International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3760–3767
DOI 10.1099/ijs.0.065961-0
Sulfitobacter geojensis sp. nov., Sulfitobacter noctilucae sp. nov., and Sulfitobacter noctilucicola sp. nov., isolated from coastal seawater Min-Jung Kwak,1,2 Jung-Sook Lee,3 Keun Chul Lee,3 Kwang Kyu Kim,3 Mi Kyung Eom,3 Byung Kwon Kim2 and Jihyun F. Kim2 Correspondence Jihyun F. Kim [email protected]
1
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), 217 Gajung-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea
2
Department of Systems Biology and Division of Life Sciences, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea
3
Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea
Four Gram-stain-negative, aerobic, rod-shaped bacterial strains, MM-124, MM-126, NB-68 and NB-77, were isolated from the coastal seawater or a region with a bloom of sea sparkle around Geoje island in Korea. The sequence similarity values of the 16S rRNA gene between the isolates and Sulfitobacter mediterraneus DSM 12244T ranged from 97.7 to 98.2 %, and phylogenetic relationships suggested that they belong to a phylogenetic branch that includes the genera Sulfitobacter and Roseobacter. The isoprenoid quinone of all three novel strains was ubiquinone10 and the major fatty acid was cis-vaccenic acid, as in other species of the genus Sulfitobacter. However, there were several differences in the morphological, physiological and biochemical characteristics among the four strains and the reference species of the genus Sulfitobacter. Moreover, the average nucleotide identity values between the three sequenced isolates and the reference strains were below 76.33, indicating that genomic variation exists between the isolates and reference strains. Chemotaxonomic characteristics together with phylogenetic affiliations and genomic distances illustrate that strains MM-124, NB-68 and NB-77 represent novel species of the genus Sulfitobacter, for which the names Sulfitobacter geojensis sp. nov. (type strain MM-124T5KCTC 32124T5JCM 18835T), Sulfitobacter noctilucae sp. nov. (type strain NB-68T5KCTC 32122T5JCM 18833T) and Sulfitobacter noctilucicola sp. nov. (type strain NB-77T5KCTC 32123T5JCM 18834T) are proposed.
The genus Sulfitobacter is a member of the family Rhodobacteraceae, comprising highly abundant heterotrophs found in coastal areas (Buchan et al., 2005). Recently, the Roseobacter clade became one of the most interesting marine bacterial groups because, apart from its abundance and adaptability, its members have diverse roles such as sulfur oxidation, dimethylsulfoniopropionate demethylation, carbon monoxide oxidation and aerobic anoxygenic photosynthesis (Brinkhoff et al., 2008). The genus Sulfitobacter was first isolated as a sulfite oxidizer in 1995 (Sorokin, 1995a), Abbreviation: ANI, average nucleotide identity. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of MM-124, NB-68 and NB-77 are KC428714, KC428716 and KC428717, respectively. The GenBank accession numbers of the genome sequences of MM-124, NB-68 and NB-77 are JASE00000000, JASC00000000 and JASD00000000, respectively. Three supplementary figures and one supplementary table are available with the online version of this paper.
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and received attention due to its ability to produce a sulfite oxidase, which can be used in biosensor systems for detecting sulfite (Muffler & Ulber, 2008). Moreover, bacteria of the genus Sulfitobacter are known to degrade dimethylsulfoniopropionate to dimethylsulfide, which is an essential organic sulfur compound in the sulfur cycle and known as cloud condensation nuclei (Berresheim et al., 1993; Curson et al., 2008; Mou et al., 2005). In this study, we characterized four isolates, strains MM-124, MM-126, NB-68 and NB-77, as potential novel members of the genus Sulfitobacter using a polyphasic taxonomic approach, which included a comparison of whole genome sequences. Strains MM-124, MM-126, NB-68 and NB-77 were isolated from seawater around Geoje island, Korea in May 2012. Water samples for strains MM-124 and MM-126 were collected from the coastal seawater around Geoje island (35u 029 87.80 N 128u 719 47.30 E), and those for strains NB-68 and NB-77 were collected from a region of sea 065961 G 2014 IUMS Printed in Great Britain
Three novel Sulfitobacter species
sparkle bloom (34u 999 30.80 N 128u 679 39.30 E). The seawater samples were spread directly onto marine agar 2216 (BD Difco) and incubated at 16 uC for 5 days.
ml21), oleandomycin (15 mg ml21), cefalotin (30 mg ml21), neomycin (30 mg ml21), erythromycin (15 mg ml21), vancomycin (30 mg ml21) and chloramphenicol (30 mg ml21).
A 27F and 1492R universal primer set was used for PCRs of the small subunit rRNA (annealing 55 uC, 30 s; extension 72 uC, 1.5 min; 30 cycles). PCR products were sequenced and used as queries for BLAST searches at the EzTaxon server (Chun et al., 2007). Phylogenetic analysis was performed using ARB (Ludwig et al., 2004), MEGA5 (Tamura et al., 2011) and PAUP (Swofford, 1993).
For fatty acid analysis, MM-124, MM-126, NB-68, NB-77 and all the reference strains were grown on marine agar at 25 uC for 3 days. Fatty acid methyl esters were extracted according to the standard protocol of the microbial identification system and identified using gas chromatographic analysis (Sasser, 1990). For polar lipid analysis, 10 mg of freeze-dried cells of MM-124, MM-126, NB-68, NB-77 and two reference strains (KCTC 12864T and KCTC 32186T) were prepared. Polar lipids were extracted and analysed by two dimensional TLC, according to a described protocol (Minnikin et al., 1977). For isoprenoid quinone analysis, 100–300 mg of freeze-dried cells of MM-124, MM-126, NB-68, NB-77 and one reference strain (KCTC 32188T) were prepared. Isoprenoid quinones were extracted using chloroform/methanol (2 : 1, v/v), purified by one dimensional TLC and identified by HPLC.
Nine type strains of the genus Sulfitobacter were obtained from the Korean Collection for Type Cultures (KCTC) to be used as references for comparison with the isolates. They were: Sulfitobacter litoralis KCTC 12521T (5Iso 3T), Sulfitobacter marinus KCTC 12738T (5SW-265T), Sulfitobacter donghicola KCTC 12864T (5DSW-25T), Sulfitobacter delicatus KCTC 32183T (5KMM 3584T), Sulfitobacter dubius KCTC 32184T (5KMM 3554T), Sulfitobacter pontiacus KCTC 32185T (5ChLG 10T), Sulfitobacter brevis KCTC 32186T (5EL162T), Sulfitobacter guttiformis KCTC 32187T (5EL-38T) and Sulfitobacter mediterraneus KCTC 32188T (5CH-B427T). All strains were routinely cultured on marine agar or in marine broth at 25 uC. Cell morphology and motility were observed using a light microscope (Carl Zeiss, Axio Lab.A1) and a transmission electron microscope (Philips, CM20). Gram staining was performed using a Gram straining kit (YD Diagnostics) and the motility test was performed by the hanging drop method (Skerman, 1967). Growth at various temperatures (4–45 uC) was observed on marine agar and growth at various NaCl concentrations (2–20 %) and pH 4.5–10 (at 0.5 pH unit intervals; buffered with Na2HPO4 and KH2PO4) was observed on marine agar at 25 uC for 7 days. For testing anaerobic growth, strains were incubated for 4 weeks in an anaerobic chamber. Catalase and oxidase activity were tested using 3 % (v/v) H2O2 and an oxidase reagent dropper (Becton Dickinson), respectively. H2S production and indole production were tested using SIM medium and an indole reagent dropper (Becton Dickinson). Haemolysis was tested on marine agar with 5 % (v/v) defibrinated sheep blood. The presence of bacteriochlorophyll a was checked with a spectrophotometer using the cell lysates of strains representing the genus Sulfitobacter after incubation in marine broth overnight in the dark. Other enzyme activities and the assimilation of substrates were tested using API 20NE and API ZYM (BioMe´rieux). GN2 microplates (Biolog) were used for the oxidation testing of 95 carbon sources with bacterial suspensions in artificial seawater. Antibiotic susceptibility was tested on marine agar using antibiotic susceptibility test discs (Oxoid) with antibiotics at the following concentrations: piperacillin (110 mg ml21), penicillin G (10 U ml21), tetracycline (30 mg ml21), polymyxin B (300 U ml21), lincomycin (15 mg ml21), novobiocin (5 mg ml21), carbenicillin (100 mg ml21), kanamycin (30 mg ml21), ampicillin (10 mg ml21), streptomycin (25 mg ml21), gentamicin (30 mg ml21), rifampicin (30 mg http://ijs.sgmjournals.org
To determine the DNA G+C content and the genome relatedness between strains, we performed paired-end genome sequencing of MM-124, NB-68 and NB-77 using the HiSeq 2000 genome sequencer of the Illumina/Solexa platform (Macrogen). Sequence trimming and de novo assembly were performed with CLC Genomics Workbench 5.1 (CLC bio). Entangled or over-collapsed contigs were resolved and gaps between contigs were filled further manually. The quality of the assembly was assured by a bell-shaped normal distribution of the paired-end reads with a mean insert size of 510 bp. Mapping of the reads to the contigs resulted in a mean of 91.34 % being pair-ended. The draft genome sequences consisted of 5 contigs with 1974-fold coverage for MM-124, 14 contigs with 600-fold coverage for NB-68, and 8 contigs with 730-fold coverage for NB-77. According to the Lander–Waterman model (Lander & Waterman, 1988), the genome coverage of each strain based on sequencing depth should approximate to 100 %. To determine the DNA relatedness between the isolates and strains of other species, we calculated the average nucleotide identity (ANI) values, as described by Goris et al. (2007) and Kim et al. (2014). For this purpose, we also conducted genome sequencing and assembly of three type strains of species of the genus Sulfitobacter. The draft genomes consisted of 8 contigs with 723-fold coverage for S. donghicola KCTC 12864T, 4 contigs with 523-fold coverage for S. guttiformis KCTC 32187T, and 31 contigs with 987-fold coverage for S. mediterraneus KCTC 32188T. MM-124T, MM-126, NB-68T and NB-77T were Gramstain-negative, rod-shaped and obligately aerobic (Fig. S1, available in the online Supplementary Material). They formed slightly yellowish colonies on marine agar within 3 days. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the four strains are affiliated with the genus Sulfitobacter. Among the species of the genus Sulfitobacter, sequence similarities of the 16S rRNA genes 3761
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of MM-124T, MM-126, NB-68T and NB-77T were highest with that of S. mediterraneus DSM 12244T (98.1 %, 97.7 %, 97.9 % and 98.2 %, respectively). On the other hand, similarity values of the 16S rRNA gene sequences of MM124T, MM-126, NB-68T and NB-77T with that of Roseobacter denitrificans OCh 114T were below 95.4 %. The similarity value of the 16S rRNA genes between MM-124T and MM126 was 99.4 %, between MM-124T and NB-68T was 98.3 %, between MM-124T and NB-77T was 98.1 %, between MM126 and NB-68T was 98.3 %, between MM-126 and NB-77T was 97.8 %, and between NB-68T and NB-77T was 98.8 %. The 16S rRNA gene sequence similarity of ¢99 % along with identical polar lipid compositions (see below) and almost identical phenotypic characteristics (data not shown) indicated that MM-124T and MM-126 are members of the same species. Phylogenetic trees based on the 16S rRNA gene demonstrated that MM-124T, MM-126, NB-68T and NB-77T form a monophyletic group with all the other species of the genus Sulfitobacter as a single sister clade (Figs 1 and S2). Conventionally, DNA–DNA hybridization experimentation has been used for the comparison of DNA relatedness between two strains. Recently, Goris and colleagues reported a correlation between the percentage DNA–DNA hybridization and ANI values (Goris et al., 2007). A value of 70 % DNA–DNA hybridization, which is the recommended cut-off for species delineation, corresponds to 95 % ANI. Moreover, Kim and colleagues reported the taxonomic consistency between ANI values of whole genomes and similarity values of the 16S rRNA genes (Kim et al., 2014). They observed a distinction in the ANI distribution between intra- and inter-species relationships at around 95 % ANI, and proposed that 98.65 % 16S rRNA gene sequence similarity can be used as the threshold for differentiating two species. Several recent taxonomic papers adopted ANI values instead of percentages of DNA–DNA hybridization for the identification of novel bacterial species (CameloCastillo et al., 2014; Dura´n et al., 2014; Lucena et al., 2014). The ANI values, based on BLASTn between MM-124T, NB68T, NB-77T and other related taxa in genera Sulfitobacter and Roseobacter were below 74.43 % (Table 1), which corresponds to less than 20 % DNA–DNA hybridization (Goris et al., 2007). The ANI values between MM-124T, NB68T, NB-77T and S. mediterraneus DSM 12244T, which is the closest related strain based on 16S rRNA gene sequence similarity, ranged from 73.32 to 74.43 %, whereas the values among the three Geoje isolates ranged from 75.17 to 76.43 %. Consistent with the observation of Kim et al. 2014, the ANI value was 96.99 or 97.04 % between EE-36 and NAS-14.1, which are the strains of S. pontiacus.
nitrate to nitrite, whereas NB68T and NB-77T do not. On the other hand, NB-77T is positive for alkaline phosphatase and urease, while MM124T and NB68T are negative. The major fatty acid of the four isolates was cis-vaccenic acid (C18 : 1v7c) and its proportions were 82.9, 89.6, 86.1 and 87.6 % in MM-124T, MM-126, NB-68T and NB-77T, respectively (Table S1). The isoprenoid quinone was ubiquinone-10 (Q-10) as in other species of the genus Sulfitobacter. The polar lipids of MM-124T and MM-126 comprised phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) (Table 2 and Fig. S3). The polar lipids of NB-68T consisted of PC, PG, PE and unknown aminophospholipids. The polar lipids of NB77T consisted of PC, PG, PE and diphosphatidylglycerol (DPG). The DNA G+C contents of MM-124T, NB-68T and NB-77T were 57.84, 58.33 and 57.06 %, respectively. Their phylogenetic position as a sister group to the other members of the genus Sulfitobacter, chemotaxonomic characteristics similar to other species of the genus Sulfitobacter, and genomic distances equivalent to less than 20 % DNA–DNA hybridization with other species of the genus Sulfitobacter indicated that MM-124T, NB-68T and NB-77T represent novel species of the genus Sulfitobacter, for which the names Sulfitobacter geojensis sp. nov., Sulfitobacter noctilucae sp. nov. and Sulfitobacter noctilucicola sp. nov., respectively, are proposed. Strain MM-126 belongs to the same species as strain MM-124T. Description of Sulfitobacter geojensis sp. nov. Sulfitobacter geojensis (ge.o.jen9sis. N.L. masc. adj. geojensis from Geoje island, the place where the type strain was isolated).
The physiological, biochemical and chemotaxonomic characteristics of MM-124T, MM-126, NB-68T and NB-77T are given in Table 2. Between the isolates, there are at least three different biochemical or physiological properties such as the presence of flagella, the activities of several enzymes such as nitrate reductase, alkaline phosphatase and urease, and the major polar lipids. MM124T has polar flagella and reduces
Cells are Gram-stain-negative, aerobic and rod shaped. Forms convex and slightly yellowish colonies within 3 days on marine agar at 25 uC. Anaerobic growth does not occur on marine agar. Possible growth temperature range is 4– 30 uC, and the optimum temperature is 25 uC. Reaches the stationary growth phase after 24 h on marine broth at 25 uC. Cells grow at pH §5.5, and the optimum is 7.0–8.0. The NaCl concentration for growth is up to 6 % (w/v). Growth does not occur on L medium, R2A medium, typticase soy agar or MacConkey agar, regardless of NaCl concentration. Utilizable carbon sources for growth (Biolog, GN2 microplate) are: adonitol, D-fructose, Dsorbitol, itaconic acid, DL-lactic acid, quinic acid, Lasparagine, L-proline, L-pyroglutamic acid and phenylethylamine. Negative for catalase, H2S production and indole production; positive for oxidase and nitrate reduction. Haemolytic activity and bacteriochlorophyll a are not detected. The results of enzyme assays (API ZYM and 20NE) for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin and agalactosidase are positive. The results of enzyme assays for alkaline phosphatase, leucine arylamidase, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase,
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Three novel Sulfitobacter species
Sulfitobacter litoralis Iso 3T (DQ097527) 0.01
Sulfitobacter pontiacus ChLG 10T (Y13155) Sulfitobacter marinus SW-265T (DQ683726) 52
Sulfitobacter brevis Ekho Lake-162T (Y16425) 100
62
Oceanibulbus indoliflex HEL-45T (AJ550939) Sulfitobacter dubius KMM 3554T (AY180102) Sulfitobacter delicatus KMM 3584T (AY180103)
Sulfitobacter porphyrae SCM-1T (AB758574) Sulfitobacter mediterraneus CH-B427T (JASH00000000)
59 56
Sulfitobacter guttiformis Ekho Lake-38T (JASG00000000)
97
Sulfitobacter donghicola DSW-25T (JASF00000000) Sulfitobacter geojensis MM-124T (JASE00000000)
99
99
99
Sulfitobacter geojensis MM-126 (KC428715) Sulfitobacter noctilucae NB-68T (JASC00000000)
93
Sulfitobacter noctilucicola NB-77T (JASD00000000) Roseobacter denitrificans OCh 114T (L01784)
100
86
Roseobacter litoralis ATCC 49566T (X78312) Sagittula stellata E-37T (U58356)
99
Antarctobacter heliothermus EL-219T (Y11552) Phaeobacter inhibens T5T (AY177712)
95
82
Phaeobacter gallaeciensis BS107T (Y13244)
58
Phaeobacter arcticus 20188T (DQ514304) 97
Leisingera aquimarina LMG 24366T (AM900415)
97
Leisingera methylohalidivorans DSM 14336T (AY005463)
50
80
Phaeobacter caeruleus LMG 24369T (AM943630)
87
Phaeobacter daeponensis TF-218T (DQ981486) 64
Ruegeria pomeroyi DSM 15171T (DQ915631) Ruegeria lacuscaerulensis DSM 11314T (DQ915630)
70 89
Ruegeria scottomollicae LMG 24367T (AM905330) 100
Ruegeria pelagia HTCC 2662T (DQ916141) Ruegeria mobilis NBRC 101030T (EU977137)
100
Octadecabacter antarcticus 307T (U14583) Octadecabacter arcticus 238T (U73725)
85
Thalassobius gelatinovorus NBRC 15761T (AB289591)
74 91
Thalassobius mediterraneus CECT 5383T (AJ878874) Thalassobius aestuarii JC2049T (AY442178)
100
Marivita cryptomonadis CL-SK44T (EU512919) Marivita litorea CL-JM1T (EU512918)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of type strains showing the phylogenetic position of MM-124T, MM-126, NB-68T, NB-77T and type strains of phylogenetically related taxa [accession numbers of the 16S rRNA gene are in parentheses; accession numbers of type JAS(X)00000000 indicate the draft genome sequences determined in this study]. The phylogenetic tree was reconstructed based on a comparison of 1199 nt and evolutionary distances were calculated using the Jukes–Cantor model. Bootstrap values (percentages of 1000 replications) greater than 50 % are shown at each node and Agrobacterium tumefaciens NCPPB 2437T (16S rRNA gene accession number D14500) was used as an out-group. Filled circles indicate that the corresponding nodes were also obtained from maximum-parsimony and maximum-likelihood trees. Open circles indicate that the corresponding nodes were also obtained from maximumparsimony or maximum-likelihood trees. Bar, 0.01 substitutions per nucleotide position.
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Table 1. Average percent nucleotide identity values among the genome sequences of strains of species of the genera Sulfitobacter and Roseobacter based on BLASTn analysis Strains: 1, MM-124T; 2, NB-68T; 3, NB-77T; 4, S. donghicola KCTC 12864T; 5, S. guttiformis KCTC 32187T; 6, S. mediterraneus KCTC 32188T; 7, S. pontiacus EE-36; 8, S. pontiacus NAS-14.1; 9, Roseobacter denitrificans OCh 114T; 10, R. litoralis Och-149T.
MM-124 NB-68 NB-77 KCTC 12864 KCTC 32187 KCTC 32188 EE-36 NAS-14.1 OCh-114 Och-149
1
2
3
4
5
6
7
8
9
10
— 75.77 76.43 71.54 71.32 74.26 73.42 73.56 71.33 71.02
75.72 — 75.17 71.18 71.19 73.96 73.15 73.38 71.16 70.67
76.40 75.25 — 71.01 70.85 73.32 72.52 72.44 70.60 70.31
71.69 71.26 71.00 — 74.05 72.03 72.10 71.94 70.21 70.03
71.42 71.05 70.83 73.94 — 71.19 71.45 71.36 69.76 69.45
74.43 74.22 73.43 71.97 71.24 — 73.89 73.80 71.52 71.05
73.36 73.17 72.35 71.90 71.46 73.54 — 96.99 71.67 71.14
73.47 73.13 72.41 71.84 71.45 73.56 97.04 — 71.69 71.26
71.48 71.44 70.74 70.36 69.90 71.52 71.83 71.90 — 87.36
71.12 71.02 70.52 70.17 69.61 71.09 71.38 71.47 87.33 —
GenBank accession numbers: S. donghicola KCTC 12864T, JASF00000000; S. guttiformis KCTC 32187T, JASG00000000; S. mediterraneus KCTC 32188T, JASH00000000. EE-36 and NAS-14.1 are strains of S. pontiacus, which is the type species of the genus (Sorokin, 1995a), and their accession numbers for the draft genome sequences are AALZ01000001–AALZ010000027 and AALV01000001–AALV010000015, respectively. OCh 114 and Och 149 are strains of the species R. denitrificans and R. litoralis, respectively. Accession numbers of the complete genome of R. denitrificans are CP000362, CP000464, CP000465, CP000466, and CP000467. Those of R. litoralis are CP002623 to CP002626.
b-glucosaminidase, a-mannosidase, a-fucosidase, arginine
dihydrolase, urease and protease are negative. Susceptible to tetracycline, polymyxin B, carbenicillin, kanamycin, streptomycin, gentamicin, rifampicin, oleandomycin and neomycin, but not piperacillin, penicillin G, lincomycin, novobiocin, ampicillin, cefalotin, erythromycin, vancomycin or chloramphenicol. The isoprenoid quinone is Q-10. The major fatty acid is C18 : 1v7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The type strain, MM-124T (5KCTC 32124T5JCM 18835T), was isolated from the coastal seawater of Geoje island, Korea. The DNA G+C content of the type strain is 57.84 mol%. Description of Sulfitobacter noctilucae sp. nov. Sulfitobacter noctilucae (noc.ti.lu9cae. N.L. gen. n. of Noctiluca scientific name of the sea sparkle from which the strain was isolated). Cells are Gram-stain-negative, aerobic, rod-shaped and non-motile. Forms convex and slightly yellowish colonies within 3 days on marine agar at 25 uC. Anaerobic growth does not occur on marine agar. Possible growth temperature range is 4–30 uC, and the optimum temperature is 30 uC. Reaches the stationary growth phase after 48 h on marine broth at 30 uC. Cells grow at pH §6.0, and the optimum pH is 7.0–8.0. The NaCl concentration for growth is up to 6 % (w/v). Growth does not occur on L medium, R2A medium, trypticase soy agar or MacConkey agar, regardless of NaCl concentration. Utilizable carbon sources for growth (Biolog, GN2 microplate) are: glycogen, 3764
Tween 80, L-arabinose, L-fucose, D-galactose, a-D-glucose, methyl b-D-glucoside, D-raffinose, L-rhamnose, sucrose, Dtrehalose, xylitol, b-hydroxybutyric acid, DL-lactic acid, propionic acid, D-alanine, L-alanine, L-asparagine, Lornithine, L-threonine, uridine, thymidine, phenylethylamine and a-D-glucose 1-phosphate. Positive for catalase and oxidase, and negative for nitrate reduction, H2S production and indole production. Haemolytic activity and bacteriochlorophyll a are not detected. The results of enzyme assays (API ZYM and 20NE) for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin and a-galactosidase are positive. The results of enzyme assays for alkaline phosphatase, leucine arylamidase, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase, urease and protease are negative. Susceptible to piperacillin, penicillin G, polymyxin B, carbenicillin, kanamycin, ampicillin, streptomycin, gentamicin, rifampicin, oleandomycin, cefalotin, neomycin, erythromycin and vancomycin, but not tetracycline, lincomycin, novobiocin or chloramphenicol. The isoprenoid quinone is Q-10. The major fatty acid is C18 : 1v7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and unknown aminophospholipids. The type strain, NB-68T (5KCTC 32122T5JCM 18833T), was isolated from a region of sea sparkle bloom in Geoje island, Korea. The DNA G+C content of the type strain is 58.33 mol%. International Journal of Systematic and Evolutionary Microbiology 64
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Table 2. Phenotypic characteristics that differentiate MM-124T, NB-68T and NB-77T from other species of the genus Sulfitobacter Strains: 1, MM-124T; 2, NB-68T; 3, NB-77T; 4, S. litoralis KCTC 12521T (Park et al., 2007); 5, S. marinus KCTC 12738T (Yoon et al., 2007a); 6, S. donghicola KCTC 12864T (Yoon et al., 2007b); 7, S. delicatus KCTC 32183T (Ivanova et al., 2004); 8, S. dubius KCTC 32184T (Ivanova et al., 2004); 9, S. pontiacus KCTC 32185T (Sorokin, 1995b); 10, S. brevis KCTC 32186T (Labrenz et al., 2000); 11, S. guttiformis KCTC 32187T (Labrenz et al., 2000); 12, S. mediterraneus KCTC 32188T (Pukall et al., 1999). According to the results obtained in this study, all strains were Gram-stain-negative and oxidase-positive. All were negative for H2S production, indole production, haemolysis activity and bacteriochlorophyll a. All were positive for esterase (C4), lipase (C14) and cytochrome oxidase, and negative for leucine arylamidase, acid phosphatase, b-glucuronidase, a-glucosidase, b-glucosidase, b-glucosaminidase, a-mannosidase, a-fucosidase and arginine dihydrolase. PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine, DPG, diphosphatidylglycerol. +, Positive; 2, negative. NA, data not available. Characteristic
1
2
3
4
Flagellum* + 2 2 + Temperature range for growth* 4–30 4–30 10–30 4–30 NaCl (%, w/v) range for growth* ƒ6 ƒ6 ƒ6 1–10 pH range for growth* §5.5 §6.0 §6.0 5.0–9.0 Nitrate reductionD + ” ” + Enzyme activitiesD Alkaline phosphatase ” ” + ”§ Trypsin + + + +d Naphthol-AS-BI2 2 ” +d phosphohydrolase Urease ” ” + + Major polar lipids* PG, PC, PE PG, PC, PE PG, PC, PE, DPG NA DNA G+C content (mol%)* 57.84 58.33 57.06 NA
5
6
7
8
9
10
11
12
+ 4–35 ƒ12 §5.0 +d
2 10–31 ƒ6 §6.0 ”§
2 12–37 1–8 6.0–10.0 +§
+ 10–30 1–12 6.0–11.0 ”d
+ 4–35 0.5–8 ”
+ 3–33.5 1–8 5.5–9.5 ”§
+ 4–32 1–4 5.3–9 ”d
+ 4–35 0.2–8 6.5–8.5 +d
+§ +d +d
”d ”§ +d
+ + +
+ + +
+ + +
” ” ”
” + +
+ + ”
Ӥ NA
57.8
”§ ” ” PG, PC, PE PG, PC, PE PG, PC, PE 56.9 60.3 63.7
+ NA
61.7–62.5
” ” PG, PC, PE, DPG PG, PC, PE 57.9–58.1 55.0–56.3
” NA
59
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*Except for MM-124T, NB-68T and NB-77T, data for other type strains used as references are from previously published papers. DResults of nitrate reduction and enzyme activities for the reference strains without d or § were not described in the original papers. dResults from this study, which do not confirm the original description. §Results from this study, which confirm the original description.
NA
M.-J. Kwak and others
Description of Sulfitobacter noctilucicola sp. nov. Sulfitobacter noctilucicola (noc.ti.lu.ci9co.la. N.L. n. Noctiluca scientific name of the sea sparkle from which the strain was isolated; L. suff. -cola a dweller; N.L. masc. n. noctilucicola dwelling on Noctiluca). Cells are Gram-stain-negative, aerobic, rod-shaped and non-motile. Forms convex and slightly yellowish colonies within 3 days on marine agar at 25 uC. Anaerobic growth does not occur on marine agar. Possible growth temperature range is 4–30 uC, and optimum temperature is 25 uC. Reaches the stationary growth phase after 48 h on marine broth at 25 uC. Cells grow at pH §6.0, and the optimum pH is 7.0–8.0. The NaCl concentration for growth is up to 6 % (w/v). Growth does not occur on L medium, R2A medium, trypticase soy agar or MacConkey agar, regardless of NaCl concentration. Utilizable carbon sources for growth (Biolog, GN2 microplate) are adonitol, D-arabitol, L-fucose, a-lactose, maltose, trehalose, glucuronamide, Dalanine and phenylethylamine. Positive for catalase and oxidase, and negative for nitrate reduction, H2S production and indole production. Haemolytic activity and bacteriochlorophyll a are not detected. The results of enzyme assays (API ZYM and 20NE) for alkaline phosphatase, urease, esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin and a-galactosidase are positive. The results of enzyme assays for leucine arylamidase, a-chymotrypsin, acid phosphatase, naphtholAS-BI-phosphohydrolase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase and protease are negative. Susceptible to piperacillin, penicillin G, tetracycline, polymyxin B, lincomycin, novobiocin, carbenicillin, kanamycin, ampicillin, streptomycin, gentamicin, rifampicin, oleandomycin, cefalotin, neomycin, erythromycin and vancomycin, but not chloramphenicol. The isoprenoid quinone is Q-10. The major fatty acid is C18 : 1v7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. The type strain, NB-77T (5KCTC 32123T5JCM 18834T), was isolated from a region of sea sparkle bloom in Geoje island, Korea. The DNA G+C content of the type strain is 57.06 mol%.
Acknowledgements This work was made possible through the financial assistance from the National Research Foundation of the Ministry of Science, ICT and Future Planning, Republic of Korea (grant no. NRF-2011-0017670 to J. F. K.). We are grateful to Bernhard Schink for his suggestion to the naming of new taxa.
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isolated from the East Sea, Korea. Int J Syst Evol Microbiol 57, 692– 695. Pukall, R., Buntefuß, D., Fru¨hling, A., Rohde, M., Kroppenstedt, R. M., Burghardt, J., Lebaron, P., Bernard, L. & Stackebrandt, E. (1999).
Sulfitobacter mediterraneus sp. nov., a new sulfite-oxidizing member of the a-Proteobacteria. Int J Syst Bacteriol 49, 513–519. Sasser, M. (1990). Identification of bacteria by gas chromatography of
cellular fatty acids, MIDI Technical Note 101 Newark. DE: MIDI Inc. Skerman, V. B. D. (1967). A Guide to the Identification of the Genera of
Bacteria, with Methods and Digests of Generic Characteristics, 2nd edn. Baltimore, MD: Williams & Wilkins.
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Sorokin, D. Y. (1995b). Sulfitobacter pontiacus gen. nov, sp. nov - a new heterotrophic bacterium from the black-sea, specialized on sulfite oxidation. Microbiology 64, 295–305. Swofford, D. L. (1993). PAUP: a computer-program for phylogenetic inference using maximum parsimony. J Gen Physiol 102, A9. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Yoon, J. H., Kang, S. J. & Oh, T. K. (2007a). Sulfitobacter marinus
sp. nov., isolated from seawater of the East Sea in Korea. Int J Syst Evol Microbiol 57, 302–305. Yoon, J-H., Kang, S-J., Lee, M-H. & Oh, T-K. (2007b). Description of
Sulfitobacter donghicola sp. nov., isolated from seawater of the East Sea in Korea, transfer of Staleya guttiformis Labrenz et al. 2000 to the genus Sulfitobacter as Sulfitobacter guttiformis comb. nov. and emended description of the genus Sulfitobacter. Int J Syst Evol Microbiol 57, 1788–1792.
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DOI 10.1099/ijs.0.065961-0
Sulfitobacter geojensis sp. nov., Sulfitobacter noctilucae sp. nov., and Sulfitobacter noctilucicola sp. nov., isolated from coastal seawater Min-Jung Kwak,1,2 Jung-Sook Lee,3 Keun Chul Lee,3 Kwang Kyu Kim,3 Mi Kyung Eom,3 Byung Kwon Kim2 and Jihyun F. Kim2 Correspondence Jihyun F. Kim [email protected]
1
Biosystems and Bioengineering Program, Korea University of Science and Technology (UST), 217 Gajung-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea
2
Department of Systems Biology and Division of Life Sciences, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749, Republic of Korea
3
Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea
Four Gram-stain-negative, aerobic, rod-shaped bacterial strains, MM-124, MM-126, NB-68 and NB-77, were isolated from the coastal seawater or a region with a bloom of sea sparkle around Geoje island in Korea. The sequence similarity values of the 16S rRNA gene between the isolates and Sulfitobacter mediterraneus DSM 12244T ranged from 97.7 to 98.2 %, and phylogenetic relationships suggested that they belong to a phylogenetic branch that includes the genera Sulfitobacter and Roseobacter. The isoprenoid quinone of all three novel strains was ubiquinone10 and the major fatty acid was cis-vaccenic acid, as in other species of the genus Sulfitobacter. However, there were several differences in the morphological, physiological and biochemical characteristics among the four strains and the reference species of the genus Sulfitobacter. Moreover, the average nucleotide identity values between the three sequenced isolates and the reference strains were below 76.33, indicating that genomic variation exists between the isolates and reference strains. Chemotaxonomic characteristics together with phylogenetic affiliations and genomic distances illustrate that strains MM-124, NB-68 and NB-77 represent novel species of the genus Sulfitobacter, for which the names Sulfitobacter geojensis sp. nov. (type strain MM-124T5KCTC 32124T5JCM 18835T), Sulfitobacter noctilucae sp. nov. (type strain NB-68T5KCTC 32122T5JCM 18833T) and Sulfitobacter noctilucicola sp. nov. (type strain NB-77T5KCTC 32123T5JCM 18834T) are proposed.
The genus Sulfitobacter is a member of the family Rhodobacteraceae, comprising highly abundant heterotrophs found in coastal areas (Buchan et al., 2005). Recently, the Roseobacter clade became one of the most interesting marine bacterial groups because, apart from its abundance and adaptability, its members have diverse roles such as sulfur oxidation, dimethylsulfoniopropionate demethylation, carbon monoxide oxidation and aerobic anoxygenic photosynthesis (Brinkhoff et al., 2008). The genus Sulfitobacter was first isolated as a sulfite oxidizer in 1995 (Sorokin, 1995a), Abbreviation: ANI, average nucleotide identity. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of MM-124, NB-68 and NB-77 are KC428714, KC428716 and KC428717, respectively. The GenBank accession numbers of the genome sequences of MM-124, NB-68 and NB-77 are JASE00000000, JASC00000000 and JASD00000000, respectively. Three supplementary figures and one supplementary table are available with the online version of this paper.
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and received attention due to its ability to produce a sulfite oxidase, which can be used in biosensor systems for detecting sulfite (Muffler & Ulber, 2008). Moreover, bacteria of the genus Sulfitobacter are known to degrade dimethylsulfoniopropionate to dimethylsulfide, which is an essential organic sulfur compound in the sulfur cycle and known as cloud condensation nuclei (Berresheim et al., 1993; Curson et al., 2008; Mou et al., 2005). In this study, we characterized four isolates, strains MM-124, MM-126, NB-68 and NB-77, as potential novel members of the genus Sulfitobacter using a polyphasic taxonomic approach, which included a comparison of whole genome sequences. Strains MM-124, MM-126, NB-68 and NB-77 were isolated from seawater around Geoje island, Korea in May 2012. Water samples for strains MM-124 and MM-126 were collected from the coastal seawater around Geoje island (35u 029 87.80 N 128u 719 47.30 E), and those for strains NB-68 and NB-77 were collected from a region of sea 065961 G 2014 IUMS Printed in Great Britain
Three novel Sulfitobacter species
sparkle bloom (34u 999 30.80 N 128u 679 39.30 E). The seawater samples were spread directly onto marine agar 2216 (BD Difco) and incubated at 16 uC for 5 days.
ml21), oleandomycin (15 mg ml21), cefalotin (30 mg ml21), neomycin (30 mg ml21), erythromycin (15 mg ml21), vancomycin (30 mg ml21) and chloramphenicol (30 mg ml21).
A 27F and 1492R universal primer set was used for PCRs of the small subunit rRNA (annealing 55 uC, 30 s; extension 72 uC, 1.5 min; 30 cycles). PCR products were sequenced and used as queries for BLAST searches at the EzTaxon server (Chun et al., 2007). Phylogenetic analysis was performed using ARB (Ludwig et al., 2004), MEGA5 (Tamura et al., 2011) and PAUP (Swofford, 1993).
For fatty acid analysis, MM-124, MM-126, NB-68, NB-77 and all the reference strains were grown on marine agar at 25 uC for 3 days. Fatty acid methyl esters were extracted according to the standard protocol of the microbial identification system and identified using gas chromatographic analysis (Sasser, 1990). For polar lipid analysis, 10 mg of freeze-dried cells of MM-124, MM-126, NB-68, NB-77 and two reference strains (KCTC 12864T and KCTC 32186T) were prepared. Polar lipids were extracted and analysed by two dimensional TLC, according to a described protocol (Minnikin et al., 1977). For isoprenoid quinone analysis, 100–300 mg of freeze-dried cells of MM-124, MM-126, NB-68, NB-77 and one reference strain (KCTC 32188T) were prepared. Isoprenoid quinones were extracted using chloroform/methanol (2 : 1, v/v), purified by one dimensional TLC and identified by HPLC.
Nine type strains of the genus Sulfitobacter were obtained from the Korean Collection for Type Cultures (KCTC) to be used as references for comparison with the isolates. They were: Sulfitobacter litoralis KCTC 12521T (5Iso 3T), Sulfitobacter marinus KCTC 12738T (5SW-265T), Sulfitobacter donghicola KCTC 12864T (5DSW-25T), Sulfitobacter delicatus KCTC 32183T (5KMM 3584T), Sulfitobacter dubius KCTC 32184T (5KMM 3554T), Sulfitobacter pontiacus KCTC 32185T (5ChLG 10T), Sulfitobacter brevis KCTC 32186T (5EL162T), Sulfitobacter guttiformis KCTC 32187T (5EL-38T) and Sulfitobacter mediterraneus KCTC 32188T (5CH-B427T). All strains were routinely cultured on marine agar or in marine broth at 25 uC. Cell morphology and motility were observed using a light microscope (Carl Zeiss, Axio Lab.A1) and a transmission electron microscope (Philips, CM20). Gram staining was performed using a Gram straining kit (YD Diagnostics) and the motility test was performed by the hanging drop method (Skerman, 1967). Growth at various temperatures (4–45 uC) was observed on marine agar and growth at various NaCl concentrations (2–20 %) and pH 4.5–10 (at 0.5 pH unit intervals; buffered with Na2HPO4 and KH2PO4) was observed on marine agar at 25 uC for 7 days. For testing anaerobic growth, strains were incubated for 4 weeks in an anaerobic chamber. Catalase and oxidase activity were tested using 3 % (v/v) H2O2 and an oxidase reagent dropper (Becton Dickinson), respectively. H2S production and indole production were tested using SIM medium and an indole reagent dropper (Becton Dickinson). Haemolysis was tested on marine agar with 5 % (v/v) defibrinated sheep blood. The presence of bacteriochlorophyll a was checked with a spectrophotometer using the cell lysates of strains representing the genus Sulfitobacter after incubation in marine broth overnight in the dark. Other enzyme activities and the assimilation of substrates were tested using API 20NE and API ZYM (BioMe´rieux). GN2 microplates (Biolog) were used for the oxidation testing of 95 carbon sources with bacterial suspensions in artificial seawater. Antibiotic susceptibility was tested on marine agar using antibiotic susceptibility test discs (Oxoid) with antibiotics at the following concentrations: piperacillin (110 mg ml21), penicillin G (10 U ml21), tetracycline (30 mg ml21), polymyxin B (300 U ml21), lincomycin (15 mg ml21), novobiocin (5 mg ml21), carbenicillin (100 mg ml21), kanamycin (30 mg ml21), ampicillin (10 mg ml21), streptomycin (25 mg ml21), gentamicin (30 mg ml21), rifampicin (30 mg http://ijs.sgmjournals.org
To determine the DNA G+C content and the genome relatedness between strains, we performed paired-end genome sequencing of MM-124, NB-68 and NB-77 using the HiSeq 2000 genome sequencer of the Illumina/Solexa platform (Macrogen). Sequence trimming and de novo assembly were performed with CLC Genomics Workbench 5.1 (CLC bio). Entangled or over-collapsed contigs were resolved and gaps between contigs were filled further manually. The quality of the assembly was assured by a bell-shaped normal distribution of the paired-end reads with a mean insert size of 510 bp. Mapping of the reads to the contigs resulted in a mean of 91.34 % being pair-ended. The draft genome sequences consisted of 5 contigs with 1974-fold coverage for MM-124, 14 contigs with 600-fold coverage for NB-68, and 8 contigs with 730-fold coverage for NB-77. According to the Lander–Waterman model (Lander & Waterman, 1988), the genome coverage of each strain based on sequencing depth should approximate to 100 %. To determine the DNA relatedness between the isolates and strains of other species, we calculated the average nucleotide identity (ANI) values, as described by Goris et al. (2007) and Kim et al. (2014). For this purpose, we also conducted genome sequencing and assembly of three type strains of species of the genus Sulfitobacter. The draft genomes consisted of 8 contigs with 723-fold coverage for S. donghicola KCTC 12864T, 4 contigs with 523-fold coverage for S. guttiformis KCTC 32187T, and 31 contigs with 987-fold coverage for S. mediterraneus KCTC 32188T. MM-124T, MM-126, NB-68T and NB-77T were Gramstain-negative, rod-shaped and obligately aerobic (Fig. S1, available in the online Supplementary Material). They formed slightly yellowish colonies on marine agar within 3 days. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the four strains are affiliated with the genus Sulfitobacter. Among the species of the genus Sulfitobacter, sequence similarities of the 16S rRNA genes 3761
M.-J. Kwak and others
of MM-124T, MM-126, NB-68T and NB-77T were highest with that of S. mediterraneus DSM 12244T (98.1 %, 97.7 %, 97.9 % and 98.2 %, respectively). On the other hand, similarity values of the 16S rRNA gene sequences of MM124T, MM-126, NB-68T and NB-77T with that of Roseobacter denitrificans OCh 114T were below 95.4 %. The similarity value of the 16S rRNA genes between MM-124T and MM126 was 99.4 %, between MM-124T and NB-68T was 98.3 %, between MM-124T and NB-77T was 98.1 %, between MM126 and NB-68T was 98.3 %, between MM-126 and NB-77T was 97.8 %, and between NB-68T and NB-77T was 98.8 %. The 16S rRNA gene sequence similarity of ¢99 % along with identical polar lipid compositions (see below) and almost identical phenotypic characteristics (data not shown) indicated that MM-124T and MM-126 are members of the same species. Phylogenetic trees based on the 16S rRNA gene demonstrated that MM-124T, MM-126, NB-68T and NB-77T form a monophyletic group with all the other species of the genus Sulfitobacter as a single sister clade (Figs 1 and S2). Conventionally, DNA–DNA hybridization experimentation has been used for the comparison of DNA relatedness between two strains. Recently, Goris and colleagues reported a correlation between the percentage DNA–DNA hybridization and ANI values (Goris et al., 2007). A value of 70 % DNA–DNA hybridization, which is the recommended cut-off for species delineation, corresponds to 95 % ANI. Moreover, Kim and colleagues reported the taxonomic consistency between ANI values of whole genomes and similarity values of the 16S rRNA genes (Kim et al., 2014). They observed a distinction in the ANI distribution between intra- and inter-species relationships at around 95 % ANI, and proposed that 98.65 % 16S rRNA gene sequence similarity can be used as the threshold for differentiating two species. Several recent taxonomic papers adopted ANI values instead of percentages of DNA–DNA hybridization for the identification of novel bacterial species (CameloCastillo et al., 2014; Dura´n et al., 2014; Lucena et al., 2014). The ANI values, based on BLASTn between MM-124T, NB68T, NB-77T and other related taxa in genera Sulfitobacter and Roseobacter were below 74.43 % (Table 1), which corresponds to less than 20 % DNA–DNA hybridization (Goris et al., 2007). The ANI values between MM-124T, NB68T, NB-77T and S. mediterraneus DSM 12244T, which is the closest related strain based on 16S rRNA gene sequence similarity, ranged from 73.32 to 74.43 %, whereas the values among the three Geoje isolates ranged from 75.17 to 76.43 %. Consistent with the observation of Kim et al. 2014, the ANI value was 96.99 or 97.04 % between EE-36 and NAS-14.1, which are the strains of S. pontiacus.
nitrate to nitrite, whereas NB68T and NB-77T do not. On the other hand, NB-77T is positive for alkaline phosphatase and urease, while MM124T and NB68T are negative. The major fatty acid of the four isolates was cis-vaccenic acid (C18 : 1v7c) and its proportions were 82.9, 89.6, 86.1 and 87.6 % in MM-124T, MM-126, NB-68T and NB-77T, respectively (Table S1). The isoprenoid quinone was ubiquinone-10 (Q-10) as in other species of the genus Sulfitobacter. The polar lipids of MM-124T and MM-126 comprised phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) (Table 2 and Fig. S3). The polar lipids of NB-68T consisted of PC, PG, PE and unknown aminophospholipids. The polar lipids of NB77T consisted of PC, PG, PE and diphosphatidylglycerol (DPG). The DNA G+C contents of MM-124T, NB-68T and NB-77T were 57.84, 58.33 and 57.06 %, respectively. Their phylogenetic position as a sister group to the other members of the genus Sulfitobacter, chemotaxonomic characteristics similar to other species of the genus Sulfitobacter, and genomic distances equivalent to less than 20 % DNA–DNA hybridization with other species of the genus Sulfitobacter indicated that MM-124T, NB-68T and NB-77T represent novel species of the genus Sulfitobacter, for which the names Sulfitobacter geojensis sp. nov., Sulfitobacter noctilucae sp. nov. and Sulfitobacter noctilucicola sp. nov., respectively, are proposed. Strain MM-126 belongs to the same species as strain MM-124T. Description of Sulfitobacter geojensis sp. nov. Sulfitobacter geojensis (ge.o.jen9sis. N.L. masc. adj. geojensis from Geoje island, the place where the type strain was isolated).
The physiological, biochemical and chemotaxonomic characteristics of MM-124T, MM-126, NB-68T and NB-77T are given in Table 2. Between the isolates, there are at least three different biochemical or physiological properties such as the presence of flagella, the activities of several enzymes such as nitrate reductase, alkaline phosphatase and urease, and the major polar lipids. MM124T has polar flagella and reduces
Cells are Gram-stain-negative, aerobic and rod shaped. Forms convex and slightly yellowish colonies within 3 days on marine agar at 25 uC. Anaerobic growth does not occur on marine agar. Possible growth temperature range is 4– 30 uC, and the optimum temperature is 25 uC. Reaches the stationary growth phase after 24 h on marine broth at 25 uC. Cells grow at pH §5.5, and the optimum is 7.0–8.0. The NaCl concentration for growth is up to 6 % (w/v). Growth does not occur on L medium, R2A medium, typticase soy agar or MacConkey agar, regardless of NaCl concentration. Utilizable carbon sources for growth (Biolog, GN2 microplate) are: adonitol, D-fructose, Dsorbitol, itaconic acid, DL-lactic acid, quinic acid, Lasparagine, L-proline, L-pyroglutamic acid and phenylethylamine. Negative for catalase, H2S production and indole production; positive for oxidase and nitrate reduction. Haemolytic activity and bacteriochlorophyll a are not detected. The results of enzyme assays (API ZYM and 20NE) for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin and agalactosidase are positive. The results of enzyme assays for alkaline phosphatase, leucine arylamidase, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, bgalactosidase, b-glucuronidase, a-glucosidase, b-glucosidase,
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International Journal of Systematic and Evolutionary Microbiology 64
Three novel Sulfitobacter species
Sulfitobacter litoralis Iso 3T (DQ097527) 0.01
Sulfitobacter pontiacus ChLG 10T (Y13155) Sulfitobacter marinus SW-265T (DQ683726) 52
Sulfitobacter brevis Ekho Lake-162T (Y16425) 100
62
Oceanibulbus indoliflex HEL-45T (AJ550939) Sulfitobacter dubius KMM 3554T (AY180102) Sulfitobacter delicatus KMM 3584T (AY180103)
Sulfitobacter porphyrae SCM-1T (AB758574) Sulfitobacter mediterraneus CH-B427T (JASH00000000)
59 56
Sulfitobacter guttiformis Ekho Lake-38T (JASG00000000)
97
Sulfitobacter donghicola DSW-25T (JASF00000000) Sulfitobacter geojensis MM-124T (JASE00000000)
99
99
99
Sulfitobacter geojensis MM-126 (KC428715) Sulfitobacter noctilucae NB-68T (JASC00000000)
93
Sulfitobacter noctilucicola NB-77T (JASD00000000) Roseobacter denitrificans OCh 114T (L01784)
100
86
Roseobacter litoralis ATCC 49566T (X78312) Sagittula stellata E-37T (U58356)
99
Antarctobacter heliothermus EL-219T (Y11552) Phaeobacter inhibens T5T (AY177712)
95
82
Phaeobacter gallaeciensis BS107T (Y13244)
58
Phaeobacter arcticus 20188T (DQ514304) 97
Leisingera aquimarina LMG 24366T (AM900415)
97
Leisingera methylohalidivorans DSM 14336T (AY005463)
50
80
Phaeobacter caeruleus LMG 24369T (AM943630)
87
Phaeobacter daeponensis TF-218T (DQ981486) 64
Ruegeria pomeroyi DSM 15171T (DQ915631) Ruegeria lacuscaerulensis DSM 11314T (DQ915630)
70 89
Ruegeria scottomollicae LMG 24367T (AM905330) 100
Ruegeria pelagia HTCC 2662T (DQ916141) Ruegeria mobilis NBRC 101030T (EU977137)
100
Octadecabacter antarcticus 307T (U14583) Octadecabacter arcticus 238T (U73725)
85
Thalassobius gelatinovorus NBRC 15761T (AB289591)
74 91
Thalassobius mediterraneus CECT 5383T (AJ878874) Thalassobius aestuarii JC2049T (AY442178)
100
Marivita cryptomonadis CL-SK44T (EU512919) Marivita litorea CL-JM1T (EU512918)
Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of type strains showing the phylogenetic position of MM-124T, MM-126, NB-68T, NB-77T and type strains of phylogenetically related taxa [accession numbers of the 16S rRNA gene are in parentheses; accession numbers of type JAS(X)00000000 indicate the draft genome sequences determined in this study]. The phylogenetic tree was reconstructed based on a comparison of 1199 nt and evolutionary distances were calculated using the Jukes–Cantor model. Bootstrap values (percentages of 1000 replications) greater than 50 % are shown at each node and Agrobacterium tumefaciens NCPPB 2437T (16S rRNA gene accession number D14500) was used as an out-group. Filled circles indicate that the corresponding nodes were also obtained from maximum-parsimony and maximum-likelihood trees. Open circles indicate that the corresponding nodes were also obtained from maximumparsimony or maximum-likelihood trees. Bar, 0.01 substitutions per nucleotide position.
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Table 1. Average percent nucleotide identity values among the genome sequences of strains of species of the genera Sulfitobacter and Roseobacter based on BLASTn analysis Strains: 1, MM-124T; 2, NB-68T; 3, NB-77T; 4, S. donghicola KCTC 12864T; 5, S. guttiformis KCTC 32187T; 6, S. mediterraneus KCTC 32188T; 7, S. pontiacus EE-36; 8, S. pontiacus NAS-14.1; 9, Roseobacter denitrificans OCh 114T; 10, R. litoralis Och-149T.
MM-124 NB-68 NB-77 KCTC 12864 KCTC 32187 KCTC 32188 EE-36 NAS-14.1 OCh-114 Och-149
1
2
3
4
5
6
7
8
9
10
— 75.77 76.43 71.54 71.32 74.26 73.42 73.56 71.33 71.02
75.72 — 75.17 71.18 71.19 73.96 73.15 73.38 71.16 70.67
76.40 75.25 — 71.01 70.85 73.32 72.52 72.44 70.60 70.31
71.69 71.26 71.00 — 74.05 72.03 72.10 71.94 70.21 70.03
71.42 71.05 70.83 73.94 — 71.19 71.45 71.36 69.76 69.45
74.43 74.22 73.43 71.97 71.24 — 73.89 73.80 71.52 71.05
73.36 73.17 72.35 71.90 71.46 73.54 — 96.99 71.67 71.14
73.47 73.13 72.41 71.84 71.45 73.56 97.04 — 71.69 71.26
71.48 71.44 70.74 70.36 69.90 71.52 71.83 71.90 — 87.36
71.12 71.02 70.52 70.17 69.61 71.09 71.38 71.47 87.33 —
GenBank accession numbers: S. donghicola KCTC 12864T, JASF00000000; S. guttiformis KCTC 32187T, JASG00000000; S. mediterraneus KCTC 32188T, JASH00000000. EE-36 and NAS-14.1 are strains of S. pontiacus, which is the type species of the genus (Sorokin, 1995a), and their accession numbers for the draft genome sequences are AALZ01000001–AALZ010000027 and AALV01000001–AALV010000015, respectively. OCh 114 and Och 149 are strains of the species R. denitrificans and R. litoralis, respectively. Accession numbers of the complete genome of R. denitrificans are CP000362, CP000464, CP000465, CP000466, and CP000467. Those of R. litoralis are CP002623 to CP002626.
b-glucosaminidase, a-mannosidase, a-fucosidase, arginine
dihydrolase, urease and protease are negative. Susceptible to tetracycline, polymyxin B, carbenicillin, kanamycin, streptomycin, gentamicin, rifampicin, oleandomycin and neomycin, but not piperacillin, penicillin G, lincomycin, novobiocin, ampicillin, cefalotin, erythromycin, vancomycin or chloramphenicol. The isoprenoid quinone is Q-10. The major fatty acid is C18 : 1v7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine. The type strain, MM-124T (5KCTC 32124T5JCM 18835T), was isolated from the coastal seawater of Geoje island, Korea. The DNA G+C content of the type strain is 57.84 mol%. Description of Sulfitobacter noctilucae sp. nov. Sulfitobacter noctilucae (noc.ti.lu9cae. N.L. gen. n. of Noctiluca scientific name of the sea sparkle from which the strain was isolated). Cells are Gram-stain-negative, aerobic, rod-shaped and non-motile. Forms convex and slightly yellowish colonies within 3 days on marine agar at 25 uC. Anaerobic growth does not occur on marine agar. Possible growth temperature range is 4–30 uC, and the optimum temperature is 30 uC. Reaches the stationary growth phase after 48 h on marine broth at 30 uC. Cells grow at pH §6.0, and the optimum pH is 7.0–8.0. The NaCl concentration for growth is up to 6 % (w/v). Growth does not occur on L medium, R2A medium, trypticase soy agar or MacConkey agar, regardless of NaCl concentration. Utilizable carbon sources for growth (Biolog, GN2 microplate) are: glycogen, 3764
Tween 80, L-arabinose, L-fucose, D-galactose, a-D-glucose, methyl b-D-glucoside, D-raffinose, L-rhamnose, sucrose, Dtrehalose, xylitol, b-hydroxybutyric acid, DL-lactic acid, propionic acid, D-alanine, L-alanine, L-asparagine, Lornithine, L-threonine, uridine, thymidine, phenylethylamine and a-D-glucose 1-phosphate. Positive for catalase and oxidase, and negative for nitrate reduction, H2S production and indole production. Haemolytic activity and bacteriochlorophyll a are not detected. The results of enzyme assays (API ZYM and 20NE) for esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin and a-galactosidase are positive. The results of enzyme assays for alkaline phosphatase, leucine arylamidase, a-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase, urease and protease are negative. Susceptible to piperacillin, penicillin G, polymyxin B, carbenicillin, kanamycin, ampicillin, streptomycin, gentamicin, rifampicin, oleandomycin, cefalotin, neomycin, erythromycin and vancomycin, but not tetracycline, lincomycin, novobiocin or chloramphenicol. The isoprenoid quinone is Q-10. The major fatty acid is C18 : 1v7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and unknown aminophospholipids. The type strain, NB-68T (5KCTC 32122T5JCM 18833T), was isolated from a region of sea sparkle bloom in Geoje island, Korea. The DNA G+C content of the type strain is 58.33 mol%. International Journal of Systematic and Evolutionary Microbiology 64
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Table 2. Phenotypic characteristics that differentiate MM-124T, NB-68T and NB-77T from other species of the genus Sulfitobacter Strains: 1, MM-124T; 2, NB-68T; 3, NB-77T; 4, S. litoralis KCTC 12521T (Park et al., 2007); 5, S. marinus KCTC 12738T (Yoon et al., 2007a); 6, S. donghicola KCTC 12864T (Yoon et al., 2007b); 7, S. delicatus KCTC 32183T (Ivanova et al., 2004); 8, S. dubius KCTC 32184T (Ivanova et al., 2004); 9, S. pontiacus KCTC 32185T (Sorokin, 1995b); 10, S. brevis KCTC 32186T (Labrenz et al., 2000); 11, S. guttiformis KCTC 32187T (Labrenz et al., 2000); 12, S. mediterraneus KCTC 32188T (Pukall et al., 1999). According to the results obtained in this study, all strains were Gram-stain-negative and oxidase-positive. All were negative for H2S production, indole production, haemolysis activity and bacteriochlorophyll a. All were positive for esterase (C4), lipase (C14) and cytochrome oxidase, and negative for leucine arylamidase, acid phosphatase, b-glucuronidase, a-glucosidase, b-glucosidase, b-glucosaminidase, a-mannosidase, a-fucosidase and arginine dihydrolase. PG, phosphatidylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine, DPG, diphosphatidylglycerol. +, Positive; 2, negative. NA, data not available. Characteristic
1
2
3
4
Flagellum* + 2 2 + Temperature range for growth* 4–30 4–30 10–30 4–30 NaCl (%, w/v) range for growth* ƒ6 ƒ6 ƒ6 1–10 pH range for growth* §5.5 §6.0 §6.0 5.0–9.0 Nitrate reductionD + ” ” + Enzyme activitiesD Alkaline phosphatase ” ” + ”§ Trypsin + + + +d Naphthol-AS-BI2 2 ” +d phosphohydrolase Urease ” ” + + Major polar lipids* PG, PC, PE PG, PC, PE PG, PC, PE, DPG NA DNA G+C content (mol%)* 57.84 58.33 57.06 NA
5
6
7
8
9
10
11
12
+ 4–35 ƒ12 §5.0 +d
2 10–31 ƒ6 §6.0 ”§
2 12–37 1–8 6.0–10.0 +§
+ 10–30 1–12 6.0–11.0 ”d
+ 4–35 0.5–8 ”
+ 3–33.5 1–8 5.5–9.5 ”§
+ 4–32 1–4 5.3–9 ”d
+ 4–35 0.2–8 6.5–8.5 +d
+§ +d +d
”d ”§ +d
+ + +
+ + +
+ + +
” ” ”
” + +
+ + ”
Ӥ NA
57.8
”§ ” ” PG, PC, PE PG, PC, PE PG, PC, PE 56.9 60.3 63.7
+ NA
61.7–62.5
” ” PG, PC, PE, DPG PG, PC, PE 57.9–58.1 55.0–56.3
” NA
59
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Three novel Sulfitobacter species
*Except for MM-124T, NB-68T and NB-77T, data for other type strains used as references are from previously published papers. DResults of nitrate reduction and enzyme activities for the reference strains without d or § were not described in the original papers. dResults from this study, which do not confirm the original description. §Results from this study, which confirm the original description.
NA
M.-J. Kwak and others
Description of Sulfitobacter noctilucicola sp. nov. Sulfitobacter noctilucicola (noc.ti.lu.ci9co.la. N.L. n. Noctiluca scientific name of the sea sparkle from which the strain was isolated; L. suff. -cola a dweller; N.L. masc. n. noctilucicola dwelling on Noctiluca). Cells are Gram-stain-negative, aerobic, rod-shaped and non-motile. Forms convex and slightly yellowish colonies within 3 days on marine agar at 25 uC. Anaerobic growth does not occur on marine agar. Possible growth temperature range is 4–30 uC, and optimum temperature is 25 uC. Reaches the stationary growth phase after 48 h on marine broth at 25 uC. Cells grow at pH §6.0, and the optimum pH is 7.0–8.0. The NaCl concentration for growth is up to 6 % (w/v). Growth does not occur on L medium, R2A medium, trypticase soy agar or MacConkey agar, regardless of NaCl concentration. Utilizable carbon sources for growth (Biolog, GN2 microplate) are adonitol, D-arabitol, L-fucose, a-lactose, maltose, trehalose, glucuronamide, Dalanine and phenylethylamine. Positive for catalase and oxidase, and negative for nitrate reduction, H2S production and indole production. Haemolytic activity and bacteriochlorophyll a are not detected. The results of enzyme assays (API ZYM and 20NE) for alkaline phosphatase, urease, esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cystine arylamidase, trypsin and a-galactosidase are positive. The results of enzyme assays for leucine arylamidase, a-chymotrypsin, acid phosphatase, naphtholAS-BI-phosphohydrolase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, b-glucosaminidase, a-mannosidase, a-fucosidase, arginine dihydrolase and protease are negative. Susceptible to piperacillin, penicillin G, tetracycline, polymyxin B, lincomycin, novobiocin, carbenicillin, kanamycin, ampicillin, streptomycin, gentamicin, rifampicin, oleandomycin, cefalotin, neomycin, erythromycin and vancomycin, but not chloramphenicol. The isoprenoid quinone is Q-10. The major fatty acid is C18 : 1v7c. The major polar lipids are phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. The type strain, NB-77T (5KCTC 32123T5JCM 18834T), was isolated from a region of sea sparkle bloom in Geoje island, Korea. The DNA G+C content of the type strain is 57.06 mol%.
Acknowledgements This work was made possible through the financial assistance from the National Research Foundation of the Ministry of Science, ICT and Future Planning, Republic of Korea (grant no. NRF-2011-0017670 to J. F. K.). We are grateful to Bernhard Schink for his suggestion to the naming of new taxa.
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