INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1990, p. 224-230 Vol. 40, No. 3 0020-7713/90/030224-07$02.00/0 Copyright 0 1990, International Union of Microbiological Societies

16s rRNA Sequence Determination for Members of the Genus Carnobacterium and Related Lactic Acid and Description of salmoninarum sp. nov.

S. WALLBANKS,l A. J. MARTINEZ-MURCIA,l J. L. FRYER,2 B. A. PHILLIPS,l AND M. D. COLLINS1* Department of Microbiology, Agricultural and Food Research Council Institute of Food Research, Reading Laboratory, Shinfield, Reading, RG2 9AT, United Kingdom,' and Department of Microbiology, Oregon State University, Corvallis, Oregon 97331 -38M2

The phylogenetic interrelationships of members of the genus Carnobacterium and some atypical lactobacilli isolated from diseased salmonid fish were investigated by using reverse transcriptase sequencing of 16s rRNA. The four species Carnobacterium piscicola, Carnobacterium divergens, Carnobacterium gallinarum, and Carnobacterium mobile exhibited a high degree of sequence similarity with each other (ca. 96 to 98%) and formed a phylogenetically coherent group that was quite distinct from all other . The sequence data clearly demonstrated that carnobacteria are phylogenetically closer to the genera Enterococcus and Vagococcus than to members of the genus Lactobacillus. The strains from fish were found to be phylogenetically related to the genus Vagococcus and represent a new species, Vagococcus salmoninarum. The type strain of Vagococcus salmoninarum is strain NCFB 2777.

The genus Carnobacterium was proposed by Collins et al. in Oregon, and strain Rangen 128-81 was isolated from an (4) to accommodate the species Lactobacillus piscicola (17) adult (Oncorhynchus mykiss) at a trout hatch- and Lactobacillus divergens (18) and some so-called atypical ery near Buhl, Idaho. Cultures were checked for purity, lactobacilli isolated from poultry meat (24). Currently, four harvested by centrifugation, and washed in distilled water. species, Carnobacterium piscicola (type species), Carno- rRNA extraction and determination of rRNA sequence. bacterium divergens, Carnobacterium gallinarum, and Car- Total cellular rRNA was extracted from ca. 2 g (wet weight) nobacterium mobile are recognized in this genus (4). Al- of cells (6). Nucleotide sequences were determined by the though these four species form a phenotypically coherent dideoxynucleotide method, using avian myeloblastosis virus group (4, 9), the separateness of the genus Carnobacterium reverse transcriptase. Most of the sequences of the oligonu- (in particular its relationship to the genus Lactobacillus) cleotide primers and their 16s rRNA target sites have been remains unclear. Members of the genus Carnobacterium described previously by Lane et al. (20) and Embley et al. differ from Lactobacillus species by their inability to grow on acetate medium (4, 9) and by their synthesis of oleic acid (6); a DNA primer having the sequence 5'-TCACCCTCT (c18:1A9,10) instead of cis-vaccenic acid (c18:1A11,12), CAGGTCGGCTA was used for the region at ca. position 300 which is produced by lactobacilli (4). At present these are (complementary to positions 287 to 306; Escherichia coli). the only criteria which distinguish the two genera. The products of the sequencing reactions were separated on Sequencing of 16s rRNA by reverse transcriptase (20, 21) 55-cm wedge-shaped (0.2- to 0.6-mm) 6% (wthol) polyacryl- is currently the most rapid and powerful technique for amide denaturing (7 M urea) gels at 55°C by using an LKB elucidating the natural relationships of microorganisms (26). model Macrophor 2010 sequencing unit operated at 50 W per This method produces long stretches of sequence (ca. 95% of gel. the total sequence), which enables precise phylogenetic Analysis of rRNA sequence data. The sequences were relationships to be determined (26). In this study we deter- aligned, and the homology values determined by using the mined 16s rRNA primary structures for members of the Microgenie program (Beckman Instruments, Inc.) (22). Nu- genus Carnobacterium by using reverse transcription in an cleotide substitution rates (K,,, values) were calculated attempt to clarify the relationship of these organisms to the (19), and an unrooted phylogenetic tree or network was genus Lactobacillus and other lactic acid bacteria. In addi- produced by using the algorithm of Fitch and Margoliash (10) tion, the phylogenetic position of two representative strains contained in a program written by Felsenstein (8) (PHYLIP belonging to a group of atypical lactobacilli isolated from version 3.1) for IBM personal computers. salmonid fish was also determined. DNA studies. DNA was prepared by using a modification (7) of the method of Garvie (11).DNA base composition was MATERIALS AND METHODS estimated by thermal denaturation in standard saline citrate Cultures and cultivation. The test strains which we used as described by Garvie (ll), using DNA from E. coli K-12 are shown in Table 1. Strains of carnobacteria, enterococci, strain NCDO 1984 (51.5 mol% guanine plus cytosine) as the lactococci, leuconostocs, pediococci, and streptococci and standard. DNA-DNA hybridizations were performed by fish isolates OS1-68T (T = type strain) and Rangen 128-81 using the membrane filter technique described previously were grown in YGPB broth (12) at 30°C. Strains OS1-68T and (13). Rangen 128-81 were isolated by R.A. Holt, Oregon Depart- Fatty acid analyses. Fatty acids were extracted from dry ment of Fish and Wildlife; strain OS1-68T was isolated in cells by acid methanolysis, and purified methyl esters were 1968 from an adult rainbow trout at the Oak Spring Hatchery examined by capillary gas liquid chromatography as de- scribed prevtously (4). The positions of double bonds in * Corresponding author. unsaturated acids were determined by dimethyl disulfide

224 TABLE 1. Homology for a 1,340-nucleotide region of 16s rRNAs from carnobacteria and other strains

% Homology with strain:

Vagococcus Strain NCDO NCFB NCFB NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO s$mz'- 2763T 2766T 2765T 2762T 2674= 596T 2369T 2160 213T 1750T 2156 2118 523T 1832T 651 573T 643 2497 NCFB 217it Bacillus subtilis NCDO 1769T 90.1 90.4 90.2 90.5 91.2 91.1 91.4 89.1 87.9 87.0 85.2 86.2 86.4 89.3 88.0 85.9 86.3 89.7 89.9 Camobacterium divergens NCDO 2763= 97.0 96.0 97.5 94.3 94.6 94.2 88.9 87.7 89.3 87.4 88.5 87.5 91.6 89.7 88.9 89.2 93.4 94.1 Camobacterium gallinarum NCFB 2766= 95.7 98.7 93.9 94.1 94.4 89.2 87.8 89.0 87.7 88.3 87.0 91.3 90.2 88.0 88.5 92.3 93.2 Camobacterium mobile NCFB 2765= 96.3 93.6 93.7 92.6 88.8 87.7 89.2 87.6 88.1 86.3 91.2 88.7 87.5 87.5 93.4 93.7 Carnobacterium piscicola NCDO 2762T 94.3 94.7 94.7 89.9 89.0 89.9 88.4 88.1 86.7 91.8 89.9 88.9 89.3 93.4 93.8 Enterococcus cecorum NCDO 2674= 96.7 96.8 89.6 88.5 90.2 90.2 89.9 88.3 91.5 91.2 89.8 90.8 94.5 93.4 N Enterococcus durans NCDO 596= 98.3 90.4 88.4 89.5 89.5 89.5 88.1 92.1 91.3 90.7 90.8 94.6 94.5 Enterococcus avium NCDO 2369= 89.6 88.6 94.3 88.9 89.5 88.0 91.6 91.0 90.0 90.9 93.7 94.0 Lactobacillus ok NCDO 2160 89.8 93.1 87.2 85.1 88.5 91.9 86.3 84.9 86.2 90.2 87.5 Lactobacillus delbrueckii NCDO 213= 89.0 86.8 85.7 87.5 89.5 86.0 84.4 84.2 88.0 87.4 Lactobacillus fermentum NCDO 1750T 87.8 85.8 87.6 92.0 87.4 86.0 87.0 89.9 87.0 Lactococcus garvieae NCDO 2156 95.0 85.9 87.9 92.3 89.9 90.9 87.3 87.8 Lactococcus lactis subsp. lacti3 NCDO 2118 84.6 89.6 91.9 91.4 91.7 87.5 88.4 Leuconostoc mesentemides NCDO 523= 88.3 87.9 86.3 86.7 87.7 86.3 Pedwcoccus damnosus NCDO 1832= 90.0 89.1 88.2 91.6 90.8 Streptococcus paraubek NCDO 651 94.5 96.5 89.3 88.9 Streptococcus thermophilus NCDO 573= 96.0 89.0 88.8 Streptococcus uberis NCDO 643 88.7 87.4 Vagococcus fluvialis NCDO 2497 96.3 226 WALLBANKS ET AL. INT.J. SYST. BACTERIOL.

C.pi. C~G~AGA~CACCAGUGGC~GACUCU~~C~C~~~ C. ga. CGGW;AAAUGCGUAGAUAUGUGGAGGAACACCACUGGCGA C.di. CG~AGUCGCGAAGGCGA~~~~CGCWGGCUCGNAAGC~~~GGAU c.m. cGGUGAAAUGCGUAGWAUGUGGAGGAACACCAmK;GCGAA V.sa. CGGUGMWX~C~GA~CU~~~UGACA~~~ FIG. 1. Primary structures of 16s rRNAs from C. piscicola (C.pi.) NCDO 2762T, C. gaffinarum(C.ga.) NCFB 2766T, C. divergens (C.di.1 NCDO 2763T, C. mobile (C.mo.) NCFB 2765T, and V. salmoninarum (V.sa) NCFB 2777T as determined by using reverse transcriptase. The first nucleotide and last nucleotide in the sequences of C. piscicola, C. galfinarum, C. divergens, and V. salmoninarum are analogous to positions 1 and 1481 of the E. cofi 16s rRNA sequence (l), respectively, The sequence for determined C. mobile runs from position 107. The first nucleotide and last nucleotide in the long stretch (1,340 bases) used to calculate K,,, values correspond to positions 107 (G) and 1433 (A) in the E. cofi sequence, respectively. N, Undetermined nucleotide. Spaces in the sequences correspond to alignment gaps. derivitization and gas chromatography-mass spectrometry RESULTS AND DISCUSSION as described by Dunkelblum et al. (5). Biochemical and physiological tests. Biochemical tests were The reverse-transcriptase-determinedsequences (contain- performed by using the API 10E and API 50CH systems ing between 1,384 and 1,491 nucleotides) of the 16s rRNAs (API-bioMerieux) according to the instructions of the man- of the type strains of C. piscicola, C. divergens, C. galli- ufacturer. YGA medium (25 g of nutrient broth no. 2 [Oxoid narum, and C. mobile and salmonid fish isolate OS1-6flT(= Ltd.], 3 g of yeast extract [Oxoid], 5 g of glucose, 15 g of agar NCFB 2777T) are shown in Fig. 1. In order to determine the no. 3 [Oxoid], 1 liter of water; pH 6.8) was used for test phylogenetic relationships of the Carnobacterium species cultures. These cultures were incubated at 25"C, and API and the fish isolate, the sequences were aligned and com- STREP basal medium was used for carbohydrate tests. pared with the sequences of 15 reference strains from eight Motility was tested by stab inoculation into YGPB medium genera (3, 15, 25; M. D. Collins, A. M. Williams, and S. modified to contain 0.1% glucose, 0.1% lactose, and 0.2% Wallbanks, FEMS Microbiol. Lett., in press; A. J. Martinez- (wthol) (Oxoid) no. 3 agar. Growth temperatures were Murcia and M. D. Collins, FEMS Microbiol. Lett., in press; determined in YGPB medium supplemented with 1% lac- A. M. Williams and M. D. Collins, J. Appl. Bacteriol., in tose; readings were made at 48 h, 7 days, and 14 days. press). Representative homology values based on a compar- VOL. 40, 1990 rRNA SEQUENCES OF LACTIC ACID BACTERIA 227

B C.pi. C.ga. C.di. C.mO. V.sa. C.pi. C.ga. C.di. C.mO. V.sa. C.pi. C.ga. C.di. C.nn0. V.sa. C.pi. C.ga. C.di. C.mO. V. sa. C.pi. C.ga. C.di. C.mO. V. sa. C.pi. C.ga. C.di. C.mO. V.sa. C.pi. C.ga. C.di. C.W. V.sa.

ison of 1,340 bases (ranging from positions 107 to 1433 on the matrix treeing program confirmed the close affinity between E. coli numbering system [l]) are shown in Table 1. An strains OS1-6gT and NCDO 2497T (Fig. 2). The fish isolate unrooted phylogenetic tree constructed from the derived and V.fluvialis formed a distinct cluster that was phyloge- K,,, (evolutionary distance) values (Table 2) is shown in netically close, but nevertheless distinct from, carnobacteria Fig. 2. The four Carnobacterium species exhibited very high and enterococci (Fig. 2). It is interesting that an analysis of levels of sequence relatedness (ca. 96 to 98%) and clearly the sequence data revealed that strains OS1-6gT and NCDO formed a single group that was phylogenetically distinct from 2497T possess a very characteristic signature in the V6 all of the other lactic acid bacteria which was examined. It is region (nomenclature of Gray et al. [14]). The V6 regions of evident from the levels of sequence homology (ca. 92 to these strains possess bases UGG at positions 457 to 459 and 94%) and the branching pattern of the tree that the closest complementary nucleotides CCA at positions 474 to 476 relatives of the genus Carnobacterium are Vagococcus (Fig. 3). This novel signature distinguishes strains OS1-68T fluvialis and the enterococci (Table 1 and Fig. 2). Notable and NCDO 2497T from all of the lactic acid bacteria exam- were the low levels of relatedness with Lactobacillus del- ined by us to date (more than 100 strains) (Collins et al., brueckii (type species of the genus) and the other lactobacilli unpublished data) and may prove to be a useful diagnostic examined (Fig. 2). The low levels of sequence relatedness marker. between carnobacteria and lactobacilli are consistent with The results of a long-chain fatty acid analysis reinforced the results of DNA-rRNA hybridization studies (2) and the the close relationship between fish strain OS1-68T and V. differences in cellular fatty acid composition (4) and clearly fluvialis. The fatty acids of strain OS1-68T are of the straight- support the recognition of the genus Carnobacterium as a chain saturated and monounsaturated types (composition: distinct taxon. 3% C14:0, 17% C16:0, 9% C18:0, 3% C14:1, 11% C16:1, 46% Salmonid isolate OS1-6gT exhibited the highest level of Clgtl, 11% C20:1. Gas chromatography-mass spectrometry sequence similarity (ca. 96%) with V.fluvialis NCDO 2497T analysis of the dimethyl disulfide adduct of the ClSt1isomer (formerly a motile group N streptococcus [16, 231) Signifi- revealed prominent ions at mlz 390 (molecular ion M+), 217 cant, albeit lower, homology values were also exhibited with (A fragment derived from the carboxylic end of the mole- carnobacteria and enterococci (ca. 92 to 94%). The distance cule), and 173 (ofragment corresponding to the aliphatic end TABLE 2. k,, values for a 1,340-nucleotide region of 16s rRNAs from carnobacteria and other strains K,,,, value with strain: Bacillus Strain subtilis NCDO NCFB NCFB NCDO NCDONCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO NCDO 2763T 2766T 2765T 2762T 2674T 59fjT 2369T 2160 213T 1750T 2156 2118 523T 1832T 651 573= 643 2497 1769T Camobacterium divergens NCDO 2763T 0.1062 Camobacterium gallinarum NCFB 2766T 0.1027 0.0306 Camobacterium mobile NCFB 2765T 0.1050 Camobacterium pkcicola NCDO 2762T 0.0411 0.0443 0.1016 0.0254 Enterococcus cecorum NCDO 2674T 0.0936 0.0131 0.0379 0.0593 0.0636 Enterococcus durans NCDO 596T 0.0947 0.0669 0.05500.0593 0.0337 Enterococcus avium NCDO 2369T 0.0560 0.0615 0.0550 0.0327 0.0172 0.0913 0.0604 0.0658 Lactobacillus oris NCDO 2160 0.1178 0.0582 0.0779 0.1085 0.1120 0.1027 0.1120 Lactobacillus delbrueckii NCDO 213T 0.1201 0.1166 0.1190 0.1248 0.1260 0.1237 0.1096 0.1320 0.1343 0.1213 Lactobacillus fermentum NCDO 1750T 0.1428 0.1331 0.1343 0.1085 0.1050 0.1131 0.0593 0.0724 0.1190 Lactococcus garvieae NCDO 2156 0.1154 0.1190 0.1260 0.1050 0.1131 0.1201 0.1404 0.1649 0.1379 0.1166 0.1452 0.1331 Lactococcus lactis subsp. lactis NCDO 2118 0.1343 0.1296 0.1085 0.1131 0.1131 0.1161 0.1525 0.1248 0.1355 0.1587 0.1574 0.0517 Leuconostoc mesenteroides NCDO 523T 0.1272 0.1296 0.1464 0.1272 0.1296 0.1308 0.1248 0.1501 0.1367 0.1367 0.1355 0.1562 0.1724 Pediococcus damnosus NCDO 1832T 0.1428 0.1513 0.0868 0.0902 0.0835 0.0891 0.0857 0.1154 0.0891 0.1131 0.0846 0.1320 0.1120 0.1272 Streptococcusparauberis NCDO 651 0.0925 0.0936 0.1085 0.0936 0.0925 0.0959 0.1513 0.1308 0.1108 0.1550 0.1379 0.0812 0.0857 0.1320 0.1073 Streptococcus thermophilus NCDO 573T 0.1050 0.1225 0.1201 0.1096 0.0993 0.1073 0.1686 0.1562 0.1201 0.1749 0.1550 0.1085 0.0913 0.1513 0.1178 0.0571 Streptococcus uberis NCDO 643 0.1308 0.1367 0.1154 0.0982 0.0982 0.0970 0.1525 0.1513 0.1166 0.1774 0.1428 0.0970 0.0880 0.1464 0.1284 0.0358 0.0411 Vagococcus fluvialis NCDO 2497 0.1248 0.1367 0.0691 0.0571 0.0560 0.0658 0.1054 0.1108 0.0691 0.1308 0.1085 0.1391 0.1367 0.1343 0.0891 0.1154 0.1190 0.1225 Vagococcus salmoniarum NCFB 2777T 0.0812 0.0691 0.0647 0.0691 0.0571 0.0625 0.1367 0.1085 0.0615 0.1379 0.1428 0.1331 0.1260 0.1513 0.0982 0.1201 0.1213 0.1379 0.0379 0.0713 0.0658 VOL.40, 1990 rRNA SEQUENCES OF LACTIC ACID BACTERIA 229

Lb. oris

Leuc.mesenteroides

L .garvieae I L. lactis V.fl~~vialis V.salmoninarum FIG. 2. Unrooted tree or network s.,owing the phylogenetic interrelationships of carnobacteria and other lactic acid bacteria. The tree is based on a comparison of 1,340 nucleotides. K,,, values and strain designations are shown in Table 2. Genus abbreviations: Lb., Lactobacillus; Leuc., Leuconostoc; B., Bacillus; C., Carnobacterium; V., Vagococcus; E., Enterococcus; L., Lactococcus; S., Streptococ- cus; P., Pediococcus of the molecule), which was consistent with the C1,:1A9,10 dance with the synthesis of oleic acid reported for V.fluvialis (oleic acid) isomer. The remaining minor unsaturated acids (23) and carnobacteria (4) and readily distinguishes these corresponded to C,,:,A5 (ions at m/z 334 [M+], 161 [A], and organisms from enterococci and lactobacilli, which synthe- 173 [o]),c1,:1A7 (ions at m/z 362 [M+], 189 [A], and 173 [o]), size predominantly cis-vaccenic acid (C18:1All,12). and C20:1A11(ions at m/z 418 [M+], 245 [A], and 173 [o]). The molecular and chemical data clearly demonstrate that The synthesis of oleic acid by strain OS1-68T is in accor- salmonid isolate OS1-68T is a member of the genus Vago- coccus. Strain OS1-68T exhibited 95% DNA homology with a second fish isolate, strain Rangen 128-81 (= NCFB 2779). AA However, these strains exhibited relatively low levels of uc DNA homology (22 to 28%) with V. fluvialis NCDO 2497T, G U A G -470 which is consistent with separate specific status. Therefore, we formally propose that rainbow trout isolates OS1-68Tand G u (C) A-U Rangen 128-81 represent a new species of the genus Vago- G-C coccus, for which the name Vagococcus salmoninarum is (U) G- c (A) proposed. (A) G- c (U) Description of Vagococcus salmoninarum sp. nov. Cells are (GI u - A (C) G-C ovoid or short rods (the shape varies with cultural condi- C tions) and occur singly, in pairs, or in short chains. Cells are A-U (C) gram positive, nonmotile, and catalase negative. Growth A-U occurs at 5 and 30°C but not at 40°C. Facultatively anaero- C-G AA bic. No gas is produced from glucose. Acid is produced from A C amygdalin, arbutin, N-acetylglucosamine, cellobiose, fruc- 450-G G tose, P-gentiobiose, glucose, maltose, mannose, a-methyl- A G D-glucoside, ribose, salicin, starch, sucrose, D-tagatose, and A U trehalose. Acid is not produced from D-arabinose, L-arabi- GA nose, D-arabitol, L-arabitol, adonitol, dulcitol, erythritol, A -U G-C D-fucose, L-fucose, galactose, gluconate, glycogen, glycerol, A - U -490 2-keto-gluconate, 5-keto-gluconate, inulin, inositol, lactose, U -A D-lyxose, melibiose, melezitose, methyl-xyloside, methyl- U -A D-mannoside, mannitol, rhamnose, raffinose, sorbose, sorb- C itol, D-turanose, D-xylose, L-xylose, and xylitol. Esculin is G -C U -A hydrolyzed. Arginine dihydrolase and urease negative. Ni- FIG. 3. Comparison of nucleotide sequences of region V6 of 16s trate is not reduced to nitrite. H2S is produced. The major rRNAs of Carnobacterium and Vagococcus spp. The nucleotides in cellular fatty acids are of the straight-chain saturated and parentheses are the nucleotides found in Carnobacterium species. monounsaturated types; the ClSt1 acid is a A9,lO isomer 230 WALLBANKS ET AL. INT.J. SYST.BACTERIOL.

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