Bacillus Crassostreae Sp. Nov., Isolated from an Oyster (Crassostrea Hongkongensis)

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1561–1566 DOI 10.1099/ijs.0.000139

Bacillus crassostreae sp. nov., isolated from an oyster (Crassostrea hongkongensis)

Jin-Hua Chen,1,2 Xiang-Rong Tian,2 Ying Ruan,1 Ling-Ling Yang,3 Ze-Qiang He,2 Shu-Kun Tang,3 Wen-Jun Li,3 Huazhong Shi4 and Yi-Guang Chen2

Correspondence 1Pre-National Laboratory for Crop Germplasm Innovation and Resource Utilization, Yi-Guang Chen Hunan Agricultural University, 410128 Changsha, PR China [email protected] 2College of Biology and Environmental Sciences, Jishou University, 416000 Jishou, PR China 3The Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan Institute of Microbiology, Yunnan University, 650091 Kunming, PR China 4Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA

A novel Gram-stain-positive, motile, catalase- and oxidase-positive, endospore-forming, facultatively anaerobic rod, designated strain JSM 100118T, was isolated from an oyster (Crassostrea hongkongensis) collected from the tidal flat of Naozhou Island in the South China Sea. Strain JSM 100118T was able to grow with 0–13 % (w/v) NaCl (optimum 2–5 %), at pH 5.5–10.0 (optimum pH 7.5) and at 5–50 6C (optimum 30–35 6C). The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant respiratory quinone was menaquinone-7 and the major cellular fatty acids were

anteiso-C15 : 0, iso-C15 : 0,C16 : 0 and C16 : 1v11c. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, an unknown glycolipid and an unknown phospholipid. The genomic DNA G+C content was 35.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 100118T belonged to the genus Bacillus, and was most closely related to Bacillus litoralis SW-211T (98.9 % 16S rRNA gene sequence similarity), Bacillus halosaccharovorans E33T (98.3 %), Bacillus niabensis 4T19T (97.8 %) and Bacillus herbersteinensis D-1,5aT (97.1 %). The combination of results from the phylogenetic analysis, DNA–DNA hybridization, and phenotypic and chemotaxonomic characterization supported the conclusion that strain JSM 100118T represents a novel species of the genus Bacillus, for which the name Bacillus crassostreae sp. nov. is proposed. The type strain is JSM 100118T (5CCTCC AB 2010452T5DSM 24486T5JCM 17523T).

The genus Bacillus in the family Bacillaceae, belonging to cellular fatty acids (Priest et al., 1988; Ash et al., 1991; the phylum Firmicutes, contains several phylogenetically Ka¨mpfer, 1994; Ta¨ubel et al., 2003; Ahmed et al., 2007; distinct groups on the basis of 16S rRNA gene sequence Zhang et al., 2010; Kosowski et al., 2014). During an analysis (Ash et al., 1991; Stackebrandt & Liesack, 1993; investigation of the diversity of the microbial population of Nielsen et al., 1994; Ventosa et al., 1998; Schlesner et al., invertebrates inhabiting Naozhou Island (20u 52–569 N 2001; Yoon et al., 2004; Carrasco et al., 2007). Members of 110u 33–389 E) in the South China Sea (Huang et al., the genus Bacillus are generally characterized to be Gram- 2009; Xiao et al., 2009, 2013), a number of novel taxa have positive, rod-shaped, endospore-forming, aerobic or facul- been isolated (Chen et al., 2009a, b, c, 2010, 2011a, b, c). tatively anaerobic, and have menaquinone-7 (MK-7) as the Here we describe the taxonomic properties of an endospore- major respiratory quinone, and phosphatidylethanolamine, forming, Gram-stain-positive bacterium, designated strain T phosphatidylglycerol and diphosphatidylglycerol as the major JSM 100118 , which was isolated from an oyster (Crassostrea polar lipids and iso-/anteiso-C15 : 0 as the predominant hongkongensis) collected from the tidal flat of Naozhou Island. Based on the results of a taxonomic study using a polyphasic approach, this strain is considered to represent a The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene novel species of the genus Bacillus. sequence of strain JSM 100118T is HQ419276. T A supplementary figure and a supplementary table are available with the Strain JSM 100118 was isolated from a homogenate of an online Supplementary Material. oyster using a standard dilution plating technique on

000139 G 2015 IUMS Printed in Great Britain 1561 J.-H. Chen and others marine agar 2216 (MA; Difco) at 30 uC for 2 weeks. After low convex with circular/slightly irregular margins and 2– primary isolation and purification, the isolate was main- 3 mm in diameter after incubation on MA (pH 7.5) at tained as serial transfers on MA slants, as lyophilized cultures 35 uC for 2–3 days. The strain grew optimally in the at 4 uC and deep-frozen at –80 uC in 20 % (v/v) glycerol. presence of 2–5 % (w/v) NaCl, at pH 7.5 and at 30–35 uC. Bacillus litoralis DSM 16303T, Bacillus halosaccharovorans Detailed phenotypic properties that differentiate strain JSM DSM 25387T, Bacillus niabensis DSM 17723T and Bacillus 100118T from related species of the genus Bacillus are herbersteinensis DSM 16534T were obtained from the included in Table 1 and the species description. Deutsche Sammlung von Mikroorganismen und Zellkulturen Genomic DNA was isolated according to Hopwood et al. (DSMZ; Braunschweig, Germany), and were used as reference (1985) and the G+C content was determined by reversed- strains for comparison in the study. Unless indicated otherwise, phase HPLC of nucleosides according to the protocol of morphological, physiological and molecular studies were Mesbah et al. (1989). The 16S rRNA gene sequence was performedwithcellsgrownonMA(pH7.5)at35uC. amplified by PCR and sequenced as described by Cui et al. In order to phenotypically characterize strain JSM 100118T, (2001). Pairwise sequence similarities were calculated using standard phenotypic tests were performed. The recom- a global alignment algorithm implemented at the EzTaxon-e mended minimal standards for describing new taxa of server (http://www.ezbiocloud.net/eztaxon; Kim et al., 2012). aerobic, endospore-forming bacteria were followed (Logan Phylogenetic analysis was performed using the software et al., 2009). Cell morphology was examined by using a package MEGA 6 (Tamura et al., 2013) after multiple align- Leica DM3000 microscope equipped with phase-contrast ment of sequence data by CLUSTAL X (Thompson et al., 1997). optics with cells grown on MA plus 10 mg MnSO4 for 1– Distances were calculated using distance options according to 6 days at 35 uC and 7–10 days at room temperature (18– Kimura’s two-parameter model (Kimura, 1980). Phylo- 20 uC). The Gram staining and the KOH lysis test were genetic trees were reconstructed using the neighbour-joining carried out according to Smibert & Krieg (1994) and (Saitou & Nei, 1987) and maximum-likelihood (Felsenstein, Gregersen (1978), respectively. Growth in the absence of 1981) algorithms integrated in the MEGA 6 software package NaCl was investigated on nutrient agar (NA) and in for phylogenetic inference. Bootstrap analysis was used to nutrient broth (NB) prepared according to the formula of evaluate the tree topology by means of 1000 resamplings Atlas (1993) except for the addition of NaCl. Tolerance of (Felsenstein, 1985). After the DNA was purified to an NaCl was tested on NA as well as in NB at different NaCl absorbance ratio of A260 nm versus A280 nm greater than 1.8, concentrations [0.1 and 0.5 % (w/v), and 1–30 % (w/v) in DNA–DNA hybridization experiments were performed accord- increments of 1 %]. Growth was tested at various tem- ing to the optical renaturation method (De Ley et al., 1970; Huß peratures (4, 5–55 uC in increments of 5 uC) and at dif- et al., 1983; Jahnke, 1992), using a UV-1206 spectrophotometer ferent pH (5.0–11.0, in increments of 0.5 pH units) on MA (Shimadzu) equipped with a TB-85 thermo-bath. and NA and in NB. The buffer solutions described by Chen The DNA G+C content of strain JSM 100118T was et al. (2007) were used for pH experiments. Methyl red and 35.9 mol%. This value is within the range for the genus Voges–Proskauer tests and determination of H S produc- 2 Bacillus and similar to that of B. litoralis (35.2 mol%; Yoon & tion from L-cysteine, hydrolysis of aesculin, indole produc- Oh, 2005) and B. herbersteinensis (36.2–36.9 mol%; Wieser tion, nitrate and nitrite reduction were performed as et al., 2005), but lower than that of B. halosaccharovorans recommended by Smibert & Krieg (1994) using media (42.6 mol%; Mehrshad et al., 2013) and B. niabensis (37.7– supplemented with 3 % NaCl. Hydrolysis of casein, 40.9 mol%; Kwon et al., 2007) (Table 1). The almost- cellulose, gelatin, starch, Tweens 20, 40, 60 and 80, and complete 16S rRNA gene sequence (1502 bp) of strain JSM urea was determined as described by Cowan & Steel (1965). 100118T was determined. Phylogenetic analysis based on 16S Growth under anaerobic conditions was determined on rRNA gene sequences revealed that strain JSM 100118T MA and NA, with or without 0.1 % (w/v) nitrate, using belonged to the genus Bacillus, and was most closely related to the GasPak Anaerobic Systems (BBL) according to the B. litoralis SW-211T (98.9 % 16S rRNA gene sequence manufacturer’s instructions. Determination of acid produc- similarity; Yoon & Oh, 2005), B. halosaccharovorans E33T tion from carbohydrates and utilization of carbon and (98.3 %; Mehrshad et al., 2013), B. niabensis 4T19T (97.8 %; nitrogen sources was performed as recommended by Ventosa Kwon et al., 2007) and B. herbersteinensis D-1,5aT (97.1 %; et al. (1982). Observation of motility and tests for catalase and Wieser et al., 2005). Sequence similarity values of equal to or oxidase activities were detected as described previously (Chen lower than 96.6 % were observed with other species of the et al., 2007). Citrate utilization and other enzymic activities genus Bacillus. In the neighbour-joining phylogenetic tree, were assayed using API 20E and API ZYM strips strain JSM 100118T and the type strain of B. litoralis formed a (bioMe´rieux) according to the manufacturer’s instructions robust sub-branch with a significant bootstrap support with 3 % (w/v) NaCl. All physiological and biochemical tests (99 %) (Fig. 1). The maximum-likelihood method resulted were repeated three times. in highly similar tree topology. DNA–DNA relatedness values Cells of strain JSM 100118T were Gram-stain-positive, between strain JSM 100118T and B. litoralis DSM 16303T, B. motile, facultatively anaerobic rods, producing endospores halosaccharovorans DSM 25387T, B. niabensis DSM 17723T which were oval and located sub-terminally in swollen and B. herbersteinensis DSM 16534T were33%,23%,24% sporangia. Colonies were creamy grey, non-transparent, and 16 %, respectively, values that are well-below the

1562 International Journal of Systematic and Evolutionary Microbiology 65 Bacillus crassostreae sp. nov.

Table 1. Characteristics used to distinguish strain JSM 100118T from closely related species of the genus Bacillus

Strains: 1, JSM 100118T;2,B. litoralis DSM 16303T;3,B. halosaccharovorans DSM 25387T;4,B. niabensis DSM 17723T;5,B. herbersteinensis DSM 16534T. All data are from this study unless indicated otherwise. All strains are endospore-forming, motile, Gram-stain-positive rods. All strains are positive for activity of catalase and oxidase and hydrolysis of aesculin and starch, but negative for H2S and indole production, hydrolysis of Tween 80, and activity of arginine dihydrolase, tryptophan deaminase and lysine and ornithine decarboxylase. +, Positive; –, negative, W, weakly positive.

Characteristic 1 2 3 4 5

Colony pigmentation Creamy grey Yellowish-white Cream Yellowish-white Cream to beige Endospore shape Oval Ellipsoidal Ellipsoidal Oval Oval Endospore position Sub-terminal Central/sub-terminal Central/sub-terminal Terminal Terminal Sporangium Swollen Swollen Swollen Swollen Unswollen NaCl for growth (%, w/v) Range 0–13 0.5–15 0.5–20 0–5 0–5 Optimum 2–5 2–5 5 0–0.5 0–0.5 pH for growth Range 5.5–10.0 6.0–11.0 5.0–10.0 6.0–10.0 6.0–10.0 Optimum 7.5 7.5–8.0 7.5–8.0 7.5–8.0 7.5 Temperature for growth (uC) Range 5–50 5–45 10–50 15–50 10–40 Optimum 30–35 35–40 35–40 30–35 30–35 Facultatively anaerobic + ––+ – Citrate utilization – + ––– Methyl red + –––– Nitrate reduction + ––+ – Voges–Proskauer test – – – W + Hydrolysis of: Casein – ++–– Gelatin ++ + – W Tween 20 ++ + +– Tween 40 ++ + +– Tween 60 + –––– Urea + ––++ Source Oyster Tidal flat sediment*a Hypersaline waterb Compostc Wall paintingd DNA G+C content (mol%) 35.9 35.2a 42.6b 40.9c 36.2d

*Data from: a, Yoon & Oh (2005); b, Mehrshad et al. (2013); c, Kwon et al. (2007); d, Wieser et al. (2005). threshold value (70 %) recommended by Wayne et al. (1987) The chemotaxonomic data for strain JSM 100118T were for the definition of members of a species. Therefore, it would consistent with the assignment of this strain to the genus appear that on the basis of phylogenetic and DNA–DNA Bacillus. Strain JSM 100118T possessed a cell-wall pepti- hybridization data, strain JSM 100118T constituted a dif- doglycan type based on meso-diaminopimelic acid as the ferent taxon separated from other species of the genus Bacillus diagnostic diamino acid. The strain contained MK-7 according to accepted criteria (Wayne et al., 1987; (96.9 %) as the predominant menaquinone, with MK-6 Stackebrandt & Goebel 1994). (1.9 %) and MK-8 (1.2 %) present in minor amounts. The polar lipids consisted of diphosphatidylglycerol, phospha- Biomasses used for chemotaxonomic analyses were obtained tidylethanolamine, phosphatidylglycerol, an unknown gly- from cultures grown in marine broth 2216 (Difco; pH 7.5) in colipid and an unknown phospholipid (Fig. S1, available in flasks on a rotary shaker (with shaking at 180 r.p.m.) at 35 uC the online Supplementary Material). The fatty acid profile for 3 days. Amino acids of whole-cell hydrolysates were of strain JSM 100118T was similar to those of the type analysed as described by Hasegawa et al. (1983). Isoprenoid strains of related species of the genus Bacillus, although quinones were analysed by HPLC as described by Groth et al. there were differences in the proportions of some fatty (1996). Polar lipids were extracted according to the method acids (Table S1; Yoon & Oh, 2005; Mehrshad et al., 2013; of Minnikin et al. (1979) and were identified by two- Kwon et al., 2007; Wieser et al., 2005). The major fatty dimensional TLC and spraying with appropriate detection acids (.10 % of the total) of the novel isolate were anteiso- reagents (Collins & Jones, 1980). Fatty acids were determined C15 : 0 (29.8 %), iso-C15 : 0 (17.6 %), C16 : 0 (14.2 %) and according to Sasser (1990) using the Microbial Identification C16 : 1v11c (10.3 %), which are characteristic of numerous System (MIDI). members of the genus Bacillus (Ka¨mpfer, 1994). http://ijs.sgmjournals.org 1563 J.-H. Chen and others

99* Bacillus crassostreae JSM 100118T (HQ419276) 85* Bacillus litoralis SW-211T (AY608605) E33T (HQ433447) 53 Bacillus halosaccharovorans 92* Bacillus niabensis 4T19T (AY998119) 0.01 Bacillus herbersteinensis D-1,5aT (AJ781029) 53 Bacillus galliciensis BFLP-1T (FM162181) Bacillus fastidiosus DSM 91T (X60615) Bacillus idriensis SMC 4352-2T (AY904033) 99* Bacillus cibi JG-30T (AY550276) 99* Bacillus indicus Sd/3T (AJ583158) Bacillus subtilis DSM 10T (AJ276351) 89 Bacillus aquimaris TF-12T (AF483625) 90* Bacillus marisflavi TF-11T (AF483624) 95* Bacillus soli LMG 21838T (AJ542513) 87* Bacillus bataviensis LMG 21833T (AJ542508) 67 Bacillus pocheonensis Gsoil 420T (AB245377) Bacillus horneckiae DSM 23495T (FR749913) 81* Bacillus kochii WCC 4582T (FN995265) 64* 52* Bacillus asahii MA001T (AB109209) LMG 20238T (AJ628748) 79* Bacillus muralis DSM 8801T (AM747813) 100* Brevibacterium frigoritolerans Bacillus simplex NBRC 15720T (AB363738) 86* Bacillus butanolivorans K9T (EF206294) Bacillus flexus NBRC 15715T (AB021185) 100* Bacillus megaterium IAM 13418T (D16273) 96 Bacillus aryabhattai B8W22T (EF114313) Bacillus horikoshii DSM 8719T (X76443) 96* Bacillus halmapalus DSM 8723T (X76447) Ureibacillus thermosphaericus DSM 10633T (AB101594)

Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain JSM 100118T and closely related members of the genus Bacillus. Asterisks indicate branches that were also obtained using the maximum- likelihood algorithm. Bootstrap values (.50 %) based on 1000 resampled datasets are shown at branch nodes. The sequence of Ureibacillus thermosphaericus DSM 10633T (GenBank accession no. AB101594) was used as outgroup. Bar, 1 substitution per 100 nt.

The results of the phylogenetic analysis and of morpho- sub-terminally in swollen sporangia. Colonies are cream– logical and chemotaxonomic investigations supported grey-pigmented, non-transparent, low convex with cir- the affiliation of strain JSM 100118T to the genus Bacillus. cular/slightly irregular margins, 2–3 mm in diameter after However, the cream–grey pigmentation and the ability to incubation on MA (pH 7.5) at 35 uC for 2–3 days. No hydrolyse Tween 60, as well as the discriminative result of the diffusible pigments are produced. Growth occurs with 0– methyl red test and the significant amount of C16 : 1v11c, 13 % (w/v) NaCl (optimum 2–5 %), at pH 5.5–10.0 differentiated the isolate clearly from its closest phylogenetic (optimum pH 7.5) and at 5–50 uC (optimum 30–35 uC). relatives (Tables 1 and S1). Also, the DNA–DNA hybridiza- Positive results for tests of methyl red and nitrate tion data clearly separated strain JSM 100118T from the most reduction, but negative results for citrate utilization, nitrite closely related species. Therefore, on the basis of these reduction, Voges–Proskauer, H2S and indole production. polyphasic taxonomic data, strain JSM 100118T should be Aesculin, gelatin, starch, Tweens 20, 40 and 60, and urea classified in the genus Bacillus as a member of a novel species, are hydrolysed, but casein, cellulose and Tween 80 are not. for which we propose the name Bacillus crassostreae sp. nov. Acids are produced from N-acetylglucosamine, L-arabinose, amygdalin, cellobiose, D-fructose, D-glucose, glycerol, Description of Bacillus crassostreae sp. nov. glycogen, lactose, D-mannitol melibiose, D-ribose, salicin and starch, but not from D-arabinose, D-galactose, maltose, Bacillus crassostreae (crass.os9tre.ae. N.L. gen. n. crassostreae D-mannose, melezitose, raffinose, L-rhamnose, sucrose, from the oyster Crassostrea honkongensis). trehalose, D-xylose, D-adonitol, dulcitol, myo-inositol or Cells are catalase- and oxidase-positive, facultatively D-sorbitol. The following compounds are utilized as sole anaerobic, motile, Gram-stain-positive rods, approximately sources of carbon and energy or sole sources of carbon, 0.6–0.8 mm wide and 2.0–4.0 mm long, occurring singly, in nitrogen and energy: L-arabinose, cellobiose, dextrin, D- pairs or short chains, and bear oval endospores that locate fructose, D-galactose, D-glucose, maltose, sucrose, trehalose,

1564 International Journal of Systematic and Evolutionary Microbiology 65 Bacillus crassostreae sp. nov. glycerol, glycogen, lactose, melibiose, D-ribose, D-xylose, D- nov., a moderately halophilic bacterium isolated from a salt mine in salicin, D-mannitol, butyrate, malate, N-acetylglucosamine Yunnan, south-west China. Int J Syst Evol Microbiol 57, 2327–2332. and L-alanine; the following are not utilized: D-arabinose, Chen, Y.-G., Zhang, Y.-Q., Shi, J.-X., Xiao, H.-D., Tang, S.-K., Liu, gentiobiose, D-mannose, melezitose, raffinose, L-rhamnose, Z.-X., Huang, K., Cui, X.-L. & Li, W.-J. (2009a). Jeotgalicoccus marinus sp. nov., a marine bacterium isolated from a sea urchin. Int J Syst Evol adonitol, D-arabitol, D-sorbitol, acetate, benzoate, gluconate, Microbiol 59, 1625–1629. L-glutamate, succinate, L-arginine, L-asparagine, L-glutamic Chen, Y.-G., Zhang, Y.-Q., Xiao, H.-D., Liu, Z.-X., Yi, L.-B., Shi, J.-X., acid, glycine, L-histidine, L-leucine, L-methionine, L-pheny- Zhi, X.-Y., Cui, X.-L. & Li, W.-J. (2009b). Pontibacillus halophilus sp. lalanine, L-proline, L-serine and L-valine. Constitutive nov., a moderately halophilic bacterium isolated from a sea urchin. enzymes expressed are acid and alkaline phosphatase, Int J Syst Evol Microbiol 59, 1635–1639. a-chymotrypsin, esterase (C4), esterase lipase (C8), b- Chen, Y.-G., Zhang, Y.-Q., Wang, Y.-X., Liu, Z.-X., Klenk, H.-P., Xiao, glucosidase and naphthol-AS-BI-phosphohydrolase; N- H.-D., Tang, S.-K., Cui, X.-L. & Li, W.-J. (2009c). Bacillus neizhouensis acetyl-b-glucosaminidase, arginine dihydrolase, cystine ary- sp. nov., a halophilic marine bacterium isolated from a sea anemone. lamidase, a-fucosidase, a- and b-galactosidase, a-glucosidase, Int J Syst Evol Microbiol 59, 3035–3039. b-glucuronidase, leucine arylamidase, lipase (C14), lysine Chen, Y.-G., Zhang, Y.-Q., Yi, L.-B., Li, Z.-Y., Wang, Y.-X., Xiao, H.-D., decarboxylase, a-mannosidase, ornithine decarboxylase, Chen, Q.-H., Cui, X.-L. & Li, W.-J. (2010). Pontibacillus litoralis sp. trypsin, tryptophan deaminase and valine arylamidase are nov., a facultatively anaerobic bacterium isolated from a sea anemone, not observed. The cell-wall peptidoglycan contains meso- and emended description of the genus Pontibacillus. Int J Syst Evol diaminopimelic acid. The predominant menaquinone is Microbiol 60, 560–565. MK-7. The major fatty acids (.10 % of the total) are anteiso- Chen, Y.-G., Hu, S.-P., Tang, S.-K., He, J.-W., Xiao, J.-Q., Zhu, H.-Y. & Li, W.-J. (2011a). 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