International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2034–2040
DOI 10.1099/ijs.0.050005-0
Polymorphobacter multimanifer gen. nov., sp. nov., a polymorphic bacterium isolated from antarctic white rock Wakao Fukuda,1 Yohzo Chino,1 Shigeo Araki,1 Yuka Kondo,2 Hiroyuki Imanaka,3 Tamotsu Kanai,2 Haruyuki Atomi2 and Tadayuki Imanaka1 Correspondence Tadayuki Imanaka [email protected]
1
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan
2
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
3
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
A Gram-stain-negative, non-spore-forming, aerobic, oligotrophic bacterium (strain 262-7T) was isolated from a crack of white rock collected in the Skallen region of Antarctica. Strain 262-7T grew at temperatures between ”4 and 30 6C, with optimal growth at 25 6C. The pH range for growth was between pH 6.0 and 9.0, with optimal growth at approximately pH 7.0. The NaCl concentration range allowing growth was between 0.0 and 1.0 %, with an optimum of 0.5 %. Strain 262-7T showed an unprecedented range of morphological diversity in response to growth conditions. Cells grown in liquid medium were circular or ovoid with smooth surfaces in the lag phase. In the exponential phase, ovoid cells with short projections were observed. Cells in the stationary phase possessed long tentacle-like projections intertwined intricately. By contrast, cells grown on agar plate medium or in liquid media containing organic compounds at low concentration exhibited short- and long-rod-shaped morphology. These projections and morphological variations clearly differ from those of previously described bacteria. Ubiquinone 10 was the major respiratory quinone. The major fatty acids were C17 : 1v6c (28.2 %), C16 : 1v7c (22.6 %), C18 : 1v7c (12.9 %) and C15 : 0 2-OH (12.3 %). The G+C content of genomic DNA was 68.0 mol%. Carotenoids were detected from the cells. Comparative analyses of 16S rRNA gene sequences indicated that strain 262-7T belongs to the family Sphingomonadaceae, and that 2627T should be distinguished from known genera in the family Sphingomonadaceae. According to the phylogenetic position, physiological characteristics and unique morphology variations, strain 262-7T should be classified as a representative of a novel genus of the family Sphingomonadaceae. Here, a novel genus and species with the name Polymorphobacter multimanifer gen. nov., sp. nov. is proposed (type strain 262-7T5JCM 18140T5ATCC BAA2413T). The novel species was named after its morphological diversity and formation of unique projections.
The climate in Antarctica, with its extreme cold, dryness, strong winds, seasonally strong UV radiation and low concentration of organic materials, makes it difficult for most organisms to sustain life. However, a great diversity of micro-organisms has been found in icy environments, such as permafrost, polar oceans, snow, lake ice, sea ice and The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 262-7T is AB649056. Three supplementary figures and one supplementary table are available with the online version of this paper.
2034
cryoconite holes (Karr et al., 2006; Prisco, 2007; Priscu et al., 1998; Rothschild & Mancinelli, 2001; Tindall, 2004). In addition, the presence of chasmolithic, hypolithic and endolithic communities have been reported (Cary et al., 2010). The rocks of Antarctica are considered to provide physical stability and to protect microbial communities from UV radiation and dryness. A white rock sample was collected by the summer party of the 46th Japanese Antarctic Research Expedition in 2004–2005 from the Skallen region in Antarctica (Yamada et al., 2011). Interestingly, the interior of the white rock was colourful (green, pink, yellow and 050005 G 2014 IUMS Printed in Great Britain
Polymorphobacter multimanifer gen. nov., sp. nov.
brown), suggesting the existence of a cryptoendolithic community containing cyanobacteria. When autotrophic bacteria were screened from the crushed rock under light conditions, a green filamentous bacterium (named strain 262-1) covered with several heterotrophic bacteria was obtained. By the cultivation of these bacteria under dark conditions, more than 10 different bacterial species including strains 262-7T and 262-8T (the type strain of Constrictibacter antarcticus) were obtained (Yamada, et al., 2011). Strain 262-7T could grow well in 0.256 LB medium (l21: 2.5 g tryptone, 1.3 g yeast extract and 5.0 g NaCl in tap water) under aerobic conditions. However, no growth was observed in 0.756 LB medium containing (l21) 7.5 g tryptone and 3.8 g yeast extract. These results indicated that strain 262-7T is an oligotroph. Strain 262-7T could also grow in 0.256 LB/ASW medium [l21: 2.5 g tryptone, 1.3 g yeast extract, 5.0 g NaCl, 0.75 g MgCl2 . 6H2O, 1.5 g MgSO4 . 7H2O, 0.25 g (NH4)2SO4, 0.050 g NaHCO3, 0.075 g CaCl2 . 2H2O, 0.13 g KCl, 0.11 g KH2PO4, 0.013 g NaBr, 0.005 g SrCl2 . 6H2O, 0.025 g ferric ammonium citrate] and quarter strength Difco Marine Broth 2216 medium (BD Difco). Strain 262-7T grew at 24–30 uC with an optimum growth temperature of 25 uC in 0.256 LB medium, while no growth was observed at temperatures above 37 uC. When strain 262-7T grew on 0.256 LB plate medium containing 20 g agar l21 at 24 uC in a freezing chamber, colony formation was observed. The pH range for growth was pH 6.0–9.0, with an optimum of approximately pH 7.0. Strain 262-7T was able to grow at NaCl concentrations of 0.0–1.0 % (w/v), with an optimum of 0.5 % (w/v). When strain 262-7T was cultivated in 0.256 LB medium under optimal growth conditions, the maximum OD660 reached approximately 0.9 after 70 h of incubation (doubling time, 5.2 h). When strain 262-7T was grown on 0.256 LB plate medium, brown, raised and circular colonies were formed with an entire margin. Strain 262-7T could not grow in 0.256 LB medium containing 5.0 g l21 of either Na2SO4 or NaNO3 under anaerobic conditions. Analyses of Gram-staining, catalase and oxidase activity, motility and spore formation were performed by methods described previously (Yamada, et al., 2011). The results revealed that strain 262-7T was a Gram-stain-negative, catalase-positive, oxidase-positive, non-spore-forming and motile bacterium. Further biochemical characteristics were analysed by using API 20NE and API ZYM kits (bioMe´rieux) and antibiotic sensitivities were examined by using an ATB VET kit (bioMe´rieux); results are given in the species description. The morphology of the cells cultivated in liquid medium was examined by a bright-field microscope (BX51; Olympus). Cells in the logarithmic and stationary phases of growth were examined, and circular and ovoid cells were observed (data not shown). The ultrastructures of cells were examined by scanning electron microscopy (S-4700; Hitachi), and it became clear that strain 262-7T showed morphology variations depending on growth phase and growth medium. http://ijs.sgmjournals.org
When cells in the lag phase were observed, circular or ovoid cells with smooth surfaces were observed (Fig. 1a). In the exponential phase, ovoid cells with short projections were observed (Fig 1b, c). Cells in the stationary phase possessed long tentacle-like projections which were intertwined intricately (Fig. 1d). It has been reported that rod-shaped and coccid cells coexist in the case of Porphyrobacter neustonensis (Hiraishi & Imhoff, 2005). However, the projections and morphology variations seen in strain 2627T are clearly distinct from those reported in other bacteria, and may represent a response to nutrient deficiency. It has been reported that Prosthecomicrobium hirschii and Ancalomicrobium adetum harbour long projections called prosthecae (Staley, 1968, 1984). In the case of A. adetum, it has been suggested that the long and straight prosthecae help the bacterium to resist ingestion by protists (Bianchi, 1989; Young, 2007). However, the straight projections of A. adetum seem quite different from those of strain 262-7T in appearance. In the case of a stalked bacterium, Caulobacter vibrioides (former name, Caulobacter crescentus), the stalk (prosthecae) affixes the organism to solid surfaces in aqueous environments, and the length of the stalk is regulated by the availability of nutrients (Young, 2007). The cells of strain 262-7T gradually became smaller in response to the progression of the projections (Fig 1c, d). It seemed that the surface area of the cell was expanded by the production of the projections, and the projections of strain 262-7T might be used for nutrient uptake in the oligotrophic environments present in the gaps inside the rock. By contrast, it could be possible that cells were connected by the projections, and the projections might be involved in biofilm formation. A tendency of 262-7T cells to aggregate in the stationary phase was observed. When cells cultivated on 0.256 LB agar medium were analysed, long-rod-shaped cells (more than 10 mm in length) were observed among short-rod-shaped cells (Fig. 1e). To distinguish live cells from dead cells, nucleic acids
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. Scanning electron micrographs (a–e) and atomic force micrograph (f) of strain 262-7T. (a) Cell in lag phase. (b) Cells in early exponential phase. (c) Cells in mid–late exponential phase. (d) Cells in stationary phase. (e) Cells cultivated on 0.25¾ LB agar medium. (f) Atomic force micrograph of strain 262-7T in midexponential phase. Bars, 1 mm (a–e) and 2 mm each (f). 2035
W. Fukuda and others
of strain 262-7T were stained with SYTO9 dye, which stains cells with intact and damaged membranes, and with propidium iodide (PI) dye, which only stains cells with damaged membranes, and the stained cells were examined by fluorescence microscopy (Fig. S1, available in the online Supplementary Material). As a result, nucleic acids of most of the long-rod-shaped cells were stained only by SYTO9 dye, suggesting that the long-rod-shaped cells were alive. A similar observation was reported for Caulobacter vibrioides, and the elongated cells displayed increased resistance to stress (Wortinger et al., 1998). Moreover, when strain 2627T was cultivated in media containing organic compounds at low concentrations [0.16 LB medium (l21: 1.0 g tryptone, 0.5 g yeast extract and 5.0 g NaCl in tap water) or 0.016 LB medium (l21: 0.1 g tryptone, 0.05 g yeast extract and 5.0 g NaCl in tap water)], the existence of rod-shaped cells (approx. 3–10 mm in length) was observed (data not shown). Strain 262-7T was isolated from oligotrophic environment, and, therefore, strain 262-7T may be a rod-shaped bacterium in the natural environment. The rod-shaped cell formation of strain 262-7T may be of advantage for adaptation to oligotrophic environments or for resistance to environmental changes. Although flagella could not be observed by scanning electron microscopy, a flagellum was observed by atomic force microscopy (OLS3500; Olympus) (Fig. 1f). When the 16S rRNA gene sequence of strain 262-7T was analysed with similarity search programs (nucleotide BLAST) provided by the National Center for Biotechnology Information (Altschul et al., 1990), related known strains were Sandarakinorhabdus limnophila so42T (Gich & Overmann, 2006) and Sphingomonas fennica K101T (Wittich et al., 2007) in the family Sphingomonadaceae, with 16S rRNA gene sequence similarities of 95.0 and 94.6 %, respectively. To examine the phylogenetic position
0.02
of strain 262-7T, a phylogenetic tree based on 16S rRNA gene sequences from bacteria belonging to the order Sphingomonadales (Fig. 2) was reconstructed with the CLUSTAL W program (Thompson et al., 1994). The order Sphingomonadales contains two families, Sphingomonadaceae and Erythrobacteraceae (Lee et al., 2005). Although some pleomorphic genera belong to the family Erythrobacteraceae (Hiraishi & Imhoff, 2005), strain 262-7T was located in the clade of the family Sphingomonadaceae. In addition, strain 262-7T was located in a branch distinct to the previously recognized genera belonging to the family Sphingomonadaceae, although strain 262-7T was located in a clade with Sandarakinorhabdus limnophila and Sandaracinobacter sibiricus. This result indicated that strain 262-7T is relatively closely related to the genera Sandarakinorhabdus and Sandaracinobacter, and suggested that strain 262-7T is a phylogenetically novel bacterium in the family Sphingomonadaceae. The G+C content of the genomic DNA, respiratory quinone, fatty acids and polar lipids pattern were analysed by previously described methods with slight modification (Fukuda et al., 2012; Yamada, et al., 2011). The fatty acids composition was analysed by the Sherlock Microbial Identification System using version 6.0 of the TSBA6 library. The alkaline-stable lipids were extracted by the method of Matsuyama et al. (2008). The chemotaxonomic characteristics of strain 262-7T and those of the genera in the family Sphingomonadaceae are shown in Table 1. The G+C content of the genomic DNA was 68.0 mol%, which was in the same range as those of members of the genera Sandaracinobacter and Sphingomonas, and is slightly higher than that of members of the other genera in the family Sphingomonadaceae. The major respiratory quinone of strain 262-7T was ubiquinone 10, which is the most
CC-TBT-3T
(EU564841) Sphingomicrobium lutaoense Sphingosinicella microcystinivorans Y2T (AB084247) Stakelama pacifica JLT832T (EU581829) Zymomonas mobilis ATCC 10988T (AF281031) Sphingobium yanoikuyae IFO 15102T (D13728)
54
Sphingopyxis macrogoltabida 203T (D13723) Parasphingopyxis lamellibrachiae JAMH 0132T (AB524074)
Sphingomonadaceae
Polymorphobacter multimanifer 262-7T (AB649056) Sandarakinorhabdus limnophila so42T (AY902680) Sandaracinobacter sibiricus RB16-17T (Y10678) Sphingomonas paucimobilis ATCC 29837T (EU37337)
81 64
81
Croceicoccus marinus E4A9T (EF623998) Altererythrobacter epoxidivorans JCS350T (DQ304436) Erythrobacter longus OCh101T (AF465835)
93 81 89
2036
Porphyrobacter neustonensis ACM 2844T (AB033327) 100 Erythromicrobium ramosum E5T (AF465867)
Erythrobacteraceae
Sphingorhabdus planktonica 585T (JN381068) Blastomonas natatoria DSM 3183T (AB024288) Novosphingobium capsulatum GIFU 11526T (D16147)
Fig. 2. Phylogenetic tree of strain 262-7T and members of the order Sphingomonadales based on 16S rRNA gene sequences. This tree was reconstructed using the neighbourjoining method provided by DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp/index. php?lang=en). Bootstrap resampling was performed 1000 times and only values observed in more than 50 % of the replicas are shown at branching points. The 16S rRNA gene sequence of Escherichia coli ATCC 11775T was used as an outgroup. Circles at nodes indicate nodes recovered with bootstrap values .50 % (filled circles) or .70 % (open circles) in a maximum-likelihood tree reconstructed using the DNAML program in the PHYLIP package (Felsenstein, 2009). Bar, 2 substitutions per 100 nucleotides.
International Journal of Systematic and Evolutionary Microbiology 64
http://ijs.sgmjournals.org
Table 1. Characteristics that differentiate strain 262-7T from the genera in the family Sphingomonadaceae Taxa: 1, Strain 262-7T; 2, Sandarakinorhabdus (data from Gich & Overmann, 2006); 3, Sandaracinobacter, (Yurkov, 2005a); 4, Sphingomonas (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 5, Sphingobium (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 6, Novosphingobium (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 7, Sphingopyxis (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 8, Parasphingopyxis (Uchida et al., 2012); 9, Blastomonas (Sly & Hugenholtz, 2005); 10, Sphingomicrobium (Ka¨mpfer et al., 2012); 11, Sphingosinicella (Maruyama et al., 2006); 12, Stakelama (Chen et al., 2010); 13, Zymomonas (Sprenger & Swings, 2005). +, Positive; 2, negative or absent; +or 2, different reaction in different species; ND, no data; Q, ubiquinone. Characteristics Pigmentation (colony colour)
1 Brown
2
Q-10 64.3
Orange–brown, yellow, white or colourless Long rod Rod + +/2 2 ND
nd
Q-9, Q-10 68.5
5
6
Yellow or Yellow or whitish-brown whitish-brown Rod
Rod
7 Yellow or whitishbrown Rod
8
9
Orange– Yellow– yellow orange
10
11
Yellow
Yellow
12
13
Gold–yellow Cream
Ovoid or Irregular Rod Rod Rod rod rod + or 2 + or 2 + + 2 + + + or 2 + or 2 / + + or 2 / + + / 2 + / + + / ND +/+ 2/+ 2/+ + 2 + 2 ND 2 + 2 C18 : 1, C16 : 0 C18 : 1v7c, C18 : 1v7c, C18 : 1v9 C18 : 1v7c C16 : 1v7c, C18 : 1v7c, C18 : 1 C16 : 0 C16 : 0 C18 : 1v7c C16 : 0
+ or 2 + or 2 / + + or 2 C18 : 1, C16 : 0, C17 : 1, C17 : 0 cyclo C14 : 0 2-OH, C15 : 0 2-OH
+ or 2 + or 2 / + 2 C18 : 1, C16 : 0
C14 : 0 2-OH
C14 : 0 2-OH
Q-10 62–68
Q-10 59–67
Q-10 62–67
C14 : 0 2-OH, C15 : 0 2-OH C16 : 0 2-OH Q-10 63.0–65.0
Rod
C16 : 0 2-OH
C14 : 0 2-OH
C18 : 1 2-OH
C14 : 0 2-OH
C14 : 0 2-OH
Q-10 60.1
Q-10 65
Q-10 63.4
Q-10 63.6–63.7
Q-10 66±0.5
2
ND
47.5–49.5
2037
Polymorphobacter multimanifer gen. nov., sp. nov.
Q-10 68.0
4
Orange–red Yellow– orange
Cell morphology Ovoid to rod Rod (Pleomorphic) Motility + 2 Oxidase/catalase +/+ 2/+ ND Nitrate reduction 2 C16 : 1v7c, Major fatty acids C17 : 1v6c, C16 : 1v7c, C18 : 1v7c C18 : 1v7c Major hydroxyl C15 : 0 2-OH iso-C14 : 0 2-OH fatty acids Major quinone DNA G+C content (mol%)
3
W. Fukuda and others
frequently identified respiratory quinone in bacteria belonging to the family Sphingomonadaceae (Yabuuchi & Kosako, 2005a). To analyse the composition of cellular fatty acids, cells of strain 262-7T were cultivated in 0.256 LB medium at 25 uC until the late exponential phase of growth. The major cellular fatty acids of strain 262-7T were C17 : 1v6c (28.2 %), C16 : 1v7c (22.6 %), C18 : 1v7c (12.9 %) and C15 : 0 2-OH (12.3 %); C14 : 0 2-OH (7.1 %), C16 : 0 (3.1 %) and C15 : 1v6c (3.1 %) were detected as minor fatty acids. Trace amounts of iso-C16 : 0 3-OH (0.9 %) were detected. The major nonpolar and 2-hydroxy fatty acids of strain 262-7T were different from those of the related genus Sandarakinorhabdus (Table 1), although fatty acid compositions of species of the genus Sphingomonas are similar to those of strain 262-7T (Takeuchi et al., 2001). The lipids pattern on two-dimensional TLC revealed the presence of phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, an unknown phospholipid and two unknown glycolipids (Fig. S2 and Table S1). The lipids pattern detected was similar to that of Sphingomonas xinjiangensis, although some lipids detected in Sphingomonas xinjiangensis were not found (An et al., 2011). Absence of phosphatidylcholine and the pattern of sphingoglycolipids were different from the lipids pattern of the closely related species Sandarakinorhabdus limnophila (Gich & Overmann, 2006). Pigment analysis was performed using cells in the mid–late exponential phase by the modified method described by Saga et al. (2005). The spectra of pigments extracted from cells showed three peaks at 428, 452 and 478 nm, similar to Sandaracinobacter sibiricus (Yurkov et al., 1997) (Fig. S3). The result indicated the presence of carotenoids in the cells of strain 262-7T. No peaks above 600 nm derived from bacteriochlorophyll a were observed, contrary to Sandaracinobacter sibiricus and genera in the family Erythrobacteraceae (Yurkov et al., 1997; Yurkov, 2005b). According to its phylogenetic position, strain 262-7T is most closely related to the genera Sandarakinorhabdus and Sandaracinobacter, and is clearly different from other genera in the family Sphingomonadaceae. Fatty acids composition and production of oxidase/catalase are different from the related genera Sandarakinorhabdus and Sandaracinobacter (Table 1). Above all, unique morphology variations and appearance of strain 262-7T were observed. Therefore, strain 262-7T should be classified as a representative of a novel species and genus in the family Sphingomonadaceae, for which the name Polymorphobacter multimanifer gen. nov., sp. nov. is proposed. Description of Polymorphobacter gen. nov. Polymorphobacter (po.ly.mor.pho.bac9ter. Gr. adj. polumorphos multiform; N.L. masc. n. bacter rod; N.L. masc. n. polymorphobacter a polymorphic rod).
rods. Cells contain carotenoids. Cells produce catalase and oxidase, and grow aerobically. Nitrate reduction is not observed. The major respiratory quinone is Q-10. The cellular fatty acids are mainly composed of C17 : 1v6c, C16 : 1v7c and C18 : 1v7c. The major 2-hydroxy fatty acid is C15 : 0 2-OH. The DNA G+C content of the type species is 68.0 mol%. The type species is Polymorphobacter multimanifer. Description of Polymorphobacter multimanifer sp. nov. Polymorphobacter multimanifer [mul.ti.ma9ni.fer. L. adj. multus many; L. n. manus hand; L. suff. -fer, (from L. v. fero) bearing, carrying, producing; N.L. masc. adj. n. multimanifer many hands bearer, referring to tentacle-like projections]. Colonies are brown, raised, circular and entire. Cells are circular or ovoid with smooth surfaces in the lag phase of growth. In the exponential growth phase, ovoid cells with short projections are observed. Cells in the stationary phase possess long tentacle-like projections intertwined intricately. By contrast, cells on 0.256 LB agar plate medium exhibit short- and long-rod-shaped morphology. Growth occurs between 24 and 30 uC (optimum, 25 uC), between pH 6.0 and 9.0 (optimum, approximately pH 7.0), and with between 0.0 and 1.0 % NaCl (optimum, 0.5 %). Cells can utilize maltose and malate as a carbon sources, and weak growth is observed in media containing glucose and mannose. Cannot utilize adipate, arabinose, caprate, citrate, gluconate, mannitol and phenylacetate. Production of acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase is observed. Activities of arginine dihydrolase N-acetyl-b-glucosaminidase, a-fucosidase, a-galactosidase, b-galactosidase, gelatinase, a-glucosidase, b-glucuronidase, lipase (C4), a-mannosidase and urease are not observed. H2S and indole are not produced. Cells are resistant to amoxicillin, amoxicillin/clavulanic acid, cefoperazone, cefalotin, colistin, cotrimoxazole, erythromycin, lincomycin, metronidazole, oxacillin, penicillin, sulfamethizole and tylosin. Cells are sensitive to apramycin, chloramphenicol, doxycycline, enrofloxacin, flumequine, fusidic acid, gentamicin, kanamycin, nitrofurantoin, oxolinic acid, pristinamycin, rifampicin, spectinomycin, streptomycin and tetracycline. The type strain is 262-7T (5JCM 18140T5ATCC BAA2413T) and was isolated from a crack of white rock in the Skallen region of Antarctica.
Acknowledgements
Member of the class Alphaproteobacteria, order Sphingomonadales, family Sphingomonadaceae. Cells are Gramstain-negative, motile, non-sporulating and polymorphic
The authors would like to express their sincere thanks to all members of the 46th Japanese Antarctic Research Expedition. This study was supported by JSPS KAKENHI (19205022 and 23657068).
2038
International Journal of Systematic and Evolutionary Microbiology 64
Polymorphobacter multimanifer gen. nov., sp. nov.
References
Saga, Y., Osumi, S., Higuchi, H. & Tamiaki, H. (2005).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410. An, H., Xu, M., Dai, J., Wang, Y., Cai, F., Qi, H., Peng, F. & Fang, C. (2011). Sphingomonas xinjiangensis sp. nov., isolated from desert
Bacteriochlorophyll-c homolog composition in green sulfur photosynthetic bacterium Chlorobium vibrioforme dependent on the concentration of sodium sulfide in liquid cultures. Photosynth Res 86, 123–130. Sly, L. I. & Hugenholtz, P. (2005). Genus II. Blastomonas Sly and
and filaments as a consequence of grazing pressure. Microb Ecol 17, 137–141.
Cahill 1997, 567VP emend. Hiraishi, Kuraishi and Kawahara 2000a, 1117. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 258–263. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Cary, S. C., McDonald, I. R., Barrett, J. E. & Cowan, D. A. (2010).
Sprenger, G. A. & Swings, J. (2005). Genus IX. Zymomonas Kluyver
sand. Int J Syst Evol Microbiol 61, 1865–1869. Bianchi, M. (1989). Unusual bloom of star-like prosthecate bacteria
On the rocks: the microbiology of Antarctic Dry Valley soils. Nat Rev Microbiol 8, 129–138.
and van Niel 1936, 399AL. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 282–286. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Chen, C., Zheng, Q., Wang, Y. N., Yan, X. J., Hao, L. K., Du, X. & Jiao, N. (2010). Stakelama pacifica gen. nov., sp. nov., a new member of the
Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new
family Sphingomonadaceae isolated from the Pacific Ocean. Int J Syst Evol Microbiol 60, 2857–2861.
Staley, J. T. (1984). Prosthecomicrobium-Hirschii, a new species in a
(phylogeny inference package), version 3.69. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Felsenstein, J. (2009).
PHYLIP
Fukuda, W., Yamada, K., Miyoshi, Y., Okuno, H., Atomi, H. & Imanaka, T. (2012). Rhodoligotrophos appendicifer gen. nov., sp. nov.,
an appendaged bacterium isolated from a freshwater Antarctic lake. Int J Syst Evol Microbiol 62, 1945–1950. Gich, F. & Overmann, J. (2006). Sandarakinorhabdus limnophila gen. nov., sp. nov., a novel bacteriochlorophyll a-containing, obligately aerobic bacterium isolated from freshwater lakes. Int J Syst Evol Microbiol 56, 847–854.
prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942. redefined genus. Int J Syst Bacteriol 34, 304–308. Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus
Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–1417. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Tindall, B. J. (2004). Prokaryotic diversity in the Antarctic: the tip of
the iceberg. Microb Ecol 47, 271–283.
Hiraishi, A. & Imhoff, J. (2005). Genus VII. Porphyrobacter Fuerst,
Uchida, H., Hamana, K., Miyazaki, M., Yoshida, T. & Nogi, Y. (2012).
Hawkins, Holms, Sly, Moore and Stackebrandt 1993, 132VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 275–279. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Parasphingopyxis lamellibrachiae gen. nov., sp. nov., isolated from a marine annelid worm. Int J Syst Evol Microbiol 62, 2224–2228.
Ka¨mpfer, P., Arun, A. B., Young, C. C., Busse, H. J., Kassmannhuber, J., Rossello´-Mo´ra, R., Geueke, B., Rekha, P. D. & Chen, W. M. (2012).
and Sphingomonas haloaromaticamans sp. nov., outliers of the genus Sphingomonas. Int J Syst Evol Microbiol 57, 1740–1746.
Sphingomicrobium lutaoense gen. nov., sp. nov., isolated from a coastal hot spring. Int J Syst Evol Microbiol 62, 1326–1330.
Wortinger, M. A., Quardokus, E. M. & Brun, Y. V. (1998). Morphological
Karr, E. A., Ng, J. M., Belchik, S. M., Sattley, W. M., Madigan, M. T. & Achenbach, L. A. (2006). Biodiversity of methanogenic and other
archaea in the permanently frozen Lake Fryxell, Antarctica. Appl Environ Microbiol 72, 1663–1666. Lee, K. B., Liu, C. T., Anzai, Y., Kim, H., Aono, T. & Oyaizu, H. (2005). The hierarchical system of the ‘Alphaproteobacteria’: descrip-
tion of Hyphomonadaceae fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov. Int J Syst Evol Microbiol 55, 1907–1919. Maruyama, T., Park, H. D., Ozawa, K., Tanaka, Y., Sumino, T., Hamana, K., Hiraishi, A. & Kato, K. (2006). Sphingosinicella microcystinivorans
gen. nov., sp. nov., a microcystin-degrading bacterium. Int J Syst Evol Microbiol 56, 85–89. Matsuyama, H., Katoh, H., Ohkushi, T., Satoh, A., Kawahara, K. & Yumoto, I. (2008). Sphingobacterium kitahiroshimense sp. nov., isolated
Wittich, R. M., Busse, H. J., Ka¨mpfer, P., Macedo, A. J., Tiirola, M., Wieser, M. & Abraham, W. R. (2007). Sphingomonas fennica sp. nov.
adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus. Mol Microbiol 29, 963–973. Yabuuchi, E. & Kosako, Y. (2005a). Family I. Sphingomonadaceae
Kosako, Yabuuchi, Naka, Fujiwara, and Kobayashi 2000b, 1953VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, p. 230– 233. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yabuuchi, E. & Kosako, Y. (2005b). Genus I. Sphingomonas
Yabuuchi, Yano, Oyaizu, Hashimoto, Ezaki and Yamamoto 1990b, 321VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 234–258. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yamada, K., Fukuda, W., Kondo, Y., Miyoshi, Y., Atomi, H. & Imanaka, T. (2011). Constrictibacter antarcticus gen. nov., sp. nov., a cryptoen-
from soil. Int J Syst Evol Microbiol 58, 1576–1579.
dolithic micro-organism from Antarctic white rock. Int J Syst Evol Microbiol 61, 1973–1980.
Prisco, G. D. (2007). Lake Vostok and subglacial lakes of Antarctica:
Young, K. D. (2007). Bacterial morphology: why have different
do they host life? In Physiology and Biochemistry of Extremophiles, pp. 145–154. Edited by C. Gerday & N. Glansdorff. New York: American Society for Microbiology.
Yurkov, V. (2005a). Genus VIII. Sandaracinobacter Yurkov, Stackebrandt,
Priscu, J. C., Fritsen, C. H., Adams, E. E., Giovannoni, S. J., Paerl, H. W., McKay, C. P., Doran, P. T., Gordon, D. A., Lanoil, B. D. & Pinckney, J. L. (1998). Perennial Antarctic lake ice: an oasis for life in
a polar desert. Science 280, 2095–2098. Rothschild, L. J. & Mancinelli, R. L. (2001). Life in extreme environments.
Nature 409, 1092–1101. http://ijs.sgmjournals.org
shapes? Curr Opin Microbiol 10, 596–600. Buss, Vermeglio, Gorlenko and Beatty 1997, 1177VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 279–282. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yurkov, V. V. (2005b). Genus VI. Erythromonas Yurkov, Stackebrandt, Buss, Vermeglio, Gorlenko and Beatty 1997, 1177VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 274–275.
2039
W. Fukuda and others Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yurkov, V., Stackebrandt, E., Buss, O., Vermeglio, A., Gorlenko, V. & Beatty, J. T. (1997). Reorganization of the genus Erythromicrobium:
description of ‘‘Erythromicrobium sibiricum’’ as Sandaracinobacter sibiricus gen. nov., sp. nov., and of ‘‘Erythromicrobium ursincola’’ as Erythromonas ursincola gen. nov., sp. nov. Int J Syst Bacteriol 47, 1172–1178.
2040
International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.050005-0
Polymorphobacter multimanifer gen. nov., sp. nov., a polymorphic bacterium isolated from antarctic white rock Wakao Fukuda,1 Yohzo Chino,1 Shigeo Araki,1 Yuka Kondo,2 Hiroyuki Imanaka,3 Tamotsu Kanai,2 Haruyuki Atomi2 and Tadayuki Imanaka1 Correspondence Tadayuki Imanaka [email protected]
1
Department of Biotechnology, College of Life Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan
2
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
3
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
A Gram-stain-negative, non-spore-forming, aerobic, oligotrophic bacterium (strain 262-7T) was isolated from a crack of white rock collected in the Skallen region of Antarctica. Strain 262-7T grew at temperatures between ”4 and 30 6C, with optimal growth at 25 6C. The pH range for growth was between pH 6.0 and 9.0, with optimal growth at approximately pH 7.0. The NaCl concentration range allowing growth was between 0.0 and 1.0 %, with an optimum of 0.5 %. Strain 262-7T showed an unprecedented range of morphological diversity in response to growth conditions. Cells grown in liquid medium were circular or ovoid with smooth surfaces in the lag phase. In the exponential phase, ovoid cells with short projections were observed. Cells in the stationary phase possessed long tentacle-like projections intertwined intricately. By contrast, cells grown on agar plate medium or in liquid media containing organic compounds at low concentration exhibited short- and long-rod-shaped morphology. These projections and morphological variations clearly differ from those of previously described bacteria. Ubiquinone 10 was the major respiratory quinone. The major fatty acids were C17 : 1v6c (28.2 %), C16 : 1v7c (22.6 %), C18 : 1v7c (12.9 %) and C15 : 0 2-OH (12.3 %). The G+C content of genomic DNA was 68.0 mol%. Carotenoids were detected from the cells. Comparative analyses of 16S rRNA gene sequences indicated that strain 262-7T belongs to the family Sphingomonadaceae, and that 2627T should be distinguished from known genera in the family Sphingomonadaceae. According to the phylogenetic position, physiological characteristics and unique morphology variations, strain 262-7T should be classified as a representative of a novel genus of the family Sphingomonadaceae. Here, a novel genus and species with the name Polymorphobacter multimanifer gen. nov., sp. nov. is proposed (type strain 262-7T5JCM 18140T5ATCC BAA2413T). The novel species was named after its morphological diversity and formation of unique projections.
The climate in Antarctica, with its extreme cold, dryness, strong winds, seasonally strong UV radiation and low concentration of organic materials, makes it difficult for most organisms to sustain life. However, a great diversity of micro-organisms has been found in icy environments, such as permafrost, polar oceans, snow, lake ice, sea ice and The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain 262-7T is AB649056. Three supplementary figures and one supplementary table are available with the online version of this paper.
2034
cryoconite holes (Karr et al., 2006; Prisco, 2007; Priscu et al., 1998; Rothschild & Mancinelli, 2001; Tindall, 2004). In addition, the presence of chasmolithic, hypolithic and endolithic communities have been reported (Cary et al., 2010). The rocks of Antarctica are considered to provide physical stability and to protect microbial communities from UV radiation and dryness. A white rock sample was collected by the summer party of the 46th Japanese Antarctic Research Expedition in 2004–2005 from the Skallen region in Antarctica (Yamada et al., 2011). Interestingly, the interior of the white rock was colourful (green, pink, yellow and 050005 G 2014 IUMS Printed in Great Britain
Polymorphobacter multimanifer gen. nov., sp. nov.
brown), suggesting the existence of a cryptoendolithic community containing cyanobacteria. When autotrophic bacteria were screened from the crushed rock under light conditions, a green filamentous bacterium (named strain 262-1) covered with several heterotrophic bacteria was obtained. By the cultivation of these bacteria under dark conditions, more than 10 different bacterial species including strains 262-7T and 262-8T (the type strain of Constrictibacter antarcticus) were obtained (Yamada, et al., 2011). Strain 262-7T could grow well in 0.256 LB medium (l21: 2.5 g tryptone, 1.3 g yeast extract and 5.0 g NaCl in tap water) under aerobic conditions. However, no growth was observed in 0.756 LB medium containing (l21) 7.5 g tryptone and 3.8 g yeast extract. These results indicated that strain 262-7T is an oligotroph. Strain 262-7T could also grow in 0.256 LB/ASW medium [l21: 2.5 g tryptone, 1.3 g yeast extract, 5.0 g NaCl, 0.75 g MgCl2 . 6H2O, 1.5 g MgSO4 . 7H2O, 0.25 g (NH4)2SO4, 0.050 g NaHCO3, 0.075 g CaCl2 . 2H2O, 0.13 g KCl, 0.11 g KH2PO4, 0.013 g NaBr, 0.005 g SrCl2 . 6H2O, 0.025 g ferric ammonium citrate] and quarter strength Difco Marine Broth 2216 medium (BD Difco). Strain 262-7T grew at 24–30 uC with an optimum growth temperature of 25 uC in 0.256 LB medium, while no growth was observed at temperatures above 37 uC. When strain 262-7T grew on 0.256 LB plate medium containing 20 g agar l21 at 24 uC in a freezing chamber, colony formation was observed. The pH range for growth was pH 6.0–9.0, with an optimum of approximately pH 7.0. Strain 262-7T was able to grow at NaCl concentrations of 0.0–1.0 % (w/v), with an optimum of 0.5 % (w/v). When strain 262-7T was cultivated in 0.256 LB medium under optimal growth conditions, the maximum OD660 reached approximately 0.9 after 70 h of incubation (doubling time, 5.2 h). When strain 262-7T was grown on 0.256 LB plate medium, brown, raised and circular colonies were formed with an entire margin. Strain 262-7T could not grow in 0.256 LB medium containing 5.0 g l21 of either Na2SO4 or NaNO3 under anaerobic conditions. Analyses of Gram-staining, catalase and oxidase activity, motility and spore formation were performed by methods described previously (Yamada, et al., 2011). The results revealed that strain 262-7T was a Gram-stain-negative, catalase-positive, oxidase-positive, non-spore-forming and motile bacterium. Further biochemical characteristics were analysed by using API 20NE and API ZYM kits (bioMe´rieux) and antibiotic sensitivities were examined by using an ATB VET kit (bioMe´rieux); results are given in the species description. The morphology of the cells cultivated in liquid medium was examined by a bright-field microscope (BX51; Olympus). Cells in the logarithmic and stationary phases of growth were examined, and circular and ovoid cells were observed (data not shown). The ultrastructures of cells were examined by scanning electron microscopy (S-4700; Hitachi), and it became clear that strain 262-7T showed morphology variations depending on growth phase and growth medium. http://ijs.sgmjournals.org
When cells in the lag phase were observed, circular or ovoid cells with smooth surfaces were observed (Fig. 1a). In the exponential phase, ovoid cells with short projections were observed (Fig 1b, c). Cells in the stationary phase possessed long tentacle-like projections which were intertwined intricately (Fig. 1d). It has been reported that rod-shaped and coccid cells coexist in the case of Porphyrobacter neustonensis (Hiraishi & Imhoff, 2005). However, the projections and morphology variations seen in strain 2627T are clearly distinct from those reported in other bacteria, and may represent a response to nutrient deficiency. It has been reported that Prosthecomicrobium hirschii and Ancalomicrobium adetum harbour long projections called prosthecae (Staley, 1968, 1984). In the case of A. adetum, it has been suggested that the long and straight prosthecae help the bacterium to resist ingestion by protists (Bianchi, 1989; Young, 2007). However, the straight projections of A. adetum seem quite different from those of strain 262-7T in appearance. In the case of a stalked bacterium, Caulobacter vibrioides (former name, Caulobacter crescentus), the stalk (prosthecae) affixes the organism to solid surfaces in aqueous environments, and the length of the stalk is regulated by the availability of nutrients (Young, 2007). The cells of strain 262-7T gradually became smaller in response to the progression of the projections (Fig 1c, d). It seemed that the surface area of the cell was expanded by the production of the projections, and the projections of strain 262-7T might be used for nutrient uptake in the oligotrophic environments present in the gaps inside the rock. By contrast, it could be possible that cells were connected by the projections, and the projections might be involved in biofilm formation. A tendency of 262-7T cells to aggregate in the stationary phase was observed. When cells cultivated on 0.256 LB agar medium were analysed, long-rod-shaped cells (more than 10 mm in length) were observed among short-rod-shaped cells (Fig. 1e). To distinguish live cells from dead cells, nucleic acids
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1. Scanning electron micrographs (a–e) and atomic force micrograph (f) of strain 262-7T. (a) Cell in lag phase. (b) Cells in early exponential phase. (c) Cells in mid–late exponential phase. (d) Cells in stationary phase. (e) Cells cultivated on 0.25¾ LB agar medium. (f) Atomic force micrograph of strain 262-7T in midexponential phase. Bars, 1 mm (a–e) and 2 mm each (f). 2035
W. Fukuda and others
of strain 262-7T were stained with SYTO9 dye, which stains cells with intact and damaged membranes, and with propidium iodide (PI) dye, which only stains cells with damaged membranes, and the stained cells were examined by fluorescence microscopy (Fig. S1, available in the online Supplementary Material). As a result, nucleic acids of most of the long-rod-shaped cells were stained only by SYTO9 dye, suggesting that the long-rod-shaped cells were alive. A similar observation was reported for Caulobacter vibrioides, and the elongated cells displayed increased resistance to stress (Wortinger et al., 1998). Moreover, when strain 2627T was cultivated in media containing organic compounds at low concentrations [0.16 LB medium (l21: 1.0 g tryptone, 0.5 g yeast extract and 5.0 g NaCl in tap water) or 0.016 LB medium (l21: 0.1 g tryptone, 0.05 g yeast extract and 5.0 g NaCl in tap water)], the existence of rod-shaped cells (approx. 3–10 mm in length) was observed (data not shown). Strain 262-7T was isolated from oligotrophic environment, and, therefore, strain 262-7T may be a rod-shaped bacterium in the natural environment. The rod-shaped cell formation of strain 262-7T may be of advantage for adaptation to oligotrophic environments or for resistance to environmental changes. Although flagella could not be observed by scanning electron microscopy, a flagellum was observed by atomic force microscopy (OLS3500; Olympus) (Fig. 1f). When the 16S rRNA gene sequence of strain 262-7T was analysed with similarity search programs (nucleotide BLAST) provided by the National Center for Biotechnology Information (Altschul et al., 1990), related known strains were Sandarakinorhabdus limnophila so42T (Gich & Overmann, 2006) and Sphingomonas fennica K101T (Wittich et al., 2007) in the family Sphingomonadaceae, with 16S rRNA gene sequence similarities of 95.0 and 94.6 %, respectively. To examine the phylogenetic position
0.02
of strain 262-7T, a phylogenetic tree based on 16S rRNA gene sequences from bacteria belonging to the order Sphingomonadales (Fig. 2) was reconstructed with the CLUSTAL W program (Thompson et al., 1994). The order Sphingomonadales contains two families, Sphingomonadaceae and Erythrobacteraceae (Lee et al., 2005). Although some pleomorphic genera belong to the family Erythrobacteraceae (Hiraishi & Imhoff, 2005), strain 262-7T was located in the clade of the family Sphingomonadaceae. In addition, strain 262-7T was located in a branch distinct to the previously recognized genera belonging to the family Sphingomonadaceae, although strain 262-7T was located in a clade with Sandarakinorhabdus limnophila and Sandaracinobacter sibiricus. This result indicated that strain 262-7T is relatively closely related to the genera Sandarakinorhabdus and Sandaracinobacter, and suggested that strain 262-7T is a phylogenetically novel bacterium in the family Sphingomonadaceae. The G+C content of the genomic DNA, respiratory quinone, fatty acids and polar lipids pattern were analysed by previously described methods with slight modification (Fukuda et al., 2012; Yamada, et al., 2011). The fatty acids composition was analysed by the Sherlock Microbial Identification System using version 6.0 of the TSBA6 library. The alkaline-stable lipids were extracted by the method of Matsuyama et al. (2008). The chemotaxonomic characteristics of strain 262-7T and those of the genera in the family Sphingomonadaceae are shown in Table 1. The G+C content of the genomic DNA was 68.0 mol%, which was in the same range as those of members of the genera Sandaracinobacter and Sphingomonas, and is slightly higher than that of members of the other genera in the family Sphingomonadaceae. The major respiratory quinone of strain 262-7T was ubiquinone 10, which is the most
CC-TBT-3T
(EU564841) Sphingomicrobium lutaoense Sphingosinicella microcystinivorans Y2T (AB084247) Stakelama pacifica JLT832T (EU581829) Zymomonas mobilis ATCC 10988T (AF281031) Sphingobium yanoikuyae IFO 15102T (D13728)
54
Sphingopyxis macrogoltabida 203T (D13723) Parasphingopyxis lamellibrachiae JAMH 0132T (AB524074)
Sphingomonadaceae
Polymorphobacter multimanifer 262-7T (AB649056) Sandarakinorhabdus limnophila so42T (AY902680) Sandaracinobacter sibiricus RB16-17T (Y10678) Sphingomonas paucimobilis ATCC 29837T (EU37337)
81 64
81
Croceicoccus marinus E4A9T (EF623998) Altererythrobacter epoxidivorans JCS350T (DQ304436) Erythrobacter longus OCh101T (AF465835)
93 81 89
2036
Porphyrobacter neustonensis ACM 2844T (AB033327) 100 Erythromicrobium ramosum E5T (AF465867)
Erythrobacteraceae
Sphingorhabdus planktonica 585T (JN381068) Blastomonas natatoria DSM 3183T (AB024288) Novosphingobium capsulatum GIFU 11526T (D16147)
Fig. 2. Phylogenetic tree of strain 262-7T and members of the order Sphingomonadales based on 16S rRNA gene sequences. This tree was reconstructed using the neighbourjoining method provided by DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp/index. php?lang=en). Bootstrap resampling was performed 1000 times and only values observed in more than 50 % of the replicas are shown at branching points. The 16S rRNA gene sequence of Escherichia coli ATCC 11775T was used as an outgroup. Circles at nodes indicate nodes recovered with bootstrap values .50 % (filled circles) or .70 % (open circles) in a maximum-likelihood tree reconstructed using the DNAML program in the PHYLIP package (Felsenstein, 2009). Bar, 2 substitutions per 100 nucleotides.
International Journal of Systematic and Evolutionary Microbiology 64
http://ijs.sgmjournals.org
Table 1. Characteristics that differentiate strain 262-7T from the genera in the family Sphingomonadaceae Taxa: 1, Strain 262-7T; 2, Sandarakinorhabdus (data from Gich & Overmann, 2006); 3, Sandaracinobacter, (Yurkov, 2005a); 4, Sphingomonas (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 5, Sphingobium (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 6, Novosphingobium (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 7, Sphingopyxis (Takeuchi, et al., 2001; Yabuuchi & Kosako, 2005b); 8, Parasphingopyxis (Uchida et al., 2012); 9, Blastomonas (Sly & Hugenholtz, 2005); 10, Sphingomicrobium (Ka¨mpfer et al., 2012); 11, Sphingosinicella (Maruyama et al., 2006); 12, Stakelama (Chen et al., 2010); 13, Zymomonas (Sprenger & Swings, 2005). +, Positive; 2, negative or absent; +or 2, different reaction in different species; ND, no data; Q, ubiquinone. Characteristics Pigmentation (colony colour)
1 Brown
2
Q-10 64.3
Orange–brown, yellow, white or colourless Long rod Rod + +/2 2 ND
nd
Q-9, Q-10 68.5
5
6
Yellow or Yellow or whitish-brown whitish-brown Rod
Rod
7 Yellow or whitishbrown Rod
8
9
Orange– Yellow– yellow orange
10
11
Yellow
Yellow
12
13
Gold–yellow Cream
Ovoid or Irregular Rod Rod Rod rod rod + or 2 + or 2 + + 2 + + + or 2 + or 2 / + + or 2 / + + / 2 + / + + / ND +/+ 2/+ 2/+ + 2 + 2 ND 2 + 2 C18 : 1, C16 : 0 C18 : 1v7c, C18 : 1v7c, C18 : 1v9 C18 : 1v7c C16 : 1v7c, C18 : 1v7c, C18 : 1 C16 : 0 C16 : 0 C18 : 1v7c C16 : 0
+ or 2 + or 2 / + + or 2 C18 : 1, C16 : 0, C17 : 1, C17 : 0 cyclo C14 : 0 2-OH, C15 : 0 2-OH
+ or 2 + or 2 / + 2 C18 : 1, C16 : 0
C14 : 0 2-OH
C14 : 0 2-OH
Q-10 62–68
Q-10 59–67
Q-10 62–67
C14 : 0 2-OH, C15 : 0 2-OH C16 : 0 2-OH Q-10 63.0–65.0
Rod
C16 : 0 2-OH
C14 : 0 2-OH
C18 : 1 2-OH
C14 : 0 2-OH
C14 : 0 2-OH
Q-10 60.1
Q-10 65
Q-10 63.4
Q-10 63.6–63.7
Q-10 66±0.5
2
ND
47.5–49.5
2037
Polymorphobacter multimanifer gen. nov., sp. nov.
Q-10 68.0
4
Orange–red Yellow– orange
Cell morphology Ovoid to rod Rod (Pleomorphic) Motility + 2 Oxidase/catalase +/+ 2/+ ND Nitrate reduction 2 C16 : 1v7c, Major fatty acids C17 : 1v6c, C16 : 1v7c, C18 : 1v7c C18 : 1v7c Major hydroxyl C15 : 0 2-OH iso-C14 : 0 2-OH fatty acids Major quinone DNA G+C content (mol%)
3
W. Fukuda and others
frequently identified respiratory quinone in bacteria belonging to the family Sphingomonadaceae (Yabuuchi & Kosako, 2005a). To analyse the composition of cellular fatty acids, cells of strain 262-7T were cultivated in 0.256 LB medium at 25 uC until the late exponential phase of growth. The major cellular fatty acids of strain 262-7T were C17 : 1v6c (28.2 %), C16 : 1v7c (22.6 %), C18 : 1v7c (12.9 %) and C15 : 0 2-OH (12.3 %); C14 : 0 2-OH (7.1 %), C16 : 0 (3.1 %) and C15 : 1v6c (3.1 %) were detected as minor fatty acids. Trace amounts of iso-C16 : 0 3-OH (0.9 %) were detected. The major nonpolar and 2-hydroxy fatty acids of strain 262-7T were different from those of the related genus Sandarakinorhabdus (Table 1), although fatty acid compositions of species of the genus Sphingomonas are similar to those of strain 262-7T (Takeuchi et al., 2001). The lipids pattern on two-dimensional TLC revealed the presence of phosphatidylglycerol, phosphatidylethanolamine, sphingoglycolipid, an unknown phospholipid and two unknown glycolipids (Fig. S2 and Table S1). The lipids pattern detected was similar to that of Sphingomonas xinjiangensis, although some lipids detected in Sphingomonas xinjiangensis were not found (An et al., 2011). Absence of phosphatidylcholine and the pattern of sphingoglycolipids were different from the lipids pattern of the closely related species Sandarakinorhabdus limnophila (Gich & Overmann, 2006). Pigment analysis was performed using cells in the mid–late exponential phase by the modified method described by Saga et al. (2005). The spectra of pigments extracted from cells showed three peaks at 428, 452 and 478 nm, similar to Sandaracinobacter sibiricus (Yurkov et al., 1997) (Fig. S3). The result indicated the presence of carotenoids in the cells of strain 262-7T. No peaks above 600 nm derived from bacteriochlorophyll a were observed, contrary to Sandaracinobacter sibiricus and genera in the family Erythrobacteraceae (Yurkov et al., 1997; Yurkov, 2005b). According to its phylogenetic position, strain 262-7T is most closely related to the genera Sandarakinorhabdus and Sandaracinobacter, and is clearly different from other genera in the family Sphingomonadaceae. Fatty acids composition and production of oxidase/catalase are different from the related genera Sandarakinorhabdus and Sandaracinobacter (Table 1). Above all, unique morphology variations and appearance of strain 262-7T were observed. Therefore, strain 262-7T should be classified as a representative of a novel species and genus in the family Sphingomonadaceae, for which the name Polymorphobacter multimanifer gen. nov., sp. nov. is proposed. Description of Polymorphobacter gen. nov. Polymorphobacter (po.ly.mor.pho.bac9ter. Gr. adj. polumorphos multiform; N.L. masc. n. bacter rod; N.L. masc. n. polymorphobacter a polymorphic rod).
rods. Cells contain carotenoids. Cells produce catalase and oxidase, and grow aerobically. Nitrate reduction is not observed. The major respiratory quinone is Q-10. The cellular fatty acids are mainly composed of C17 : 1v6c, C16 : 1v7c and C18 : 1v7c. The major 2-hydroxy fatty acid is C15 : 0 2-OH. The DNA G+C content of the type species is 68.0 mol%. The type species is Polymorphobacter multimanifer. Description of Polymorphobacter multimanifer sp. nov. Polymorphobacter multimanifer [mul.ti.ma9ni.fer. L. adj. multus many; L. n. manus hand; L. suff. -fer, (from L. v. fero) bearing, carrying, producing; N.L. masc. adj. n. multimanifer many hands bearer, referring to tentacle-like projections]. Colonies are brown, raised, circular and entire. Cells are circular or ovoid with smooth surfaces in the lag phase of growth. In the exponential growth phase, ovoid cells with short projections are observed. Cells in the stationary phase possess long tentacle-like projections intertwined intricately. By contrast, cells on 0.256 LB agar plate medium exhibit short- and long-rod-shaped morphology. Growth occurs between 24 and 30 uC (optimum, 25 uC), between pH 6.0 and 9.0 (optimum, approximately pH 7.0), and with between 0.0 and 1.0 % NaCl (optimum, 0.5 %). Cells can utilize maltose and malate as a carbon sources, and weak growth is observed in media containing glucose and mannose. Cannot utilize adipate, arabinose, caprate, citrate, gluconate, mannitol and phenylacetate. Production of acid phosphatase, alkaline phosphatase, cystine arylamidase, esterase (C4), esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin and valine arylamidase is observed. Activities of arginine dihydrolase N-acetyl-b-glucosaminidase, a-fucosidase, a-galactosidase, b-galactosidase, gelatinase, a-glucosidase, b-glucuronidase, lipase (C4), a-mannosidase and urease are not observed. H2S and indole are not produced. Cells are resistant to amoxicillin, amoxicillin/clavulanic acid, cefoperazone, cefalotin, colistin, cotrimoxazole, erythromycin, lincomycin, metronidazole, oxacillin, penicillin, sulfamethizole and tylosin. Cells are sensitive to apramycin, chloramphenicol, doxycycline, enrofloxacin, flumequine, fusidic acid, gentamicin, kanamycin, nitrofurantoin, oxolinic acid, pristinamycin, rifampicin, spectinomycin, streptomycin and tetracycline. The type strain is 262-7T (5JCM 18140T5ATCC BAA2413T) and was isolated from a crack of white rock in the Skallen region of Antarctica.
Acknowledgements
Member of the class Alphaproteobacteria, order Sphingomonadales, family Sphingomonadaceae. Cells are Gramstain-negative, motile, non-sporulating and polymorphic
The authors would like to express their sincere thanks to all members of the 46th Japanese Antarctic Research Expedition. This study was supported by JSPS KAKENHI (19205022 and 23657068).
2038
International Journal of Systematic and Evolutionary Microbiology 64
Polymorphobacter multimanifer gen. nov., sp. nov.
References
Saga, Y., Osumi, S., Higuchi, H. & Tamiaki, H. (2005).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410. An, H., Xu, M., Dai, J., Wang, Y., Cai, F., Qi, H., Peng, F. & Fang, C. (2011). Sphingomonas xinjiangensis sp. nov., isolated from desert
Bacteriochlorophyll-c homolog composition in green sulfur photosynthetic bacterium Chlorobium vibrioforme dependent on the concentration of sodium sulfide in liquid cultures. Photosynth Res 86, 123–130. Sly, L. I. & Hugenholtz, P. (2005). Genus II. Blastomonas Sly and
and filaments as a consequence of grazing pressure. Microb Ecol 17, 137–141.
Cahill 1997, 567VP emend. Hiraishi, Kuraishi and Kawahara 2000a, 1117. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 258–263. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Cary, S. C., McDonald, I. R., Barrett, J. E. & Cowan, D. A. (2010).
Sprenger, G. A. & Swings, J. (2005). Genus IX. Zymomonas Kluyver
sand. Int J Syst Evol Microbiol 61, 1865–1869. Bianchi, M. (1989). Unusual bloom of star-like prosthecate bacteria
On the rocks: the microbiology of Antarctic Dry Valley soils. Nat Rev Microbiol 8, 129–138.
and van Niel 1936, 399AL. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 282–286. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Chen, C., Zheng, Q., Wang, Y. N., Yan, X. J., Hao, L. K., Du, X. & Jiao, N. (2010). Stakelama pacifica gen. nov., sp. nov., a new member of the
Staley, J. T. (1968). Prosthecomicrobium and Ancalomicrobium: new
family Sphingomonadaceae isolated from the Pacific Ocean. Int J Syst Evol Microbiol 60, 2857–2861.
Staley, J. T. (1984). Prosthecomicrobium-Hirschii, a new species in a
(phylogeny inference package), version 3.69. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Felsenstein, J. (2009).
PHYLIP
Fukuda, W., Yamada, K., Miyoshi, Y., Okuno, H., Atomi, H. & Imanaka, T. (2012). Rhodoligotrophos appendicifer gen. nov., sp. nov.,
an appendaged bacterium isolated from a freshwater Antarctic lake. Int J Syst Evol Microbiol 62, 1945–1950. Gich, F. & Overmann, J. (2006). Sandarakinorhabdus limnophila gen. nov., sp. nov., a novel bacteriochlorophyll a-containing, obligately aerobic bacterium isolated from freshwater lakes. Int J Syst Evol Microbiol 56, 847–854.
prosthecate freshwater bacteria. J Bacteriol 95, 1921–1942. redefined genus. Int J Syst Bacteriol 34, 304–308. Takeuchi, M., Hamana, K. & Hiraishi, A. (2001). Proposal of the genus
Sphingomonas sensu stricto and three new genera, Sphingobium, Novosphingobium and Sphingopyxis, on the basis of phylogenetic and chemotaxonomic analyses. Int J Syst Evol Microbiol 51, 1405–1417. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Tindall, B. J. (2004). Prokaryotic diversity in the Antarctic: the tip of
the iceberg. Microb Ecol 47, 271–283.
Hiraishi, A. & Imhoff, J. (2005). Genus VII. Porphyrobacter Fuerst,
Uchida, H., Hamana, K., Miyazaki, M., Yoshida, T. & Nogi, Y. (2012).
Hawkins, Holms, Sly, Moore and Stackebrandt 1993, 132VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 275–279. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer.
Parasphingopyxis lamellibrachiae gen. nov., sp. nov., isolated from a marine annelid worm. Int J Syst Evol Microbiol 62, 2224–2228.
Ka¨mpfer, P., Arun, A. B., Young, C. C., Busse, H. J., Kassmannhuber, J., Rossello´-Mo´ra, R., Geueke, B., Rekha, P. D. & Chen, W. M. (2012).
and Sphingomonas haloaromaticamans sp. nov., outliers of the genus Sphingomonas. Int J Syst Evol Microbiol 57, 1740–1746.
Sphingomicrobium lutaoense gen. nov., sp. nov., isolated from a coastal hot spring. Int J Syst Evol Microbiol 62, 1326–1330.
Wortinger, M. A., Quardokus, E. M. & Brun, Y. V. (1998). Morphological
Karr, E. A., Ng, J. M., Belchik, S. M., Sattley, W. M., Madigan, M. T. & Achenbach, L. A. (2006). Biodiversity of methanogenic and other
archaea in the permanently frozen Lake Fryxell, Antarctica. Appl Environ Microbiol 72, 1663–1666. Lee, K. B., Liu, C. T., Anzai, Y., Kim, H., Aono, T. & Oyaizu, H. (2005). The hierarchical system of the ‘Alphaproteobacteria’: descrip-
tion of Hyphomonadaceae fam. nov., Xanthobacteraceae fam. nov. and Erythrobacteraceae fam. nov. Int J Syst Evol Microbiol 55, 1907–1919. Maruyama, T., Park, H. D., Ozawa, K., Tanaka, Y., Sumino, T., Hamana, K., Hiraishi, A. & Kato, K. (2006). Sphingosinicella microcystinivorans
gen. nov., sp. nov., a microcystin-degrading bacterium. Int J Syst Evol Microbiol 56, 85–89. Matsuyama, H., Katoh, H., Ohkushi, T., Satoh, A., Kawahara, K. & Yumoto, I. (2008). Sphingobacterium kitahiroshimense sp. nov., isolated
Wittich, R. M., Busse, H. J., Ka¨mpfer, P., Macedo, A. J., Tiirola, M., Wieser, M. & Abraham, W. R. (2007). Sphingomonas fennica sp. nov.
adaptation and inhibition of cell division during stationary phase in Caulobacter crescentus. Mol Microbiol 29, 963–973. Yabuuchi, E. & Kosako, Y. (2005a). Family I. Sphingomonadaceae
Kosako, Yabuuchi, Naka, Fujiwara, and Kobayashi 2000b, 1953VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, p. 230– 233. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yabuuchi, E. & Kosako, Y. (2005b). Genus I. Sphingomonas
Yabuuchi, Yano, Oyaizu, Hashimoto, Ezaki and Yamamoto 1990b, 321VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 234–258. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yamada, K., Fukuda, W., Kondo, Y., Miyoshi, Y., Atomi, H. & Imanaka, T. (2011). Constrictibacter antarcticus gen. nov., sp. nov., a cryptoen-
from soil. Int J Syst Evol Microbiol 58, 1576–1579.
dolithic micro-organism from Antarctic white rock. Int J Syst Evol Microbiol 61, 1973–1980.
Prisco, G. D. (2007). Lake Vostok and subglacial lakes of Antarctica:
Young, K. D. (2007). Bacterial morphology: why have different
do they host life? In Physiology and Biochemistry of Extremophiles, pp. 145–154. Edited by C. Gerday & N. Glansdorff. New York: American Society for Microbiology.
Yurkov, V. (2005a). Genus VIII. Sandaracinobacter Yurkov, Stackebrandt,
Priscu, J. C., Fritsen, C. H., Adams, E. E., Giovannoni, S. J., Paerl, H. W., McKay, C. P., Doran, P. T., Gordon, D. A., Lanoil, B. D. & Pinckney, J. L. (1998). Perennial Antarctic lake ice: an oasis for life in
a polar desert. Science 280, 2095–2098. Rothschild, L. J. & Mancinelli, R. L. (2001). Life in extreme environments.
Nature 409, 1092–1101. http://ijs.sgmjournals.org
shapes? Curr Opin Microbiol 10, 596–600. Buss, Vermeglio, Gorlenko and Beatty 1997, 1177VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 279–282. Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yurkov, V. V. (2005b). Genus VI. Erythromonas Yurkov, Stackebrandt, Buss, Vermeglio, Gorlenko and Beatty 1997, 1177VP. In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2, pp. 274–275.
2039
W. Fukuda and others Edited by D. J. Brenner, N. R. Kreig, J. T. Staley & G. M. Garrity. New York: Springer. Yurkov, V., Stackebrandt, E., Buss, O., Vermeglio, A., Gorlenko, V. & Beatty, J. T. (1997). Reorganization of the genus Erythromicrobium:
description of ‘‘Erythromicrobium sibiricum’’ as Sandaracinobacter sibiricus gen. nov., sp. nov., and of ‘‘Erythromicrobium ursincola’’ as Erythromonas ursincola gen. nov., sp. nov. Int J Syst Bacteriol 47, 1172–1178.
2040
International Journal of Systematic and Evolutionary Microbiology 64
