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The BaeSR Two-Component Regulatory System Activates Transcription of the yegMNOB (mdtABCD) Transporter Gene Cluster in Escherichia coli and Increases Its Resistance to Novobiocin and Deoxycholate. Natalya Baranova, 2002.Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible  acrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR . When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the
acrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold) . Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux . The genes baeSR are part of a putative operon, yegMNOB baeSR . Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay . The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance . It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate . We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter) . Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance .
Alcaligin Siderophore Production by Bordetella bronchiseptica Strain RB50 Is Not Repressed by the BvgAS Virulence Control System. Timothy J. Brickman, 2002.A previous study found that alcaligin siderophore production by Bordetella bronchiseptica strain RB50 is Bvg repressed . In contrast, we report that alcaligin production by RB50 does not require Bvg phenotypic phase modulation and that isogenic Bvg(Con) and Bvg- phase-locked mutants both produce alcaligin in response to iron starvation . Transcription and Analysis of Polymorphism in a Cluster of Genes Encoding Surface-Associated Proteins of Clostridium difficile. Marie-Pierre Savariau-Lacomme, 2003.Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins . Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin . To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases . It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli . Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots . The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream . The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence . No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains . The cwp84 gene is strongly transcribed as a monocistronic message . This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C . difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster . Expression of Primary Sigma Factor (PSF) and PSF-Like Sigma Factors in the Cyanobacterium Synechocystis sp . Strain PCC 6803. Ilona Tuominen, 2003.Large amounts of sigA mRNA, encoding the primary  factor (PSF) in Synechocystis sp . strain PCC 6803, accumulated under standard growth conditions, while stress conditions like heat or high salinity led to a rapid decrease in sigA mRNA content . The sigB, sigC, sigD, and sigE genes, encoding PSF-like
factors, were under strict physiological control .
Prospecting for Novel Biocatalysts in a Soil Metagenome. S. Voget, 2003.The metagenomes of complex microbial communities are rich sources of novel biocatalysts . We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy . A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value . Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes . Interestingly, nine of these agarase genes probably originated from gene duplications . Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an  -amylase (amyA), a 1,4--glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119) . Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system . The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined . Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium .
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