We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.Vibrios are gram-negative, usually motile rods, are mesophilic and chemoorganotrophic, and have a facultatively fermentative metabolism (5). They are generally able to grow on marine agar and on the selective medium thiosulfate-citratebile salt-sucrose agar and are mostly oxidase positive. Vibrios belong to the Gammaproteobacteria according to 16S rRNA gene sequence analysis. These bacteria are found abundantly in aquatic habitats and in association with eukaryotes. Associations established by vibrios range from mutualistic, e.g., Vibrio fischeri-bobtail squid (26), to pathogenic, e.g., V. cholerae-humans (45). Probiotic Vibrio strains for fish and shellfish have also been documented (44).The current family Vibrionaceae comprises the genera Enterovibrio (2 species), Grimontia (1 species), Photobacterium (7 species), Salinivibrio (1 species), and Vibrio (64 species). The novel species Photobacterium rosenbergii and Enterovibrio coralii have recently been proposed to encompass isolates associated with coral bleaching (41). Several new Vibrio species, mainly in the phylogenetic neighborhood of V. harveyi, V. halioticoli, V. splendidus, V. tubiashii, and V. fluvialis, have been described in the last few years, with V. neonatus, V. ezurae (28), and V. ponticus (22) being the most recent ones. V. harveyi, V. splendidus, and V. tubiashii are frequently associated with disease in different spe...

The aim of this study was to evaluate the use of RNA polymerase a subunit (rpoA) and phenylalanyl-tRNA synthase (pheS) gene sequences as species identification tools for enterococci. Ninety-six representative strains comprising all currently recognized Enterococcus species were examined. rpoA gene sequences generated a robust classification into species groups similar to the one based on 16S rRNA gene sequence analysis. On the other hand, the pheS gene is a fast-evolving clock even better suited for species delineation than the rpoA gene, but not for recognition of species groups within Enterococcus as determined by both rpoA and 16S rRNA genes. All enterococcal species were clearly differentiated on the basis of their rpoA and pheS sequences. Evaluation of intraspecies variation showed that both rpoA and pheS genes have a high degree of homogeneity among strains of the same species. Strains of the same enterococcal species have at least 99 % rpoA and 97 % pheS gene sequence similarity, whereas, different enterococcal species have at maximum 97 % rpoA and 86 % pheS gene sequence similarity. It was concluded that both genes can be used as reliable tools for identification of clinical and environmental species of Enterococcus and are efficient screening methods for the detection of novel species. The sequence data obtained in this study were compared to the available atpA and 16S rRNA gene sequences. The MLSA approach to Enterococcus taxonomy provides portable, highly reproducible data with lower costs for rapid identification of all enterococcal species.

The aim of this study was to evaluate the use of the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) partial gene sequences for species identification of members of the genus Lactobacillus. Two hundred and one strains representing the 98 species and 17 subspecies were examined. The pheS gene sequence analysis provided an interspecies gap, which in most cases exceeded 10 % divergence, and an intraspecies variation of up to 3 %. The rpoA gene sequences revealed a somewhat lower resolution, with an interspecies gap normally exceeding 5 % and an intraspecies variation of up to 2 %. The combined use of pheS and rpoA gene sequences offers a reliable identification system for nearly all species of the genus Lactobacillus. The pheS and rpoA gene sequences provide a powerful tool for the detection of potential novel Lactobacillus species and synonymous taxa. In conclusion, the pheS and rpoA gene sequences can be used as alternative genomic markers to 16S rRNA gene sequences and have a higher discriminatory power for reliable identification of species of the genus Lactobacillus.

The diversity of a collection of 102 lactococcus isolates including 91 Lactococcus lactis isolates of dairy and nondairy origin was explored using partial small subunit rRNA gene sequence analysis and limited phenotypic analyses. A subset of 89 strains of L. lactis subsp. cremoris and L. lactis subsp. lactis isolates was further analyzed by (GTG) 5 -PCR fingerprinting and a novel multilocus sequence analysis (MLSA) scheme. Two major genomic lineages within L. lactis were found. The L. lactis subsp. cremoris type-strain-like genotype lineage included both L. lactis subsp. cremoris and L. lactis subsp. lactis isolates. The other major lineage, with a L. lactis subsp. lactis type-strain-like genotype, comprised L. lactis subsp. lactis isolates only. A novel third genomic lineage represented two L. lactis subsp. lactis isolates of nondairy origin. The genomic lineages deviate from the subspecific classification of L. lactis that is based on a few phenotypic traits only. MLSA of six partial genes (atpA, encoding ATP synthase alpha subunit; pheS, encoding phenylalanine tRNA synthetase; rpoA, encoding RNA polymerase alpha chain; bcaT, encoding branched chain amino acid aminotransferase; pepN, encoding aminopeptidase N; and pepX, encoding X-prolyl dipeptidyl peptidase) revealed 363 polymorphic sites (total length, 1,970 bases) among 89 L. lactis subsp. cremoris and L. lactis subsp. lactis isolates with unique sequence types for most isolates. This allowed high-resolution cluster analysis in which dairy isolates form subclusters of limited diversity within the genomic lineages. The pheS DNA sequence analysis yielded two genetic groups dissimilar to the other genotyping analysis-based lineages, indicating a disparate acquisition route for this gene.Lactococcus lactis is the primary constituent of many industrial and artisanal starter cultures used to ferment dairy products, especially hard and semihard cheeses. These starter cultures play a key role in determining shelf-life, preservation, and organoleptic quality and thus influence the quality and safety of these fermented products (35). As a result of the industrial importance of L. lactis, it has been the subject of numerous studies, which have resulted in detailed knowledge of its physiology and molecular biology. Moreover, due to the availability of a vast molecular toolbox and three whole genome se-

Coral reefs are very important ecosystems for the marine environment as they harbour a great diversity of organisms. They are also relevant for the economy of several countries via tourism and fishing. In addition, coral reefs provide protection to coastal areas and may be a source of new bioactive compounds. The bacterial biodiversity associated with corals is poorly known (but see Rosenberg & Loya, 2004;Rosenberg & Falkovitz, 2004;Rohwer & Kelley, 2004). Rohwer et al. (2001Rohwer et al. ( , 2002 examined the bacterial community associated with different species of apparently healthy corals from Panama and Bermuda by both culture-dependent and culture-independent techniques. These authors found a high diversity of bacteria, including representative species of Bacillus, Clostridium, cyanobacteria, the Cytophaga-Flavobacterium-Bacteroides group and Proteobacteria. More than 80 % of the 1178 cloned 16S rRNA gene sequences originating from coral sources had not been allocated to recognized bacterial species as they had less than 93 % sequence similarity (Rohwer et al., 2002).Coral reefs have experienced a tremendous decline in recent decades (Hoegh-Guldberg, 2004). Global climate changes, sea-water pollution as a result of aquaculture, oil spills and urban sewage, coral bleaching and other infectious diseases are the main causes of this decline (Hoegh-Guldberg, 2004;Hughes et al., 2003;Knowlton & Rohwer, 2003;Rosenberg & Loya, 2004;Rosenberg & Ben-Haim, 2002;Sutherland et al., 2004). Kushmaro et al. (1996) suggested that bleaching is, in fact, the result of an infectious disease. Bacterial infections of corals caused by Vibrio shilonii (=Vibrio mediterranei) (Kushmaro et al., 2001) and Vibrio The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, recA and rpoA gene sequences determined in this study are shown in Table 1.Neighbour-joining trees showing relationships among Vibrio-like species on the basis of rpoA and recA gene sequence analysis are available as supplementary material in IJSEM Online. G 2005 IUMS Printed in Great Britain