|
|
|
|
|
|
Regulation of Vibrio anguillarum empA Metalloprotease Expression and Its Role in Virulence. Steven M. Denkin, 2004.Atlantic salmon (Salmo salar) were challenged with Vibrio anguillarum strains M93Sm and NB10 and empA null mutants M99 and NB12 . Both wild types were virulent when administered by intraperitoneal (i.p.) injection or anal intubation . NB12 was avirulent via either route of infection . M99 virulence was attenuated when delivered by intubation, but fully virulent by i.p . injection . Northern blot analysis revealed empA expression in M93Sm and NB10 cells incubated in mucus, while incubation in Luria-Bertani broth plus 2% NaCl (LB20) induced empA expression only in NB10 . Nucleotide differences between M93Sm and NB10 empA sequences were found in regions located 207 and 229 bp upstream of the empA translational start . Reverse transcription-PCR and 5' rapid amplification of cDNA ends revealed the empA transcriptional start site 85 bp upstream of the translational start for both strains . A putative  S-dependent promoter was identified upstream of the transcriptional start in both strains . Site-directed mutagenesis was used to create rpoS mutants of M93Sm and NB10 . Neither rpoS mutant exhibited protease activity . Since empA is expressed during stationary phase, the effects of conditioned medium on protease activity were examined . M99 conditioned LB20 supernatants stimulated protease activity in NB10 while allowing M93Sm to produce protease in LB20 . Neither acyl homoserine lactones nor AI-2 induced protease activity . Conditioned LB20 supernatant from a V . anguillarum luxS mutant caused a more rapid induction of protease activity in wild-type cells . Our data show that expression of empA is differentially regulated in V . anguillarum strains NB10 and M93Sm and requires
S, quorum-sensing molecules, and gastrointestinal mucus .
Lactococcus lactis Lytic Bacteriophages of the P335 Group Are Inhibited by Overexpression of a Truncated CI Repressor. Evelyn Durmaz, 2002.Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations . DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group . The P335 lytic phage  31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny . When the putative cI repressor gene of phage
31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp . lactis NCK203, indicating that the wild-type CI repressor was dysfunctional . Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful . The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein . In the presence of the truncated CI construct, pTRKH2::CI-per1, phage
31 was inhibited to an efficiency of plaquing (EOP) of 10-6 in NCK203 . A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the
31 EOP to <10-7 . Phage
31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding . Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI . Twelve of 16 lytic P335 group phages failed to form plaques on L . lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies . Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the
31 CI repressor . This study demonstrated that a number of different P335 phages, lytic for L . lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor .
The Bacillus thuringiensis Linear Double-Stranded DNA Phage Bam35, Which Is Highly Similar to the Bacillus cereus Linear Plasmid pBClin15, Has a Prophage State. Nelli J. Strömsten, 2003.Bam35, a 15-kbp double-stranded DNA phage, infects Bacillus thuringiensis . Recently, sequencing of the related Bacillus cereus revealed a 15.1-kbp linear plasmid, pBClin15 . We show that pBClin15 closely resembles Bam35 and demonstrate conversion of Bam35 to a prophage . This state is common, as several B . thuringiensis strains release Bam35-related viruses . Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10. Irena Pastar, 2003.A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized . The prtR gene and flanking regions were cloned and sequenced . The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence . Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci . Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein . According to its  S1- and ß-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L . rhamnosus strains tested . Proteinase extracts of the BGT10 strain obtained with Ca2+-free buffer at pH 6.5 were proteolytically active . The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions . The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription . This is in correlation with the catalytic activity of the PrtR proteinase .
|
© 2005
Transgalactic Ltd (manufacturer of Bioscreen C software) |
Privacy Statement | P.O. Box
1393, 00101 Helsinki, Finland,
Last modified: May 25, 2005
| ||||||
