International Journal of Systematic and Evolutionary Microbiology (2014), 64, 1729–1735
DOI 10.1099/ijs.0.057059-0
Jeotgalibaca dankookensis gen. nov., sp. nov., a member of the family Carnobacteriaceae, isolated from seujeot (Korean traditional food) Dong-Geol Lee,1 Martha E. Trujillo,2 Heecheol Kang3 and Tae-Young Ahn1 Correspondence
1
Tae-Young Ahn
2
[email protected]
Department of Microbiology, Dankook University, Cheonan 330-714, Republic of Korea Departamento de Microbiologı´a y Gene´tica, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
3
604 GFC, B-Dong, Digital-Empire 980-3, Yeongtong-Dong, Yeongtong-Gu, Suwon-City, Gyeonggi-Do 443-720, Republic of Korea
A novel, Gram-stain-positive bacterium, designated strain EX-07T, was isolated from seujeot (Korean traditional food). The strain was aerobic, halotolerant and non-motile; it formed cocci that grouped into tetrads and sarcinae or formed irregular conglomerates. Growth occurred at pH 7–9, at 10–37 6C and with up to 9 % NaCl. Isolate EX-07T was catalase- and oxidase-negative and used sugars and organic acids as carbon sources. The 16S rRNA gene sequence of the novel strain showed 94.2–94.5 % similarity with the type strains of Trichococcus pasteurii, Trichococcus patagoniensis, Trichococcus collinsii, Trichococcus flocculiformis and Trichococcus palustris and only 92.2 % with representatives of the genera Bavariicoccus, Carnobacterium and Granulicatella. Sequence similarities based on the groEL gene ranged from 81.3 to 82.8 % between the novel isolate and the type strains of all species of the genus Trichococcus, and only 74.2 and 75.3 % with type strains of members of the genera Bavariicoccus and Granulicatella, respectively. The G+C content of the genomic DNA was 39.6 mol%. The predominant fatty acids were C16 : 1v9c, C18 : 1v9c, C16 : 0 and C14 : 0. The polar lipid profile was very complex and included phosphatidylethanolamine and several unidentified aminolipids, glycolipids and phospholipids. Based on the genotypic and phenotypic results obtained in this study, it is proposed that isolate EX-07T represents a novel species of a new genus in the family Carnobacteriaceae for which the name Jeotgalibaca dankookensis gen. nov., sp. nov. is proposed. The type strain of Jeotgalibaca dankookensis is EX-07T (5KCCM 90229T5JCM 19215T).
Jeotgal or jeot, a traditional Korean salted and fermented food, is made by adding 20–30 % (w/w) salt to various types of seafood. Seujeot is a type of jeotgal, the name deriving from the Korean words seu (shrimp) and jeot. Among other seafoods, salted shrimp (25 % w/v), seawater and other ingredients are used for its preparation and subsequent fermentation. Salt-resistant aerobic and anaerobic bacteria
exist in most jeotgals (Lee, 1993) and recent studies have reported the isolation of many novel species and genera from this food (Yoon et al., 2003). This paper describes the isolation and characterization of strain EX-07T isolated from seujeot at Cheon-an Dankook University, South Korea. According to the results obtained it is proposed that the strain represents a novel species of a new genus within the family Carnobacteriaceae (Ludwig et al., 2009).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and groEL sequences of strain EX-07T are GU317945 and KJ160234, respectively. The GenBank/EMBL/DDBJ accession numbers for the groEL sequences of Trichococcus patagoniensis PmagG1T, Bavariicoccus seileri WCC 4188T, Trichococcus collinsii 37AN3*T, Trichococcus flocculiformis DSM 2094T, Trichococcus palustris DSM 9172T and Trichococcus pasteurii KoTa2T are KJ160232–KJ160233, and KJ160235–KJ160238, respectively.
Members of the family Carnobacteriaceae, which, at the time of writing, comprises 17 genera, are Gram-stain-positive rods or cocci that do not form endospores and may be motile. They are usually facultatively anaerobic, but some species grow aerobically or microaerophilically. The diamino acids lysine, ornithine or meso-diaminopimelic acid may be present in the cell wall. These bacteria may be psychrotolerant, halotolerant or alkaliphilic (Ludwig et al., 2009).
Five supplementary figures are available with the online version of this paper.
057059 G 2014 IUMS
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The genomic DNA of strain EX-07T was extracted using the InstaGene Matrix kit (Bio-Rad). PCR amplification (BioRad C1000) of the 16S rRNA gene was carried out using the universal primer pair 27F–1492R (Lane, 1991). The sequence reactions were performed on an ABI PRISM 3730XL analyser (Applied Biosystems) using the BigDye terminator v3.0 cycle sequencing kit as supplied by the manufacturer (Applied Biosystems). Identification of phylogenetic neighbours were identified and pairwise 16S rRNA gene sequence similarities were calculated using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). The sequences of strain EX-07T and related taxa were retrieved from the GenBank database and aligned using the BioEdit software (Hall, 1999). Phylogenetic distances were calculated with Kimura’s two-parameter model (Kimura, 1980) and evolutionary trees based on partial deletion were inferred using the maximum-parsimony (Fitch, 1971), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood methods (Felsenstein, 1981). These analyses were performed using the software package MEGA5 (Tamura et al., 2011). Bootstrap analysis (1000 replications) was performed to determine the stability of the branches. Amplification and sequencing of the groEL gene were performed following the procedure described by Goh et al. (1996). Phylogenetic analyses were performed as described for the 16S rRNA gene (see above). All groEL sequences were obtained in this study except that of Granulicatella adiacens, which was retrieved from the GenBank database.
The sampled seujeot had a pH of 7.3 and a total aerobic microbial cell count of 1.66103 c.f.u. ml21. For isolation, serially diluted seujeot samples were spread onto the following agars (all from Difco): nutrient agar (NA), R2A agar, trypticase soy agar (TSA), marine agar (MA) and potato dextrose agar (PDA). Plates were incubated at 25 uC for 5 days under aerobic conditions after which colonies were formed on TSA. One colony was picked and subcultured on modified TSA (per litre distilled water: 17.0 g tryptone, 3.0 g soytone, 2.5 g glucose, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate; pH 7.3). Cell morphology of strain EX-07T grown on TSA for 5 days at 25 uC was observed at 61000 magnification with an Olympus microscope (GX71). Motility was investigated by using the hanging-drop technique with fresh cells grown in trypticase soy broth (Bernardet et al., 2002). Electron microscopy samples were prepared as described by Lee et al. (2009). Cells were then critically point dried in CO2 and sputtered with gold (SC502; Polaron) and the sample was observed with a scanning electron microscope (Hitachi S4300N; installed in the Korea Basic Science Institute). The Gram-reaction was determined using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Strain EX-07T stained Gram-positive and consisted of non-motile, non-spore-forming, coccus-shaped cells which grouped into tetrads, sarcinae and conglomerates (Fig. S1, available in the online Supplementary Material). This cell arrangement differed from that reported for strains of the genus Trichococcus (Table 1). Isolate EX-07T did not form chains as reported for members of the genus Granulicatella (Collins & Lawson, 2000).
The partial 16S rRNA gene sequence of strain EX-07T obtained had a length of 1447 bp. Sequence similarity
Table 1. Differential characteristics between strain EX-07T and the type species of phylogenetically related genera Taxa: 1, strain EX-07T; 2, Trichococcus flocculiformis; 3, Bavariicoccus seileri; 4, Granulicatella adiacens; 5, Atopobacter phocae. All data are from the present study except as noted. ND, No data; 2, not detected. Characteristic Cell morphology
Oxygen requirement Maximum salt tolerance (%) Major cellular fatty acids Polar lipids* Lipids Glycolipids Aminolipids Phospholipids Peptidoglycan type DNA G+C content (mol%)
1
2
3
4
5
Cocci, tetrads, sarcinae
Cocci
Cocci; single, pairs or short chains
Rods
Aerobic
Spherical to ovoid, olive-shaped; single or paired cells Aerotolerant
Aerotolerant
Facultatively anaerobic
Facultatively anaerobic
9 C16 : 1v9c, C18 : 1v9c, C16 : 0, C14 : 0
1 C16 : 1, C18 : 1v9c, C16 : 0,
11 C16 : 0, C18 : 1v9c
ND
ND
C16 : 0, C18 : 1v9c
C16 : 0, C18 : 1v9c
3 (L1, 2, 3) 3 (GL1, 2, 3) 2 (AL1, 2) 1 (PL1) A4a (L-Lys–D-Glu) 39.6
2 1 (GL1) 3 (AL1, 2, 3) 3 (PL1, 2, 3,) A4a (L-Lys–D-Asp) 47–49
4 (L1, 2, 3, 4) 2 (GL1, 2) 3 (AL1, 2, 3) 3 (PL1, 2, 3) A4a (L-Lys–D-Asp) 38–39
2 5 (GL1, 2, 3, 4, 5) 2 2 A3a 36–37.5
5 (L1, 3, 5, 6, 7) 5 (GL1, 2, 3, 4, 5) 2 3 (PL1, 2, 3) A4b ND
*Polar lipid data for strain EX-07T and T. flocculiformis DSM 2094T obtained in this study. Data for B. seileri, G. adiacens and A. phocae obtained from Schmidt et al. (2009). 1730
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Jeotgalibaca dankookensis gen. nov., sp. nov.
calculations indicated that strain EX-07T is a member of the phylum Firmicutes and belongs to the family Carnobacteriaceae, with Trichococcus patagoniensis PmagG1T (94.5 % similarity), Trichococcus pasteurii KoTa2T (94.5 %), Trichococcus collinsii 37AN3*T (94.4 %), Trichococcus flocculiformis DSM 2094T (94.4 %) and Trichococcus palustris DSM 9172T (94.1 %) being the closest cultured relatives. Members of the remaining genera in the family Carnobacteriaceae showed less than 92.2 % 16S rRNA gene sequence similarity. A BLAST search against the NCBI database yielded a list of over 100 uncultured bacterial clones with 96–99 % 16S rRNA gene sequence similarity to strain EX-07T that have been detected in diverse environments. The phylogenetic tree reconstructed with the maximum-likelihood method and Kimura’s two-parameter model showed that strain EX07T was recovered in an independent branch that joined the cluster formed by the five species of the genus Trichococcus (Fig. 1; see also Fig. S2). This relationship was supported by a bootstrap value of 98 %. Additional trees based on the neighbour-joining and maximum-parsimony methods also supported this topology (Figs S3 and S4). Levels of DNA sequence similarity of the groEL DNA contig (465 bp) of strain EX-07T were 81.3–82.8 % when compared with the type strains of all species of the genus Trichococcus and less than 76 % with the type strains of species of the genera Bavariicoccus and Granulicatella. Fig. 2 shows the phylogenetic relationship between strain EX07T and its closest relatives based on groEL sequences.
100 98 52 88
98
99
Analysis of the cell-wall peptidoglycan was performed by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen following the methods of Schleifer & Kandler (1972) and Schleifer (1985). Isolate EX-07T contained the amino acids lysine, glutamic acid, aspartic acid and alanine, indicating a peptidoglycan type A4a with an interpeptide bridge L-Lys–D-Glu (A11.33, Schumann 2011). A peptidoglycan type A4a has also been reported for the genera Bavariicoccus and Trichococcus, although strains of members of these genera have an interpeptide bridge L-Lys–D-Asp (A11.31, Schumann
Trichococcus flocculiformis DSM 2094T (AJ306611) Trichococcus pasteurii KoTa2T (X87150) Trichococcus palustris DSM 9172T (AJ296179) Jeotgalibaca dankookensis EX-07T (GU317945) Granulicatella adiacens ATCC 49175T (AY123705) Atopobacter phocae CCUG 42358T (FM177903) Bavariicoccus seileri WCC 4188T (FM177901) Vagococcus fluvialis CCUG 32704T (Y18098) Carnobacterium divergens DSM 20623T (M58816) Isobaculum melis M577-94T (AJ302648)
59
Desemzia incerta ATCC 8363T (Y17300) Pisciglobus halotolerans C01T (GU459067) Lacticigenium naphtae MIC1-18T (AB430339) Atopococcus tabaci CCUG 48253T (AJ34917) Alkalibacterium olivapovliticus WW2-SN4aT (AF143511) Marinilactibacillus psychrotolerans M13-2T (AB083406)
56
99
98 92
63
To measure the G+C content, the genomic DNA of strain EX-07T was extracted and purified as described by Price et al. (1978) and enzymically degraded to nucleotides. The DNA G+C content was determined as described by Mesbah et al. (1989), using reversed-phase HPLC. The value of 39.6 mol% obtained was different from that reported for members of the genus Trichococcus (45–49 mol%, Liu et al., 2002), but similar to the values reported for members of the genera Bavariicoccus (38 mol%, Schmidt et al., 2009) and Granulicatella (36–37 mol%, Collins & Lawson, 2000).
93 Trichococcus patagoniensis PmagG1T (AF394926) T 87 Trichococcus collinsii 37AN3* (AJ306612)
0.01
98
The novel isolate formed an independent branch supported by a 100 % bootstrap value and confirmed the moderate relationship with all members of the genus Trichococcus. The combined results of the 16S rRNA gene and groEL sequence analyses support the classification of strain EX07T within a new genus.
100
Alloiococcus otitis 7760T (X59765) Dolosigranulum pigrum R91/1468T (X70907) Allofustis seminis 01-570-1T (AJ410303) Atopostipes suicloacalis PPC79T (AF445248)
Fig. 1. Maximum-likelihood tree based on 16S rRNA gene sequences of strain EX-07T and related taxa. Evolutionary distances were computed using Kimura’s two-parameter method. Bootstrap values .50 % are indicated. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org
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T 100 Trichococcus palustris DSM 9172 (KJ160237)
0.05
81
Trichococcus pasteurii KoTa2T (KJ160238) Trichococcus collinsii 37AN3*T (KJ160235)
100 100
87
Trichococcus flocculiformis DSM 2094T (KJ160236) Trichococcus patagoniensis PmagG1T (KJ160232) Jeotgalibaca dankookensis EX-07T (KJ160234)
100
Granulicatella adiacens ATCC 49175T (AY123705) Bavariicoccus seileri WCC 4188T (KJ160233)
Fig. 2. Maximum-likelihood tree based on groEL sequences of strain EX-07T and related taxa. Evolutionary distances were computed using Kimura’s two-parameter method. Bootstrap values .50 % are indicated. Bar, 0.05 substitutions per nucleotide position.
2011). A peptidoglycan type A3a is reported for the genus Granulicatella (Schmidt et al., 2009). Strain EX-07T and the type strains of recognized species of the genus Trichococcus were analysed for their polar lipid composition. Cells were cultivated on TSA and R2A agar media, respectively, for 72 h at 30 uC. Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Komagata & Suzuki, 1987). As with other members of the family Carnobacteriaceae, the major polar lipid profile of strain EX-07T was rather complex and included phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and the following unidentified lipids: three glycolipids, two aminolipids, one phospholipid and three lipids. This profile showed several differences when compared with the profiles obtained for species of the genus Trichococcus as listed in Table 1 (Fig. S5). The polar lipid pattern of strain EX-07T also differed from that of Granulicatella adiacens and Bavariicoccus seileri based on the number of unknown lipids or the presence of glycolipids (Schmidt et al., 2009). Preparation of quinones was performed as described by Altenburger et al. (1996) but these cellular compounds were not detected. Similar results were reported for B. seileri (Schmidt et al., 2009).
The overall fatty acid profile of isolate EX-07T is given in Table 2 with C16 : 1v9c (35.1 %), C18 : 1v9c (26.1 %), C16 : 0 (18.5 %) and C14 : 0 (6.9 %) being the major components. This pattern differed from the general profile presented by species of the genus Trichococcus, especially in the amount of C16 : 1v9c which was either absent or present in minor amounts (,2 %, Table 2). The fatty acids C16 : 0 and
Table 2. Fatty acid composition of strain EX-07T and type strains of species of the genus Trichococcus, Granulicatella adiacens and Bavariicoccus seileri Strains: 1, EX-07T; 2, T. patagoniensis PmagG1T; 3, T. pasteurii KoTa2T; 4, T. collinsii 37AN3*T; 5, T. flocculiformis DSM 2094T; 6, T. palustris DSM 9172T; 7, G. adiacens ATCC 49175T; 8, B. seileri DSM 19936T. Fatty acids that amount to ,1.0 % of the total fatty acids in all strains are not shown. 2, Not detected. All data are from the present study except for B. seileri (Schmidt et al., 2009). Fatty acid C10 : 0 C12 : 0 C14 : 0 C16 : 0 C16 : 1 C18 : 0 C16 : 1v9c C18 : 1v7c C18 : 1v9c C20 : 1v9c Summed features* 3 5
1
2
3
4
5
1.4 2 2 2 2 1.3 2.0 1.4 4.8 1.2 6.9 12.1 18.0 42.9 10.7 18.5 16.3 13.0 23.1 18.2 2 2 2 20.6 41.4 2.4 3.2 1.1 0.4 5.3 35.1 2 2 2.1 1.0 2 2 2 2 2 26.1 24.7 20.3 4.2 17.1 1.0 2 2 2 2
6
7
8
2 2 17.9 11.4 24.1 2.2 1.0 1.0 19.5 2
2 2 21.0 15.0 20.0 4.0 2 2.0 22.0 2
2 0.3 4.3 21.3 2 9.0 6.3 2 53.0 1.6
1.2 2
2 2
2 2
The fatty acid profiles of isolate EX-07T, the type strains of all species of the genus Trichococcus and G. adiacens ATCC 49175T were determined under identical conditions. Cells were grown on TSA for 5 days at 25 uC and collected from the third streak quadrant to obtain cells of the same physiological age. Samples were saponified, methylated and extracted according to the standard protocol of the Sherlock Microbial Identification System (Sasser, 1990). The fatty acids were analysed by GC (model 6890; Hewlett Packard) and identified using the TSBA40 database of the Microbial Identification software package (MIDI, Version 4.5). All experiments were performed in triplicate.
*Summed features represent groups of two or more fatty acids that could not be separated by the Microbial Identification System. Summed feature 3 comprises C16 : 1v6c and/or C16 : 1v7c; summed feature 5 comprises C18 : 2v6,9c or anteiso-C18 : 0.
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5.5 40.6 45.0 2 2 2
0.8 2
0.9 4.2
Jeotgalibaca dankookensis gen. nov., sp. nov.
Table 3. Differential phenotypic characteristics between strain EX-07T and related phylogenetic neighbours Strains: 1, EX-07T; 2, T. patagoniensis PmagG1T; 3, T. pasteurii KoTa2T, 4, T. collinsii 37AN3*T, 5, T. flocculiformis DSM 2094T; 6, T. palustris DSM 9172T; 7, G. adiacens ATCC 49175T; 8, B. seileri DSM 19936T. +, Positive; 2, negative; ND, not determined. All data are from the present study except for G. adiacens (Collins & Lawson, 2000) and B. seileri (Schmidt et al., 2009). Characteristic Pigmentation Production of: Chymotrypsin Tyrosinase Amylase Esterase b-Galactosidase Growth at 37 uC Minimum growth temperature (uC) Growth on: D-Ribose D-Galactose Trehalose D-Arabinose Arbutin Sucrose Inosine D-Gluconic acid
1
2
3
4
5
6
7
8
Yellow–orange
White–pink
White
White
White–opaque
White
None
None
+ 2 2 2 + + 10
2 + + + 2 2 25
2 2 2 + + 2 0
2 2 2 + 2 2 25
2 2 2 2 2 + 4
2 2 2 2 2 + 0
ND
ND
ND
ND
+ + + 2 + 2 + +
+ 2 2 + + + + 2
2 + + 2 2 + 2 2
+ + + 2 2 + 2 2
2 + + 2 + + 2 2
2 2 2 + + + 2 2
C18 : 1v9c have also been reported as major components of the genera Bavariicoccus and Granulicatella (Schmidt et al., 2009). Strain EX-07T and type strains of all species of the genus Trichococcus were studied in parallel for the following phenotypic characteristics: growth at 4, 10, 25, 30, 37 and 40 uC and at pH 4.5–8.5 (at 0.5 pH unit intervals). The pH of the medium was adjusted as described by Qu & Yuan (2008). Tolerance to salinity was tested in trypticase soy broth supplemented with 0.5–20.0 % (w/v) NaCl at 0.5 % intervals. All results were recorded after 7 days of incubation. Growth on NA, R2A agar, MA and PDA was evaluated at 25 uC for 7 days. Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2, and oxidase activity was determined using 1 % (w/v) tetramethyl phenylenediamine. Tests in the commercial systems GN2 MicroPlates (Biolog), API 20NE, API 50CH, API ID32 GN and API ZYM (bioMe´rieux) were performed in triplicate according to the manufacturers’ instructions. The API ZYM tests were read after 4 h of incubation at 28 uC and the other API tests were read after 48 h at 28 uC. Anaerobic growth was tested in serum bottles by adding sodium thioglycollate (1 g l21) to trypticase soy broth and substituting the upper airspace with nitrogen gas. Tests for the degradation of DNA, casein, chitin, starch, Tween 80 (Kouker & Jaeger, 1987) and carboxymethylcellulose (Ten et al., 2004) were performed and evaluated after 5 days of incubation at 28 uC. Good growth was obtained on TSA but not on NA, PDA or R2A agar, whereas it grew weakly on half-strength MA. The strain was heterotrophic and aerobic; no anaerobic growth http://ijs.sgmjournals.org
2
2
ND
ND
2 + .10
2 + 10
2
+
2 + + 2 + 2
ND
ND
ND
ND
ND
2 2 ND
was detected under the conditions tested. Unlike species of the genus Trichococcus, strain Ex-07T utilized D-gluconic acid as a carbon source and showed chymotrypsin activity. Other physiological characteristics of strain EX-07T are summarized in the species description while comparison of selected characteristics with those of related phylogenetic species is given in Table 3. The results of 16S rRNA gene and groEL sequencing studies, polar lipid profiles, peptidoglycan type, cellular fatty acid composition and DNA G+C content showed that strain EX-07T is only moderately related to the genera Trichococcus, Granulicatella and Bavariicoccus. On the basis of genotypic and phenotypic data, strain EX-07T is inferred to represent a novel species of a new genus in the family Carnobacteriaceae, for which the name Jeotgalibaca dankookensis gen. nov., sp. nov. is proposed. Description of Jeotgalibaca gen. nov. Jeotgalibaca [Je.ot.ga.li.ba9ca. N.L. n. jeotgalum (from Korean n. jeotgal) jeotgal, traditional Korean food; L. fem. n. baca a grain or berry, and in bacteriology a coccus; N.L. fem. n. Jeotgalibaca, coccus from jeotgal]. Cells are Gram-stain-positive cocci which group in tetrads, sarcinae or irregular conglomerates. Non-spore-forming and non-motile. Aerobic and chemoheterotrophic. Oxidaseand catalase-negative. The major fatty acids are C16 : 1v9c, C18 : 1v9c, C16 : 0 and C14 : 0. Polar lipids include phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and several unknown glycolipids, aminolipids and 1733
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phospholipids. Peptidoglycan type A4a. Phylogenetically positioned in the family Carnobacteriaceae, phylum Firmicutes. The type species is Jeotgalibaca dankookensis.
gen. nov., Granulicatella adiacens comb. nov., Granulicatella elegans comb. nov. and Granulicatella balaenopterae comb. nov. Int J Syst Evol Microbiol 50, 365–369. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
Description of Jeotgalibaca dankookensis sp. nov. Jeotgalibaca dankookensis (dan.ko.ok9en.sis. N.L. fem. adj. dankookensis of or belonging to Dankook University). Presents the following properties in addition to those given in the genus description. Cells are 1.0–1.3 mm in diameter. Good growth occurs on TSA but not on NA, PDA or R2A agar; weak growth occurs on one-tenth-strength MA. Colonies on TSA are circular, convex and pale orange. Growth occurs at 10–37 uC (optimum, 28 uC), at pH 7.0– 9.0 and on TSA supplemented with up to 9 % NaCl. Nitrate is not reduced. DNA, casein, chitin, starch, Tween 80 and carboxymethylcellulose are not degraded. Results based on the commercial systems Biolog GN2, API 20NE, API 50CH and API ID32 GN are as follows: positive for assimilation of D-ribose, D-glucose, D-fructose, D-galactose, maltose, Dmannose, arbutin and salicin; positive for aesculin hydrolysis and b-galactosidase; negative for indole production, glucose fermentation and urease. Assimilates (Biolog GN2) D-fructose, D-galactose, D-glucose, D-psicose, trehalose, D-mannose, maltose, N-acetylglucosamine, pyruvic acid, D-gluconic acid, a-ketobutyric acid, inosine, uridine, thymidine and glycerol. According to the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), chymotrypsin, acid phosphatase and naphthol-AS-BIphosphohydrolase activities are present; N-acetyl-b-glucosaminidase, b-glucosidase, a-fucosidase, leucine arylamidase, lipase (C14), valine arylamidase, cystine arylamidase, a-glucosidase, a-galactosidase, b-glucuronidase, b-glucosidase, a-mannosidase and trypsin activities are absent. The type strain, EX-07T (5KCCM 90229T5JCM 19215T), was isolated from seujeot (traditional Korean food) in Cheon-an Dankook University, South Korea. The DNA G+C content of the type strain is 39.6 mol%.
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of Trichococcus collinsii sp. nov., and reclassification of Lactosphaera pasteurii as Trichococcus pasteurii comb. nov. and of Ruminococcus palustris as Trichococcus palustris comb. nov. in the low-G+C grampositive bacteria. Int J Syst Evol Microbiol 52, 1113–1126. Ludwig, W., Schleifer, K.-H. & Whitman, W. (2009). Family III.
Acknowledgements This work was supported by the Small & Medium Business Administration, Republic of Korea.
Carnobacteriaceae fam. nov. In Bergey’s Manual of Systematic Bacteriology, vol. 3, p. 549. Edited by P. de Vos, G. M. Garrity, D. Jones, N. R. Krieg, W. Ludwig, F. A. Rainey, K.-H. Schleifer & W. B. Whitman. New York: Springer. Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
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Schmidt, V. S., Mayr, R., Wenning, M., Glo¨ckner, J., Busse, H. J. & Scherer, S. (2009). Bavariicoccus seileri gen. nov., sp. nov., isolated
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http://ijs.sgmjournals.org
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DOI 10.1099/ijs.0.057059-0
Jeotgalibaca dankookensis gen. nov., sp. nov., a member of the family Carnobacteriaceae, isolated from seujeot (Korean traditional food) Dong-Geol Lee,1 Martha E. Trujillo,2 Heecheol Kang3 and Tae-Young Ahn1 Correspondence
1
Tae-Young Ahn
2
[email protected]
Department of Microbiology, Dankook University, Cheonan 330-714, Republic of Korea Departamento de Microbiologı´a y Gene´tica, Edificio Departamental, Campus Miguel de Unamuno, Universidad de Salamanca, 37007 Salamanca, Spain
3
604 GFC, B-Dong, Digital-Empire 980-3, Yeongtong-Dong, Yeongtong-Gu, Suwon-City, Gyeonggi-Do 443-720, Republic of Korea
A novel, Gram-stain-positive bacterium, designated strain EX-07T, was isolated from seujeot (Korean traditional food). The strain was aerobic, halotolerant and non-motile; it formed cocci that grouped into tetrads and sarcinae or formed irregular conglomerates. Growth occurred at pH 7–9, at 10–37 6C and with up to 9 % NaCl. Isolate EX-07T was catalase- and oxidase-negative and used sugars and organic acids as carbon sources. The 16S rRNA gene sequence of the novel strain showed 94.2–94.5 % similarity with the type strains of Trichococcus pasteurii, Trichococcus patagoniensis, Trichococcus collinsii, Trichococcus flocculiformis and Trichococcus palustris and only 92.2 % with representatives of the genera Bavariicoccus, Carnobacterium and Granulicatella. Sequence similarities based on the groEL gene ranged from 81.3 to 82.8 % between the novel isolate and the type strains of all species of the genus Trichococcus, and only 74.2 and 75.3 % with type strains of members of the genera Bavariicoccus and Granulicatella, respectively. The G+C content of the genomic DNA was 39.6 mol%. The predominant fatty acids were C16 : 1v9c, C18 : 1v9c, C16 : 0 and C14 : 0. The polar lipid profile was very complex and included phosphatidylethanolamine and several unidentified aminolipids, glycolipids and phospholipids. Based on the genotypic and phenotypic results obtained in this study, it is proposed that isolate EX-07T represents a novel species of a new genus in the family Carnobacteriaceae for which the name Jeotgalibaca dankookensis gen. nov., sp. nov. is proposed. The type strain of Jeotgalibaca dankookensis is EX-07T (5KCCM 90229T5JCM 19215T).
Jeotgal or jeot, a traditional Korean salted and fermented food, is made by adding 20–30 % (w/w) salt to various types of seafood. Seujeot is a type of jeotgal, the name deriving from the Korean words seu (shrimp) and jeot. Among other seafoods, salted shrimp (25 % w/v), seawater and other ingredients are used for its preparation and subsequent fermentation. Salt-resistant aerobic and anaerobic bacteria
exist in most jeotgals (Lee, 1993) and recent studies have reported the isolation of many novel species and genera from this food (Yoon et al., 2003). This paper describes the isolation and characterization of strain EX-07T isolated from seujeot at Cheon-an Dankook University, South Korea. According to the results obtained it is proposed that the strain represents a novel species of a new genus within the family Carnobacteriaceae (Ludwig et al., 2009).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and groEL sequences of strain EX-07T are GU317945 and KJ160234, respectively. The GenBank/EMBL/DDBJ accession numbers for the groEL sequences of Trichococcus patagoniensis PmagG1T, Bavariicoccus seileri WCC 4188T, Trichococcus collinsii 37AN3*T, Trichococcus flocculiformis DSM 2094T, Trichococcus palustris DSM 9172T and Trichococcus pasteurii KoTa2T are KJ160232–KJ160233, and KJ160235–KJ160238, respectively.
Members of the family Carnobacteriaceae, which, at the time of writing, comprises 17 genera, are Gram-stain-positive rods or cocci that do not form endospores and may be motile. They are usually facultatively anaerobic, but some species grow aerobically or microaerophilically. The diamino acids lysine, ornithine or meso-diaminopimelic acid may be present in the cell wall. These bacteria may be psychrotolerant, halotolerant or alkaliphilic (Ludwig et al., 2009).
Five supplementary figures are available with the online version of this paper.
057059 G 2014 IUMS
Printed in Great Britain
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D.-G. Lee and others
The genomic DNA of strain EX-07T was extracted using the InstaGene Matrix kit (Bio-Rad). PCR amplification (BioRad C1000) of the 16S rRNA gene was carried out using the universal primer pair 27F–1492R (Lane, 1991). The sequence reactions were performed on an ABI PRISM 3730XL analyser (Applied Biosystems) using the BigDye terminator v3.0 cycle sequencing kit as supplied by the manufacturer (Applied Biosystems). Identification of phylogenetic neighbours were identified and pairwise 16S rRNA gene sequence similarities were calculated using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). The sequences of strain EX-07T and related taxa were retrieved from the GenBank database and aligned using the BioEdit software (Hall, 1999). Phylogenetic distances were calculated with Kimura’s two-parameter model (Kimura, 1980) and evolutionary trees based on partial deletion were inferred using the maximum-parsimony (Fitch, 1971), neighbour-joining (Saitou & Nei, 1987) and maximum-likelihood methods (Felsenstein, 1981). These analyses were performed using the software package MEGA5 (Tamura et al., 2011). Bootstrap analysis (1000 replications) was performed to determine the stability of the branches. Amplification and sequencing of the groEL gene were performed following the procedure described by Goh et al. (1996). Phylogenetic analyses were performed as described for the 16S rRNA gene (see above). All groEL sequences were obtained in this study except that of Granulicatella adiacens, which was retrieved from the GenBank database.
The sampled seujeot had a pH of 7.3 and a total aerobic microbial cell count of 1.66103 c.f.u. ml21. For isolation, serially diluted seujeot samples were spread onto the following agars (all from Difco): nutrient agar (NA), R2A agar, trypticase soy agar (TSA), marine agar (MA) and potato dextrose agar (PDA). Plates were incubated at 25 uC for 5 days under aerobic conditions after which colonies were formed on TSA. One colony was picked and subcultured on modified TSA (per litre distilled water: 17.0 g tryptone, 3.0 g soytone, 2.5 g glucose, 5.0 g sodium chloride, 2.5 g dipotassium hydrogen phosphate; pH 7.3). Cell morphology of strain EX-07T grown on TSA for 5 days at 25 uC was observed at 61000 magnification with an Olympus microscope (GX71). Motility was investigated by using the hanging-drop technique with fresh cells grown in trypticase soy broth (Bernardet et al., 2002). Electron microscopy samples were prepared as described by Lee et al. (2009). Cells were then critically point dried in CO2 and sputtered with gold (SC502; Polaron) and the sample was observed with a scanning electron microscope (Hitachi S4300N; installed in the Korea Basic Science Institute). The Gram-reaction was determined using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Strain EX-07T stained Gram-positive and consisted of non-motile, non-spore-forming, coccus-shaped cells which grouped into tetrads, sarcinae and conglomerates (Fig. S1, available in the online Supplementary Material). This cell arrangement differed from that reported for strains of the genus Trichococcus (Table 1). Isolate EX-07T did not form chains as reported for members of the genus Granulicatella (Collins & Lawson, 2000).
The partial 16S rRNA gene sequence of strain EX-07T obtained had a length of 1447 bp. Sequence similarity
Table 1. Differential characteristics between strain EX-07T and the type species of phylogenetically related genera Taxa: 1, strain EX-07T; 2, Trichococcus flocculiformis; 3, Bavariicoccus seileri; 4, Granulicatella adiacens; 5, Atopobacter phocae. All data are from the present study except as noted. ND, No data; 2, not detected. Characteristic Cell morphology
Oxygen requirement Maximum salt tolerance (%) Major cellular fatty acids Polar lipids* Lipids Glycolipids Aminolipids Phospholipids Peptidoglycan type DNA G+C content (mol%)
1
2
3
4
5
Cocci, tetrads, sarcinae
Cocci
Cocci; single, pairs or short chains
Rods
Aerobic
Spherical to ovoid, olive-shaped; single or paired cells Aerotolerant
Aerotolerant
Facultatively anaerobic
Facultatively anaerobic
9 C16 : 1v9c, C18 : 1v9c, C16 : 0, C14 : 0
1 C16 : 1, C18 : 1v9c, C16 : 0,
11 C16 : 0, C18 : 1v9c
ND
ND
C16 : 0, C18 : 1v9c
C16 : 0, C18 : 1v9c
3 (L1, 2, 3) 3 (GL1, 2, 3) 2 (AL1, 2) 1 (PL1) A4a (L-Lys–D-Glu) 39.6
2 1 (GL1) 3 (AL1, 2, 3) 3 (PL1, 2, 3,) A4a (L-Lys–D-Asp) 47–49
4 (L1, 2, 3, 4) 2 (GL1, 2) 3 (AL1, 2, 3) 3 (PL1, 2, 3) A4a (L-Lys–D-Asp) 38–39
2 5 (GL1, 2, 3, 4, 5) 2 2 A3a 36–37.5
5 (L1, 3, 5, 6, 7) 5 (GL1, 2, 3, 4, 5) 2 3 (PL1, 2, 3) A4b ND
*Polar lipid data for strain EX-07T and T. flocculiformis DSM 2094T obtained in this study. Data for B. seileri, G. adiacens and A. phocae obtained from Schmidt et al. (2009). 1730
International Journal of Systematic and Evolutionary Microbiology 64
Jeotgalibaca dankookensis gen. nov., sp. nov.
calculations indicated that strain EX-07T is a member of the phylum Firmicutes and belongs to the family Carnobacteriaceae, with Trichococcus patagoniensis PmagG1T (94.5 % similarity), Trichococcus pasteurii KoTa2T (94.5 %), Trichococcus collinsii 37AN3*T (94.4 %), Trichococcus flocculiformis DSM 2094T (94.4 %) and Trichococcus palustris DSM 9172T (94.1 %) being the closest cultured relatives. Members of the remaining genera in the family Carnobacteriaceae showed less than 92.2 % 16S rRNA gene sequence similarity. A BLAST search against the NCBI database yielded a list of over 100 uncultured bacterial clones with 96–99 % 16S rRNA gene sequence similarity to strain EX-07T that have been detected in diverse environments. The phylogenetic tree reconstructed with the maximum-likelihood method and Kimura’s two-parameter model showed that strain EX07T was recovered in an independent branch that joined the cluster formed by the five species of the genus Trichococcus (Fig. 1; see also Fig. S2). This relationship was supported by a bootstrap value of 98 %. Additional trees based on the neighbour-joining and maximum-parsimony methods also supported this topology (Figs S3 and S4). Levels of DNA sequence similarity of the groEL DNA contig (465 bp) of strain EX-07T were 81.3–82.8 % when compared with the type strains of all species of the genus Trichococcus and less than 76 % with the type strains of species of the genera Bavariicoccus and Granulicatella. Fig. 2 shows the phylogenetic relationship between strain EX07T and its closest relatives based on groEL sequences.
100 98 52 88
98
99
Analysis of the cell-wall peptidoglycan was performed by the Identification Service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen following the methods of Schleifer & Kandler (1972) and Schleifer (1985). Isolate EX-07T contained the amino acids lysine, glutamic acid, aspartic acid and alanine, indicating a peptidoglycan type A4a with an interpeptide bridge L-Lys–D-Glu (A11.33, Schumann 2011). A peptidoglycan type A4a has also been reported for the genera Bavariicoccus and Trichococcus, although strains of members of these genera have an interpeptide bridge L-Lys–D-Asp (A11.31, Schumann
Trichococcus flocculiformis DSM 2094T (AJ306611) Trichococcus pasteurii KoTa2T (X87150) Trichococcus palustris DSM 9172T (AJ296179) Jeotgalibaca dankookensis EX-07T (GU317945) Granulicatella adiacens ATCC 49175T (AY123705) Atopobacter phocae CCUG 42358T (FM177903) Bavariicoccus seileri WCC 4188T (FM177901) Vagococcus fluvialis CCUG 32704T (Y18098) Carnobacterium divergens DSM 20623T (M58816) Isobaculum melis M577-94T (AJ302648)
59
Desemzia incerta ATCC 8363T (Y17300) Pisciglobus halotolerans C01T (GU459067) Lacticigenium naphtae MIC1-18T (AB430339) Atopococcus tabaci CCUG 48253T (AJ34917) Alkalibacterium olivapovliticus WW2-SN4aT (AF143511) Marinilactibacillus psychrotolerans M13-2T (AB083406)
56
99
98 92
63
To measure the G+C content, the genomic DNA of strain EX-07T was extracted and purified as described by Price et al. (1978) and enzymically degraded to nucleotides. The DNA G+C content was determined as described by Mesbah et al. (1989), using reversed-phase HPLC. The value of 39.6 mol% obtained was different from that reported for members of the genus Trichococcus (45–49 mol%, Liu et al., 2002), but similar to the values reported for members of the genera Bavariicoccus (38 mol%, Schmidt et al., 2009) and Granulicatella (36–37 mol%, Collins & Lawson, 2000).
93 Trichococcus patagoniensis PmagG1T (AF394926) T 87 Trichococcus collinsii 37AN3* (AJ306612)
0.01
98
The novel isolate formed an independent branch supported by a 100 % bootstrap value and confirmed the moderate relationship with all members of the genus Trichococcus. The combined results of the 16S rRNA gene and groEL sequence analyses support the classification of strain EX07T within a new genus.
100
Alloiococcus otitis 7760T (X59765) Dolosigranulum pigrum R91/1468T (X70907) Allofustis seminis 01-570-1T (AJ410303) Atopostipes suicloacalis PPC79T (AF445248)
Fig. 1. Maximum-likelihood tree based on 16S rRNA gene sequences of strain EX-07T and related taxa. Evolutionary distances were computed using Kimura’s two-parameter method. Bootstrap values .50 % are indicated. Bar, 0.01 substitutions per nucleotide position. http://ijs.sgmjournals.org
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D.-G. Lee and others
T 100 Trichococcus palustris DSM 9172 (KJ160237)
0.05
81
Trichococcus pasteurii KoTa2T (KJ160238) Trichococcus collinsii 37AN3*T (KJ160235)
100 100
87
Trichococcus flocculiformis DSM 2094T (KJ160236) Trichococcus patagoniensis PmagG1T (KJ160232) Jeotgalibaca dankookensis EX-07T (KJ160234)
100
Granulicatella adiacens ATCC 49175T (AY123705) Bavariicoccus seileri WCC 4188T (KJ160233)
Fig. 2. Maximum-likelihood tree based on groEL sequences of strain EX-07T and related taxa. Evolutionary distances were computed using Kimura’s two-parameter method. Bootstrap values .50 % are indicated. Bar, 0.05 substitutions per nucleotide position.
2011). A peptidoglycan type A3a is reported for the genus Granulicatella (Schmidt et al., 2009). Strain EX-07T and the type strains of recognized species of the genus Trichococcus were analysed for their polar lipid composition. Cells were cultivated on TSA and R2A agar media, respectively, for 72 h at 30 uC. Polar lipids were extracted using the procedures described by Minnikin et al. (1984) and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Komagata & Suzuki, 1987). As with other members of the family Carnobacteriaceae, the major polar lipid profile of strain EX-07T was rather complex and included phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine and the following unidentified lipids: three glycolipids, two aminolipids, one phospholipid and three lipids. This profile showed several differences when compared with the profiles obtained for species of the genus Trichococcus as listed in Table 1 (Fig. S5). The polar lipid pattern of strain EX-07T also differed from that of Granulicatella adiacens and Bavariicoccus seileri based on the number of unknown lipids or the presence of glycolipids (Schmidt et al., 2009). Preparation of quinones was performed as described by Altenburger et al. (1996) but these cellular compounds were not detected. Similar results were reported for B. seileri (Schmidt et al., 2009).
The overall fatty acid profile of isolate EX-07T is given in Table 2 with C16 : 1v9c (35.1 %), C18 : 1v9c (26.1 %), C16 : 0 (18.5 %) and C14 : 0 (6.9 %) being the major components. This pattern differed from the general profile presented by species of the genus Trichococcus, especially in the amount of C16 : 1v9c which was either absent or present in minor amounts (,2 %, Table 2). The fatty acids C16 : 0 and
Table 2. Fatty acid composition of strain EX-07T and type strains of species of the genus Trichococcus, Granulicatella adiacens and Bavariicoccus seileri Strains: 1, EX-07T; 2, T. patagoniensis PmagG1T; 3, T. pasteurii KoTa2T; 4, T. collinsii 37AN3*T; 5, T. flocculiformis DSM 2094T; 6, T. palustris DSM 9172T; 7, G. adiacens ATCC 49175T; 8, B. seileri DSM 19936T. Fatty acids that amount to ,1.0 % of the total fatty acids in all strains are not shown. 2, Not detected. All data are from the present study except for B. seileri (Schmidt et al., 2009). Fatty acid C10 : 0 C12 : 0 C14 : 0 C16 : 0 C16 : 1 C18 : 0 C16 : 1v9c C18 : 1v7c C18 : 1v9c C20 : 1v9c Summed features* 3 5
1
2
3
4
5
1.4 2 2 2 2 1.3 2.0 1.4 4.8 1.2 6.9 12.1 18.0 42.9 10.7 18.5 16.3 13.0 23.1 18.2 2 2 2 20.6 41.4 2.4 3.2 1.1 0.4 5.3 35.1 2 2 2.1 1.0 2 2 2 2 2 26.1 24.7 20.3 4.2 17.1 1.0 2 2 2 2
6
7
8
2 2 17.9 11.4 24.1 2.2 1.0 1.0 19.5 2
2 2 21.0 15.0 20.0 4.0 2 2.0 22.0 2
2 0.3 4.3 21.3 2 9.0 6.3 2 53.0 1.6
1.2 2
2 2
2 2
The fatty acid profiles of isolate EX-07T, the type strains of all species of the genus Trichococcus and G. adiacens ATCC 49175T were determined under identical conditions. Cells were grown on TSA for 5 days at 25 uC and collected from the third streak quadrant to obtain cells of the same physiological age. Samples were saponified, methylated and extracted according to the standard protocol of the Sherlock Microbial Identification System (Sasser, 1990). The fatty acids were analysed by GC (model 6890; Hewlett Packard) and identified using the TSBA40 database of the Microbial Identification software package (MIDI, Version 4.5). All experiments were performed in triplicate.
*Summed features represent groups of two or more fatty acids that could not be separated by the Microbial Identification System. Summed feature 3 comprises C16 : 1v6c and/or C16 : 1v7c; summed feature 5 comprises C18 : 2v6,9c or anteiso-C18 : 0.
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International Journal of Systematic and Evolutionary Microbiology 64
5.5 40.6 45.0 2 2 2
0.8 2
0.9 4.2
Jeotgalibaca dankookensis gen. nov., sp. nov.
Table 3. Differential phenotypic characteristics between strain EX-07T and related phylogenetic neighbours Strains: 1, EX-07T; 2, T. patagoniensis PmagG1T; 3, T. pasteurii KoTa2T, 4, T. collinsii 37AN3*T, 5, T. flocculiformis DSM 2094T; 6, T. palustris DSM 9172T; 7, G. adiacens ATCC 49175T; 8, B. seileri DSM 19936T. +, Positive; 2, negative; ND, not determined. All data are from the present study except for G. adiacens (Collins & Lawson, 2000) and B. seileri (Schmidt et al., 2009). Characteristic Pigmentation Production of: Chymotrypsin Tyrosinase Amylase Esterase b-Galactosidase Growth at 37 uC Minimum growth temperature (uC) Growth on: D-Ribose D-Galactose Trehalose D-Arabinose Arbutin Sucrose Inosine D-Gluconic acid
1
2
3
4
5
6
7
8
Yellow–orange
White–pink
White
White
White–opaque
White
None
None
+ 2 2 2 + + 10
2 + + + 2 2 25
2 2 2 + + 2 0
2 2 2 + 2 2 25
2 2 2 2 2 + 4
2 2 2 2 2 + 0
ND
ND
ND
ND
+ + + 2 + 2 + +
+ 2 2 + + + + 2
2 + + 2 2 + 2 2
+ + + 2 2 + 2 2
2 + + 2 + + 2 2
2 2 2 + + + 2 2
C18 : 1v9c have also been reported as major components of the genera Bavariicoccus and Granulicatella (Schmidt et al., 2009). Strain EX-07T and type strains of all species of the genus Trichococcus were studied in parallel for the following phenotypic characteristics: growth at 4, 10, 25, 30, 37 and 40 uC and at pH 4.5–8.5 (at 0.5 pH unit intervals). The pH of the medium was adjusted as described by Qu & Yuan (2008). Tolerance to salinity was tested in trypticase soy broth supplemented with 0.5–20.0 % (w/v) NaCl at 0.5 % intervals. All results were recorded after 7 days of incubation. Growth on NA, R2A agar, MA and PDA was evaluated at 25 uC for 7 days. Catalase activity was determined by assessing bubble production in 3 % (v/v) H2O2, and oxidase activity was determined using 1 % (w/v) tetramethyl phenylenediamine. Tests in the commercial systems GN2 MicroPlates (Biolog), API 20NE, API 50CH, API ID32 GN and API ZYM (bioMe´rieux) were performed in triplicate according to the manufacturers’ instructions. The API ZYM tests were read after 4 h of incubation at 28 uC and the other API tests were read after 48 h at 28 uC. Anaerobic growth was tested in serum bottles by adding sodium thioglycollate (1 g l21) to trypticase soy broth and substituting the upper airspace with nitrogen gas. Tests for the degradation of DNA, casein, chitin, starch, Tween 80 (Kouker & Jaeger, 1987) and carboxymethylcellulose (Ten et al., 2004) were performed and evaluated after 5 days of incubation at 28 uC. Good growth was obtained on TSA but not on NA, PDA or R2A agar, whereas it grew weakly on half-strength MA. The strain was heterotrophic and aerobic; no anaerobic growth http://ijs.sgmjournals.org
2
2
ND
ND
2 + .10
2 + 10
2
+
2 + + 2 + 2
ND
ND
ND
ND
ND
2 2 ND
was detected under the conditions tested. Unlike species of the genus Trichococcus, strain Ex-07T utilized D-gluconic acid as a carbon source and showed chymotrypsin activity. Other physiological characteristics of strain EX-07T are summarized in the species description while comparison of selected characteristics with those of related phylogenetic species is given in Table 3. The results of 16S rRNA gene and groEL sequencing studies, polar lipid profiles, peptidoglycan type, cellular fatty acid composition and DNA G+C content showed that strain EX-07T is only moderately related to the genera Trichococcus, Granulicatella and Bavariicoccus. On the basis of genotypic and phenotypic data, strain EX-07T is inferred to represent a novel species of a new genus in the family Carnobacteriaceae, for which the name Jeotgalibaca dankookensis gen. nov., sp. nov. is proposed. Description of Jeotgalibaca gen. nov. Jeotgalibaca [Je.ot.ga.li.ba9ca. N.L. n. jeotgalum (from Korean n. jeotgal) jeotgal, traditional Korean food; L. fem. n. baca a grain or berry, and in bacteriology a coccus; N.L. fem. n. Jeotgalibaca, coccus from jeotgal]. Cells are Gram-stain-positive cocci which group in tetrads, sarcinae or irregular conglomerates. Non-spore-forming and non-motile. Aerobic and chemoheterotrophic. Oxidaseand catalase-negative. The major fatty acids are C16 : 1v9c, C18 : 1v9c, C16 : 0 and C14 : 0. Polar lipids include phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and several unknown glycolipids, aminolipids and 1733
D.-G. Lee and others
phospholipids. Peptidoglycan type A4a. Phylogenetically positioned in the family Carnobacteriaceae, phylum Firmicutes. The type species is Jeotgalibaca dankookensis.
gen. nov., Granulicatella adiacens comb. nov., Granulicatella elegans comb. nov. and Granulicatella balaenopterae comb. nov. Int J Syst Evol Microbiol 50, 365–369. Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.
Description of Jeotgalibaca dankookensis sp. nov. Jeotgalibaca dankookensis (dan.ko.ok9en.sis. N.L. fem. adj. dankookensis of or belonging to Dankook University). Presents the following properties in addition to those given in the genus description. Cells are 1.0–1.3 mm in diameter. Good growth occurs on TSA but not on NA, PDA or R2A agar; weak growth occurs on one-tenth-strength MA. Colonies on TSA are circular, convex and pale orange. Growth occurs at 10–37 uC (optimum, 28 uC), at pH 7.0– 9.0 and on TSA supplemented with up to 9 % NaCl. Nitrate is not reduced. DNA, casein, chitin, starch, Tween 80 and carboxymethylcellulose are not degraded. Results based on the commercial systems Biolog GN2, API 20NE, API 50CH and API ID32 GN are as follows: positive for assimilation of D-ribose, D-glucose, D-fructose, D-galactose, maltose, Dmannose, arbutin and salicin; positive for aesculin hydrolysis and b-galactosidase; negative for indole production, glucose fermentation and urease. Assimilates (Biolog GN2) D-fructose, D-galactose, D-glucose, D-psicose, trehalose, D-mannose, maltose, N-acetylglucosamine, pyruvic acid, D-gluconic acid, a-ketobutyric acid, inosine, uridine, thymidine and glycerol. According to the API ZYM gallery, alkaline phosphatase, esterase (C4), esterase lipase (C8), chymotrypsin, acid phosphatase and naphthol-AS-BIphosphohydrolase activities are present; N-acetyl-b-glucosaminidase, b-glucosidase, a-fucosidase, leucine arylamidase, lipase (C14), valine arylamidase, cystine arylamidase, a-glucosidase, a-galactosidase, b-glucuronidase, b-glucosidase, a-mannosidase and trypsin activities are absent. The type strain, EX-07T (5KCCM 90229T5JCM 19215T), was isolated from seujeot (traditional Korean food) in Cheon-an Dankook University, South Korea. The DNA G+C content of the type strain is 39.6 mol%.
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