Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed in Escherichia coli and in the nonproteolytic species Aeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of the Pseudomonas aeruginosa elastase (52% identity), Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant of A. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.Aeromonas hydrophila is a gram-negative opportunistic pathogen in humans and several fish species, causing soft tissue wound infections and diarrhea in the former (1, 18, 21) and fatal hemorrhagic septicemia in the latter (2,12,15,37). It has been speculated that A. hydrophila virulence could involve several extracellular enzymes including proteases, hemolysins, enterotoxins, and acetylcholinesterase. Some of the toxins have been biochemically characterized, but their precise roles in the pathogenicity of A. hydrophila have not yet been determined (8,29,35,41,42). The two major extracellular proteolytic activities of A. hydrophila that have been described so far, a 38-kDa thermostable metalloprotease (29, 41) and a 68-kDa temperature-labile serine protease (30,42), are present in most A. hydrophila culture supernatants. In addition, a 19-kDa zinc proteinase was found in the growth medium of a strain of A. hydrophila isolated from the intestinal tract of the leech Hirudo medicinalis (31), and a 22-kDa serine proteinase, which is stable at 56°C for 10 min, was purified from A. hydrophila strain B 32 culture supernatant (43). Several strategies have been used to examine the role of some A. hydrophila proteases in virulence, including Tn5-induced protease-deficient mutants of A. hydrophila (29) and direct inoculation of purified 22-kDa serine protease in rainbow trout (43), but with conflicting results. Two major secretion products of A. salmonicida, an extracellular serine protease (AspA) and a glycerophospholipid:cholesterol acyltransferase (SatA), had previous...
Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12 Staphylococcus spp. by PCR. In the present study, AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity.
Yersinia ruckeri possesses a quorum‐sensing system detected by cross‐streaking against the white mutant Chromobacterium voilaceum CV0blu. Quorum sensing, which occurs in a number of Gram‐negative pathogens, is known to control virulence gene expression through cell to cell communication. There are two genes required for quorum sensing which are luxR/luxI homologues and there is an N‐acylhomoserine lactone (AHL, commonly called autoinducer) synthesized by the product of luxI homologue which interacts with a response regulator (the product of luxR homologue). The Y. ruckeri quorum sensing system, termed yruR/yruI, was cloned from a gene library constructed in pUC18 plasmid vector. Nucleotide sequence analysis of Y.ruckeri yruR and yruI revealed convergent transcription with overlapped 3′ ends and two open reading frames (ORFs) of 247 and 217 amino acids, respectively. Two pairs of synthetic oligonucleotide primers of 24 bases were used in a PCR assay to amplify yruR/yruI genes with short flanking sequences as well as an internal DNA fragment within yruR/yruI genes. DNA fragments of 1900 and 1000 bp were amplified from both sources, lysed Y. ruckeri cells and isolated DNA. Amplified sequences were detected in ethidium‐bromide agarose gels or by Southern blot analysis with an internal 336‐bp fragment as a hybridization probe. Detection of Y. ruckeri by PCR amplification of yruR/yruI genes has great potential for rapid identification of this fish pathogen bacterium as it has proved to be highly specific.
The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.
The aroA gene of Yersinia ruckeri, which encodes 5-enolpyruvylshikimate 3-phosphate synthase, was insertionally inactivated with a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of Y. ruckeri 21102 O1 by means of the suicide vector pIVET8. The Y. ruckeri aroA::Kan(r) mutant was highly attenuated when inoculated intraperitoneally into rainbow trout, with a 50% lethal dose of >5 x 10(7) CFU. The mutants were not recoverable from the internal organs 48 h post-inoculation or later. The vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection (relative percentage survival = 90%) against the pathogenic wild-type strain of Y. ruckeri.
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