Biomedical subjects

M A Cervin

Publications and source records attributed to M A Cervin.

5 recordsLinked to original sources

PubMed2000

A role for Asp75 in domain interactions in the Bacillus subtilis response regulator Spo0A.

Spo0A is a two-domain response regulator required for sporulation initiation in Bacillus subtilis. Studies on response regulators have focused on the activity of each domain, but very little is known about the mechanism by which the regulatory domain inhibits the activator domain. In this study, we created a single amino acid substitution in the regulatory domain, D75S, which resulted in a dramatic decrease in sporulation in vivo. In vitro studies with the purified Spo0AD75S protein demonstrated that phosphorylation and DNA binding were comparable with wild type Spo0A. However, the mutant was unable to stimulate transcription by final sigma(A)-RNA polymerase from the Spo0A-dependent spoIIG operon promoter. We suggest that the amino acid Asp(75) and/or the region within which it resides, the alpha3-beta4 loop, are involved in the inhibitory interaction between the regulatory and activator domains of Spo0A.

Asparagine↗

PubMed1999

The Spo0A sof mutations reveal regions of the regulatory domain that interact with a sensor kinase and RNA polymerase.

Spo0A is a two-domain response regulator required for the initiation of sporulation in Bacillus subtilis. Spo0A is activated by phosphorylation of its regulatory domain by a multicomponent phosphorelay. To define the role of the regulatory domain in the activation of Spo0A, we have characterized four of the sof mutations in vitro. The sof mutations were identified previously as suppressors of the sporulation-negative phenotype resulting from a deletion of the gene for one of the phosphorelay components, spo0F. Like wild-type Spo0A, the transcription stimulation properties of all of the Sof proteins were dependent upon phosphorylation. Sof mutants from two classes were improved substrates for direct phosphorylation by the KinA sensor kinase, providing an explanation for their suppression properties. Two other Sof proteins showed a phosphorylation-dependent enhancement of the stability of the Sof approximately P-RNA polymerase-DNA complex. One of these mutants, Sof114, increased the stability of the Sof114 approximately P-RNAP-DNA complex without increasing its own affinity for the spoIIG promoter. A comparison of the location of the sof mutations with mutations in CheY suggests that phosphorylation of Spo0A results in the exposure of a region in the regulatory domain that interacts with RNA polymerase, thereby contributing to the signal transduction mechanism.

Bacillus subtilis↗

PubMed1998

The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase.

Initiation of sporulation in Bacillus subtilis is controlled by several regulators which affect activation by phosphorylation of the key response regulator Spo0A or transcription of Spo0A-P-dependent genes. In vivo overexpression of one of these regulators, sinR , results in suppression of transcription from the Spo0A-P-dependent promoters of spo0A , spoIIA , spoIIE and spoIIG and in vitro SinR binds to the promoters of the spoIIA operon and the spo0A gene. In this study we have demonstrated that in vitro SinR directly repressed Spo0A- P-dependent transcription by B.subtilis RNA polymerase from the spoIIG operon promoter. SinR inhibited transcription prior to formation of heparin-resistant complexes but did not displace RNA polymerase from the spoIIG promoter. DNase I protection studies demonstrated that SinR protected a large region of the spoIIG promoter and induced DNase I hypersensitive sites, particularly around the 0A boxes, at the same positions as those induced by zinc. Since binding of zinc induces bends in the DNA, we concluded that SinR binding also altered the conformation of the spoIIG promoter. We propose that SinR-induced conformational changes in Spo0A-dependent promoters prevent activation of trans-cription by Spo0A-P.

Bacillus subtilis↗

PubMed1998

A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis.

The SpoOJA and SpoOJB proteins of Bacillus subtilis are similar to the ParA and ParB plasmid-partitioning proteins, respectively, and mutation of spoOJB prevents the expression of stage II genes of sporulation. This phenotype is a consequence of SpoOJA activity in the absence of SpoOJB, and its basis was unknown. In the studies reported here, SpoOJA was found specifically to dissociate transcription initiation complexes formed in vitro by the phosphorylated sporulation transcription factor SpoOA and RNA polymerase with the spollG promoter. This repressor-like activity is likely to be the basis for preventing the onset of differentiation in vivo. SpoOJB is known to neutralize SpoOJA activity in vivo and also to interact with a mitotic-like apparatus responsible for chromosome partitioning. These data suggest that SpoOJA and SpoOJB form a regulatory link between chromosome partition and development gene expression.

Bacillus subtilis↗

PubMed1994

Differences in eucaryotic cell binding of Pseudomonas.

The lungs of cystic fibrosis (CF) patients are frequently chronically colonized by Pseudomonas aeruginosa. Recently there has been an increase in colonization by another pathogen Pseudomonas cepacia, which can cause a rapid decline in clinical condition or death of the patient. The nature of the factor(s) which predispose CF patients to colonization by one or both of these opportunistic pathogens is unknown. It has been suggested that the genetic defect in CF patients results in an increase in the number of epithelial cell receptors available to P. aeruginosa in the lung, thus rendering CF patients more susceptible to bacterial colonization than non-CF individuals. In this study, we have examined adherence of several strains of P. aeruginosa and P. cepacia to a variety of continuous cell lines, as well as primary cultures of CF and non-CF nasal polyp cells. The results suggested that there may be a decrease in the number of receptors available to both strains of Pseudomonas on cells of canine origin compared to human cells. Both strains appear to use pili as the primary adhesin, but there is also evidence that non-pilus adhesins contribute significantly to eucaryotic cell binding. P. cepacia exhibited microcolony formation on all cell types, which is typical of the localized adherence pattern characteristic of the enteropathogenic Escherichia coli. However, we were unable to demonstrate, with either P. cepacia or P. aeruginosa, a significant increase in adherence to CF compared to non-CF nasal polyp cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗