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Bacillus Alkalilacus Sp. Nov., Isolated from a Sediment Sample From

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Bacillus Alkalilacus Sp. Nov., Isolated from a Sediment Sample From Author Version of : International Journal of Systematic and Evolutionary Microbiology, vol.68(5);2018;1665-1671 Formatted: Font: 10.5 pt, Not Bold, Italic, Complex Script 14-03-2018 Font: 10.5 pt Bacillus alkalilacus sp. nov., isolated from a sediment sample from Sambhar lake, Formatted: Justified, Indent: Left: 0", First line: 0" Jaipur, Rajasthan, India Formatted: Font: 10.5 pt, Complex Script Font: 10.5 pt Formatted: Font: 10.5 pt, Not Bold, Italic, Complex Script Font: 10.5 pt Harjodh Singh1, 2, 3, Manpreet Kaur1, 2, 3, Shivani Sharma2, Lakhwinder Kaur2 Sunita Mishra3 Formatted: Font: 10.5 pt, Not Bold, Italic, Complex Script Font: 10.5 pt Naga Radha Srinivas Tanuku4, Anil Kumar Pinnaka.*1, 2 Formatted: Left: 0.75", Right: 0.75", Width: 8.27", Height: 11.69" Formatted: Font: 10.5 pt, Complex Script Font: 10.5 pt, Not Bold, Italic 1 Academy of Scientific and Innovative Research, (AcSIR), CSIR Campus, Chennai, India Formatted: Font: 13 pt, Not Italic, Complex Script Font: 13 pt 2MTCC-Microbial Type Culture Collection & Gene Bank, CSIR-Institute of Microbial Technology, Chandigarh-160036, India, Formatted: Font: 13 pt, Complex Script Font: 13 pt *[email protected] 3Council of Scientific and Industrial Research (CSIR) Central Scientific Instruments Organisation, Formatted: Block Text, Right: 0", Space After: 6 pt Chandigarh-160030, India Formatted: Font: Bold 4 CSIR-National Institute of Oceanography, Regional Centre, 176, Lawsons Bay Colony, Formatted: Centered, Indent: Left: 0", Space After: 6 pt, Visakhapatnam-530017, India Line spacing: single The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AK73T is Formatted: Font: Bold LT844663. Formatted: Centered, Indent: Left: 0" Address for correspondence *Dr. P. Anil Kumar Formatted: Font: 10 pt, Complex Script Font: 10 pt MTCC-Microbial Type Culture Collection & Gene Bank, CSIR- Formatted: Centered, Indent: Left: 0", Right: -0.03", Line spacing: single Institute of Microbial Technology, Chandigarh-160036, India Formatted: Font: 10 pt, Complex Script Font: 10 pt E-mail: [email protected] Formatted: Font: 9 pt Telephone: +91-172-6665728 Formatted: Font: 10 pt Running title: Bacillus alkalilacus sp. nov. Formatted: Font: 9 pt, Complex Script Font: 10 pt Formatted: Font: 10 pt, Complex Script Font: 10 pt Formatted: Font: 10 pt, Complex Script Font: 10 pt Subject category New taxa (Firmicutes) Formatted: Font: 10 pt, Complex Script Font: 10 pt Formatted: Font: 5 pt Abstract Formatted: Font: 11 pt, Complex Script Font: 11 pt The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain AK73T is Formatted: Font: 11 pt, Complex Script Font: 11 pt LT844663. Formatted: Font: 6 pt, Complex Script Font: 11 pt An aerobic, endospore forming, haloalkali tolerant, Gram-positive-staining, motile, rod shaped Formatted: Font: 11 pt, Complex Script Font: 11 pt bacterium, designated strain AK73T, was isolated from a sediment sample collected from Sambhar Formatted: Indent: Left: 0", First line: 0" lake, Jaipur, Rajasthan, India. Colonies were circular, 1-2 mm in diameter, glossy, smooth, yellowish Formatted: Font: Bold and convex with entire margin after 48 h growth on Marine Agar at pH 9 and 37oC. Growth occurred Formatted: Space After: 6 pt, Line spacing: single at 15-42 oC, 0-10% (w/v) NaCl and pH of 7-12. Strain AK73T was positive for catalase and arginine dihydrolase 2 activities and hydrolysis of Tween 20/40/80 and negative for esculinase, caseinase, gelatinase, -galactosidase, lipase (Tween 60) and urease activities. The fatty acids were dominated by branched with iso-, anteiso-, saturated fatty acids with a high abundance of iso-C15:0, anteiso-C15:0, C16:0 and anteiso-C17:0; MK-7 is the major menaquinone; and the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, four unidentified phospholipids and three unidentified lipids. The DNA G+C content of strain AK73T was 54 mol%. Analysis based on comparative 16S rRNA gene sequence indicated that Bacillus alcalophilus was the nearest phylogenetic neighbor, with a pair-wise sequence similarity of 96.0%. Phylogenetic analysis showed that strain AK73T formed a separate lineage but 1 was loosely associated with a peripheral cluster of organisms that contained Bacillus gibsonii, Bacillus murimartini and Bacillus plakortidis with similarity values of 93.6, 93.5 and 93.4 respectively. Based on its phenotypic characteristics and on phylogenetic inference, strain AK73T represents a novel species of the genus Bacillus, for which the name Bacillus alkalilacus sp. nov. is proposed. The type strain is AK73T (= JCM 32184T = MTCC 12637T = KCTC 33880T). Key words: Bacillus alkalilacus; 16S rRNA gene sequence based phylogeny; chemotaxonomy. Formatted: Indent: First line: 0" Formatted: Indent: First line: 0" Introduction Bacillus is a genus of Gram-stain-positive, rod-shaped bacteria and a member of the family Formatted: Space After: 6 pt, Line spacing: 1.5 lines Bacillaceae, phylum Firmicutes [1, 2]. Bacillus species can be obligate aerobes or facultative anaerobes and positive for catalase with DNA G+C content is from 32-66 mol%) [2]. Species of the genus Bacillus are ubiquitous in nature, Bacillus includes both free-living (non-parasitic) and parasitic pathogenic species. During unfavaourable conditions, several species of the genus Bacillus can produce endospores that can reduce themselves to and remain in a dormant state for very longer periods. During the recent past, dramatic taxonomic revision has taken place with respect to the genus and using polyphasic approaches many species were transferred to the new genera or existing genera of the phylum Firmicutes such as Aeribacillus, Alicyclobacillus, Alkalibacillus, Alteribacillus, Anaerobacillus, Aneurinibacillus, Brevibacillus, Bhargavaea, Falsibacillus, Fictibacillus, Geobacillus, Gracilibacillus, Hydrogenibacillus, Jeotgalibacillus, Kyrpidia, Lysinibacillus, Paenibacillus, Psychrobacillus, Pullulanibacillus, Rummeliibacillus, Salibacterium, Salimicrobium, Salipaludibacillus, Sporosarcina, Solibacillus, Sporolactobacillus, Ureibacillus, Viridibacillus and Virgibacillus [3]. Bacillus axarquiensis, Bacillus malacitensis and Bacillus velezensis are later heterotypic synonyms of Bacillus mojavensis, Bacillus mojavensis and Bacillus amyloliquefaciens, respectively [3]. At the time of writing the genus Bacillus comprises 242 valid species and 7 subspecies [3]. Bacillus subtilis is the type species of the genus [4]. In the present study, the characterization and classification of a strain AK73T isolated from sediment sample of Sambhar lake, was performed by polyphasic taxonomic approaches [5]. The organism represents a new species of the genus Bacillus for which the name Bacillus alkalilacus sp. nov. is proposed. 2 Strain AK73T was isolated from the sediment sample collected from haloalkaline Sambhar lake, Jaipur, Rajasthan, India. The GPS coordinates of the sampling point is 26º55.520’N 075o11.827’E. The temperature and pH of the sediment sample was 35 oC and 10, respectively. For isolation of the strain AK73T, the sediment sample was serially diluted (up to 10 fold dilutions) in 2% (w/v) NaCl solution and 100 µl of each dilution was spread-plated on Zobell’s Marine Agar (MA; HIMEDIA) plates, and incubated at 37 °C for 5 days. Out of different colony morphotypes that appeared one pale yellowish-coloured colony was selected and characterized. Sub-cultivation of the isolate was carried out on Zobell’s Marine Agar (MA; HIMEDIA) at 37 °C. A stock culture of the isolate in Nutrient broth (NB; HIMEDIA) with 20% glycerol was preserved at -80 °C. Strain AK73T was characterized simultaneously with reference strains with Bacillus alcalophilus MTCC 7263T, Bacillus gibsonii MTCC 7262T and Bacillus subtilis MTCC 121T. Colony morphology was examined following growth of the strain on MA at 37C for 1 day. Cell morphology was investigated by light microscopy (Nikon) at x 1000 magnification and also by transmission electron microscope (Jeol JEM 2100) at operating voltage of 200 kV. The Gram reaction was determined by using the HIMEDIA Gram Staining Kit according to manufacturer’s protocol. Endospore formation was determined by observing under phase contrast microscope and malachite-green staining of isolate grown on MA for a week [6]. Motility was assessed on Motility- Indole-Lysine HiVegTM medium (HIMEDIA) with agar 2 g l-1 and also under light microscopy using hanging drop method. Growth at 4, 10, 15, 20, 25, 30, 37, 42, 45, 50, 55 and 60 °C was ascertained using Marine Broth (MB; HIMEDIA) and salt tolerance [0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15 and 20% (w/v) NaCl] was ascertained using Nutrient Broth containing peptone (5 g l-1) and beef extract (3 g l-1). Growth of strain AK73T at pH 4, 5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11 and 12 was assessed on MB buffered either with acetate or citrate 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). Anaerobic growth on MA was determined after incubation in an anaerobic Jar (Anoxomat [USA]), after giving an anaerobic cycle using Anoxomat (Mart, USA). Different biochemical tests listed in description of species and as well as in Table 1 were carried out using cultures grown at 37 oC on MA medium as described by Lányί [7] (catalase, oxidase activities, nitrate reduction, indole
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