Streptococcus salivarius is a lactose-and galactose-positive bacterium that is phylogenetically closely related to Streptococcus thermophilus, a bacterium that metabolizes lactose but not galactose. In this paper, we report a comparative characterization of the S. salivarius and S. thermophilus gal-lac gene clusters. The clusters have the same organization with the order galR (codes for a transcriptional regulator and is transcribed in the opposite direction), galK (galactokinase), galT (galactose-1-P uridylyltransferase), galE (UDP-glucose 4-epimerase), galM (galactose mutarotase), lacS (lactose transporter), and lacZ (␤-galactosidase). An analysis of the nucleotide sequence as well as Northern blotting and primer extension experiments revealed the presence of four promoters located upstream from galR, the gal operon, galM, and the lac operon of S. salivarius. Putative promoters with virtually identical nucleotide sequences were found at the same positions in the S. thermophilus gal-lac gene cluster. An additional putative internal promoter at the 3 end of galT was found in S. thermophilus but not in S. salivarius. The results clearly indicated that the gal-lac gene cluster was efficiently transcribed in both species. The Shine-Dalgarno sequences of galT and galE were identical in both species, whereas the ribosome binding site of S. thermophilus galK differed from that of S. salivarius by two nucleotides, suggesting that the S. thermophilus galK gene might be poorly translated. This was confirmed by measurements of enzyme activities.Streptococcus salivarius is an oral bacterium that is phylogenetically closely related to Streptococcus thermophilus, which is used in food fermentation (16,17,25,29). Both species were initially placed in the S. salivarius taxon as S. salivarius subsp. salivarius and S. salivarius subsp. thermophilus (7) but were regarded as distinct species by Schleifer et al. (28) on the basis of both genetic and phenetic criteria. These two species, together with Streptococcus vestibularis, form a distinct cluster within the streptococcal phylogenetic tree (13,16,25). Lactose, the principal energy source used by S. thermophilus for growth in milk, is transported into the cell by a permease (LacS) belonging to the glycoside-pentoside-hexuronide-cation symporter family (23). Lactose is hydrolyzed within the cell into glucose and galactose by ␤-galactosidase. Glucose is metabolized to lactic acid via the glycolytic, Embden-Meyerhof-Parnas pathway, whereas in most strains galactose cannot be metabolized and is expelled into the external medium (11,14). The organization of the galactose operon coding for the Leloir pathway enzymes in S. thermophilus has recently been elucidated (5, 24, 36), indicating that the inability of S. thermophilus to metabolize galactose is not caused by the absence of the genetic information required for the synthesis of suitable metabolic pathways. Moreover, the activities of the enzymes involved in the Leloir pathway (galactokinase, galactose-1-P uridylyltransferase, and...

The ubiquitous water-borne Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a worldwide disease in fish farms. Plasmids carrying antibiotic resistance genes have already been described for this bacterium. The aim of the present study was to identify and characterize additional multidrug resistance plasmids in A. salmonicida subsp. salmonicida. We sequenced the plasmids present in two multiple antibiotic-resistant isolates using highthroughput technologies. We also investigated 19 other isolates with various multidrug resistance profiles by genotyping PCR and assessed their resistance to tetracycline. We identified variants of the pAB5S9 and pSN254 plasmids that carry several antibiotic resistance genes and that have been previously reported in bacteria other than A. salmonicida subsp. salmonicida, which suggests a high level of interspecies exchange. Genotyping analyses and the antibiotic resistance profiles of the 19 other isolates support the idea that multiple versions of pAB5S9 and pSN254 exist in A. salmonicida subsp. salmonicida. We also identified variants of the pRAS3 plasmid. The present study revealed that A. salmonicida subsp. salmonicida harbors a wide variety of plasmids, which suggests that this ubiquitous bacterium may contribute to the spread of antibiotic resistance genes in the environment. The Gram-negative bacterium Aeromonas salmonicida subsp. salmonicida is an opportunistic fish pathogen (1). It is the etiological agent of furunculosis, a disease that especially affects salmonids in fish farms (2). While antibiotics are commonly used to treat A. salmonicida subsp. salmonicida infections, multidrug-resistant isolates have been frequently detected (3-5), preventing the effective treatment of furunculosis.Many fully characterized plasmids from A. salmonicida subsp. salmonicida have provided antibiotic resistance to this species (2). All the known plasmids in A. salmonicida subsp. salmonicida harboring antibiotic resistance genes include at least a tetracycline resistance gene. The vast majority of the plasmids bearing antibiotic resistance genes confer multiple types of resistance to A. salmonicida subsp. salmonicida, including the large (167-kb) plasmid pAsa4, which provides resistance against chloramphenicol, spectinomycin, streptomycin, sulfonamides, tetracycline, mercury, and quaternary ammonium compounds (6). A plasmid bearing multiple resistance genes that is similar to the large pSN254 plasmid in Salmonella enterica (7) has been partially described in A. salmonicida subsp. salmonicida (3). This pSN254-like plasmid can be transferred via conjugation from A. salmonicida subsp. salmonicida to multiple receivers, including Escherichia coli, Edwardsiella tarda, and Aeromonas hydrophila (3).Plasmid variants appear to be relatively frequent in A. salmonicida subsp. salmonicida. The best example is the pRAS3 plasmid. To date, two variants of this plasmid (pRAS3.1 and pRAS3.2) have been described (8). The differences between them are very s...

Aeromonas salmonicida , a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS 11 s from the IS 256 family encircle the rearranged regions. To confirm that these IS 11 s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS 11 -dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS 11 -dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS 11 . Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS 11 . This is the first study showing that ISs are involved in plasmid instability in A. salmonicida .

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BackgroundStreptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2) are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor.ResultsTwo mutants (G6G and M3G) possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757) that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9%) and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%), which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200), motif II (His239), and motif III (Ser568). SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly) at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p < 0.001).ConclusionIn summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.