International Journal of Systematic and Evolutionary Microbiology (2010), 60, 2409–2414 DOI 10.1099/ijs.0.

016295-0

Oceanobacillus neutriphilus sp. nov., isolated from

activated sludge in a bioreactor
Jun-Yi Yang,1,2 Ying-Yi Huo,3 Xue-Wei Xu,1,2 Fan-Xu Meng,1,2 Min Wu3
and Chun-Sheng Wang1,2
Correspondence 1
Laboratory of Marine Ecosystem and Biogeochemistry, State Oceanic Administration,
Chun-Sheng Wang Hangzhou 310012, People’s Republic of China
[email protected] 2
Second Institute of Oceanography, State Oceanic Administration, Hangzhou 310012,
People’s Republic of China
3
College of Life Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China

A Gram-stain-positive, neutrophilic, rod-shaped bacterium, strain A1gT, was isolated from

activated sludge of a bioreactor and was subjected to a polyphasic taxonomic characterization.
The isolate grew in the presence of 0–17.0 % (w/v) NaCl and at pH 6.0–9.0; optimum growth
was observed in the presence of 3.0–5.0 % (w/v) NaCl and at pH 7.0. Strain A1gT was motile,
formed cream-coloured colonies, was catalase- and oxidase-positive and was able to hydrolyse
aesculin, Tween 40 and Tween 60. Chemotaxonomic analysis revealed menaquinone-7 as the
predominant respiratory quinone and anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0 as
major fatty acids. The genomic DNA G+C content of strain A1gT was 36.3 mol%. Comparative
16S rRNA gene sequence analysis revealed that the new isolate belonged to the genus
Oceanobacillus and exhibited closest phylogenetic affinity to the type strains of Oceanobacillus
oncorhynchi subsp. incaldanensis (97.9 % similarity) and O. oncorhynchi subsp. oncorhynchi
(97.5 %), but less than 97 % sequence similarity with respect to the type strains of other
recognized Oceanobacillus species. Levels of DNA–DNA relatedness between strain A1gT and
reference strains O. oncorhynchi subsp. incaldanensis DSM 16557T, O. oncorhynchi subsp.
oncorhynchi JCM 12661T and Oceanobacillus iheyensis DSM 14371T were 29, 45 and 38 %,
respectively. On the basis of phenotypic and genotypic data, strain A1gT is considered to
represent a novel species of the genus Oceanobacillus, for which the name Oceanobacillus
neutriphilus sp. nov. is proposed. The type strain is A1gT (5CGMCC 1.7693T 5JCM 15776T).

The genus Oceanobacillus was proposed by Lu et al. (2001, Kim et al. 2007), mural paintings [O. picturae (Heyrman
2002) with the description of Oceanobacillus iheyensis as et al. 2003) Lee et al. 2006], freshwater fish (O. oncorhynchi
the type species; the description of the genus was later subsp. oncorhynchi Yumoto et al. 2005), algae (O.
emended by Yumoto et al. (2005) and Lee et al. (2006). The oncorhynchi subsp. incaldanensis Romano et al. 2006), an
genus Oceanobacillus comprises aerobic, Gram-positive, insect (O. chironomi Raats and Halpern 2007), activated
motile, rod-shaped bacteria that are characterized chemo- sludge (O. caeni Nam et al. 2008) and food (O. kapialis
taxonomically by the presence of menaquinone-7 as the Namwong et al. 2009). The type strains of O. oncorhynchi
major isoprenoid quinone and anteiso-C15 : 0 as the subsp. oncorhynchi and O. oncorhynchi subsp. incaldanensis
predominant cellular fatty acid (Lee et al., 2006). share high 16S rRNA gene sequence similarity (99.5 %) but
At the time of writing, the genus comprises seven can be differentiated on the basis of phenotypic traits. The
recognized species, including two subspecies. Members of former is sporulating, facultatively aerobic and obligately
the genus have been isolated from diverse sources such as alkaliphilic (pH 9–10), whereas the latter is non-sporulat-
deep-sea sediment (O. iheyensis Lu et al. 2002; O. profundus ing, obligately aerobic and alkalitolerant (pH 6.5–9.5)
(Romano et al., 2006).
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain A1gT is EU709018. The aim of the present study was to determine the exact
T
Detailed fatty acid profiles of strain A1g and related type strains and
taxonomic position of a novel Oceanobacillus-like strain by
maximum-parsimony and maximum-likelihood 16S rRNA gene using a polyphasic approach that included analysis of
sequence-based phylogenetic trees are available as supplementary phenotypic properties and phylogenetic analysis based on
material with the online version of this paper. 16S rRNA gene sequences and DNA–DNA relatedness data.

016295 G 2010 IUMS Printed in Great Britain 2409

J.-Y. Yang and others

Strain A1gT was isolated from activated sludge of a Fatty acid methyl esters were obtained from cells grown on
sequential batch reactor treating salt-containing wastewater MA for 2 days at 35 uC and were analysed by using GC/MS
taken from a preserved Szechuan pickle factory. The (Kuykendall et al., 1988). Isoprenoid quinones were
reactor had been operational for 9 months at the time analysed as described by Komagata & Suzuki (1987) by
when the sludge was sampled. The average in situ tem- using reversed-phase HPLC. Cell-wall peptidoglycan was
perature was 30 uC and the pH was approximately 7.4. The prepared and hydrolysed according to the methods given
sludge sample was suspended in sterile wastewater and by Kawamoto et al. (1981) and the amino acid composi-
vortexed for 15 min. A portion of the suspension was tion was analysed with an automatic amino acid analyser
spread directly on sterile wastewater agar plates, which (Hitachi L-8900). Genomic DNA was obtained by using
contained (per litre wastewater) 1.0 g glucose, 1.0 g the method described by Marmur (1961). Purified DNA
peptone (BD) and 15.0 g agar (pH 7.0). The plates were was hydrolysed with P1 nuclease and the nucleotides were
incubated at 30 uC for several days. Single colonies on the dephosphorylated with calf intestine alkaline phosphatase;
plates were picked out and strain A1gT was obtained by the G+C content of the resulting deoxyribonucleosides
repeated restreaking. This isolate was routinely cultured on was determined by reversed-phase HPLC and was calcu-
marine agar 2216 (MA; BD) and was maintained as a lated from the ratio of deoxyguanosine to thymidine
glycerol suspension (30 % v/v) at 280 uC. (Mesbah & Whitman, 1989).
The optimal conditions for growth were determined in The 16S rRNA gene was amplified and analysed as
PY broth (Lu et al., 2001) with different NaCl con- described by Xu et al. (2007). PCR products were cloned
centrations (0, 0.5, 1, 3, 5, 7.5, 10, 12.5, 15, 16, 17, 18, 19, into pMD 19-T vector (TaKaRa) and then sequenced. An
20, 22.5 and 25 %, w/v). The pH range for growth was almost-complete 16S rRNA gene sequence of strain A1gT
determined by adding MES (pH 5.0–6.0), PIPES (1484 nt) was obtained and was compared with closely
(pH 6.5–7.0), Tricine (pH 7.5–8.5), CAPSO (pH 9.0– related sequences of reference organisms from the EzTaxon
10.0) or CAPS (pH 10.5) to PY broth supplemented with service (Chun et al., 2007). Sequence data were aligned
3 % (w/v) NaCl. The temperature range for growth was with CLUSTAL W 1.8 (Thompson et al., 1994). Phylogenetic
determined in PY broth (pH 7.0) at 4, 10, 15, 20, 25, 30, trees were constructed by using the neighbour-joining
35, 37, 40, 42, 45 and 48 uC. Cell morphology and (Saitou & Nei, 1987) and maximum-parsimony (Fitch,
motility were examined by optical microscopy (Olympus 1971) methods with the MEGA 4 program package (Tamura
BX40) and electron microscopy (Hitachi H-7650 and et al., 2007) and by using the maximum-likelihood method
JEOL JEM-1230). (Felsenstein, 1981) with the TreePuzzle 5.2 program.
Oxidase activity was determined based on oxidation of 1 % Evolutionary distances were calculated according to the
p-aminodimethylaniline oxalate and catalase activity was algorithm of Kimura’s two-parameter model (Kimura,
determined based on bubble production in 3 % (v/v) H2O2 1980) for the neighbour-joining method. Bootstrap
solution (Dong & Cai, 2001). Biochemical characteristics analysis was used to evaluate the tree topology by means
were determined according to the methods described by of 1000 resamplings.
Dong & Cai (2001) and Romano et al. (2006). Single Cells of strain A1gT were Gram-stain-positive, sporulating
carbon source assimilation tests were performed by using rods that were motile by means of polar flagella (Fig. 1).
medium 2 supplemented with 3 % (w/v) NaCl (Romano The NaCl concentration, pH and temperature ranges for
et al., 2006). The corresponding filter-sterilized sugar growth in PY broth were 0–17 % (w/v), pH 6.0–9.0 and
(0.2 %), alcohol (0.2 %), organic acid (0.1 %) or amino 10–45 uC. The cell-wall diamino acid was meso-diamino-
acid (0.1 %) was added to liquid medium. Acid production pimelic acid. The isoprenoid quinone of strain A1gT was
was tested by using MOF medium supplemented with MK-7 and the DNA G+C content was 36.3 mol%. These
1.0 % sugars or alcohols (Leifson, 1963; Xu et al., 2008). chemotaxonomic characteristics were in accordance with
Susceptibility to antibiotics was determined on agar plates those given for the genus Oceanobacillus (Yumoto et al.,
by using antibiotic discs with the following compounds 2005; Lee et al., 2006). Detailed results are given in the
(amounts in mg unless otherwise stated): amoxicillin (10), species description below and in Table 1.
ampicillin (10), carbenicillin (100), cefotaxime (30),
cefoxitin (30), chloramphenicol (30), erythromycin (15), 16S rRNA gene sequence comparisons showed that strain
kanamycin (30), neomycin (30), nitrofurantoin (300), A1gT should be placed within the genus Oceanobacillus, being
novobiocin (30), nystatin (100), penicillin (10), polymyxin related most closely to the type strains of O. oncorhynchi
B (300 IU), rifampicin (5), streptomycin (10) and tetra- subsp. incaldanensis (97.9 % similarity), O. oncorhynchi
cycline (30). Additional enzyme activities and biochemical subsp. oncorhynchi (97.5 %) and O. iheyensis (96.3 %); levels
characteristics were determined by using API 20E, API 20 of 16S rRNA gene sequence similarity with respect to the type
NE, API 50 CH and API ZYM kits as recommended by the strains of other recognized Oceanobacillus species were 94.2–
manufacturer (bioMérieux). O. iheyensis DSM 14371T, O. 95.3 %. Phylogenetic analysis based on 16S rRNA gene
oncorhynchi subsp. oncorhynchi JCM 12661T and O. sequences showed that strain A1gT had closest affinity with
oncorhynchi subsp. incaldanensis DSM 16557T were used the type strains of O. oncorhynchi and O. iheyensis with high
as controls in these tests. levels of bootstrap support (Fig. 2). The topologies of the

2410 International Journal of Systematic and Evolutionary Microbiology 60

 Oceanobacillus neutriphilus sp. nov.

iso-C15 : 0 (6.8 %). This profile was different from those

of O. iheyensis DSM 14371T and O. oncorhynchi subsp.
incaldanensis DSM 16557T (Supplementary Table S1). The
iso-C14 : 0 content of strain A1gT (3.8 %) was lower than
that of O. oncorhynchi subsp. incaldanensis DSM 16557T
(17.5 %) and O. iheyensis DSM 14371T (17.0 %). Addi-
tionally, strain A1gT could be differentiated from recog-
nized Oceanobacillus species on the basis of several
phenotypic characteristics, such as cultural conditions,
nitrate reduction, hydrolysis of substrates, acid production
from sugars or alcohols, susceptibility to antibiotics and
enzyme activities (Table 1).
On the basis of the genotypic and phenotypic data
presented in this study, strain A1gT should be assigned to
a novel species within the genus Oceanobacillus, for which
the name Oceanobacillus neutriphilus sp. nov. is proposed.

Description of Oceanobacillus neutriphilus

sp. nov.
Oceanobacillus neutriphilus (neu.tri.phi9lus. L. adj. neuter
-tra -trum neither, used to refer to neutral pH; Gr. adj.
philos loving; N.L. masc. adj. neutriphilus preferring neutral
pH).
Cells are Gram-positive-staining, aerobic rods, 0.7–1.2 mm
wide and 1.5–2.5 mm long with rounded ends, that are
motile by means of polar flagella and produce ellipsoidal
spores in a central position. Colonies on MA are 1–2 mm
in diameter, of low convexity, smooth, circular with regular
borders and cream-coloured after 48 h. Growth occurs in
PY medium at NaCl concentrations of 0–17.0 % (w/v) with
optimum growth at 3.0–5.0 %. The pH and temperature
ranges for growth are 6.0–9.0 and 10–45 uC (optimum
Fig. 1. Transmission electron micrographs of cells of strain A1gT. growth at pH 7.0 and 37 uC). No growth is detected below
(a) Exponentially growing cell, showing polar flagella. Bar, 0.5 mm. pH 5.5 or above pH 9.5. Positive for oxidase and catalase.
(b) Ultrathin section showing outer membrane of the cell. PG, Hydrolyses aesculin, Tween 40 and Tween 60, but not
Peptidoglycan; OL, outer layer. Bar, 0.2 mm. casein, DNA, gelatin, starch, Tween 80 or tyrosine. Nitrate
is not reduced to nitrite. H2S is not produced from
thiosulfate. Positive for o-nitrophenyl-b-D-galactopyrano-
phylogenetic trees built by using the maximum-parsimony sidase. Negative for arginine dihydrolase, indole produc-
and maximum-likelihood methods also supported the tion, lysine decarboxylase, ornithine decarboxylase,
notion that strain A1gT formed a stable clade with the type tryptophan deaminase and urease. Utilizes D-fructose, D-
strains of O. oncorhynchi and O. iheyensis (Supplementary glucose, maltose, mannitol, D-mannose and sucrose as sole
Fig. S1 in IJSEM Online). carbon and energy sources, but not acetate, L-alanine, L-
arabinose, citrate, L-cysteine, ethanol, formate, fumarate,
DNA–DNA hybridization experiments were performed by
glutamate, L-glutamine, glycine, L-histidine, isoleucine,
the thermal denaturation and renaturation method of De
lactate, lactose, malate, malonate, L-methionine, myo-
Ley et al. (1970) as modified by Huß et al. (1983), by using
inositol, L-ornithine, propionate, pyruvate, raffinose, L-
a Beckman DU 800 spectrophotometer. Levels of DNA–
serine, L-sorbitol, L-sorbose, starch, succinate or L-valine.
DNA relatedness between strain A1gT and O. oncorhynchi
Acid is produced from D-glucose, maltose, D-mannose and
subsp. incaldanensis DSM 16557T, O. oncorhynchi subsp.
sucrose, but not from L-arabinose, ethanol, myo-inositol,
oncorhynchi JCM 12661T and O. iheyensis DSM 14371T
lactose, raffinose, L-sorbitol or L-sorbose. Susceptible to (mg
were 29, 45 and 38 %, respectively, significantly below the
per disc unless indicated otherwise) amoxicillin (10),
value of 70 % which is considered to be the threshold for
ampicillin (10), carbenicillin (100), cefotaxime (30),
the delineation of species (Wayne et al., 1987).
chloramphenicol (30), erythromycin (15), kanamycin
The major fatty acids of strain A1gT were anteiso-C15 : 0 (30), neomycin (30), nitrofurantoin (300), novobiocin
(37.7 %), anteiso-C17 : 0 (18.9 %), iso-C16 : 0 (15.8 %) and (30) and rifampicin (5), but not to cefoxitin (30), nystatin

http://ijs.sgmjournals.org 2411
J.-Y. Yang and others

Table 1. Differential characteristics between strain A1gT and the type strains of related members of
Oceanobacillus
Strains: 1, A1gT; 2, O. oncorhynchi subsp. incaldanensis DSM 16557T; 3, O. oncorhynchi subsp. oncorhynchi JCM
12661T; 4, O. iheyensis DSM 14371T. Data were obtained in the present study unless indicated. +, Positive;
2, negative; W, weakly positive.

Characteristic 1 2 3 4

Spore position* C NS T T
NaCl range for growth (%, w/v) 0–17.0 0–20.0 0–22.5 0–18.0
NaCl optimum (%, w/v) 3.0–5.0 10.0 7.5–10.0 3.0–5.0
Growth temperature range (uC) 10–45 10–45 10–45 15–42
pH optimum 7.0 9.0 9.5 8.0–8.5
Nitrate reduction 2 + + 2
o-Nitrophenyl-b-D-galactopyranosidase + 2 2 2
Hydrolysis of:
Casein 2 2 2 +
Gelatin 2 2 2 +
Tween 60 + 2 2 +
Urea 2 2 + 2
Acid production from:
Arbutin + + + 2
Cellobiose W + + 2
Gentiobiose 2 + + 2
Glycerol W 2 2 +
D-Mannitol + + 2 W
Raffinose 2 2 + 2
Salicin + + + 2
Sucrose + + + 2
D-Tagatose + + + 2
Trehalose + + + 2
Sensitivity to:
Cefotaxime (30 mg) + + 2 +
Cefoxitin (30 mg) 2 + + +
Nitrofurantoin (300 mg) + + 2 +
Penicillin G (10 mg) 2 + + +
Streptomycin (10 mg) 2 2 + 2
API ZYM results
a-Chymotrypsin + + + 2
a-Glucosidase + + + 2
b-Glucosidase + + + 2
DNA G+C content (mol%) (HPLC) 36.3 40.1aD 38.5b 35.8c

*C, Central; NS, not sporulated; T, terminal.

DData from: a, Romano et al. (2006); b, Yumoto et al. (2005); c, Lu et al. (2001).

Fig. 2. Neighbour-joining tree based on 16S

rRNA gene sequences, showing the phyloge-
netic relationships between strain A1gT and
related taxa. Bootstrap values at nodes are
based on 1000 replicates; only values .50 %
are shown. Filled circles indicate nodes recov-
ered with bootstrap values .50 % in the
maximum-parsimony and maximum-likelihood
trees. Bar, 0.02 substitutions per nucleotide
position.

2412 International Journal of Systematic and Evolutionary Microbiology 60

 Oceanobacillus neutriphilus sp. nov.

(100), penicillin (10), polymyxin B (300 IU), streptomycin Kawamoto, I., Oka, T. & Nara, T. (1981). Cell wall composition of
(10) or tetracycline (30). The following constitutive enzyme Micromonospora olivoasterospora, Micromonospora sagamiensis, and
related organisms. J Bacteriol 146, 527–534.
activities are detected in API ZYM tests: acid phosphatase,
alkaline phosphatase, a-chymotrypsin, esterase (C4), ester- Kim, Y.-G., Choi, D. H., Hyun, S. & Cho, B. C. (2007). Oceanobacillus
profundus sp. nov., isolated from a deep-sea sediment core. Int J Syst
ase lipase (C8), a-glucosidase, b-glucosidase and naphthol-
Evol Microbiol 57, 409–413.
AS-BI-phosphohydrolase. N-Acetyl-b-glucosaminidase, cystine
Kimura, M. (1980). A simple method for estimating evolutionary rates
arylamidase, a-fucosidase, a-galactosidase, b-galactosidase,
of base substitutions through comparative studies of nucleotide
b-glucuronidase, lipase (C14), leucine arylamidase, a- sequences. J Mol Evol 16, 111–120.
mannosidase, trypsin and valine arylamidase activities are
Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in
not observed. Oxidative acid production in API 50 CH bacterial systematics. Methods Microbiol 19, 161–207.
tests is positive for N-acetylglucosamine, arbutin, aesculin,
Kuykendall, L. D., Roy, M. A., O’Neill, J. J. & Devine, T. E. (1988). Fatty
D-fructose, D-glucose, maltose, D-mannitol, salicin, suc-
acids, antibiotic resistance, and deoxyribonucleic acid homology
rose, D-tagatose and trehalose and weakly positive for groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361.
cellobiose and glycerol. Principal fatty acids (.5 %) are Lee, J.-S., Lim, J.-M., Lee, K. C., Lee, J.-C., Park, Y.-H. & Kim, C.-J.
anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The (2006). Virgibacillus koreensis sp. nov., a novel bacterium from a salt
isoprenoid quinone is MK-7. The cell-wall diamino acid is field, and transfer of Virgibacillus picturae to the genus Oceanobacillus
meso-diaminopimelic acid. The DNA G+C content of the as Oceanobacillus picturae comb. nov. with emended descriptions. Int
type strain is 36.3 mol%. J Syst Evol Microbiol 56, 251–257.
Leifson, E. (1963). Determination of carbohydrate metabolism of
The type strain, A1gT (5CGMCC 1.7693T 5JCM 15776T), marine bacteria. J Bacteriol 85, 1183–1184.
was isolated from activated sludge of a bioreactor treating
Lu, J., Nogi, Y. & Takami, H. (2001). Oceanobacillus iheyensis gen. nov.,
salt-containing wastewater. sp. nov., a deep-sea extremely halotolerant and alkaliphilic species
isolated from a depth of 1050 m on the Iheya Ridge. FEMS Microbiol
Lett 205, 291–297.
Acknowledgements
Lu, J., Nogi, Y. & Takami, H. (2002). Oceanobacillus iheyensis gen. nov.,
This work was supported by grants from the Ministry of Science and sp. nov. In Validation of the Publication of New Names and New
Technology of China (973 Program, 2004CB719604-3; 863 Program, Combinations Previously Effectively Published Outside the IJSEM, List
2007AA021305), the National Natural Science Foundation of China no. 85. Int J Syst Evol Microbiol 52, 685–690.
(40806066), the China Ocean Mineral Resources R & D Association Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
(COMRA) Special Foundation (DYXM-115-01-3-01, DYXM-115-01- acid from microorganisms. J Mol Biol 3, 208–218.
3-02) and the Key Project of Zhejiang Science and Technology
(2006C13053). Mesbah, M. & Whitman, W. B. (1989). Measurement of deoxyguano-
sine/thymidine ratios in complex mixtures by high-performance
liquid chromatography for determination of the mole percentage
guanine + cytosine of DNA. J Chromatogr 479, 297–306.
References
Nam, J.-H., Bae, W. & Lee, D.-H. (2008). Oceanobacillus caeni sp. nov.,
Chun, J., Lee, J.-H., Jung, Y., Kim, M., Kim, S., Kim, B. K. & Lim, Y.-W. isolated from a Bacillus-dominated wastewater treatment system in
(2007). EzTaxon: a web-based tool for the identification of Korea. Int J Syst Evol Microbiol 58, 1109–1113.
prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Namwong, S., Tanasupawat, S., Lee, K. C. & Lee, J.-S. (2009).
Evol Microbiol 57, 2259–2261. Oceanobacillus kapialis sp. nov., from fermented shrimp paste in
De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative Thailand. Int J Syst Evol Microbiol 59, 2254–2259.
measurement of DNA hybridization from renaturation rates. Eur J Raats, D. & Halpern, M. (2007). Oceanobacillus chironomi sp. nov., a
Biochem 12, 133–142. halotolerant and facultatively alkaliphilic species isolated from a
Dong, X.-Z. & Cai, M.-Y. (2001). Determinative Manual for Routine chironomid egg mass. Int J Syst Evol Microbiol 57, 255–259.
Bacteriology. Beijing: Scientific Press. Romano, I., Lama, L., Nicolaus, B., Poli, A., Gambacorta, A. &
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a Giordano, A. (2006). Oceanobacillus oncorhynchi subsp. incaldanensis
maximum likelihood approach. J Mol Evol 17, 368–376. subsp. nov., an alkalitolerant halophile isolated from an algal mat
collected from a sulfurous spring in Campania (Italy), and emended
Fitch, W. M. (1971). Toward defining the course of evolution:
description of Oceanobacillus oncorhynchi. Int J Syst Evol Microbiol 56,
minimum change for a specific tree topology. Syst Zool 20, 406– 805–810.
416.
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
Heyrman, J., Logan, N. A., Busse, H.-J., Balcaen, A., Lebbe, L.,
method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
Rodriguez-Diaz, M., Swings, J. & De Vos, P. (2003). Virgibacillus
carmonensis sp. nov., Virgibacillus necropolis sp. nov. and Virgibacillus Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007).MEGA4: molecular

picturae sp. nov., three novel species isolated from deteriorated mural evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol
paintings, transfer of the species of the genus Salibacillus to Evol 24, 1596–1599.
Virgibacillus, as Virgibacillus marismortui comb. nov. and Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W:
Virgibacillus salexigens comb. nov., and emended description of the improving the sensitivity of progressive multiple sequence alignment
genus Virgibacillus. Int J Syst Evol Microbiol 53, 501–511. through sequence weighting, position-specific gap penalties and
Huß, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the weight matrix choice. Nucleic Acids Res 22, 4673–4680.
spectrophotometric determination of DNA hybridization from Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler,
renaturation rates. Syst Appl Microbiol 4, 184–192. O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. &

http://ijs.sgmjournals.org 2413
J.-Y. Yang and others

other authors (1987). International Committee on Systematic Xu, X.-W., Wu, Y.-H., Wang, C.-S., Yang, J.-Y., Oren, A. & Wu, M.
Bacteriology. Report of the ad hoc committee on reconciliation of (2008). Marinobacter pelagius sp. nov., a moderately halophilic
approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464. bacterium. Int J Syst Evol Microbiol 58, 637–640.
Xu, X.-W., Wu, Y.-H., Zhou, Z., Wang, C.-S., Zhou, Y.-G., Zhang, H.-B., Yumoto, I., Hirota, K., Nodasaka, Y. & Nakajima, K. (2005).
Wang, Y. & Wu, M. (2007). Halomonas saccharevitans sp. nov., Oceanobacillus oncorhynchi sp. nov., a halotolerant obligate alkaliphile
Halomonas arcis sp. nov. and Halomonas subterranea sp. nov., isolated from the skin of a rainbow trout (Oncorhynchus mykiss), and
halophilic bacteria isolated from hypersaline environments of China. emended description of the genus Oceanobacillus. Int J Syst Evol
Int J Syst Evol Microbiol 57, 1619–1624. Microbiol 55, 1521–1524.

2414 International Journal of Systematic and Evolutionary Microbiology 60