Analysis of CenKR essentiality in Sinorhizobium meliloti and its activity at a target gene promoter in vivo.

The two-component regulatory system CenK-CenR has recently emerged as a regulator of cell envelope and cell division processes in the alpha-proteobacteria. In Sinorhizobium meliloti, CenK-CenR regulates the expression of SrlA, a thioredoxin-domain protein of unknown function. Deletion of srlA causes sensitivity to salt and oxidizing agents on solid growth medium. In this work, we report that the response regulator CenR, but not the histidine kinase CenK, is essential for cell viability in S. meliloti. We also demonstrate that phosphorylation of the target residue D55 is not required for viability, suggesting that the unphosphorylated transcription factor sufficiently regulates expression of one or more essential genes in the genome. Using transcription assays and phenotype testing we examine CenK-CenR-dependent activation of the srlA promoter and demonstrate its absolute dependence on phosphoryl-CenR for activity and that the CenR substitution D55E acts as a phosphomimetic that partially restores activity at the srlA promoter in the absence of phosphorylation by CenK. Finally, we report a mutational analysis of the CenR binding site in the srlA promoter required for transcriptional activation.

The two-component regulatory system CenK-CenR regulates expression of a previously uncharacterized protein required for salinity and oxidative stress tolerance in Sinorhizobium meliloti.

Genes of unknown function constitute a considerable fraction of most bacterial genomes. In a Tn5-based search for stress response genes in the nitrogen-fixing facultative endosymbiont Sinorhizobium (Ensifer) meliloti, we identified a previously uncharacterized gene required for growth on solid media with increased NaCl concentrations. The encoded protein carries a predicted thioredoxin fold and deletion of the gene also results in increased sensitivity to hydrogen peroxide and cumene hydroperoxide. We have designated the gene srlA (stress resistance locus A) based on these phenotypes. A deletion mutant yields phenotypic revertants on high salt medium and genome sequencing revealed that all revertants carry a mutation in genes homologous to either cenK or cenR. srlA promoter activity is abolished in these revertant host backgrounds and in a strain carrying a deletion in cenK. We also observed that the srlA promoter is autoregulated, displaying low activity in a wildtype (wt) host background and high activity in the srl deletion mutant background. The srlA promoter includes a conserved inverted repeat directly upstream of the predicted -35 subsequence. A mutational analysis demonstrated that the site is required for the high promoter activity in the srlA deletion background. Electromobility shift assays using purified wildtype CenR response regulator and a D55E phosphomimetic derivative suggest this protein acts as a likely Class II activator by binding promoter DNA. These results document the first identified CenK-CenR regulon member in S. meliloti and demonstrate this two-component regulatory system and gene srlA influences cellular growth and persistence under certain stress-inducing conditions.

Polymerase Chain Reaction Assay for Detection of Stenotrophomonas maltophilia in Cheese Samples Based on the smeT Gene.

Stenotrophomonas maltophilia is an emerging opportunistic pathogen linked not only to bacteremia, sepsis, and pneumonia but also to severe chronic enteritis. Persons with the impaired immune system are prone to be infected by S. maltophilia since its pathogenicity seems to be more associated with the host immune system than with the acquisition of specific virulence genes. In the dairy chain, S. maltophilia is linked to clinical and subclinical bovine mastitis in dairy cows, and it has been identified in cheese, and raw and pasteurized milk. There are reports of misidentification of S. maltophilia by commercial systems and PCR assays using primers based on the 23S rRNA and smeD genes, so the smeT gene is an alternative to identifying S. maltophilia by PCR due to its specificity to the S. maltophilia species. The present study reports an alternative species-specific PCR assay based on the smeT gene designed to identify S. maltophilia in cheese samples. We performed in silico and in vitro analyses to check the specificity of the primer pair. In silico analysis showed specificity of the primer pair to the species level. In vitro analysis was performed by testing the primer pair against pools of bacteria grown from 33 fresh Minas cheese samples acquired in the city of Rio de Janeiro, Brazil, without unspecific amplification.