The establishment of genetic competence in Bacillus subtilis is controlled by a vast signal transduction network involving the products of genes that function in several postexponential-phase processes. Two of these proteins, SinR and DegU, serve as molecular switches that influence a cell's decision to undergo either sporulation or genetic competence development. In order to determine the roles of SinR and DegU in competence control, multicopy suppression experiments with plasmid-amplified comS, sinR, and degU genes were undertaken. Multicopy comS was found to elevate competence gene transcription and transformation efficiency in both wild-type and sinR mutant cells but not in degU mutant cells. Multicopy degU failed to suppress comS or sinR mutations. No suppression of comS or degU by multicopy sinR was observed. The expression of a comS::lacZ translational fusion and srf-lacZ operon fusion was examined in sinR cells and cells bearing plasmid-amplified sinR. The expression of comS::lacZ gene fusion was reduced by the sinR mutation, but both comS::lacZ and srf-lacZ were repressed by multicopy sinR. Cells bearing plasmid-amplified sinR were poorly competent. These results suggest that sinR is required for optimal comS expression but not transcription from the srf promoter and that SinR at high concentrations represses srf transcription initiation.
Lipopolysaccharide (LPS) is a known inducer of numerous pro-inflammatory events including the production of platelet-activating factor (PAF). PAF released in response to LPS is a major contributor to the pathological events associated with endotoxemia. The present study demonstrates that dexmethasone (DEX) inhibited the LPS-induced early plasma PAF raise in a dose- and time-dependent manner. In addition, DEX prevented the subsequent PAF-mediated pathological phenomena such as anaphylactic shock-like symptoms, symptoms of disseminated intravascular coagulation and hemorrhage in renal medullae. DEX or the PAF antagonist BN 50739 significantly inhibited LPS-induced NF-kappaB activation. The inhibition of NF-kappaB activation by DEX was overcome by the injection of exogenous PAF. Administration of PAF or LPS resulted in a rapid loss of IkappaBalpha protein. The LPS-induced degradation of IkappaBalpha was prevented by pretreatment with BN 50739, suggesting that PAF is a critical intermediate in the LPS-triggered degradation of IkappaBalpha protein. DEX prevented the LPS-induced IkappaBalpha degradation, which was also reversed by exogenous PAF. Administration of DEX or BN 50739 caused an increase in cytoplasmic IkappaBalpha level. Our results indicate that DEX inhibits IkappaBalpha degradation and subsequent NF-kappaB activation through blocking the initial release of PAF.
The expression of the srf operon of Bacillus subtilis, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in srf expression and surfactin production. This apparent pH-dependent induction of srf expression requiredspo0K, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates srf expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent srf transcription, were required for optimal expression of srf at low and high pHs, but ComP was not required for pH-dependent srf induction. The known negative regulators of srf, RapC and CodY, were found not to function significantly in pH-dependent srf expression. Late-growth culture supernatants at low pH were not active in inducingsrf expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the srf-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction ofsrf-lacZ at neutral pH. The results suggest thatsrf expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.
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