No abstract

Pseudomonas aeruginosa controls several genes in a cell density-dependent manner through a phenomenon termed quorum sensing. The transcriptional activator protein of the las quorum-sensing system is encoded for by the lasR gene, which is at the top of a quorum-sensing hierarchy. The activation of LasR as a transcriptional activator induces the expression of multiple genes that code for factors important for virulence, and rhlR, which encodes the transcriptional activator protein of the P. aeruginosa rhl quorum-sensing system. Elucidating the method of lasR regulation is crucial to understanding P. aeruginosa quorum sensing. In this report, we present studies on the transcriptional control of lasR. We identified two distinct transcriptional start sites for lasR that were located 201 bp (transcript T1) and 231 bp (transcript T2) upstream from the lasR start of translation. With the use of transcriptional lasRp-lacZ fusions, we showed that in P. aeruginosa, lasR expression is cell density dependent. This gene was expressed at a basal level until it was induced during the second half of log-phase growth, with expression becoming maximal during stationary-phase growth. We also showed that lasR expression was regulated through the cyclic AMP receptor protein (CRP)-binding consensus sequence in its promoter region. Our results from P. aeruginosa mutant studies and gel retardation assays indicated that this regulation was mediated by Vfr, a homolog of the Escherichia coli CRP.The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to regulate specific genes in a cell densitydependent manner. Quorum-sensing systems are found in many gram-negative bacteria, the first of which was the marine symbiont Vibrio fischeri, and consist of a transcriptional activator protein and a small diffusible chemical molecule termed autoinducer (see reference 14 for a review). P. aeruginosa has two distinct quorum-sensing systems, las and rhl. The las quorum-sensing system consists of the transcriptional activator protein LasR and the Pseudomonas autoinducer PAI-1 [N-(3-oxododecanoyl)-L-homoserine lactone] (16, 32, 33). At high cell densities, PAI-1 reaches a threshold concentration and forms a complex with LasR, thereby converting the protein into a transcriptional activator that has been shown to be important for transcription of lasI, lasB, lasA, apr, toxA, and rhlR (17, 23, 32, 36, 42, 48). The rhl quorum-sensing system consists of the transcriptional activator protein RhlR and the autoinducer PAI-2 (N-butyryl-L-homoserine lactone; formerly named factor 2 [14]) (30,31,34). So far, rhl quorum sensing has been shown to control the transcription of rhlA, rhlI, lasB, and rpoS (4, 23, 29, 35).In P. aeruginosa, a quorum-sensing hierarchy exists in which las quorum sensing is dominant. The las quorum-sensing system regulates rhlR at both the transcriptional and translational levels, which means that the quorum-sensing chain of events begins with lasR transcription (23, 36). Therefore, determining the manner in which lasR is regul...

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.

Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.

Page 2874, Acknowledgments, lines 1 and 2: "We thank the members of the University of Wisconsin genomics team for expert technical assistance" should read "We thank the members of the University of Wisconsin genomics team for expert technical assistance, especially Sean Phillips and Nicholas Hermersmann whose contributions were outstanding."