Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli

The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein-induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP-CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non-responsive to glucose repression. The involvement of cAMP-CRP complex in the activation of the algD promoter in E. coli has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP-like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

Regulation of exotoxin A synthesis in Pseudomonas aeruginosa: characterization of toxA-lacZ fusions in wild-type and mutant strains

A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild-type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. beta-galactosidase synthesis in the strains (cointegrates) carrying the toxA-lacZ fusions was regulated like ETA synthesis is in P. aeruginosa. Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of beta-galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of beta-galactosidase and ETA synthesis were abolished.     

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.     

nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.     

铜绿假单胞菌随机启动子库的构建及受铁调控基因的筛选和鉴定

[目的]针对铁这种几乎所有微生物都必需的,对病原菌尤为重要的营养元素,在全基因组水平上检测铜绿假单胞菌的铁感应基因.[方法]采用含LuxCDABE报道子的质粒pMS402为载体,构建了铜绿假单胞菌的随机启动子库,建立了特定条件下研究铜绿假单胞菌全基因组基因表达变化的高通量平台.[结果]验证了2个已知的受铁调控的基因,并发现了尚未报道的二氢叶酸还原酶,磷酸葡萄糖脱水酶和柠檬酸铁转运蛋白受铁调控的现象,发现与铁相关的四个功能未知的基因和3个假定蛋白的基因.[结论]研究结果对于阐明铁在铜绿假单胞菌中的调控作用,揭示铁对细菌生理及致病性的作用机制提供了理论基础.随机启动子库的构建也为细菌基因组水平研究基因表达提供了一个有用的工具.     

Components of intestinal epithelial hypoxia activate the virulence circuitry of Pseudomonas

We have previously shown that a lethal virulence trait in Pseudomonas aeruginosa, the PA-I lectin, is expressed by bacteria within the intestinal lumen of surgically stressed mice. The aim of this study was to determine whether intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression. A fusion construct was generated to express green fluorescent protein downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P. aeruginosa. Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1-0.3% O2) for 1 h, with or without a recovery period of normoxia (21% O2) for 2 h, and then inoculated with P. aeruginosa containing the PA-I reporter construct. Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 h; P < 0.001). Similar activation of PA-I was also induced by cell-free apical, but not basal, media from hypoxic Caco-2 monolayers. PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia. We conclude that the virulence circuitry of P. aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.