Biomedical subjects

J M Reyrat

Publications and source records attributed to J M Reyrat.

23 records · Page 2Linked to original sources

PubMed1995

The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin.

The ureABC genes of Mycobacterium tuberculosis were cloned. By using a set of degenerate primers corresponding to a conserved region of the urease enzyme (EC 3.5.1.5), a fragment of the expected size was amplified by PCR and was used to screen a M. tuberculosis cosmid library. Three open reading frames with extensive similarity to the urease genes from other organisms were found. The locus was mapped on the chromosome, using an ordered M. tuberculosis cosmid library. A suicide vector containing a ureC gene disrupted by a kanamycin marker (aph) was used to construct a urease-negative Mycobacterium bovis bacillus Calmette-Guérin mutant by allelic exchange involving replacement of the ureC gene with the aph::ureC construct. To our knowledge, allelic exchange has not been reported previously in the slow-growing mycobacteria. Homologous recombination will be an invaluable genetic tool for deciphering the mechanisms of tuberculosis pathogenesis, a disease that causes 3 x 10(6) deaths a year worldwide.

Alleles↗

PubMed1994

Phosphorylation of the Rhizobium meliloti FixJ protein induces its binding to a compound regulatory region at the fixK promoter.

The FixJ protein is a member of the regulator class of two-component systems involved in the transcriptional activation of nitrogen fixation genes in Rhizobium meliloti. Phosphorylation of FixJ was previously demonstrated to dramatically enhance its transcriptional activity at the nifA and fixK promoters. Here we show that the isolated carboxyl-terminal domain of FixJ, FixJC, binds the fixK promoter, whereas binding of the full-length FixJ protein requires its phosphorylation. By analyzing the DNase I and Exonuclease III protection patterns of the wild-type and a mutant fixK promoter, we have identified two overlapping binding regions for both phosphorylated FixJ and FixJC. A higher affinity region is located between positions -69 and -44 relative to the transcription start site, and a lower affinity region, between positions -57 and -31, overlaps the -35 region of the promoter.

Bacterial Proteins↗

PubMed1994

Intramolecular signal transduction within the FixJ transcriptional activator: in vitro evidence for the inhibitory effect of the phosphorylatable regulatory domain.

FixJ is a phosphorylatable 'response regulator' controlling the transcription of the key nitrogen fixation genes nifA and fixK in Rhizobium meliloti. Sequence and genetic analyses indicated that FixJ comprises an N-terminal phosphorylatable regulatory domain, FixJN, and a C-terminal transcriptional activator domain, FixJC. We have now overexpressed and purified the FixJC protein and show that it is fully active in an in vitro transcription system with purified RNA polymerase. FixJC appeared to act synergistically with RNA polymerase at the nifA promoter. Furthermore FixJC was more active in vitro than the full-length dephosphorylated FixJ protein. Therefore activity of FixJC is inhibited by FixJN within the FixJ protein. This inhibition is relieved by phosphorylation of FixJN. Such a negative mode of intramolecular signal transduction may be generalizable to other response regulators.

Bacterial Proteins↗

PubMed1994

FixL of Rhizobium meliloti enhances the transcriptional activity of a mutant FixJD54N protein by phosphorylation of an alternate residue.

In Rhizobium meliloti, transcription of nitrogen fixation genes is induced in oxygen-depleted conditions under the control of the two-component regulatory system FixLJ. FixJ is a transcriptional activator whose activity is dramatically enhanced by phosphorylation, whereas FixL is a hemoprotein kinase that controls the level of phosphorylated FixJ in response to oxygen availability. We have found that a mutant FixJ protein, FixJD54N, in which the presumed site of phosphorylation (aspartate 54) was changed to an asparagine, is strongly affected for phosphorylation by FixL and is not detectably phosphorylated from the low-molecular-weight phosphate donor, acetyl-phosphate. Unexpectedly, FixL strongly enhances the transcriptional activity of the FixJD54N protein both in vivo and in vitro. We present evidence that FixJD54N transcriptional activity is enhanced by phosphorylation of an alternate residue in a reaction that requires FixL and ATP and is not affected by oxygen. We also demonstrate the key role of Asp-54 of FixJ in oxygen signal transduction.

Bacterial Proteins↗

PubMed1993

Oxygen-regulated in vitro transcription of Rhizobium meliloti nifA and fixK genes.

Oxygen concentration regulates the expression of nitrogen fixation genes in the symbiotic bacterium Rhizobium meliloti. We demonstrate that two proteins, FixL and FixJ, that belong to the two-component family of regulatory proteins are necessary and sufficient for oxygen-regulated in vitro transcription of the two key regulatory genes, nifA and fixK. We show directly that FixJ is a transcriptional activator, working in conjunction with the RNA polymerase sigma 70 holoenzyme. Addition of FixL122, a soluble form of the sensor FixL protein, to the transcription assay enhanced FixJ transcriptional activity in response to low oxygen concentration. This enhancement of FixJ activity was correlated with FixJ phosphorylation.

Bacterial Proteins↗