces nitrate to nitrite.Does not produce nicotine blue.

Description of
The type strain is ATCC 14358 T 5BCRC (formerly CCRC) 11573 T 5CCUG 17757 T 5CIP 107005 T 5DSM 20119 T 5 HAMBI 1857 T 5IFO (now NBRC) 12138 T 5IMET 10684 T 5 JCM 2521 T 5LMG 3816 T 5NCIMB 9333 T 5NRIC 0154 T 5 VKM Ac-1114 T .The G+C content of the genomic DNA of the type strain is 63.1 mol% (HPLC; Kodama et al., 1992).The description is as provided by Kallimanis et al. (2009) for Arthrobacter phenanthrenivorans with the following additional properties.In contrast to the genus description, the quinone system consists predominantly of MK-8 and relatively large amounts of MK-9(H 2 ); the polar lipid profile contains phosphatidylethanolamine and glycolipids are absent.
The type strain is Sphe3 T (5DSM 18606 T 5JCM 16027 T 5 LMG 23796 T ).The description is as provided by Huang et al. (2005) for Arthrobacter scleromae with the following additional properties.In contrast to the genus description, the major menaquinone is MK-8(H 2 ).The polar lipid profile contains the major glycolipids dimannosylglyceride and monogalactosyldiacylglycerol, but the presence of other lipids has not been studied (Pas ´ciak et al., 2010).

2013.
The description is as provided by SantaCruz-Calvo et al. (2013) for Arthrobacter siccitolerans with the following additional properties.Shares the peptidoglycan type, quinone system and fatty acid profile listed in the genus description, but the polar lipid profile is unknown.
The type strain is 4J27 T (5CECT 8257 T 5LMG 27359 T ).The G+C content of the genomic DNA of the type strain is 65.3 mol% (HPLC).The description is as provided by Borodina et al. (2002) for Arthrobacter sulfonivorans with the following additional properties.The peptidoglycan type, quinone system and fatty acid profile are in agreement with the genus description, but the polar lipid profile is unknown.
Description of Glutamicibacter gen.nov.
Based on available data for the type species and other species assigned to the genus, the peptidoglycan type is A4a (Lys-Ala-Glu; A11.35).The quinone system contains exclusively unsaturated menaquinones (MK-8 and/or MK-9).The polar lipid profile is composed of the major compounds diphosphatidylglycerol, phosphatidylglycerol and dimannosyldiacylglycerol.Monogalactosyldiacylglycerol and minor amounts of trimannosyldiacylglycerol may also be present.Phosphatidylinositol is absent.The major fatty acid is anteiso-C 15 : 0 .Relatively large amounts of iso-C 15 : 0 , iso-C 16 : 0 , anteiso-C 17 : 0 and C 16 : 0 may also be present.The G+C content of the genomic DNA is in the range 55-67 mol%.The type species is Glutamicibacter protophormiae.
Basonyms: Brevibacterium protophormiae Lysenko 1959; Arthrobacter protophormiae Stackebrandt et al. 1984.The description is as provided by Stackebrandt et al. (1983) for Arthrobacter protophormiae with the following additional properties.Shares the characteristics listed in the genus description (Collins & Kroppenstedt, 1983).According to Osorio et al. (1999), the type strain is negative for urease and b-galactosidase.Positive for nitrate reduction and pyrazinamidase.Assimilates cellobiose, glycerol, maltose and 5-ketogluconate.Amygdalin, arbutin, D-arabitol, galactose, D-mannose, turanose, D-xylose, L-arabinose, inositol, mannitol, melibiose, rhamnose, ribose, The description is as provided by Irlinger et al. (2005) for Arthrobacter arilaitensis with the following additional properties.The peptidoglycan composition is in accordance with the genus description, but the quinone system and polar lipid and fatty acid profiles are unknown.
Glutamicibacter bergerei (ber.ge9re.i.N.L. gen.n. bergerei of Berge `re, to honour Jean-Louis Berge `re, a French microbiologist).The description is as provided by Irlinger et al. (2005) for Arthrobacter bergerei with the following additional properties.The peptidoglycan composition is in accordance with the genus description, but the quinone system and polar lipid and fatty acid profiles are unknown.
The type strain is SYB2 T (5KCTC 19291 T 5DSM 19449 T ).The description is as provided by Stackebrandt et al. (1983) for Arthrobacter uratoxydans with the following additional properties.The peptidoglycan composition and quinone system are in accordance with the genus description; the fatty acid profile is in agreement with the genus description, consisting of the major fatty acid anteiso-C 15 : 0 , followed by iso-C 15 : 0 , anteiso-C 17 : 0 and iso-C 17 : 0 .Minor fatty acids are iso-C 16 : 0 , iso-C 14 : 0 , C 16 : 0 and C 18 : 0 (Funke et al., 1996).According to Osorio et al. (1999), the type strain is negative for b-galactosidase.Positive for nitrate reduction, urease and pyrazinamidase.Assimilates glycerol, ribose and N-acetylglucosamine. Amygdalin, arbutin, cellobiose, D-arabitol, galactose, maltose, D-mannose, turanose, D-xylose, L-arabinose, mannitol, melibiose, rhamnose, salicin, starch, sucrose, trehalose, xylitol, gentiobiose and 5-ketogluconate are not assimilated.The peptidoglycan type is A4a (Lys-Glu; A11.54).The quinone system contains exclusively unsaturated menaquinones, with MK-9 or MK-10 predominating.The polar lipid profile is composed of the major compounds diphosphatidylglycerol, phosphatidylglycerol and digalactosyldiacylglycerol.Phosphatidylinositol is absent.The major fatty acid is anteiso-C 15 : 0 .Large amounts of iso-C 15 : 0 , iso-C 16 : 0 , anteiso-C 17 : 0 and C 16 : 0 may also be present.The G+C content of the genomic DNA is in the range 58-68 mol%.The type species is Paeniglutamicibacter sulfureus.The description is as provided by Pindi et al. (2010) for Arthrobacter antarcticus with the following additional properties.Shares the quinone system and fatty acid profile listed in the genus description but, in contrast to the genus description, the polar lipid profile is reported to be composed only of phosphatidylethanolamine and diphosphatidylglycerol and not to contain any glycolipid.

Description of
The type strain is SPC26 T (5LMG 24542 T 5NCCB 100228 T ).
Additional information.The information on the peptidoglycan composition given by Pindi et al. (2010) has not been added to the species description because it may cause confusion.In the species description for Arthrobacter antarcticus, the authors state 'The peptidoglycan diamino acids are lysine and alanine and the acyl type is glutamic acid (A4a variation)'.Firstly, alanine is not a diamino acid.Secondly, from the presence of the amino acids lysine, alanine and glutamic acid, the peptidoglycan variation A4a cannot be concluded because the majority of bacteria with the diamino acid lysine also contain glutamic acid at position two of the peptide side chain.In order to reach conclusions on the peptidoglycan type, at least relative amounts of the different amino acids must be provided.Thirdly, the acyl type is never glutamic acid, but is either acetyl or glycolyl (Uchida & Seino, 1997).The description is as provided by Ganzert et al. (2011) for Arthrobacter cryotolerans with the following additional properties and modifications.The peptidoglycan composition and quinone system are in accordance with the genus description.In contrast to the genus description, C 18 : 0 is the second most abundant fatty acid, and relatively large amounts of C 16 : 0 , C 18 : 2 and C 18 : 1 v9c (5-11 %) are present.Contrary to the species description for Arthrobacter cryotolerans, the polar lipid profile contains diphosphatidylglycerol, phosphatidylglycerol, digalactosyldiacylglycerol and two minor lipids (Fig. 3j).
Basonym: Arthrobacter gangotriensis Gupta et al. 2004.The description is as provided by Gupta et al. (2004) for Arthrobacter gangotriensis with the following additional properties.The peptidoglycan structure, fatty acid profile and quinone system are in accordance with the genus description.The polar lipid profile is unknown.
The type strain is Lz1y T (5CIP 108630 T 5DSM 15796 T 5 JCM 12166 T ).The description is as provided by Gupta et al. (2004) for Arthrobacter kerguelensis with the following additional properties.The peptidoglycan structure, fatty acid profile and quinone system are in accordance with the genus description.The polar lipid profile is unknown.

Description of
The type strain is KGN15 T (5CIP 108629 T 5DSM 15797 T 5 JCM 12165 T ).The description is as provided by Margesin et al. (2004) for Arthrobacter psychrophenolicus, except that the polar lipid profile is composed of diphosphatidylglycerol, phosphatidylglycerol and digalactosyldiacylglycerol and phosphatidylinositol is absent (Fig. 3i).

Description of
The type strain is AG31 T (5CIP 108593 T 5DSM 15454 T 5 IAM 15315 T 5JCM 135685LMG 21914 T ).The peptidoglycan type is A4a (Lys-Ala-Glu, A11.35; or Lys-Ser-Glu, A11.58).The quinone system contains predominantly menaquinone MK-8(H 2 ).The polar lipid profile is more complex than in other arthrobacters.It is composed of the major compounds diphosphatidylglycerol, phosphatidylglycerol and dimannosyldiacylglycerol.Phosphatidylinositol, monogalactosyldiacylglycerol, trimannosyldiacylglycerol and significant amounts of five unidentified phospholipids are also present.The major fatty acid is anteiso-C 15 : 0 .Relatively large amounts of iso-C 16 : 0 and anteiso-C 17 : 0 are also present.The G+C content of the genomic DNA of the type strain of the type species is 60 mol%.The type species is Pseudoglutamicibacter cumminsii.
Pseudoglutamicibacter cumminsii (cum.min9si.i.N.L. gen.masc.n. cumminsii of Cummins, to honour Cecil S. Cummins, a prominent American microbiologist and a pioneer of chemotaxonomy).The description is as provided by Funke et al. (1996Funke et al. ( , 1998) ) for Arthrobacter cumminsii with the following additional properties.Shares the polar lipid profile and quinone system listed in the genus description.The peptidoglycan structure is A4a (L-Lys-L-Ser-L-Glu; A11.58).
Description of Pseudoglutamicibacter albus comb.nov.
Pseudoglutamicibacter albus (al9bus.L. masc.adj.albus white, because of the white colonies of the organism).The description is as provided by Wauters et al. (2000) for Arthrobacter albus with the following additional properties.Shares the polar lipids and quinone system listed in the genus description.The peptidoglycan structure is A4a (L-Lys-L-Ala-L-Glu; A11.35).

Status of the remaining species of the genus Arthrobacter
The data discussed in this study strongly suggest that the genus Arthrobacter sensu stricto should be restricted to the four species A. globiformis, A. pascens, A. oryzae and A. humicola, which share high 16S rRNA gene sequence similarities, a quinone system with MK-9(H 2 ) predominant and a peptidoglycan with Lys-Ala 2-3 (A3a; A11.5, A11.6).However, such a proposal would exclude numerous species from the genus Arthrobacter without the option to reclassify them in other genera, since stable diagnostic traits are not yet available.Hence, the description of Arthrobacter sensu stricto should await the availability of data allowing the assignment to other genera of species that differ from the genus description of Arthrobacter sensu stricto.
For this reason, a description of the genus Arthrobacter sensu lato is provided in order to cover species belonging to groups of the genus Arthrobacter that, so far, cannot be reclassified in novel genera.The description of Arthrobacter sensu lato is provided to replace the original description of Conn & Dimmick (1947) and the description given by Keddie (1974) because these descriptions are based exclusively on cultural, morphological and physiological characteristics that have to be considered not to be sufficiently conserved to characterize a genus.Cells exhibit a rod-coccus cycle but exclusively coccoid cells may occur.The quinone system usually contains MK-9(H 2 ) as the predominant compound, but almost equal amounts of MK-9(H 2 ) and MK-8(H 2 ) or MK-8(H 2 ) as the major compound may occur.The peptidoglycan type is A3a [variations Lys-Ala 1-4 , Lys-Thr-Ala 1-3 , Lys-Ala-Ser-Ala 3 , Lys-Gly-Ala 3 and Lys-Ala 2 -Gly 2-3 -Ala(Gly)]; peptidoglycan type A4a may also be present, as reported for Arthrobacter woluwensis (Lys-D-Asp) or in combinations with quinone system MK-9(H 2 ) in Arthrobacter rhombi and Arthrobacter halodurans (Lys-Ala-D-Glu; Chen et al., 2009).The polar lipid profile contains predominantly diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and dimannosylglyceride.The fatty acid profile is dominated by anteiso-and iso-methyl-branched acids.The major fatty acid is usually anteiso-C 15 : 0 .Fatty acids iso-C 15 : 0 , iso-C 16 : 0 and anteiso-C 17 : 0 often contribute significantly to the profile.The type species is Arthrobacter globiformis.

Emended description of the genus Arthrobacter
Emended description of Arthrobacter roseus Reddy et al. 2002 The description is as provided by Reddy et al. (2002), except that the polar lipid profile does not contain phosphatidylethanolamine.In addition to phosphatidylglycerol and diphosphatidylglycerol, phosphatidylinositol, International Journal of Systematic and Evolutionary Microbiology 66 The type strain is CMS 90r T (5CIP 107726 T 5CMC 90or T 5CMS 90or T 5DSM 14508 T 5JCM 11881 T 5MTCC 3712 T 5NCIMB 14039 T ).

Fig. 1 .
Fig.1.Maximum-likelihood tree based on 16S rRNA gene sequences (1224 nt).The 16S rRNA gene sequence of Microbacterium lacticum DSM 20427 T was used as an outgroup.Bootstrap values .60 based on 100 replicates are given at nodes.Bar, 0.1 substitutions per nucleotide position.The names of groups of the genus Arthrobacter, as defined byBusse et al. (2012), are provided in shaded rectangles.Filled squares indicate that the same branching node with bootstrap support .60 % was found after analyses using the maximum-parsimony and neighbour-joining algorithms.Open circles indicate that the same branching node with bootstrap support .60 % was found after analyses using either the maximum-parsimony or neighbour-joining algorithm.Type species of genera are shown in bold face.

Fig. 2 .
Fig. 2. Maximum-likelihood tree based on recA gene sequences (381 nt).The corresponding gene sequence of Microbacterium lacticum DSM 20427 T was used as outgroup.Bootstrap percentages .60 % based on 200 replicates are given at nodes.Bar, 0.1 substitutions per nucleotide position.

Table 1
Keddie et al. (1986))Bacteriology except that the two groups of Arthrobacter differ with respect to their lipid composition, citation of this paper concerning the presence of DMDG and PI in both groups is apparently due to an error.This information is also not provided in the following chapter in Bergey's Manual of Systematic Bacteriology dealing with the genus Arthrobacter(Keddie et al., 1986).These latter authors indicated that the majority of species of Arthrobacter analysed for polar lipids, including two species of the 'A.nicotianae group', namely A. nicotianae and A. protophormiae, contain DMDG, whereas a single representative of the 'A.nicotianae group', A. sulfureus, instead contains DGDG.Keddie et al. (1986)also emphasized the absence of PI in the representatives of the 'A.nicotianae group'.Hence, the information that both groups of the genus Arthrobacter are characterized by the presence of DMDG and PI in the polar lipid profile is misleading, Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Mon, 04 Feb 2019 19:10:17 concerning lipids is provided in the cited chapter of Bergey's because the members of the 'A.nicotianae group' lack PI and contain either DMDG or DGDG.It is important to point out this error because, to date, Stackebrandt et al.
Stackebrandt et al. (1983)ally separate from the 'A.globiformis group' but without bootstrap support for the branching node(Zhang et al., 2012; Fig. 1), the recently described species A. cupressi might be considered another member of this group.The type strain shares the highest 16S rRNA gene sequence similarity with two members of the group, A. oryzae (97.7 %) and A. humicola (97.3 %), and 96.9 and 96.8 % similarity with the type strains of A. pascens and A. globiformis, respectively.Like in species of the 'A.globiformis group', its peptidoglycan structure shows the interpeptide bridge Lys-Ala 2 , but the presence of a glycine bound to the a-carboxyl group of D-gluta- mic acid distinguishes it from other members of the group.thespecies A. ardleyensis, A. arilaitensis, A. bergerei, A. mysorens, A. nicotianae, A. protophormiae, A. soli and A. uratoxydans, which share 96.7-99.7 % 16S rRNA gene sequence similarity.Except A. soli, which was not analysed for this trait, all species show peptidoglycan type A4a (Lys-Ala-Glu) corresponding to peptidoglycan structure A11.35.According toStackebrandt et al. (1983), the glutamic acid in the interpeptide bridge of A. nicotianae, A. protophormiae and A. uratoxydans occurs in the L-configuration.The quinone system contains exclusively completely unsaturated menaquinones, always with MK-8 as a major component (no data are available for A. arilaitensis, A. bergerei and A. soli).A. rhombi and A. creatinolyticus had been assigned to this group because they share 16S rRNA gene sequence similarity of .96.5 % with at least one representative of the group and show the presence of the group-specific peptidoglycan composition and/or quinone system.However, the presence of a quinone system menaquinone MK-9(H 2 ) The 'A. aurescens group' is defined based on high 16S rRNA gene sequence similarity among its members (.97.5 %), the presence of the major respiratory menaquinone MK-9(H 2 ) and peptidoglycan of type A3a (Lys-Ala-Thr-Ala) corresponding to peptidoglycan structure A11.17.The group comprises six species, A. aurescens, A. histidinolovorans, A. ilicis, A. nicotinovorans, Arthrobacter nitroguajacolicus and A. ureafaciens.The 'A.oxydans group' contains the species A. chlorophenolicus, A. defluvii, A. niigatensis, A. oxydans, A. phenanthrenivorans, A. polychromogenes, A. scleromae and Arthrobacter sulfonivorans, which share high 16S rRNA gene sequence similarities (.97 %), MK-9(H 2 ) as a major quinone [except A. scleromae, which was reported to contain predominantly MK-8(H 2 )] and a peptidoglycan type with Lys-Ser-Thr-Ala in the interpeptide corresponding to peptidoglycan structure A11.23 chain (A.phenanthrenivorans was not analysed for its peptidoglycan type).thesamecharacteristic peptidoglycan structure and quinone system and shares high 16S rRNA gene sequence similarity with species of the 'A.oxydans group' (.98 %).Species of the'A.protophormiae group' (including A. rhombi) were reported to share 95.5-99.7 % 16S rRNA gene sequence similarity.The core of this group is composed International Journal of Systematic and Evolutionary Microbiology 66 Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Mon, 04 Feb 2019 19:10:17 of suggest that they generally contain a core polar lipid profile that is composed of DPG, PG and at least one glycolipid (either a International Journal of Systematic and Evolutionary Microbiology 66 from www.microbiologyresearch.orgby IP: 54.70.40.11On: Mon, 04 Feb 2019 19:10:17