[Keywords: Circadian; Neurospora; frq; PEST; post-translational] Supplemental material is available at http://www.genesdev.org.
Burkholderia cepacia has emerged as an important pulmonary pathogen in immunocompromised patients and in patients with cystic fibrosis (CF). Little is known about the virulence factors and pathogenesis of B. cepacia, although the persistent and sometimes invasive infections caused by B. cepacia suggest that the organism possesses mechanisms for both cellular invasion and evasion of the host immune response. In this study, cultured human cells were used to analyze the invasion and intracellular survival of B. cepacia J2315, a highly transmissible clinical isolate responsible for morbidity and mortality in CF patients. Quantitative invasion and intracellular growth assays demonstrated that B. cepacia J2315 was able to enter, survive, and replicate intracellularly in U937-derived macrophages and A549 pulmonary epithelial cells. Transmission electron microscopy of infected macrophages confirmed the presence of intracellular B. cepacia and showed that intracellular bacteria were contained within membrane-bound vacuoles. An environmental isolate of B. cepacia, strain J2540, was also examined for its ability to invade and survive intracellularly in cultured human cells. J2540 entered cultured macrophages with an invasion frequency similar to that of the clinical strain, but it was less invasive than the clinical strain in epithelial cells. In marked contrast to the clinical strain, the environmental isolate was unable to survive or replicate intracellularly in either cultured macrophages or epithelial cells. Invasion and intracellular survival may play important roles in the ability of virulent strains of B. cepacia to evade the host immune response and cause persistent infections in CF patients.
High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase 32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus 32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli 32 and cross-reacts with a monoclonal antibody to E. coli 32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in 32 levels after a temperature shift from 30 to 42؇C similar to that described for E. coli. In addition, increasing the cellular content of 32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30؇C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and 32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to 32 expression in this organism. Both the rpoH gene and 32 protein were expressed constitutively throughout the cell cycle at 30؇C. The isolation of rpoH provides an important tool for future studies of the role of 32 in the normal physiology of C. crescentus.Heat shock proteins (HSPs) are generally either molecular chaperones that act to maintain the proper folding of cell proteins, such as DnaK or Hsp70 and GroEL or Hsp60, or proteases, such as Clp and Lon. This group of proteins, found in prokaryotes and eukaryotes, functions both at physiological temperature and during cellular responses to high temperature and other environmental stresses. Distinct modes of transcriptional induction of the HSPs have been described for Escherichia coli and Saccharomyces cerevisiae (reviewed in reference 27). In E. coli, heat shock enhances the level of the 32 subunit of RNA polymerase which then recognizes the promoters of the heat shock genes and increases their levels of transcription (10,22,24). By contrast, in S. cerevisiae a transcriptional activator, HSF, binds constitutively to a highly conserved DNA repeat, or heat shock element, in the promoter region. Elevated temperature induces a conformational change in HSF which enables it to activate the transcription of heat shock genes (27).Although the general heat shock response in bacteria is highly conserved, its regulation by 32 has previously been documented in bacteria that are members of the ␥ subdivision of proteobacteria. Only recently have 32 homologs been reported in ␣ proteobacteria (32). The role of 32 in the heat shock response is best understood for E. coli (reviewed in references 7, 19, 21, and 50). In this organism, 32 levels increase 15-to 20-fold in response to a sudden rise in temperature (25,44 promoters and a E promoter ...
The role of several regulatory elements in environmental modulation of mucoidy in Pseudomonas aeruginosa was studied. Transcriptional activation of algD, necessary for the mucoid phenotype, was found to depend on FUS, the newly identified far-upstream sites of the algD promoter. The FUS were delimited to a region spanning nucleotides -432 to -332 relative to the algD mRNA start site. Insertional inactivation of algR in PA0568 abolished the algD promoter response to nitrogen availability and greatly diminished but did not completely eliminate reactivity to changes in salt concentration. Insertional inactivation of rpoN (ntrA) in PA0568 did not affect algR and algD transcription.
Activation ofalgD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely
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