Hyperosmotic shock induces the sigma32 and sigmaE stress regulons of Escherichia coli.

The rise in the levels of sigmaS that accompanies hyperosmotic shock plays an important role in Escherichia coli survival by increasing the transcription of genes involved in the synthesis and transport of osmoprotectants. To determine if other stress regulons collaborate with sigmaS in dealing with high osmolality, we used single copy fusions of lacZ to representative promoters induced by protein misfolding in the cytoplasm (dnaK and ibp ), extracytoplasmic stress [P3rpoH and htrA(degP )] and cold shock (cspA). Both the sigma32-dependent, dnaK and ibp, promoters, and the sigmaE-dependent, P3rpoH and htrA, promoters were rapidly but transiently induced when mid-exponential phase cells were treated with 0.464 M sucrose. The cspA promoter, however, did not respond to the same treatment. Overproduction of the cytoplasmic domain of the sigmaE anti-sigma factor, RseA, reduced the magnitude of osmotic induction in lambdaphi(P3rpoH:lacZ ) lysogens, but had no effect on the activation of the dnaK and ibp promoters. Similarly, induction of the dnaK:lacZ and ibp:lacZ fusions was not altered in either rpoS or ompR genetic backgrounds. Osmotic upshift led to a twofold increase in the enzymatic activity of the lambdaTLF247 rpoH:lacZ translational fusion whether or not the cells were treated with rifampicin, indicating that both heat shock and exposure to high osmolality trigger a transient increase in rpoH translation. Our results suggest that the sigma32, sigmaE and sigmaS regulons closely co-operate in the managment of hyperosmotic stress. Induction of the sigma32 and sigmaE regulons appears to be an emergency response required to repair protein misfolding and facilitate the proper folding of proteins that are rapidly synthesized following loss of turgor, while providing a mechanism to increase the activity of sigmaS, the primary stress factor in osmoadaptation.

Stress responses as a tool To detect and characterize the mode of action of antibacterial agents.

Single-copy gene fusions between the lacZ reporter gene and Escherichia coli strains containing promoters induced by cold shock (cspA), cytoplasmic stress (ibp), or protein misfolding in the cell envelope (P3rpoH) were constructed and tested to determine their ability to detect antibacterial agents while simultaneously providing information on their cellular targets. Antibiotics that affect prokaryotic ribosomes selectively induced the cspA::lacZ or ibp::lacZ gene fusion, depending on their mode of action. The membrane-damaging peptide polymyxin B induced both the P3rpoH::lacZ and ibp::lacZ fusions, while the beta-lactam antibacterial agent carbenicillin activated only the P3rpoH promoter. Nalidixic acid, a compound that causes DNA damage, downregulated beta-galactosidase synthesis from P3rpoH but had little effect on expression of the reporter enzyme from either the cspA or ibp promoter. All model antibiotics could be identified over a wide range of sublethal concentrations with signal-to-noise ratios between 2 and 11. A blue halo assay was developed to rapidly characterize the modes of action of antibacterial agents by visual inspection, and this assay was used to detect chloramphenicol secreted into the growth medium of Streptomyces venezuelae cultures. This simple system holds promise for screening natural or combinatorial libraries of antimicrobial compounds.

Characterization of the rat neurofibromatosis 2 gene and its involvement in asbestos-induced mesothelioma.

The neurofibromatosis 2 (NF2) tumor suppressor gene was recently implicated in the genesis of human mesothelioma. To investigate the role of this tumor suppressor gene in rat asbestos-induced mesothelioma, a commonly used model for the human disease, we characterized the rat homologue of NF2 and examined rat chrysotile-induced primary mesotheliomas and cell lines derived from chrysotile- and crocidolite-induced mesotheliomas for alterations in this gene. The coding sequence obtained for the rat NF2 gene had 90% nucleotide homology with the human NF2 gene. The rat NF2 gene was ubiquitously expressed as a 4.4-kb transcript in normal rat tissues as well as in rat mesothelioma cell lines. Reverse transcription-polymerase chain reaction analysis to examine splicing of NF2 exons in mesothelioma cells indicated that the exon splicing pattern was similar in normal and neoplastic cells. To determine if mutations had occurred in the NF2 coding region in rat mesotheliomas, single-strand conformation polymorphism analysis and direct sequencing were used to screen 10 primary tumors and six tumor cell lines. No DNA sequence alterations were observed in any of the rat mesothelioma samples examined. These findings contrast with data reported previously for human mesotheliomas, in which the NF2 gene was found to be mutated in 40% of cases. Taken together, these data suggest that the role of NF2 in the development of rodent asbestos-induced mesothelioma may differ significantly from the role in the human disease.