International Journal of Systematic and Evolutionary Microbiology (2009), 59, 2618–2623 DOI 10.1099/ijs.0.002691-0

Bhargavaea cecembensis gen. nov., sp. nov., isolated from the Chagos–Laccadive ridge system in the Indian Ocean

3 3 R. Manorama, P. K. Pindi, G. S. N. Reddy and S. Shivaji

Correspondence Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India S. Shivaji [email protected]

A novel Gram-positive, rod-shaped, non-motile, non-spore-forming bacterium, strain DSE10T, was isolated from a deep-sea sediment sample collected at a depth of 5904 m from the Chagos– Laccadive ridge system in the Indian Ocean. Cells of strain DSE10T were positive for catalase,

oxidase, urease and lipase activities and contained iso-C14 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-

C15 : 0 as the major fatty acids. The major respiratory quinones were MK-6 and MK-8 and the major lipids were phosphatidylglycerol and diphosphatidylglycerol. The cell-wall peptidoglycan contained diaminopimelic acid as the diagnostic diamino acid. A BLAST sequence similarity search based on 16S rRNA gene sequences indicated that the genera Planococcus, Planomicrobium, and Geobacillus were the nearest phylogenetic neighbours to the novel isolate with gene sequence similarities ranging from 94.9 to 95.2 %. Phylogenetic analyses using neighbour- joining, minimum-evolution and maximum-parsimony methods indicated that strain DSE10T formed a deeply rooted lineage distinct from the clades represented by the genera Planococcus, Planomicrobium, Bacillus and Geobacillus. Further, strain DSE10T could be distinguished from the above-mentioned genera based on the presence of signature nucleotides G, A, C, T, C, A, G, C and T at positions 182, 444, 480, 492, 563, 931, 1253, 1300 and 1391, respectively, in the 16S rRNA gene sequence. Based on the phenotypic and phylogenetic characteristics determined in this study, strain DSE10T was assigned as the type of a new genus, Bhargavaea gen. nov., as Bhargavaea cecembensis sp. nov. The type strain of Bhargavaea cecembensis gen. nov., sp. nov. is DSE10T (5LMG 24411T5JCM 14375T). The genomic DNA G+C content of strain DSE10T is 59.5±2.5 mol%.

Many novel deep-sea bacterial species (close to 100 species) et al., 2006; Kaneko et al., 2007; Kimura et al., 2007; Shivaji representing 28 new genera and belonging to the et al., 2007), deep-sea carbonate crusts (Heijs et al., 2006) and Proteobacteria (12 genera), Aquificae (3 genera), seawater (Lauro & Bartlett, 2008). In the present study, we Deinococcus–Thermus group (3 genera), Cytophaga– describe another novel bacterium belonging to a new genus, Flavobacterium–Bacteroidetes (CFB) group (2 genera), Bhargavaea gen. nov., a member of the . Deferribacter (1 genus), Thermotogae (1 genus) and Strain DSE10T was isolated from a deep-sea sediment Firmicutes (6 genera) have been identified. The majority of sample collected from the Chagos–Laccadive ridge system these taxa have been isolated from hydrothermal vents (111060 S, 721310 E), which is a part of the Chagos (Nakagawa et al., 2004; Prokofeva et al., 2005; Miroshni- Archipelago in the Indian Ocean. The Chagos Archipelago chenko & Bonch-Osmolovskaya, 2006; Naganuma et al., is a group of seven atolls comprising more than 64 2007; Reysenbach et al., 2006), deep-sea-sediments (Arakawa individual tropical islands about 300 miles south of the Maldives. The sediment sample was collected as described 3These authors contributed equally to this work. previously (Raghukumar et al., 2004). In brief, the Abbreviations: ME, minimum-evolution; NJ, neighbour-joining; TEM, sediment core (length 460 cm and diameter 10 cm) was transmission electron microscopy. collected at a depth of 5904 m using a gravity core made The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene from polyvinyl carbonate (PVC). The core was then sequence of Bhargavaea cecembensis gen. nov., sp. nov. DSE10T is extruded into a plastic bag under sterile conditions and AM286423. sectioned into 30 cm pieces from the top to the bottom. Two additional phylogenetic trees based on 16S rRNA gene sequences For isolation of , a 100 mg sediment sample from and a table detailing the signature nucleotides of Bhargavaea gen. nov. the centre of the 30 cm was recovered and suspended in are available with the online version of this paper. 0.3 % saline solution (Shivaji et al., 2007) and vortexed for

2618 002691 G 2009 IUMS Printed in Great Britain Bhargavaea cecembensis gen. nov., sp. nov.

30 min. The suspension was allowed to settle and 100 ml independent experiments is given. The variation between supernatant was plated on ZoBell marine agar medium the experiments was less than 2 %. For 16S rRNA gene (ZoBell, 1941) and incubated at room temperature for sequencing, DNA was prepared using the MoBio microbial 15 days. The viable bacterial cell count obtained was DNA isolation kit (MoBio Laboratories Inc.) and approximately 2.5–7.56105 c.f.u. Based on colony mor- sequenced as described previously (Reddy et al., 2000). phology, a brown-coloured dominating morphotype was The resulting almost complete 16S rRNA gene sequence selected and characterized in the present study. contained 1510 nucleotides [26–1536, according to the Escherichia coli 16S rRNA gene (GenBank accession no. Cell morphology was studied using light and transmission J01695) numbering]. The 16S rRNA gene sequence was electron microscopy (TEM). For TEM, cells were negatively aligned with closely related sequences belonging to stained with 0.5 % (w/v) uranyl acetate and mounted on organisms of the genera Bacillus, Planococcus, Planomicro- Formvar-coated copper grids. The grids were examined in bium and Geobacillus using CLUSTAL W (Thompson et al., TEM mode on a JEOL JEM-1010 electron microscope 1994). Pairwise evolutionary distances were computed operated at 60 kV (Reddy et al., 2006). Motility was using the DNADIST program with the Kimura two- assessed on 0.4 % TSA agar plates and also by light parameter model as developed by Kimura (1980). microscopy. Spore staining was performed using a Phylogenetic trees were constructed using three tree- Schaeffer & Fulton’s spore staining kit (K006-1KT, making algorithms, the minimum-evolution (ME), neigh- HiMedia) according to the manufacturer’s protocols. bour-joining (NJ) and maximum-parsimony (DNAPARS) Growth at different temperatures, tolerance to salt, methods, using the MEGA3 software package (Kumar et al., biochemical characteristics, carbon assimilation, H2S 2004) and the stability among the clades was assessed by production and the sensitivity of the cultures to different employing 1000 replicate datasets. antibiotics were determined by previously described T methods (Lanyi, 1987; Smibert & Krieg, 1994; Reddy Strain DSE10 grew well on ZoBell marine broth 2216 et al., 2006). Biochemical characteristics were also double- (ZoBell, 1941) and on TSA medium. The major cellular checked with a Hi25 Enterobacteriaceae identification kit fatty acids were (%): anteiso-C15 : 0 (31.2), iso-C15 : 0 (12.8), (KB003; HiMedia) and HiCarbohydrate kit parts A, B and iso-C16 : 0 (10.7), iso-C14 : 0 (8.5), anteiso-C17 : 0 (6.2), C16 : 0 C (KB009; HiMedia) according to the manufacturer’s (4.7), C15 : 1v5c (4.7) and C16 : 1v7c alcohol (4.5). The T protocol. Growth at different pH values was checked using menaquinones were MK-6 and MK-8. Strain DSE10 TSA medium buffered either with citric acid–NaOH (for contained phosphatidylglycerol and diphosphatidylglycerol pH 5 and 6), phosphate (for pH 7 and 8), glycine–NaOH as the major lipids. The cell-wall peptidoglycan contained the amino acids alanine, glutamic acid and diaminopimelic (for pH 9 and 10) or Tris (for pH 11 and 12) buffer. To T check the tolerance of strain DSE10T to pressure, cells were acid in a ratio of 2 : 1 : 1. Cells of strain DSE10 could subjected to 2500 p.s.i. under sterile conditions in a French tolerate a pressure of 2500 p.s.i. Additional characteristics press for 30 min and then plated on TSA medium. of the novel strain are given in the species description. A BLAST sequence similarity search, based on the 16S rRNA Fatty acid methyl esters were prepared and analysed by T Sherlock Microbial Identification System (MIDI) accord- gene sequence of strain DSE10 resulted in the identifica- ing to the protocol described by Agilent Technologies. For tion of various strains of cultivable bacterial strains without this purpose, strain DSE10T was grown on TSA medium validly published names and environmental sequences of the genus Bacillus with similarity of more than 97 %. The (pH 7.5) at 25 uC for two days. Polar lipids were extracted T and analysed according to the method described by phylogenetic neighbours of strain DSE10 with validly Komagata & Suzuki (1987). Menaquinones were extracted published names included Planococcus rifietoensis (Romano by following a modified method. In this modified method, et al., 2003), Planococcus maritimus (Yoon et al., 2003), the cell pellet obtained from 500 ml freshly grown broth Planococcus citreus (Farrow et al., 1992), Bacillus bataviensis culture was suspended in a chloroform/methanol (2 : 1) (Heyrman et al., 2004), Bacillus fumarioli (Logan et al., mixture and homogenized for 5 min on ice. The lower 2000), Bacillus fortis (Scheldeman et al., 2004), Bacillus layer containing quinones was collected by spinning the foraminis (Tiago et al., 2006), Bacillus novalis (Heyrman et suspension at 6000 g for 10 min and dried with nitrogen al., 2004), Bacillus niacini (Nagel & Andreesen, 1991), gas. This process was repeated and 20 ml acetone was Bacillus methanolicus (Arfman et al., 1992), Brevibacterium added to the remaining pellet, which was then homo- frigoritolerans (Delaporte & Sasson, 1967) and Geobacillus thermoglucosidasius (Nazina et al., 2001), which had gene genized. The upper acetone layer was collected and dried sequence similarities ranging from 94.9 % to 95.2 %. The with nitrogen gas. The quinones obtained were initially BLAST results indicated that the novel strain could be a separated by TLC (Collins et al., 1977) and further member of the genera Planococcus, Planomicrobium, separated on HPLC using the isocratic solvent system of Bacillus or Geobacillus. However, phylogenetic analyses, methanol/isopropyl ether (3 : 1) (Tamaoka et al., 1983). using NJ, ME and DNAPARS, resulted in the positioning of Isolation of DNA and the determination of the DNA G+C strain DSE10T as a deeply rooted lineage distinct from the content were conducted as described previously (Marmur, clades represented by the genera Planococcus and 1961; Marmur & Doty, 1962). The mean value from two Planomicrobium, with a bootstrap value of 100 (Fig. 1 http://ijs.sgmjournals.org 2619 R. Manorama and others

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the relationships between strain DSE10T and other related reference micro-organisms. Bootstrap values (expressed as percentages of 1000 replications) .50 % are indicated at nodes. Bar, nucleotide substitutions per 100 nucleotides. and Supplementary Figs S1a and S1b in IJSEM Online). Geobacillus. It is worth mentioning that the genera The ambiguous clustering and less than 95 % 16S rRNA Planococcus and Planomicrobium also share 16S rRNA gene sequence similarity with the nearest phylogenetic gene sequence similarity of more than 95 %, justifying the neighbours (Ludwig et al., 1998) clearly supported the creation of a new genus to accommodate strain DSE10T. creation of a higher taxon to accommodate strain DSE10T. In addition to the above genotypic and phylogenetic Further evidence in support of genus status for strain differences, strain DSE10T can be differentiated from DSE10T comes from an analysis based on a comparison of members of the genera Planococcus and Planomicrobium the 16S rRNA gene sequences. This indicated that strain in that strain DSE10T is non-motile, contains diaminopi- DSE10T exhibited a difference of 93, 102, 97 and 56 melic acid as the diagnostic diamino acid of the cell-wall nucleotides with the genera Bacillus, Planococcus, peptidoglycan and has MK-6 and MK-8 as the predom- Planomicrobium and Geobacillus, respectively, in the highly inant menaquinones (Table 1). Further, strain DSE10T can variable region of the 16S rRNA gene. It is also interesting be differentiated from the genera Bacillus and Geobacillus to note that strain DSE10T possessed nine unique with respect to sporulation, fatty acid methyl esters and nucleotides in the highly conserved region of the 16S menaquinones (Table 1). Thus, the combined differences rRNA gene. The bases G, A, C, T, C, A, G, C and T at that strain DSE10T exhibits when compared with the above positions 182, 444, 480, 492, 563, 931, 1253, 1300 and 1391, genera unambiguously support the creation of a new genus respectively (positions with respect to the E. coli 16S rRNA and novel species, for which the name Bhargavaea gene sequence with GenBank accession number X80725) cecembensis gen. nov., sp. nov. is proposed. were present only in strain DSE10T and not in members of the genera Planococcus, Planomicrobium, Bacillus or Description of Bhargavaea gen. nov. Geobacillus (see Supplementary Table S1 in IJSEM Bhargavaea (Bhar.ga.va9e.a. N.L. fem. n. Bhargavaea named Online). The conserved bases in strain DSE10T may be in honour of Pushpa Mittra Bhargava, the renowned considered as the signature nucleotides that separate it Indian biologist). from the genera Planococcus, Planomicrobium, Bacillus and Geobacillus. In addition, strain DSE10T shared the highest Cells are Gram-positive, aerobic, non-spore-forming rods 16S rRNA gene sequence similarity of 95 % with species of that give a positive result in tests for catalase, oxidase, the genera Bacillus, Planococcus, Planomicrobium and urease and lipase activities. The major fatty acids are iso-

2620 International Journal of Systematic and Evolutionary Microbiology 59 Bhargavaea cecembensis gen. nov., sp. nov.

Table 1. Characteristics that differentiate the genus Bhargavaea gen. nov. from other closely related genera

Genera: 1, Bhargavaea gen. nov.; 2, Planococcus (data from Hao & Komagata, 1985; Yoon et al., 2001); 3, Planomicrobium (Yoon et al., 2001); 4, Bacillus (Claus & Berkeley, 1986; Nazina et al., 2001); 5, Geobacillus (Nazina et al., 2001). +, Positive; 2, negative; +/2, positive or negative; V, variable; NR, not reported; D, differs from species to species; PG, phosphatidylglycerol; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; m-DAP, meso-diaminopimelic acid.

Characteristic 1 2 3 4 5

Sporulation* Non-spore-forming Non-spore-forming Non-spore-forming Spore-forming Spore-forming Cell shape Rods Cocci Cocci/short rods/rods Rods Rods Gram-staining ++ V + V Motility Non-motile Motile Motile Motile Motile/non-motile Oxidase + 22V +/2 Urease + 22D 2 Gelatinase 2 ++D +/2 Major isoprenoid quinones MK-6, MK-8 MK-7, MK-8 MK-6, MK-7, MK-8 MK-7 MK-7

Major cellular fatty acids iso-C14 : 0, iso-C14 : 0, anteiso-C15 : 0, iso-C14 : 0, iso-C15 : 0, iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0,C16 : 1v7c anteiso-C15 : 0, iso-C16 : 0, anteiso-C15 : 0 iso-C16 : 0, iso-C15 : 0, iso-C16 : 0 alcohol C16 : 1v7c alcohol iso-C17 : 0 Lipids PG, DPG PG, DPG, PE PE, PG, DPG PE, PG NR Peptidoglycan type m-DAP L-Lys–D-Glu L-Lys–D-Glu m-DAP NR DNA G+C content (mol%) 59.5 39–52 35247 32–69 49–58

*Sporulation medium (M1018; HiMedia) and sporulation medium in combination with TSA were used for induction of sporulation.

C14 : 0, iso-C15 : 0, iso-C16 : 0 and anteiso-C15 : 0. MK-6 and Voges–Proskauer reactions are negative. Tween 60 is MK-8 are the predominant respiratory quinones. The cell- hydrolysed but Tween 80, starch, aesculin and gelatin are wall peptidoglycan contains diaminopimelic acid, lysine not hydrolysed. Nitrate is reduced to nitrite and H2S gas is and glutamic acid in the ratio of 1 : 2 : 1. The type strain of produced. Cells do not produce either acid or gas from T the type species, DSE10 , contains the signature nucleo- adonitol, D- arabinose, dulcitol, erythritol, D-fructose, D- tides G, A, C, T, C, A, G, C and T at positions 182, 444, galactose, D-glucose, inositol, lactose, maltose, mannitol, D- 480, 492, 563, 931, 1253, 1300 and 1391, respectively, in the mannose, raffinose, L-rhamnose, salicin, sucrose, D-sorbi- 16S rRNA gene sequence. The G+C content of the tol, trehalose or xylose even after incubation at an genomic DNA is 59.5±2.5 mol%. The genus is affiliated to optimum temperature for 1 week. Assimilates adonitol, the family of the order of the class D-fructose, melezitose and maltose, but not N-acetylglu- Firmicutes. The type species is Bhargavaea cecembensis. cosamine, D-arabinose, L-arabinose, arbutin, cellobiose, citric acid, dulcitol, galactose, D-glucose, glycerol, glyoxylic Description of Bhargavaea cecembensis sp. nov. acid, inositol, inulin, lactose, D-mannose, mannitol, melibiose, methyl a-D-glucoside, polyethylene glycol, [ Bhargavaea cecembensis ce.cem.ben9sis. N.L. fem. adj. acetate, raffinose, L-rhamnose, ribose, salicin, gluconate, cecembensis pertaining to CCMB, arbitrary adjective sorbitol, L-sorbose, sucrose, trehalose, xylitol or D-xylose. formed from the acronym of the Centre for Cellular and Molecular Biology (CCMB), where the taxonomic studies on this novel species were performed]. Has the following characteristics in addition to those given for the genus above. Cells are non-motile and rod-shaped (2.0 mm in length66.0 mm in width) and occur singly, in pairs or in groups (Fig. 2). Colonies on TSA medium are convex, 1–2 mm in diameter, smooth, regular and opaque. Cells grow from 15 to 55 uC with an optimum temperature of 37 uC and tolerate up to 6.0 % (w/v) NaCl. Growth occurs over a pH range of 7–7.5. The doubling time is 5 h. Tests for catalase, oxidase and urease activities are positive, but tests for phosphatase, b-galactosidase, arginine dihy- drolase, ornithine decarboxylase, lysine decarboxylase and Fig. 2. TEM images of negatively stained cells (a) and thin tryptophan deaminase are negative. Tyrosine is not sectioned cells (b) of strain DSE10T. Bars, 0.5 mm (a) and 200 nm degraded, indole is not produced, The methyl red and (b). http://ijs.sgmjournals.org 2621 R. Manorama and others

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