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Author version: Int. J. Systemat. Evolut. Microbiol., vol.63; 2013; 274-279

Silanimonas mangrovi sp. nov., a novel bacterium of the family isolated from mangrove sediment and emended description of the genus Silanimonas

Srinivas, T. N. R2♣., Kailash T. B1, and Anil Kumar, P1*♣

1Microbial Type Culture Collection and Gene bank, Institute of Microbial Technology (CSIR), Sector 39A, Chandigarh - 160 036, INDIA

2National Institute of Oceanography (CSIR), Regional centre, P B No. 1913, Dr. Salim Ali Road, Kochi - 682018 (Kerala), INDIA

Address for correspondence* Dr. P. Anil Kumar Microbial Type Culture Collection and Gene bank, Institute of Microbial Technology (CSIR), Sector 39A, Chandigarh - 160 036, INDIA Email: [email protected] Telephone: 00-91-172-6665170

Running title Silanimonas mangrovi sp. nov.

Subject category New taxa ()

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AK13T is HE573746.

♣These authors contributed equally to this work.

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A novel Gram-negative, rod shaped, motile bacterium, designated strain AK13T, was isolated

from a sediment sample collected from mangrove of Namkhana, Sunderbans, West Bengal, India. Strain

AK13T was positive for oxidase, DNase and lipase activities and negative for catalase, gelatinase,

ornithine decarboxylase, lysine decarboxylase, nitrate reductase, aesculinase and urease, activities. The

fatty acids were dominated by iso-C11 : 0, iso-C11 : 0 3OH, iso-C15 : 0, iso-C16 : 0, iso-C17 : 1 ω9c and summed

T in feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2OH). Strain AK13 contained Q-8 as the major respiratory

quinone and diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol,

phosphatidylserine, two unidentified aminolipids, one unidentified glycolipid and one unidentified lipid

as polar lipids. The DNA G + C content of the strain AK13T was 55.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence of strain AK13T indicated Silanimonas lenta of the family

Xanthomonadaceae (phylum ) as the closest related with sequence similarity of

95.2%. Other members of the family showed sequence similarities <94.4%. Based on the phenotypic

characteristics and on phylogenetic inference, strain AK13T is proposed as a novel species of the genus

Silanimonas as Silanimonas mangrovi sp. nov. and the type strain is AK13T (= MTCC 11082T = DSM

24914T)

Key words: Silanimonas mangrovi, 16S rRNA gene sequence based phylogeny and chemotaxonomy.

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Mangrove forests are one of the most dynamic and bio-diverse wetlands on our globe. However, these distinctive coastal tropical forests are amongst the most threatened habitats on the earth. These habitats may be diminishing further rapidly than inland tropical rain forests. Growing in the estuary mouths and inter-tidal regions, mangroves offer significant environment for a varied marine and terrestrial flora and fauna. The health of the marine ecology depends on the healthy mangrove forests. The Sundarbans, covering some 10,000 sq. km. of mangrove forest and water (of which some 40% is in India and the rest in Bangladesh), is part of the world’s largest delta (80,000 sq. km.). With limited efforts a number of novel (47) belonging to different phyla (Actinobacteria, Firmicutes and Proteobacteria) were isolated from different mangrove environments from past few years.

The genus Silanimonas was proposed by Lee et al. (2005), with the type species Silanimonas lenta. Phylogenetically, the genus belongs to the family Xanthomonadaceae within the class ‘Gammaproteobacteria’. Silanimonas lenta was isolated from a hot spring at Baekdoo Mountain in Korea (Lee et al., 2005). In the course of bacterial diversity studies from mangrove samples, a bacterial strain, AK13T, was isolated from a mangrove sediment sample collected near Namkhana, Sunderbans, West Bengal, India. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain AK13T was closely related to the genus Silanimonas of the family Xanthomonadaceae. In this study, by using a polyphasic taxonomic approach including genotypic, phenotypic including chemotaxonomic characterizations, the strain AK13T was demonstrated to represent a novel species of the genus Silanimonas.

Strain AK13T was isolated from a sediment sample collected from mangrove of Namkhana (GPS Positioning 21o45’39.35” N 88o13’48.05” E), Sunderbans, West Bengal, India. The sample that yielded strain AK13T had a pH of 8.0, temperature of 32 oC. For isolation of bacteria, 1 g of the sediment sample was serially diluted in 1 % sterile saline water and from each dilution 100 µl was plated on ZoBell Marine agar (MA) & Nutrient agar (NA)(HIMEDIA) plates, and incubated at 30 oC for 15 days. Out of different morphotypes, one yellow colored colony was selected and characterized. Subcultivation of the isolate was carried out on NA plates at 30 °C. Stock culture of the isolate in nutrient broth with 20% glycerol was preserved at -80 °C.

Colony morphology was studied after 48 h growth of the strain on NA at 30 °C. Cell morphology was investigated by light microscopy (Zeiss) at X 1000. Gram reaction was determined by

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using the HIMEDIA Gram Staining Kit according to manufacturer’s protocol. Endospore formation was determined after malachite-green staining of isolate grown on R2A (HIMEDIA) plates for a week. Motility was also assessed on Motility-Indole-Lysine HiVegTM medium (cat. no. MV847; HIMEDIA) with agar 2 g l-1 and also under phase contrast microscope.

Growth was tested on MA, NA and Tryptone Soya Agar (TSA; HIMEDIA). Growth at 4, 10, 18, 25, 30, 35, 37, 40, 45, 47, 50, 55 and 60 °C was ascertained using nutrient broth medium and salt tolerance [0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 and 20% (w/v) NaCl] were ascertained using nutrient broth without NaCl. Growth of strain AK13T at pH 5, 6, 7, 7.5, 8, 8.5, 9, 10, 11 and 12 was assessed on

Tryptone Soya medium buffered with acetate buffer (pH 4-6), 100 mM NaH2PO4/Na2HPO4 buffer (pH

7-8), 100 mM NaHCO3/Na2CO3 buffer (pH 9-10) or 100 mM Na2CO3/NaOH buffer (pH 11-12). Biochemical characteristics such as activity of oxidase, lysine decarboxylase, ornithine decarboxylase, nitrate reduction, hydrolysis of aesculin, gelatin, ONPG, starch, Tween 20, 40, 60, 80, carbon source

assimilation, H2S production and the sensitivity to 18 different antibiotics using the disc diffusion method with commercially available discs (HIMEDIA) were determined by previously described methods (Lanyi, 1987; Smibert & Krieg, 1994). Biochemical and enzymatic characterization of the strain AK13T was also performed using Vitek 2 GN (bioMérieux), according to the manufacturer’s protocol, except that sterile 2.0 % (w/v) NaCl was used to prepare the inoculum.

For cellular fatty acids analysis, strains AK13T and Silanimonas lenta DSM 16282T were grown on NA plates at 37 ºC for 24 h. Standardization of the physiological age of strain AK13T and the type strain was done based on the protocol (http://www.microbialid.com/PDF/TechNote_101.pdf) given by Sherlock Microbial Identification System (MIDI, USA). Cellular fatty acid methyl esters (FAMEs) were obtained from cells by saponification, methylation and extraction following the protocol of Sherlock Microbial Identification System (MIDI, USA). Cellular FAMEs were separated by Gas Chromatograph (6890), identified and quantified with Sherlock Microbial Identification System Software (version.6.0, using the aerobe TSBA6 method and TSBA6 database). Polar lipids and quinones were analysed by taking freeze-dried cells. Cells grown on marine agar 2216 (Difco) at 30 ºC for 3 days under aerobic condition were extracted for the polar lipid analysis (Bligh & Dyer, 1959) and analyzed by two dimensional thin layer chromatograph followed by spraying with appropriate detection reagents (Komagata & Suzuki, 1987). Isoprenoid quinones were extracted as described by Collins et al. (1977) and analyzed by high performance liquid chromatograph (Groth et al., 1997). The DNA of strain AK13T

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was isolated according to the procedure of Marmur (1961) and the G + C content was determined from melting point (Tm) curves (Sly et al., 1986) obtained by using a Lambda 2 UV-Vis spectrophotometer (Perkin Elmer) equipped with the Templab 2.0 software package (Perkin Elmer). strain DH5-α DNA was used as a standard in determining the DNA G + C content.

For 16S rRNA gene sequencing, DNA was prepared using the Mo Bio microbial DNA isolation kit (Mo Bio Laboratories Inc.). PCR amplification of 16S rRNA gene was performed with bacterial universal primers 27f (5´-AGA GTT TGA TCC TGG CTC AG-3´) and 1492r (5´-TAC GGY TAC CTT GTT ACG ACT T-3´) Escherichia coli 16S rRNA gene sequence numbering system (Brosius et al., 1978). Reaction mixture contained 100 ng of chromosomal DNA, 1 U of Deep Vent DNA polymerase, 1 X Thermopol reaction buffer, 200 µM of each deoxynucleoside triphosphate (New England Biolabs) and 20 pmol of each primer (BioBasic Inc). PCR cycling parameters included an initial denaturation at 95 ºC for 5 min, followed by 29 cycles of denaturation at 95 ºC for 1 min, annealing at 55 ºC for 1 min and extension at 72 ºC for 2 min and a final extension for 10 min at 72 oC. PCR product was separated and 1.5 kb fragment detected and later eluted by Qiaquick gel extraction kit (Qiagen). The 16S rRNA gene was sequenced using the forward and the reverse PCR primers (27f and 1492r) and in addition one forward and reverse primers, viz. 530f and 907r (Johnson, 1994), following the dideoxy chain- termination method as described earlier (Pandey et al., 2002). The 16S rRNA gene sequence of the isolate was subjected to BLAST sequence similarity search (Altschul et al., 1990) to identify the nearest taxa. In EzTaxon a search is performed against a database of 16S rRNA gene sequences of type strains only using the algorithm of Myers & Miller (1988). The 16S rRNA gene sequences of closely related validly published taxa belonging to the family Xanthomonadaceae were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov) and aligned using the CLUSTAL_X program (Thompson et al., 1997) and the alignment was then corrected manually by deleting gaps and missing data. There were a total of 1386 positions in the final dataset. Phylogenetic tree was reconstructed using the maximum likelihood method using the PhyML program (Guindon et al., 2005) and Neighbor-Joining method (Saitou & Nei, 1987) using the MEGA4 package (Tamura et al., 2007) and the tree topologies were evaluated by bootstrap analysis based on 100 and 1000 replicates respectively. The evolutionary distances were computed using the Kimura 2-parameter method Kimura, (1980) and are in the units of the number of base substitutions per site.

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Cells of strain AK13T were Gram-negative, non-endospore forming, motile rods, 0.3-0.5 µm wide and 2.0-3.0 µm long. Colonies were circular, 2-3 mm in diameter, smooth, translucent, bright yellowish and raised with entire margins after 2 days growth on NA plates at 30 oC. Strain AK13T can be differentiated from Silanimonas lenta DSM 16282T using colony characteristics like colour, margin and texture (Silanimonas lenta DSM 16282T produces pale yellow, irregular, sticky colonies).

The strain was positive for oxidase activity and negative for catalase, lysine decarboxylase, ornithine decarboxylase and arginine dihydrolase activities. Strain AK13T do not reduce nitrate. Strain AK13T hydrolyse casein, DNA, Tween 20, 40 and 60 but did not hydrolyse aesculin, gelatin, starch, Tween 80 and urea. It was negative for indole production. Growth was observed at 10-40 °C with optimum growth at 30-37 °C, at 0-8 % (w/v) NaCl, with optimum growth at 0-2 % (w/v) and at pH 6- 12, with optimum growth at pH 7-8.5. The strain AK13T can be differentiated from Silanimonas lenta DSM 16282T using different physiological and biochemical characteristics like salinity, temperature, pH growth range, optimum salinity and pH. Other differentiating characteristics between the strain AK13T and the type strain Silanimonas lenta DSM 16282T were presented in Table 1.

The cellular fatty acid composition of strain AK13T showed a spectrum of 25 fatty acids with a

pronounced dominance of (>5 %) of iso-C11 : 0, iso-C11 : 0 3OH, iso-C15 : 0, iso-C16 : 0, iso-C17 : 1 ω9c and C16

: 1 ω7c and/or iso-C15 : 0 2OH (Summed Feature 3) (Supplementary Table 1). The fatty acids were dominated by branched with iso-, saturated and unsaturated fatty acids. Compared with Silanimonas lenta (17 fatty acids in the present study and 14 fatty acids in Lee et al., 2005) the number of fatty acids detected in strain AK13T was higher and the composition differed considerably (Supplementary Table 1). The respiratory quinone present in strain AK13T was ubiquinone 8 (Q-8). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, two unidentified aminolipids, one unidentified glycolipid and one unidentified lipid as polar lipids (Fig. 2a). The type strain Silanimonas lenta DSM 16282T comprises diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, two unidentified aminophospholipids, one unidentified aminolipid, one unidentified glycolipid and one unidentified lipid (Fig. 2b). Strain AK13T had a DNA G + C content of 55.2 ± 0.5 mol%, higher than Silanimonas lenta DSM 16282T (50.7 mol%) (51.2 mol% from present data)(Table 1).

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The phylogenetic relationships of strain AK13T was ascertained based on the 16S rRNA gene sequence similarity with other strains using BLAST sequence similarity search (NCBI-BLAST). The 16S rRNA gene sequence analysis placed strain AK13T within the family Xanthomonadaceae. The results indicated that at the 16S rRNA gene sequence level, strain AK13T was close to the phylogenetic neighbor Silanimonas lenta DSM 16282T with pairwise sequence similarity of 95.2%. Phylogenetic analysis based on maximum likelihood tree further revealed clear affiliations of the novel isolate with the Silanimonas lenta DSM 16282T, and placed the novel strain within the family Xanthomonadaceae. The strain AK13T clustered with the type species of the genus Silanimonas, Silanimonas lenta DSM 16282T and together clustering with the species of the genus , Thermomonas, , Xylella, , and .

Strain AK13T was close to Silanimonas lenta based on phylogenetic analysis and several other physiological & biochemical characterisitics (acid production and Vitek), major fatty acids, quinone and polar lipid composition. The DNA G + C (mol%) is also close to the genus Silanimonas (50.7) compared to the other nearest genera and Lysobacter (65.4 to 70.4 and 61.7 to 69.3 mol% respectively). But strain AK13T also exhibits a number of differences with the type species of Silanimonas lenta DSM 16282T at phenotypic and genotypic level (Table 1). Thus, the cumulative differences that strain AK13T exhibits from the Silanimonas lenta unambiguously support the proposal of a new species to accommodate the strain AK13T, for which the name Silanimonas mangrovi sp. nov. is proposed.

Description of Silanimonas mangrovi sp. nov.

Silanimonas mangrovi (man.gro’vi. N.L. gen. n. mangrovi, pertaining to mangrove, of Namkhana, Sunderbans, West Bengal, India, referring to the isolation of the type strain from a mangrove forest).

Cells are Gram-negative, motile, non-endospore forming, strictly aerobic, rod shaped, oxidase positive. Cells are 0.3-0.5 µm wide and 2.0-3.0 µm long occurs singly. Colonies on NA plates were circular, 2-3 mm in diameter, smooth, bright yellowish, translucent and raised with entire margins. Grows at 10 to 40 oC with an optimum temperature of 30-37 oC and tolerates up to 8 % NaCl (w/v) with optimum growth of 0-2 % NaCl (w/v). Grows at pH 6-12, with optimum growth at pH 7-8.5. Arginine dihydrolase, catalase, lysine decarboxylase, ornithine decarboxylase and β-galactosidase activities are

absent. Nitrate is not reduced and H2S gas and indole are not produced. The methyl red and Voges-

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Proskauer’s reactions are negative. Casein, DNA (weak), Tween 20, 40 and 60 are hydrolysed but, aesculin, agar, gelatin, starch, Tween 80 and urea are not hydrolysed. Produced acid from fructose, arabinose, cellobiose, glucose, xylose, rhamnose, mannose and salicin and do not produce acid from mannitol, inulin, mannose, mellibiose, sucrose, galactose, sorbitol, lactose, trehalose, raffinose, adonitol

and meso-inositol. Positive for Ala-Phe-Pro-arylamidase, γ-glutamyl-transferase, L-proline arylamidase, lipase, tyrosine arylamidase, α-glucosidase, phosphatase and glu-gly-arg-arylamidase activities but negative for L-pyrrolydonyl-arylamidase, β-N-acetylglucosaminidase, glutamyl arylamidase pNA, β- glucosidase, β-xylosidase, β-alanine arylamidase pNA, β-N-acetylglucosaminidase, α-galactosidase, glycine arylamidase and β-glucoronidase activities. Do not assimilate any of the substrate used individually includes adonitol, L-arabitol, D-cellobiose, D-glucose, D-maltose, D-mannitol, D-mannose, palatinose, D-sorbitol, sucrose, D-tagatose, D-trehalose, citrate, malonate, 5-keto-D-gluconate, L- histidine, courmarate, L-malate and L-lactate. Negative for L-lactate and succinate alkalinisation, fermentation of glucose and ELLMAN reaction. Susceptible to antibiotics (µg per disc unless indicated) spectinomycin (100), kanamycin (30), gentamicin (10), novobiocin (30), ceftazidime (30), polymyxin (300), neomycin (30), ciprofloxacin (30), chloramphenicol (30) and resistant to anitibiotics bacitracin (8), ampicillin (25), cephadroxil (30), penicillin-G (2 units), tetracyclin (30), amoxycillin (30),

vancomycin (30), chlortetracycline (30), lincomycin (2). The major fatty acids are iso-C11 : 0, iso-C11 : 0

3OH, iso-C15 : 0, iso-C16 : 0, iso-C17 : 1 ω9c and summed in feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2OH). Q-8 is the predominant respiratory quinone. The polar lipids include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, two unidentified aminolipids, one unidentified glycolipid and one unidentified lipid. The DNA G + C content of the type strain is 55.2 ± 0.5 mol%.

The type strain, AK13T (= MTCC 11082T = DSM 24914T) was isolated from sediment sample collected from mangrove of Namkhana, Sunderbans, West Bengal, India.

Emended description of the genus Silanimonas Lee et al., 2005

The description is as given previously (Lee et al., 2005) with the following modifications. Catalase activity is variable. The polar lipids include diphosphotidylglycerol, phosphotidylethanolamine, phosphotidylglycerol, phosphotidylserine, unidentified aminolipids, unidentified aminophospholipids, unidentified glycolipid and unidentified lipid. The DNA G + C content is 50.7-55.2 mol%.

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Acknowledgements

We thank Dr. J. Euzeby for his expert suggestion for the correct species epithet and Latin etymology. We also thank Council of Scientific and Industrial Research (CSIR) and Department of Biotechnology, Government of India for financial assistance. We would like to thank Mr. Deepak for his excellent help in 16S rRNA gene sequencing. The laboratory facility was extended by MMRF of NIO, RC, Kochi funded by the Ministry of Earth Sciences, New Delhi. TNRS is thankful to CSIR SIP projects (SIP1302, SIP1308, GAP2131) for providing facilities and funding respectively.

References

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Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111-120.

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Komagata, K. & Suzuki, K. (1987). Lipid and cell wall analysis in bacterial systematics. Methods Microbiol 19, 161-206.

Lanyi, B. (1987). Classical and rapid identification methods for medically important bacteria. Methods Microbiol 19, 1-67.

Lee, E. M., Jeon, C. O., Choi, I., Chang, K. S. & Kim, C. J. (2005). Silanimonas lenta gen. nov., sp. nov., a slightly thermophilic and alkaliphilic gammaproteobacterium isolated from a hot spring. Int J Syst Evol Microbiol 55, 385-389.

Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208-218.

Myers, E. W. & Miller, W. (1988). Optimal alignments in linear space. Comput Appl Biosci 4, 11-17.

Pandey, K. K., Mayilraj, S. & Chakrabarti, T. (2002). Pseudomonas indica sp. nov., a novel butane- utilizing species. Int J Syst Evol Microbiol 52, 1559-1567.

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Legends to Figures

Fig. 1. Maximum likelihood phylogenetic tree, based on 16S rRNA gene sequences, showing the

distant relationships between strain AK13T and representatives of the family

Xanthomonadaceae. Numbers at the nodes are bootstrap values >50%. Beggiatoa alba

B18LDT was used as an out group. Bar, 0.02 substitutions per nucleotide position.

Fig. 2. Two-dimensional thin-layer chromatogram of total polar lipids of the strain AK13T (a) and

Silanimonas lenta DSM 16282T (b). Detection of total lipids by molybdatophosphoric acid.

Abbreviations: DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PG,

phosphatidylglycerol; PS, phosphatidylserine; AL, unidentified aminolipid; APL, unidentified

aminophospholipid; GL, unidentified glycolipid; L, unidentified lipid.

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Table 1. Features that distinguish strain AK13T from the closely related species Silanimonas lenta

Data for both taxa is from the present study. Both strains were motile, rod-shaped, positive for oxidase, Ala-Phe-Pro- arylamidase, γ-glutamyl-transferase, L-proline arylamidase, lipase, tyrosine arylamidase, phosphatase and glu-gly-arg- arylamidase activities and negative for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, L-pyrrolydonyl- arylamidase, β-galactosidase, β-N-acetylglucosaminidase, glutamyl arylamidase pNA, β-glucosidase, β-xylosidase, β-alanine arylamidase pNA, β-N-acetylgalactosaminidase, -galactosidase, glycine arylamidase and β-glucoronidase activities and negative for nitrate reduction, H2S and indole production, methyl red, Voges-Proskauer‘s tests, fermentation of glucose, ELLMAN reaction, L-lactate and succinate alkalinisation. Both strains were positive for hydrolysis of Tween 20, 40 and 60 and negative for hydrolysis of aesculin, agar and urea. Both strains did not utilize adonitol, citrate, L-arabitol, D-cellobiose, D- glucose, D-maltose, D-mannitol, D-mannose, palatinose, D-sorbitol, sucrose, D-tagatose, D-trehalose, malonate, 5-keto-D- gluconate, L-histidine, L-malate, L-lactate and courmarate. Both strains produced acid from fructose, arabinose, cellobiose, glucose, xylose, mannose and salicin and do not produce acid from mannitol, inulin, mannose, mellibiose, sucrose, galactose, sorbitol, lactose, trehalose, raffinose, adonitol and meso-inositol. Both strains contain Q-8 as the respiratory quinone. +, positive; -, negative; w, weak.

Characteristic Silanimonas mangrovi Silanimonas lenta AK13T DSM 16282T Cell size (µm) 0.3-0.5 x 2.0-3.0 0.3-0.5 x 0.8-1.8 Colony colour Bright yellow Pale yellow Salinity growth range (%, w/v) 0-8 0-4 Optimum salinity (%, w/v) 0-2 2 Temperature growth range (oC) 10-40 25-50 Temperature optimum (oC) 30-37 37-47 pH growth range 6-12 6-10 Optimum pH 7-8.5 8-9 Catalase - + α-glucosidase + - Hydrolysis of: Casein w + Gelatin - + Starch - + DNA w + Tween 80 - + Acid production from rhamnose + - Major fatty acids (> 5%) iso-C11 : 0, iso-C11 : 0 3OH, iso-C15 : 0, iso-C16 : 0, iso-C15 : 0, iso-C16 : 0, iso- iso-C17 : 1 ω9c and C17 : 1 ω9c and summed in summed in feature 3 feature 3 (C16 : 1 ω7c and/or (C16 : 1 ω7c and/or iso- iso-C15 : 0 2OH) C15 : 0 2OH) Polar lipids DPG, PE, PG, PS, AL1, DPG, PE, PG, PS, AL1, AL2, GL1, L1 APL1, APL2, GL1, L2 DNA G + C content (mol%) 55.2 51.2

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Supplementay Table 1. Comparison of the fatty acid composition of strain AK13T from the closely related species Silanimonas lenta

Data for both the taxa are from the present study. AK13T and Silanimonas lenta DSM 16282T were grown on NA plates at 37 ºC for 24 h and were of the same physiological age. Results are presented as a percentage of the total fatty acids. Fatty acids amounting to 5% or more of the total fatty acids are in bold. ND, not detected. Data in the parenthesis is from Lee et al., 2005.

Fatty acid Silanimonas mangrovi Silanimonas lenta AK13T DSM 16282T C10 : 0 0.9 0.7 (ND) C10 : 0 3OH 0.8 0.6 (ND) iso-C10 : 0 0.3 ND (0.8) iso-C11 : 0 8.1 1.4 (6.6) iso-C11 : 0 3OH 8.6 4.3 (16.7) iso-C12 : 0 0.3 ND (ND) iso-C12 : 0 3OH ND ND (0.6) iso-C13 : 0 0.2 ND (0.4) C14 : 0 0.8 1.7 (0.6) iso-C14 : 0 1.1 2.2 (1.9) C15 : 0 ND ND (0.2) iso-C15 : 0 19.7 19.4 (34.9) anteiso-C15 : 0 1.7 3.1 (0.7) iso-C15 : 1 F 0.2 ND (ND) C16 : 0 3.5 8.9 (1.7) C16 : 0 N alcohol 4.0 4.4 (ND) iso-C16 : 0 16.0 21.6 (19.6) iso-C16 : 1 H 1.1 2.9 (ND)

C16 : 1 ω5c 0.6 1.0 (ND) C16 : 1 ω7c alcohol 1.2 ND (ND) iso-C17 : 0 3.6 3.5 (6.7) anteiso-C17 : 0 0.6 1.4 (ND) C17 : 0 10-methyl 0.4 ND (ND)

iso-C17 : 1 ω9c 15.4 9.1 (3.8) C18 : 1 ω7c 0.3 ND (ND) Summed Feature 3# 10.2 13.9 (ND) Summed Feature 4# 0.7 ND (ND) #Summed features represent groups of two or three fatty acids that cannot be separated by GLC with the MIDI system.

Summed Feature 3 contains iso-C15 : 0 2OH and/or C16 : 1 ω7c; Summed Feature 4 contains iso-C17 : 1 I and/or anteiso-C17 : 1 B.

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Fig. 1

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Fig. 2