Bhargavaea ullalensis sp. nov., isolated from coastal

International Journal of Systematic and Evolutionary Microbiology (2013), 63, 2450–2456

DOI 10.1099/ijs.0.045062-0

Bhargavaea ullalensis sp. nov., isolated from coastal sand Stefanie P. Glaeser,1 A. B. Arun,2 P. D. Rekha,2 Sudharshan Prabhu,2 Hans-Ju¨rgen Busse3 and Peter Ka¨mpfer1 Correspondence Peter Ka¨mpfer peter.kaempfer@umwelt. uni-giessen.de

1

Institut fu¨r Angewandte Mikrobiologie, Justus-Liebig-Universita¨t Giessen, D-35392 Giessen, Germany

2

Yenepoya Research Center, Yenepoya University, Mangalore-18, Karnataka State, India

3

Institut fu¨r Bakteriologie, Mykologie und Hygiene, Veterina¨rmedizinische Universita¨t, A-1210 Wien, Austria

A Gram-positive-staining, aerobic, non-endospore-forming bacterium, isolated from Ullal coastal sand, Mangalore, Karnataka, India, on marine agar 2216, was studied in detail for its taxonomic position. Based on 16S rRNA gene sequence similarity comparisons, strain ZMA 19T was grouped into the genus Bhargavaea with high 16S rRNA gene sequence similarities to all currently described species of the genus Bhargavaea, Bhargavaea cecembensis (99.3 %), Bhargavaea beijingensis (98.8 %) and Bhargavaea ginsengi (98.6 %). GyrB amino acid sequence-based analysis supported the phylogenetic position and also distinguished strain ZMA 19T from the three other species of the genus Bhargavaea. Amino acid sequence similarities were only 85.6 to 89.5 % between strain ZMA 19T and the type strains of members of the genus Bhargavaea, which shared higher similarities among each other (93.0 to 96.2 %). The chemotaxonomic characterization supported the allocation of the novel strain to the genus Bhargavaea. The major menaquinone was MK-8. The polar lipid profile contained predominantly diphosphatidylglycerol and moderate amounts of phosphatidylglycerol. The diagnostic peptidoglycan diamino acid was lysine and the polyamine pattern contained spermidine and spermine. The major fatty acids were iso- and anteiso-branched fatty acids. DNA–DNA hybridization with the types strains Bhargavaea cecembensis LMG 24411T, Bhargavaea beijingensis DSM 19037T and Bhargavaea ginsengi DSM 19038T resulted in values (reciprocal values in parentheses) of 26 % (29 %), 18 % (15 %) and 21 % (12 %), respectively. The results of physiological and biochemical tests allowed phenotypic differentiation of strain ZMA 19T from all other species of the genus Bhargavaea. Thus, ZMA 19T represents a novel species of this genus, for which the name Bhargavaea ullalensis sp. nov. is proposed, with ZMA 19T (5LMG 27071T 5CCM 8429T) as the type strain.

The genus Bhargavaea was proposed by Manorama et al. (2009) with one species, Bhargavaea cecembensis, to accommodate Gram-positive, rod-shaped, non-motile, non-endospore-forming bacteria. Chemotaxonomically, the genus is characterized by a cell-wall peptidoglycan type A4a with L-lysine as the diagnostic diamino acid and the presence of the major polar lipids diphosphatidylglycerol and phosphatidylglycerol and the major isoprenoid quinone type MK-8 (Verma et al., 2012). On the basis of 16S rRNA sequence comparisons and chemotaxonomic characteristics, this genus was different from the lineages represented by the genera Planococcus, Planomicrobium, The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences of strain ZMA 19T are JX144975 and JX144976, respectively.

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Bacillus, and Geobacillus. In the same year, two species of the genus Bacillus, Bacillus beijingensis and Bacillus ginsengi, were reported by Qiu et al. (2009) and showed high similarities to Bhargavaea cecembensis; consequently these were later transferred to the genus Bhargavaea by Verma et al. (2012). Here, we report a novel species of the genus Bhargavaea represented by one strain (ZMA 19T), which was isolated from coastal sand collected from Ullal coast, Mangalore, Karnataka State, south west coast of India. The sand sample was collected from the low tide zone of Ullal coast (12u 479 38. 40 N 74u 509 57.80 E) during November 2010 to evaluate the culturable bacterial diversity of coastal sand dunes from Ullal coastal sand. Sand (100 g) was collected aseptically from a depth of about 2 cm and transferred to

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Bhargavaea ullalensis sp. nov.

laboratory. Sterile water (50 ml) was added to 1 g sand, agitated for 30 min and allowed to settle. Carefully, the supernatant was transferred to a tube, serially diluted and plated on Marine agar 2216 (MA; Difco), and incubated at 32 uC for 48 h. Differently coloured colonies were picked and subcultured on Marine agar 2216 for purification and further preservation at 280 uC. Subcultivation was performed on tryptone soy agar, TSA (Oxoid) at 25 uC for 24 h. Cell morphology and motility was observed under a Zeiss light microscope at a magnification of 61000, using cells that had been grown for 3 days at 25 uC on TSA. Gram-staining was performed by the modified Hucker method according to Gerhardt et al. (1994). KOH test was carried out according to Moaledj (1986). Endospores were not observed microscopically. The physiological characterization was carried out according to the methods described by Ka¨mpfer et al. (1991) and Ka¨mpfer (1990). In addition, the presence of urease was tested on urea agar (Merck) supplemented with 2 % urea according to the manufacturer’s instructions (Christensen, 1946). Indole and sulphide production was tested in SIM agar according to the instructions of the manufacturer (Merck). Hydrolysis of casein, gelatin (plate method), DNA, Tween 80, starch and tyrosine was performed according to the methods described by Smibert & Krieg (1994). The results of physiological characterization are listed in the species description (below) and differentiating features from the most closely related species are listed in Table 1. In order to test susceptibility to heating, a freshly inoculated 2 ml culture of strain ZMA 19T was heated at 80 uC for 10 min. After incubation overnight at 28 uC, growth was unambiguously visible as indicted by turbidity of the medium, indicating that cells of strain ZMA 19T are not killed by this procedure. Similar behaviour was reported for Bhargavaea beijingensis and Bhargavaea ginsengi (Qiu et al., 2009). DNA isolation for phylogenetic analysis was performed with a commercial DNA extraction kit (GenElute Plant Genomic DNA kit; Sigma). Universal primers 27F (59GAGTTTGATCMTGGCTCAG-39) and 1492R (59-GAGTTTGATCMTGGCTCAG-39) (Lane, 1991) were used for PCR amplification and Sanger sequencing of the 16S rRNA gene. Phylogenetic analysis was performed in ARB release 5.2 (Ludwig et al., 2004) using the ‘All-Species Living Tree’ Project (LTP; Yarza et al., 2008) database release LTPs106 (August 2011). Sequences not included in the LTP database were aligned with SINA (v1.2.9) according to the SILVA seed alignment (http://www.arb-silva.de; Pruesse et al., 2007) and implemented in the ARB database. The alignment was checked manually based on secondary structure information. Pairwise sequence similarities were calculated in ARB without the use of an evolutionary substitution model. Phylogenetic trees were reconstructed with the maximumlikelihood method using RAxML v7.04 (Stamatakis, 2006) with GTR-GAMMA and rapid bootstrap analysis, and the http://ijs.sgmjournals.org

neighbour-joining method with the Jukes–Cantor correction (Jukes & Cantor, 1969). Phylogenetic trees were calculated with 100 resamplings (bootstrap analysis; Felsenstein, 1985) and based on 16S rRNA gene sequences between positions 96 and 1432 (according to Escherichia coli numbering; Brosius et al., 1978). The sequenced 16S rRNA gene of strain ZMA 19T was a continuous stretch of 1430 unambiguous nucleotides (positions 50 to 1471, E. coli numbering). Strain ZMA 19T shared 98.7 to 99.3 % 16S rRNA gene sequences similarity to the type strains of all species of the genus Bhargavaea, with highest sequence similarity to Bhargavaea cecembensis (99.3 %), followed by Bhargavaea beijingensis (98.8 %) and Bhargavaea ginsengi (98.6 %). The 16S rRNA gene sequence similarities to type strains of the next closest related genera were below 94.5 %. The reconstruction of phylogenetic trees showed, independently of the treeing method used, that strain ZMA 19T is placed within the monophyletic cluster of the genus Bhargavaea (Fig. 1). In order to further distinguish strain ZMA 19T from the other species of the genus Bhargavaea, gyrB amino acid sequence-based analysis was performed according to Verma et al. (2012). The partial gyrB sequence of strain ZMA 19T was amplified and sequenced according to Verma et al. (2012) and analysis was performed in MEGA5 (Tamura et al., 2011). The gyrB nucleotide sequences were translated into the amino acid sequences by using the fulllength gyrB sequence (LGAS_0005) from the genomesequenced strain Lactobacillus gasseri ATCC 33323T to obtain the correct open reading frame. Amino acid sequences were aligned using CLUSTAL W (Thompson et al., 1994). Pairwise amino acid sequence similarities were calculated without the use of an evolutionary model. Phylogenetic trees were reconstructed with the neighbourjoining and maximum-likelihood methods using the JTT model (Jones et al., 1992) and 100 bootstraps. In total, 330 amino acids were included in the analysis. Phylogenetic reconstruction based on partial gyrB amino acid sequences confirmed the placement of strain ZMA 19T into the monophyletic cluster of the genus Bhargavaea (Fig. 2); however gyrB amino acid sequence similarities clearly showed that strain ZMA 19T could be differentiated from the other species of the genus Bhargavaea because strain ZMA 19T shared only 85.6 to 89.5 % amino acid sequence similarity with those. In contrast, all other type strains representing members of the genus Bhargavaea shared higher amino acid sequence similarities (93 to 96.2 %) among each other. Biomass subjected to analyses of the diagnostic diamino acid, polyamines, quinones and polar lipids was grown on 3.36 PYE broth (1.0 % peptone from caseine, 1.0 % yeast extract, pH 7.2) at 28 uC. The diagnostic peptidoglycan diamino acid was extracted and analysed as reported by Schleifer (1985). Quinones and polar lipids were extracted and analysed applying the integrated procedure reported by Tindall (1990 a,b) and Altenburger et al. (1996).

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Table 1. Differential characteristics of strain ZMA 19T and members of the genus Bhargavaea Strains: 1, ZMA 19T, 2, Bhargavaea cecembensis LMG 24411T, 3, Bhargavaea beijingensis DSM 19037T; 4, Bhargavaea ginsengi DSM 19038T. Data for all species from this study. All strains were Gram-positive, non-motile, non-spore-forming, positive for oxidase, catalase, caseinase*, gelatinase, beta-haemolysis, nitrate and nitrite reduction, but negative for indole production, Simmons’ citrate, methyl red and Voges–Proskauer reaction, phenylalanine deaminase, glucose fermentation and H2S production in TSI. +, Positive; 2, negative. Characteristic Cell shape Cell dimensions Optimum growth temperature (uC) pH range for growth NaCl tolerance (%, w/v) Biochemical tests Urease Substrate utilization Acetate Propionate cis-Aconitate Citrate Fumarate Glutarate DL-3-hydroxybutyrate DL-Lactate L-Malate 2-Oxoglutarate Pyruvate L-Alanine L-Proline

1

2

3

4

Rod/single/pairs/ short chains 1.062.0–3.0 mm 30 5.5–11 0–12

Rod/single/pairs/ short chains 1.062.0–8.0 mm 37 7–7.5 0–10

Coccoid rod/ single/pairs 1.061.2 mm 30 5.5–11 0–8.0

Coccoid rod/single/ pairs/short chains 1.061.2–2.0 mm 30 6–11 0–12

+

+

+

2

+ 2 2 + + + 2 + + + + + 2

+* + + 2 2 2 + 2 +* +* +* +* +

2 2 + + 2 2 2 + +* 2 + 2* +

2 2 + 2 2 2 2 + 2* + 2* 2 +

*Data in congruence with those reported by Verma et al. (2012).

Polyamines were extracted and analysed from cells harvested at the late exponential growth phase as reported by Busse & Auling (1988). HPLC analysis was carried out using the equipment described by Stolz et al. (2007). Fatty acids were extracted and analysed as described by Ka¨mpfer & Kroppenstedt (1996). Strains were grown under identical conditions and the cells for extractions were taken from colonies of the same size. Fatty acids were identified with Sherlock version 2.11, TSBA40 Rev. 4.1. The diagnostic diamino acid of the peptidoglycan was lysine. The quinone system of strain ZMA 19T consisted of menaquinones MK-8 (93 %), MK-7 (5 %), MK-9 (2 %) and traces of MK-6. The polar lipid profile of strain ZMA 19T showed the major lipid diphosphatidylglycerol, moderate amounts of phosphatidylglycerol and minor amounts of two lipids not stainable with any of the specific detection reagents, indicating that these lipids do not contain a free amino group, phosphate or a sugar moiety (Fig. 3). These traits, lysine in the peptidoglycan, menaquinone MK-8 as the major quinone and a polar lipid profile with the major compounds diphosphatidylglycerol and phoshatidylglycerol, are in excellent agreement with the emended description of the genus Bhargavaea (Verma et al., 2012). 2452

The polyamine pattern of strain ZMA 19T contained spermidine [20.8 mmol (g dry weight)21], spermine [3.7 mmol (dry weight)-1] and traces of putrescine. No polyamine data are accessible for the recognized species of the genus Bhargavaea, but this pattern is in agreement with those of other mesophilic bacilli (Hamana, 1999; Hamana & Niitsu, 1999; Hamana et al., 1989; Ka¨mpfer et al., 2010). The fatty acids of strain ZMA 19T comprised mainly iso- and anteiso-branched fatty acids and was very similar to the most closely related species of the genus Bhargavaea. The detailed fatty acid profile obtained from cells grown on TS medium after 72 h of incubation at 28 uC is shown in Table 2. The results of the physiological characterization, performed using methods described previously (Ka¨mpfer, 1990; Ka¨mpfer et al., 1991), are given in Table 1 and in the species description. DNA–DNA hybridization (according to Ziemke et al., 1998) with the types strains of Bhargavaea cecembensis LMG 24411T, Bhargavaea beijingensis DSM 19037T and Bhargavaea ginsengi DSM 19038T resulted in hybridization values (reciprocal values in parentheses) of 26 % (29 %), 18 % (15 %) and 21 % (12 %), respectively. Based on these results, as well as phenotypic traits, we describe a novel species of the genus Bhargavaea, for which

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80*

0.001 84*

*

76

Lysinibacillus parviboronicapiens BAM-582T (AB300598) Lysinibacillus sphaericus DSM 28T (AJ310084)

Lysinibacillus boronitolerans 10aT (AB199591) Lysinibacillus odysseyi 34hs1T (AF526913) 82* Kurthia gibsonii NBRC 15534T (AB271738) 100 Kurthia zopfii NBRC 101529T (AB271740)

Kurthia sibirica DSM 4747T (AJ605774) Sporosarcina koreensis F73T (DQ073393) Sporosarcina soli I80T (DQ073394) 100 Planococcus citreus NCIMB 1493T (X62172) Planococcus rifietoensis M8T (AJ493659) * 100* Planococcus maritimus TF-9T (AF500007) Planomicrobium koreense JG07T (AF144750) 89* Planomicrobium psychrophilum CMS 53orT (AJ314746) 83* Jeotgalibacillus alimentarius YKJ-13T (AF281158) 100* Jeotgalibacillus salarius ASL-1T (EU874389) Jeotgalibacillus marinus DSM 1297T (AJ237708) 96* Bhargavaea beijingensis ge10T (EF371374) 90* Bhargavaea cecembensis DSE10T (AM286423) 100

100

Bhargavaea ullalensis ZMA 19T (JX144975) Bhargavaea ginsengi ge14T (EF37137) 81 Bacillus drentensis LMG 21831T (AJ542506) 78 Bacillus niacini IFO15566T (AB021194) Bacillus soli LMG 21838T (AJ542513) Bacillus novalis LMG 21837T (AJ542512)

90

* 86

100

Bacillus fumarioli LMG 17489T (AJ250056) 67 Bacillus subtilis subsp. subtilis DSM 10T (AJ276351) 99 Bacillus tequilensis 10bT (HQ223107) 99 Bacillus methylotrophicus CBMB205T (EU194897) Bacillus pumilus ATCC 7061T (AY876289) Bacillus boroniphilus T-15ZT (AB198719) Bacillus selenatarsenatis SF-1T (AB262082) Bacillus thioparans BMP-1T (DQ371431)

Bacillus methanolicus NCIMB 13113T (AB112727) Geobacillus stearothermophilus IFO12550T (AB021196) Geobacillus subterraneus 34T (AF276306) Escherichia coli ATCC 11775T (X80725)

Fig. 1. Maximum-likelihood tree showing the phylogenetic position of strain ZMA 19T among members of the genus Bhargavaea based on 16S rRNA gene sequence analysis. The tree was generated in ARB using RAxML (GTR-GAMMA, rapid bootstrap analysis, 100 bootstraps) and based on 16S rRNA gene sequences between positions 96 and 1432 (E. coli numbering; Brosius et al., 1978). GenBank accession numbers are given in parentheses. Numbers at branch nodes refer to bootstrap values .70 % (100 replicates). E. coli ATCC 11775T was used as an outgroup. Bar, 0.001 substitutions per site. Asterisks indicate nodes supported by high bootstrap values in the neighbour-joining analysis performed in parallel.

the name Bhargavaea ullalensis is proposed. The type strain is ZMA 19T. Description of Bhargavaea ullalensis sp. nov. Bhargavaea ullalensis (ul.lal.en9sis. N.L. fem. n. ullalensis of or belonging to Ullal, the place from where this bacteria was isolated. Ullal is a small coastal town in Mangalore City, Karnataka, India). Cells are Gram-positive in staining and KOH tests, strictly aerobic rods (2.0–3.0 mm long and 0.8–1.0 mm wide) and non-motile. No endospores are formed. Cells are not killed http://ijs.sgmjournals.org

by heating at 80 uC for 10 min. Colonies grown on TSA are circular, convex and beige. No growth on MacConkey agar. Optimum temperature for growth is 30 uC; growth occurs at 15–50 uC but not at 10 uC or 55 uC. Optimal pH for growth is 7–8; growth occurs at pH 5.5–11. Tests for catalase, oxidase and gelatinase activities are positive. Tests for production of indole and sulphide, b-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and tryptophan deaminase are negative. No acid formation from the sugars or sugar related compounds Dglucose, lactose, sucrose, D-mannitol, dulcitol, salicin, Dadonitol, D-sorbitol, L-arabinose, raffinose, L-rhamnose,


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S. P. Glaeser and others

Bacillus subtilis subsp. spizizenii BCRC 17366T (DQ309299)

0. 1

98 98*

* *

Bacillus vallismortis BCRC 17183T (DQ309298) Bacillus subtilis subsp. subtilis BCRC 10255T (DQ309293) B acillus mojavensis BCRC 17124T (DQ309297) Bacillus amyloliquefaciens BCRC 11601T (DQ309294) Bacillus atrophaeus BCRC 17123T (DQ309296)

97

Bacillus safensis FO-036bT (AY167867) Bacillus licheniformis BCRC 11702T (DQ309295) 99 Bacillus sonorensis BCRC 17416T (DQ309300) 74 100 95*

Bhargavaea beijingensis ge10T (JF261159) Bhargavaea cecembensis DSE10T (JF261161) Bhargavaea ginsengi ge14T (JF261158)

Bhargavaea ullalensis ZMA 19T (JX144976) T 100* Sporosarcina saromensis HG645 (AB243078) 99* Sporosarcina sp. HG644 (AB243077) 100* Sporosarcina sp. YM9-109 (AB243075) *

Geobacillus thermocatenulatus VKM B-1259T (GU323952) Geobacillus kaustophilus HTA426 (GK0005)

100

Geobacillus jurassicus DSM 15726T (GU459228) Geobacillus subterraneus K (GU459227) Lactobacillus gasseri ATCC 33323T (LGAS_0005)

Fig. 2. Maximum-likelihood tree showing the phylogenetic position of strain ZMA 19T among members of the genus Bhargavaea based on partial GyrB amino acid sequence analysis. The tree was generated in MEGA5 (Tamura et al., 2011) by using the JTT matrix for the calculation of the maximum-likelihood tree. In total, 330 amino acids were included in the analysis. GenBank accession numbers of nucleotide sequences, on which amino acid sequence alignments are based, are given in parentheses. Numbers at branch nodes refer to bootstrap values .70 % (100 replicates). Lactobacillus gasseri ATCC 33323T was used as an outgroup. Bar, 0.1 substitutions per site. Asterisks indicate nodes supported by high bootstrap values in the neighbourjoining analysis performed in parallel.

DPG

2nd Dimension

↑

L1 PG L2

1st Dimension →

Fig. 3. Polar lipid profile of strain ZMA 19T separated by twodimensional TLC and detected with molybdatophosphoric acid. DPG, diphosphatidylglycerol; PG, phosphatidylglycerol; L1, L2, unidentified polar lipids not reacting with the spray reagents specific for free amino groups, phosphate or sugars moieties. 2454

maltose, D-xylose, trehalose, cellobiose, erythritol, melibiose, D-arabitol or D-mannose could be observed. Urease production and citrate utilization were weakly positive. Several carbohydrates were utilized (all weakly) by strain ZMA 19T according to the method of Ka¨mpfer et al. (1991): acetate, citrate, fumarate, glutarate, DL-lactate, Lmalate and pyruvate. N-Acetyl-D-glucosamine, L-arabinose, arbutin, cellobiose, D-fructose, D-glucose, D-galactose, gluconate, maltose, L-rhamnose, sucrose, salicin, trehalose, D-xylose, myo-inositol, D-maltitol, D-mannitol, D-mannose, D-sorbitol, D-adonitol, melibiose, ribose, putrescine, propionate, trans-aconitate, cis-aconitate, adipate, 4-aminobutyrate, azelate, itaconate, 2-oxoglutarate and mesaconate are not utilized as sole carbon source. The diagnostic diamino acid of the peptidoglycan is lysine. The polyamine pattern is composed of the major polyamine spermidine. The quinone system consists of predominant menaquinone MK-8 and lesser amounts of MK-7, MK-9 and MK-6. The polar lipid profile contains predominantly diphosphatidylglycerol, moderate amounts of phosphatidylglycerol and minor amounts of two unidentified polar lipids. Major fatty acids are anteiso-C15 : 0, anteiso-C17 : 0,



Table 2. Cellular fatty acid composition (%) of strain ZMA 19T and members of the genus Bhargavaea Strains: 1, ZMA 19T; 2, Bhargavaea cecembensis LMG 24411T; 3, Bhargavaea beijingensis DSM 19037T; 4, Bhargavaea ginsengi DSM 19038T. Data for taxa 1 and 2 from this study. Strains were grown on TSA at 28 uC for 48 h. Data shown in parentheses from Verma et al. (2012). tr, Trace amount (,1 %). Fatty acid Straight chain saturated C14 : 0 C16 : 0 Branched saturated iso-C14 : 0 iso-C15 : 0 iso-C16 : 0 iso-C17 : 0 anteiso-C15 : 0 anteiso-C17 : 0 Monosaturated C16 : 1v11c C18 : 1v9c C16 : 1v9c alcohol iso-C17 : 1v10c Summed feature 4*

1

2

3

4

Felsenstein, J. (1985). Confidence limits of phylogenies: an approach

using the bootstrap. Evolution 39, 783–791. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (editors) (1994). Methods for General and Molecular Bacteriology. Washington,

DC: American Society for Microbiology. Hamana, K. (1999). Polyamine distribution catalogues of clostridia,

acetogenic anaerobes, actinobacteria, bacilli, heliobacteria and haloanaerobes within the Gram-positive eubacteria: distribution of spermine and agmatine in thermophiles and halophiles. Microbiol Cult Collect 15, 9–28. Hamana, K. & Niitsu, M. (1999). Production of 2-phenylethylamine by

– 2.3

– (1.0) 3.9 (3.9)

(tr) (2.1)

(1.1) (3.7)

5.6 8.1 10.3 – 56.8 15.8

4.3 35.9 6.9 3.9 23.9 9.8

(8.9) (18.5) (14.0) (1.9) (33.4) (9.5)

(4.2) (37.4) (5.9) (3.6) (27.0) (11.6)

(4.9) (29.0) (8.6) (3.0) (32.6) (9.9)

– – – – –

3.8 – 4.8 – 2.7

(1.5) (1.1) (4.8) (tr) (1.5)

(1.6) (1.0) (2.2) (1.2) (2.2)

(1.4) (1.0) (2.5) (1.0) (1.5)

*Summed features represent groups of two fatty acids that could not be separated by GLC with the MIDI system. Summed feature 4 contains iso-C17 : 1 I and/or anteiso-C17 : 1 B.

iso-C15 : 0 and iso-C16 : 0. In addition iso-C14 : 0 and C16 : 0 are detected. The type strain ZMA 19T (5LMG 27071T 5CCM 8429T) was isolated from Ullal coastal sand on Marine agar 2216. Ullal is the name of the small town in coastal Dakshina Kannada district in the Indian state of Karnataka.

decarboxylation of L-phenylalanine in alkaliphilic Bacillus cohnii. J Gen Appl Microbiol 45, 149–153. Hamana, K., Akiba, T., Uchino, F. & Matsuzaki, S. (1989).

Distribution of spermine in bacilli and lactic acid bacteria. Can J Microbiol 35, 450–455. Jones, D. T., Taylor, W. R. & Thornton, J. M. (1992). The rapid

generation of mutation data matrices from protein sequences. Comput Appl Biosci 8, 275–282. Jukes, T. H. & Cantor, C. R. (1969). Evolution of the protein molecules. In Mammalian Protein Metabolism, vol. 3, pp. 21–132. Edited by H. N. Munro. New York: Academic Press. Ka¨mpfer, P. (1990). Evaluation of the Titertek-Enterobac-Automated

System (TTE-AS) for identification of members of the family Enterobacteriaceae. Zentralbl Bakteriol 273, 164–172. Ka¨mpfer, P. & Kroppenstedt, R. M. (1996). Numerical analysis of

fatty acid patterns of coryneform bacteria and related taxa. Can J Microbiol 42, 989–1005. Ka¨mpfer, P., Steiof, M. & Dott, W. (1991). Microbiological

characterisation of a fuel-oil contaminated site including numerical identification of heterotrophic water and soil bacteria. Microb Ecol 21, 227–251. Ka¨mpfer, P., Falsen, E., Lodders, N., Langer, S., Busse, H.-J. & Schumann, P. (2010). Ornithinibacillus contaminans sp. nov., an

endospore-forming species. Int J Syst Evol Microbiol 60, 2930–2934. Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid

Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, Buchner, A., Lai, T., Steppi, S. & other authors (2004). ARB: a soft-

ware environment for sequence data. Nucleic Acids Res 32, 1363–1371. Manorama, R., Pindi, P. K., Reddy, G. S. N. & Shivaji, S. (2009).

Acknowledgements We thank Gundula Will, Maria Sowinsky and Katja Grebing for excellent technical assistance and Dr Jean Euze´by for nomenclatural advice.

Bhargavaea cecembensis gen. nov., sp. nov., isolated from the ChagosLaccadive ridge system in the Indian Ocean. Int J Syst Evol Microbiol 59, 2618–2623. Moaledj, K. (1986). Comparison of Gram-staining and alternate methods,

KOH test and aminopeptidase activity in aquatic bacteria: their application to numerical taxonomy. J Microbiol Methods 5, 303–310.

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