DOI: 10.15468/hdeben
title: Bacillus maritimus sp. nov., a novel member of the genus Bacillus isolated from marine sediment

description: This dataset contains the digitized treatments in Plazi based on the original journal article Pal, Deepika, Mathan Kumar, Rajendran, Kaur, Navjot, Kumar, Narender, Kaur, Gurwinder, Singh, Nitin Kumar, Krishnamurthi, Srinivasan, Mayilraj, Shanmugam (2017): Bacillus maritimus sp. nov., a novel member of the genus Bacillus isolated from marine sediment. International Journal of Systematic and Evolutionary Microbiology 67 (1): 60-66, DOI: 10.1099/ijsem.0.001569, URL: http://dx.doi.org/10.1099/ijsem.0.001569AbstractThe taxonomic position of a Gram-stain-positive, endospore-forming bacterium, strain KS16-9 T, isolated from a marine sediment sample collected from Kovalam, Kanyakumari coastal region of the Indian Ocean, India, was analysed by a polyphasic approach. Strain KS16-9 T had typical phenotypic characteristics and chemotaxonomic features (menaquinones, fatty acids and lipids) that were consistent with the genus Bacillus. omparative analysis of the 16S rRNA gene sequence of the strain with previously published Bacillus type strains confirmed that it belongs to the genus Bacillus and is moderately related to Bacillus persicus B48 T (98.42 % similarity), followed by Bacillus foraminis CV 53 T (97.67 %) and Bacillus rigiliprofundi (97.61 %). Other species in the genus Bacillus shared &lt;97.6 % 16S rRNA gene sequence similarity. Strain KS16-9 T contained MK-7 as the predominant menaquinone, meso -diaminopimelic acid as the diagnostic cell-wall diamino acid, iso-C 15: 0 and anteiso-C 15: 0 as major fatty acids, and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the major phospholipids. The DNA G+C content of strain KS16-9 T was 45.4 mol%. Based on data from this polyphasic taxonomic study, strain KS16-9 T represents a novel species of the genus Bacillus, for which the name Bacillus maritimus sp. nov. is proposed. The type strain is KS16-9 T (= MTCC 12305 T = DSM 100413 T = KCTC 33834 T).Members of the genus Bacillus have been isolated from a wide variety of aquatic and terrestrial environments, including the marine environment [1]. At the time of writing, the genus Bacillus comprises more than 189 recognized species and seven subspecies (http://www.bacterio.net/bacillus. html). Ash et al. [2] were one of the first to envisage the genus as highly heterogenic and placed the species into five different groups with all of them related to each other at least at the level of different genera. In the next few years new genera were erected from these five groups and several Bacillus species have been reclassified into novel genera, such as Paenibacillus (rRNA group 3, [3]), Aneurinibacillus and Brevibacillus [4], Virgibacillus [5]), Gracilibacillus and Salibacillus [6], Geobacillus [7], Sporosarcina [8], Viridibacillus [9], Lysinibacillus [10], Salimicrobium [11], Rummeliibacillus [12], Solibacillus [13], Falsibacillus [14], Psychrobacillus [15], Anaerobacillus [16], Caldibacillus [17], Alteribacillus [18] and Hydrogenibacillus [19]. Species such as Bacillus marinus ,, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus firmus and Bacillus lentus have been isolated from marine habitats [20 – 22]. There have, however, been only a few reports of isolation of Bacillus species from marine environments of India, Bacillus okhensis from a salt pan in Port Okha, Jamnagar, Gujarat [23], Bacillus encimensis from a sediment sample off the Veraval coast, Gujarat [24], and Bacillus campisalis from a solar saltern in Kanyakumari district, coastal region of the Bay of Bengal [25]. Here, we report a detailed polyphasic taxonomic characterization of strain KS16-9 T, which was isolated from a marine sediment sample collected during September 2011 from Kovalam, Kanyakumari coastal region of the Indian Ocean, India (8 Ǫ 05′ 04.13′′ N 77 Ǫ 31′ 15.40′′ E). Isolation used the serial dilution plating method on Zobell marine agar (ZMA; HiMedia; per litre distilled water: peptic digest of animal tissue 5 g; yeast extract 1 g; ferric citrate 0.10 g; NaCl 19.45 g; MgCl 2 8.80 g; Na 2 SO 43.24 g; CaCl 21.80 g; KCl 0.55 g; NaHCO 3 0.16 g; KBr 0.08 g; SrCl 20.034 g; boric acid 0.022 g; Na 2 SiO 20.004 g; NaF 0.0024 g; NH 4 NO 30.0016 g; Na 2 HPO 40.008 g; agar 15 g) as well as on tryptic soy agar (TSA; HiMedia; per litre distilled water: pancreatic digest of casein 15 g; papaic digest of soyabean meal 5 g; NaCl 5 g; agar 15 g) and incubated at 25 and 30 Ǫ C. Sub-culturing and maintenance of the strain were on ZMA and TSA at 4 Ǫ C and as glycerol suspensions (15 %, v/v) at ―80 Ǫ C. Polyphasic taxonomic characterization of strain KS16-9 T was performed according to the minimal standards for describing new taxa of aerobic, endospore-forming bacteria [26]. Reference strains Bacillus persicus CECT 8001 T, Bacillus foraminis MTCC 8568 T (=CIP 108889 T) and Bacillus rigiliprofundi LMG 28275 T were obtained from Spanish Type Culture Collection (CECT), Spain, Microbial Type Culture Collection and Gene Bank (MTCC), India, and Belgian Coordinated Collections of Micro-organisms (BCCM/ LMG), Belgium, respectively.Author affiliations: 1 MTCC – Microbial Type Culture Collection & Gene Bank, CSIR – Institute of Microbial Technology, Chandigarh 160 036, India; 2 Division of Protein Science and Engineering, CSIR – Institute of Microbial Technology, Chandigarh 160 036, India.*Correspondence: Srinivasan Krishnamurthi, kmurthi@imtech.res.in; Shanmugam Mayilraj, mayil@imtech.res.inKeywords: Bacillus maritimus; 16 S rRNA; fatty acid methyl esters.Abbreviation: MALDI-TOF, matrix-assisted laser desorption time-of-flight.†These authors contributed equally to this work.The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain KS16-9 T is KP317497.Three supplementary figures are available with the online Supplementary Material.Phenotypic characterization of strain KS16-9 T along with the reference strains was performed according to standard protocols [27]. Morphology, size and pigmentation were observed on TSA and ZMA plates after 24 h of incubation. Oxidase and catalase activities, nitrate reduction, hydrolysis of gelatin, starch and casein, production of H 2 S and indole, and methyl red and Voges–Proskauer tests were performed as mentioned by Smibert and Krieg [28]. Growth tolerance of NaCl was determined using basal medium of TSA (without NaCl) supplemented with 2.0, 5.0, 7.0 and 10.0 % (w/v) NaCl. Growth was tested at pH 5.0, 6.5, 7.0, 8.0, 9.0 and 10.0 (pH was set using biological buffers, [29]) and at different 12, 15, 20, 25, 30, 37 and 42 Ǫ C. Utilization of different compounds as sole carbon, nitrogen and energy sources was determined by using VITEK 2 GP cards as well as by manual methods using minimal medium supplemented with 1.0 % of the respective carbon (arabinose, D- mannitol, glucose, salicin, fructose, mannose, myo -inositol) and nitrogen sources (valine, phenylalanine, serine, cysteine, threonine, hydroxyproline, arginine and metheonine). Acid production from carbohydrates was studied according to the method of [30]. Resistance to antibiotics was examined based on the zone of inhibition around the antibiotic disc placed on TSA agar medium according to CLSI standards. Other tests performed in this study are described in Table 1.Genomic DNA from strain KS16-9 T was extracted using the Bacterial/Fungal DNA MiniPrep kit (Zymo Research), according to the manufacturer’ s instructions. To determine the phylogenetic status of the strain, the 16S rRNA gene was amplified by PCR using universal primers 27F and 1492R and methods as described by Mayilraj et al. [31]. The 16S rRNA gene sequences of closely related species were retrieved from the EzTaxon server [32] for sequence alignment and phylogenetic analysis completed using MEGA version 6.0 [33]. Phylogenetic trees were reconstructed using the neighbour-joining, maximum-likelihood and maximum-parsimony algorithms. Bootstrap analysis was performed to assess the confidence limits of the branching.For matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) analysis, protein extraction, sample preparation and mass spectral analysis were performed using a Microflex III instrument (Bruker Daltonik) as described by [34]. To study chemotaxonomic features, strain KS16-9 T and the reference strains were grown in triptic soy broth (TSB) for 24 h (late log phase) at 30 Ǫ C and freeze dried cell mass was prepared for the analysis of quinones, lipids and cell-wall diagnostic amino acids. Preparation of the cell wall and determination of the diagnostic diamino acid were performed using the methods described by Staneck and Roberts [35]. Total hydrolysates (4.0 M HCl, 100 Ǫ C, 16 h) of the peptidoglycan for amino acid and peptide analyses were separated by two-dimensional ascending TLC as described by [36]. Isoprenoid quinones were extracted and purified according to Collins et al. [37] and menaquinones were purified and determined by HPLC [38]. Polar lipids were extracted and examined by two-dimensional TLC according to the methods of Minnikin et al. [39]. Total lipids were detected and identified by spraying with 10 % (w/v) molybdatophosphoric acid in 95 % ethanol. The fatty acid profile was determined by growing cells on TSA at 30 Ǫ C for 24 h. Fatty acid extraction and identification were done using the standard Microbial Identification System (MIDI; [40, 41]). The DNA G+C content was determined using the thermal denaturation method [42].Cells of strain KS16-9 T were Gram-stain-positive, rodshaped, endospore-forming (central spores with bulging sporangia) and aerobic. After incubation on ZMA and TSA at 30 Ǫ C for 48 h, colonies were circular (0.5–0.8 mm), flat, smooth and pale yellow. NaCl was tolerated at up to 7 % (w/ v) with optimum growth at 5 % NaCl. Growth occurred at 12–42 Ǫ C (optimum 30 Ǫ C) and at pH 7.0–10.0 (optimum 8.0). The strain was negative for hydrolysis of casein, starch and gelatin. Nitrate was reduced to nitrite and H 2 S was not produced. Acid was produced from fructose, lactose and melibiose but not from adonitol, dulcitol, dextrose, galactose, inositol, inulin, manitol, mannose, maltose, rhamnose, raffinose, sucrose, salicin, sorbitol, trehalose or xylose. Other physiological properties that separated strain KS16-9 T from its closest phylogenetic neighbours are shown in Table 1 and the detailed characteristic features of strain KS16-9 T are given in the species description.The only menaquinone was determined to be MK-7. Major fatty acids (&gt;10 % of the total) were iso-C 15: 0 (18.3 %) and anteiso-C 15: 0 (26.0 %), and moderate amounts of (&gt;5 % but &lt;10 % of the total) iso-C 14: 0 (6.5 %) and summed feature 4 (iso-C 17: 1 I and/or anteiso-C 17: 1 B; 6.2 %) were also found. Significant distinctions were thus observed in the fatty acid composition between strain KS16-9 T and the reference strains B. persicus CECT 8001 T, B. foraminis MTCC 8568 T and B. rigiliprofundi LMG 28275 T. The detailed comparative fatty acid profiles are presented in Table 2. meso -Diaminopimelic acid was the diagnostic cell-wall diamino acid (Fig. S1, available in the online Supplementary Material), and the peptidoglycan type was A1 Ƴ (meso -diaminopimelic acid– D-Glu 2). The polar lipid profile of strain KS16-9 T was found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and five unknown phospholipids. Comparative analysis between strain KS16-9 T and the reference strains clearly revealed that the lipid pattern differed only in the presence or absence of unknown phospholipids (Fig. S2) The DNA G+C content of strain KS16-9 T was 45.4 mol%.Table 1. Differential characteristics between strain KS16-9T and closely related members of the genus Bacillus Strains: 1, KS16-9T; 2, B. persicus CECT 8001T; 3, B. foraminis MTCC 8568T; 4, B. subtilis subsp. subtilis MTCC 121T; 5, B. rigiliprofundi LMG 28275T. All data are from the present study except where indicated. All the strains are negative for casein and gelatin hydrolysis, and indole production; positive for catalase and oxidase. Acid is not produced from arabinose, adonitol, dulcitol, mannitol, mannose or xylose. In tests using VITEK 2-GP cards, the strains are negative for D- amygdalin, phosphatidylinositol phospholipase C, D- xylose, Ala–Phe–Pro arylamidase, cyclodextrin, Oi -mannosidase, phosphatase, leucine arylamidase, L- pyrrolidonyl-arylamidase, β -glucuronidase, β -galactosidase, alanine arylamidase, tyrosine arylamidase, D- sorbitol, polymixin B resistance, D- galactose, D- ribose, lactose, N -acetyl-D- glucosamine, bacitracin resistance, novobiocin resistance, D- mannitol, D- mannose, methyl β -D- glucopyranoside, pullulan and o/129 resistance (comp.vibrio.). Positive for utilization of nitrogen sources such as threonine and serine and carbon sources such as mannose and arabinose; negative for myo -inositol and salicin.Characteristic 1 2 3 4 5 Endospore formation + + ― + + Growth temperature (Ǫ C): Range 12–42 10–45 15–45 10–50 7–45 Optimum 30 37 37 37 30 pH for growth: Range 7–10 6.0–10.0 6.5–10 6.0–10.0 6.0–8.5 Optimum 8.0 7.5–8.0 7.0–8.5 7.5–8.0 7.5 Tolerance of NaCl (%, w/v) 0–7.0 0.5–10.0 0–5 0–10 0–12 Starch hydrolysis ― + ― + ― Citrate utilization ― + ― + ― Methyl red + + ― + ― H2S production ― ― + ― ― Nitrate reduction + + ― + ― Acid production from: Dextrose ― ― + ― ― Fructose + ― + + + Galactose ― ― + ― ― Inositol ― ― + + ― Inulin ― + + + ― Lactose + ― + ― ― Melibiose + ― + + ― Maltose ― ― + + ― Rhamnose ― ― + ― ― Raffinose + ― + + ― Sucrose ― ― + + ― Salicin ― ― + + ― Trehalose ― ― + + ― Enzyme activity using VITEK 2-GP cards: β -Glucosidase ― ― + ― ― Oi -Glucosidase + ― + ― ― Arginine dihydrolase 1 + + + + ― Arginine dihydrolase 2 + + + + ― Urease + + + + ― L- Aspartate arylamidase ― ― ― ― + β -Galactopyranosidase ― ― + ― ― L- Proline arylamidase + ― + ― ― Oi -Galactosidase + ― + ― ― L- Lactate alkalinization + + ― + ― Maltose + ― ― ― ― Raffinose + ― ― ― ― Salicin ― ― + + ― Sucrose + ― + + ― Trehalose + ― + + ― Optochine resistance ― ― + + ―Table 1. cont.Characteristic 1 2 3 4 5 Utilization of carbon and nitrogen sources: D- Mannitol ― + ― + ― Glucose + + ― + ― Fructose + ― ― ― + Valine ― ― ― + ― Phenylalanine + ― ― ― ― Cysteine + ― ― + ― Hydroxy proline + + ― + ― Arginine + + ― + ― Metheonine ― + ― ― ― DNA G+C content (mol%) 45.4 40.1* 43.1† 43.0‡ 40.5§*Data from Didari et al. [46]. †Data from Tiago et al. [47]. ‡Data from McCarthy and Bolton [48]. §Data from Sylvan et al. [49].Table 2. Fatty acid profiles of strain KS16-9 T and closely related members of the genus BacillusStrains: 1, KS16-9 T; 2, B. persicus CECT 8001 T; 3, B. foraminis MTCC 8568 T; 4, B. subtilis subsp. subtilis MTCC 121 T; 5, B. rigiliprofundi LMG 28275 T. All data are from the present study. Values are percentrages of the total fatty acids. Fatty acids amounting to &lt;0.5 % of the total fatty acids are shown as TR (trace amount).Phylogenetic analysis confirmed that strain KS16-9 T belongs to the genus Bacillus. The analysis revealed that strain KS16-9 T showed highest 16S rRNA gene sequence similarity to the type strains of B. persicus (98.42 %), followed by B. foraminis (97.67 %) and B. rigiliprofundi (97.61 %). 16S rRNA gene sequence analysis indicated that strain KS16-9 T differs from all other species of Bacillus by at least 1.58 %. Phylogenetic trees were reconstructed by the neighbour-joining as well as maximum-parsimony and maximum-likelihood algorithms. In the neighbour-joining phylogenetic tree, strain KS16-9 T formed a separate branch along with B. persicus, B. foraminis and B. rigiliprofundi (Fig. 1). DNA–DNA relatedness values between strain KS16-9 T and other closely related strains were not determined as species showing 1.4 % dissimilarity in 16S rRNA gene sequence analysis from related species of the genus can be excluded from DNA–DNA hybridization studies [43–454445].Fatty acid 1 2 3 4 5 C14:0 0.5 TR 3.9 TR 3.1 C16:0 1.7 2.0 15.2 3.6 12.5 C17:0 1.0 0.8 2.9 TR 1.4 C18:0 0.5 0.6 5.2 TR 2.5 iso-C14: 0 6.5 15.1 7.1 0.9 9.0 iso-C15: 0 18.3 31.2 9.9 24.1 20.2 iso-C16: 0 4.6 8.5 2.6 2.4 1.9 iso-C17: 0 3.2 2.6 1.2 11.5 0.9 iso-C18: 0 1.1 TR TR TR TR anteiso-C15: 0 26.0 14.5 18.6 38.4 19.1 anteiso-C17: 0 6.4 3.1 1.9 12.5 0.89 C16:1 Ɯ 11 c 3.3 2.2 14.4 1.7 20.1 C16:1 Ɯ 7 c alcohol 6.6 8.1 TR TR 1.4 C16:1 Ɯ 7 c TR TR 1.9 TR TR C17:1 Ɯ 9 c 2.5 1.0 2.4 1.3 TR C17:1 Ɯ 10 c 3.0 3.1 TR 2.4 0.6 C18:1 Ɯ 9 c 0.6 TR 5.1 0.7 2.3 iso I-C17: 1 6.2 2.5 TR 0.9 TR Summed feature 3* TR TR 1.9 TR 1.0 Summed feature 4* 6.2 2.5 TR 1.2 TR Summed feature 8* TR TR 0.6 TR TRMALDI-TOF measurements were made for strain KS16-9 T and the most closely related species B. persicus (CECT 8001 T) using a Microflex III instrument (Bruker Daltonik) for the automatic acquisition of mass spectra in the linear positive mode within a range of 2–20 kDa, according to the manufacturer’ s instructions. The peak from the generated mass spectra and their matching against the reference spectra of the integrated database was performed using MALDI Biotyper Software (Bruker Daltonics). The MALDI-TOF MS profile of strain KS16-9 T showed unique peaks represented in the red colour band at positions m / z 6634.845 and 7719.968 which were very much evident from the gel view (Fig. S3).*Summed features represent groups of two or three fatty acids that could not be separated by GC with the MIDI system. Summed feature 3 contained C16: 1 Ɯ 7 c /C16: 1 Ɯ 7 c; summed feature 4 contained iso-C17: 1 I and/or anteiso-C17: 1B; summed feature 8 contained C18: 1 Ɯ6c /C18: 1 Ɯ 7 c.Chemotaxonomic features such as predominant menaquinone, polar lipid profile and fatty acid compositions together with physiological, biochemical features and phylogenetic analysis strongly support the suggestion that strain KS16-9 T should be considered as representing a novel species of the genus Bacillus. The differential biochemical characteristic features between strains KS16-9 T, B. persicus CECT 8001 T, B. foraminis MTCC 8568 T and B. rigiliprofundi LMG 28275 T along with the type species of the genus Bacillus are depicted in Table 1. Therefore, on the basis of data from the present polyphasic taxonomic study, it is evident that strain KS16-9 T represents a novel species within the genus Bacillus, for which the name Bacillus maritimus sp. nov. is proposed.