Virulent Yersinia species possess a common plasmid that encodes essential virulence determinants (Yops) which are regulated by the extracellular stimuli Ca2+ and temperature. The V antigen operon was recently shown to be involved in the Ca21_regulated negative pathway (A. Forsberg and H. Wolf-Watz, Mol. Microbiol. 2:121-133, 1988). We show here that the V antigen-containing operon of Yersinia pseudotuberculosis is a polycistronic operon having the gene order IcrGVH-yopBD. DNA sequencing analysis of lcrGVH revealed a high homology to the corresponding genes of Yersinia pestis. LcrG was conserved and LcrH showed only one amino acid difference, while LcrV showed only 96.6% identity. The amino acid substitutions of LcrV occurred in the central domain of the protein, while the two ends of the protein were conserved. Northern (RNA) blotting experiments showed that the operon is regulated at the transcriptional level by the extracellular stimuli temperature and calcium. One 4.6-kb transcriptional product of the operon was identified. This mRNA is rapidly processed at its 5' end, resulting in different mRNA species of variable stability. By genetic analysis, the kcrV and kcrH gene products were found to be regulatory proteins having important roles in the Ca2 -controlled regulation of Yop expression. The activity of LcrH is modulated by a gene product of the operon that inhibits the negative action of LcrH on yop transcription in the absence of Ca2+.
The aetiology and pathogenesis of chronic painful Achilles tendinosis are unknown. This investigation aimed to use cDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique to study tendinosis and control tissue samples. Five patients (females mean age 57.1 i 4.3 (years k SD)) with chronic painful Achilles tendinosis were included. From all patients, one biopsy was taken from the area with tendinosis and one from a clinically normal area (control) of the tendon. The tissue samples were immediately immersed in RNAlater and frozen at -80 "C until RNA extraction. Portions of pooled RNA from control and tendinosis sites, respectively, were transcribed to cDNA, radioactively labelled ("P), hybridized to cDNA expression arrays, and exposed to phosphoimager screens over night. Expressions of specific genes, shown to be regulated in the cDNA array analysis, were analyzed in the individual samples using real-time PCR. cDNA arrays showed that gene expressions for matrixrnetalloproteinase-2 (MMP-2), fibronectin subunit B (FNRB), vascular endothelial growth factor (VEGF), and mitogen-activated protein kinase p38 (MAPKp38) were up-regulated, while matrix-metalloproteinase-3 (MMP-3) and decorin were down-regulated, in tendinosis tissue comprrred with control tissue. Using real-time PCR, 415 and 3/5 patients showed up-regulation of MMP-2 and FNRB mRNA. respectively. For decorin, VEGF, and MAPKp38. real-time PCR revealed a great variability among patients. Interestingly, the niRNAs for several cytokines and cytokine receptors were not regulated, indicating the absence of an inflammatory process in chronic painful Achilles tendinosis. In conclusion, cDNA-arrays and real-time PCR can be used to study differences in gene expression levels between tendinosis and control tendon tissue.
We further characterize a novel plasmid function preventing SOS induction called Psi (Plasmid SOS Inhibition). We show that Psi function is expressed by psiB, a gene located at coordinate 54.9 of plasmid R6-5 and near oriT, the origin of conjugal transfer. Deletions and amber mutations of the psiB gene permitted us to demonstrate that PsiB polypeptide (apparent molecular weight, 12 kDa) is responsible for Psi function. PsiB protein prevents recA730-promoted mutagenesis and intra-chromosomal recombination but not recombination following conjugation. Overproduction of PsiB protein sensitizes the host cell to UV irradiation. We propose that PsiB polypeptide has an anti-SOS action by inhibiting activation of RecA protein, thus preventing the occurrence of LexA-controlled functions.
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