Amphibacillusmarinus Sp. Nov., a New Member of the Genus Amphibacillus
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IJSEM Papers in Press. Published August 3, 2012 as doi:10.1099/ijs.0.045807-0 1 Amphibacillus marinus sp. nov., a new member of the genus 2 Amphibacillus isolated from the South China Sea 3 Biao Ren1,3†, Na Yang1,3†, Jian Wang1, Xiao-Long Ma1, Qian Wang1,3, Feng Xie1,3, 4 Hui Guo1,3, Zhi-Heng Liu1, Benoît Pugin4, Li-Xin Zhang1,2* 5 6 (1) Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, 7 Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People's Republic 8 of China. 9 (2) South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, 10 P. R. China 11 (3) Graduate School of Chinese Academy of Sciences, Beijing, 100049, P. R. China 12 (4) Laboratorio de Microbiología Molecular, Departamento de Biología, Facultad de Química y 13 Biología, Universidad de Santiago de Chile, Santiago, Chile 14 15 * Author for Correspondence: Li-Xin Zhang；[email protected] 16 17 Running title: Amphibacillus marinus sp. nov 18 Category: New taxa-Firmicutes and Related Organisms 19 † These authors contributed equally to this work. 20 The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of 21 strain J1T is GU213062. 22 23 A Gram-positive, spore-forming, rod-shaped bacterium, designated J1T was 24 isolated from deep sea mud collected from the South China Sea, and subjected to 25 polyphasic taxonomic investigation. Phylogenetic analysis based on 16S rRNA 26 gene sequences revealed that J1T clustered with the type strains of Amphibacillus 27 cookii, Amphibacillus sediminis and Amphibacillus jilinensis，and exhibited the 28 range of similarity of 93.9%-97.0% to the species in genus Amphibacillus. The 29 DNA G+C content was 36.7%. Chemotaxonomic analysis showed no quinones, 30 and the cell wall contained meso-diaminopimelic acid as the diagnostic diamino T 31 acid for strain J1 . The major cellular fatty acids were iso-C15:0 and anteiso-C15:0. 32 The strain J1T was positive for catalase activity and negative for oxidase activity. 33 On the basis of phylogenetic position and phenotypic properties, strain J1T 34 represents a new species of the genus Amphibacillus and the name Amphibacillus 35 marinus sp. nov. is proposed. The type strain is J1T (=CGMCC 1.10434T = JCM 36 17099T). 37 38 Exploration of the microbial diversity in marine environment showed an intriguing 39 picture for that many bioactive compounds isolated from marine-derived 40 microorganisms (Demain & Zhang, 2005). The main interest of our group is to 41 construct a high quality marine microbial natural products library to screen bioactive 42 metabolites by high throughput techniques (Bian et al., 2008). Based on the previous 43 microbial diversity research on the South China Sea sediments, many new species 44 have been isolated in our lab, such as Amycolatopsis marina (Bian et al., 2009), 45 Verrucosispora sediminis (Dai et al., 2010) and Prauserella marina (Wang et al., 46 2010) among which the Prauserella marina had the anti-BCG (Bacille 47 Calmette-Guérin) activity while Verrucosispora sediminis had the antifungal and 48 antibacterial activities. Recently, another new bio-surfactant-producing strain 49 belonging to the genus Amphibacillus was isolated by using an alkaline medium. 50 The genus Amphibacillus was established by Niimura et al. (1990) and to date, only 51 seven species have been described, namely, Amphibacillus xylanus (Niimura et al., 52 1990), Amphibacillus fermentum (Zhilina et al., 2001), Amphibacillus tropicus 53 (Zhilina et al., 2001), Amphibacillus sediminis (An et al., 2007), Amphibacillus 54 jilinensis (Wu et al., 2010), Amphibacillus cookii (Pugin et al., 2011) and 55 Amphibacillus indicireducens (Hirota et al., 2012). None of these species were 56 isolated from the marine environment as strain J1T was in this genus. The genus 57 Amphibacillus was characterized by spore-forming, rod-shaped, straight or slightly 58 curved and motile, cells which grew at pH 7.0 and 12.0, containing 59 meso-diaminopimelic acid in the cell wall, with anteiso- and iso- branched and 60 straight-chain acids as the major cellular fatty acids, lacking isoprenoid quinones and 61 oxidase activity, variable catalase activity and with the G+C mol% between 36-42% 62 (An et al., 2007; Hirota et al., 2012; Niimura et al., 1990). 63 Marine derived strain J1T was originally isolated from a deep sea mud [GPS 64 coordinates for the sample site are 114º34’58.314” E, 17º53’59.545” N, at a depth of 65 3601 meters], after 4 weeks incubation in Horikoshi medium (Horikoshi, 1999) 66 (glucose 10.0 g, yeast extract 5.0 g, polypeptone 5.0 g, KH2PO4 1.0 g, MgSO4 0.2 g, 67 Na2CO3 10.0 g, NaCl 50.0 g, distilled water 1000 mL, natural pH value) at 28 °C. The 68 isolate was maintained on Horikoshi or DSMZ medium 529 slants at 4 °C and as 69 suspensions of clones in glycerol (25%, v/v) at -70 °C. Biomass for chemical and 70 molecular studies was obtained by cultivation in shaking flasks (200 r.p.m) with 71 DSMZ medium 529 broth at 28 °C for 3 days. 72 Genomic DNA extraction, PCR amplification and 16S rRNA gene sequencing of 73 isolate J1T were carried out according to the procedures described by Kim et al. 74 (1998). The initial taxonomic classification of the 16S rRNA sequence was carried out 75 by using the IDENTIFY program of the online sever of the EzTaxon 76 (http;//147.47.212.35:8080/) (Chun et al., 2007). Sequences longer than 1300nt or 77 without ambiguous nucleotides of the most closely related strains were downloaded 78 from the DDBJ/EMBL/GenBank. Multiple alignments with all the cited 16S rRNA 79 sequences and calculations of levels of sequence similarity were carried out using 80 CLUSTAL W (Thompson et al., 1994). The phylogenetic tree was constructed using 81 three methods, including the neighbor-joining (NJ) tree (Saitou & Nei, 1987) using 82 the software package Mega version 4.0 (Tamura et al., 2007); the 83 maximum-parsimony (MP) tree using the software package PHYLIP version 3.6 84 (Felsenstein, 2002); the maximum-likelihood (ML) tree using the online version of 85 PhyML (Guindon et al., 2010). The topology of the phylogenetic trees was evaluated 86 by bootstrap re-sampling method with 1000 replicates (Felsenstein, 1985). The 87 phylogenetic tree shown in Fig. 1 indicated that strain J1T belonged to the genus 88 Amphibacillus. Genomic DNA for the determination of the G+C content was prepared 89 according to the method of Marmur (1961) and was determined by the thermal 90 denaturation (Tm) method (Mandel et al., 1968) with Escherichia coli K-12 (CGMCC 91 1.748) as the reference strain using the PerkinElmer’s LAMBDA 35 UV/Vis 92 spectrophotometer fitted with a thermal controller. 93 The highest degree of 16S rRNA gene sequence similarity of strain J1T (1490 nt) was 94 found with A. cookii (97.0%), A. sediminis (96.9%), followed by A. jilinensis (96.7%). 95 Phylogenetic analysis based on 16S rRNA sequence analysis revealed that strain J1T 96 formed a cluster with the three most closely related species A. cookii, A. jilinensis and 97 A. sediminis (Fig. 1). MP and ML trees were similar to the NJ tree. All the trees 98 supported that isolate J1T belonged to the genus Amphibacillus. The DNA G+C 99 content of strain J1T was 36.7%. 100 Chemosystematic studies were carried out to compare J1T chemical profile and that of 101 A. jilinensis Y1T (=CGMCC 1.5123T), A. sediminis Shu-P-Ggiii25-2T (=JCM 23213T), 102 A. xylanus Ep01T (=DSM 6626T), A. fermentum Z-7984T (=DSM 13869T), A. tropicus 103 Z-7792T (=DSM 13870T), A. cookii JW/BP-GSL-QDT(=DSM 23721T) which were 104 supplied by Xufen Zhu from Zhejiang University, the Japan Collection of 105 Microorganisms (JCM), and the German Resource Centre for Biological Material 106 (DSMZ). Fatty acids were extracted, purified, methylated and quantified by gas 107 chromatography (Sasser, 1990) using the standard Microbial Identification System 108 (MIDI Inc; Microbial ID) after cultivation in TSB (tryptic soy broth, BD BactoTM, pH 109 9.0) for 2 days at 28 °C, and were identified by TSBA6 database /peak naming table. 110 Polar lipids were extracted and examined by two-dimensional TLC (Solvent system I: 111 Chloroform: methanol: water=65: 25: 4 (v/v); Solvent system II: Chloroform: acetic 112 acid: methanol: water=80:18:12:5 (v/v); stained with Molybdenum blue reagent, 113 anisaldehyde reagent, ninhydrin reagent, and draggendorff reagent) and identified by 114 using published procedures of Minnikin et al. (1980). Halolactibacillus alkaliphilus 115 CGMCC 1.6843T (Cao et al., 2008), H. miurensis DSM 17074T and H. halophilus 116 JCM 21694T (Ishikawa et al., 2005) were also used as reference strains in polar lipids 117 analysis. The analysis of cell-wall peptidoglycan was modified from the methods of 118 Schleifer & Kandler (1972) and Hasegawa et al. (1983). Generally, about two loops 119 of strains were put in an ampoule tube, and then 0.2 mL 6 M HCl was added. Sealed 120 the tube by alcohol torch and incubated at 120 oC for about 4 h until the color of the 121 hydrolysate turn to dark brown. After cooling, 5 μL hydrolysate was directly spotted 122 on a thin cellulose plate (microcrystalline powder, Merck). 1μL standard solution 123 contained DD-, meso- and LL-diaminopimelic acid were spotted on the same plate. 124 Spread the plate twice with the solvent solution (methanol : pyridine : acetic acid : 125 water = 10 : 1 : 0.25 : 5, v/v) after air dried the TLC plates, 0.4% ninhydrin solution 126 was sprayed on and heated at 110 oC for 2-3 min.