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.

A benchmark survey of plankton, fish and benthic composition in Poblacion and Kadurong Reefs in Liloan, Cebu, Philippines.

BACKGROUND: Coral reefs offer valuable ecosystem goods and services, such as coastal protection, erosion regulation, fishery, biodiversity, habitat and nursery grounds. However, they face threats from anthropogenic activities, including poor water quality, global warming, coastal development and unsustainable fisheries. Marine Protected Areas (MPAs) provide a structured and holistic approach in addressing these threats. Regular monitoring and assessment of these MPAs are crucial components in evaluating the MPAs design and effectiveness. Two coral reefs (i.e. Poblacion and Kadurong Reefs) were established as MPAs in Liloan, Cebu, Philippines to protect crucial habitat and biodiversity with the hope of improving fisheries by avoiding fish stock disintegration. These coral reefs provide shelter to many commercially-significant fish species, supporting subsistence and livelihood in the community. These MPAs are not only biologically rich, but they also support socio-economic stability. Hence, management and protection of the coral reefs in the MPAs of Liloan, Cebu is of paramount importance. To formulate conservation and applicable management measures, research and monitoring should be in place. This paper presents the data collected from the short term monitoring in the Poblaction and Kadurong Reefs. The paper describes an important set of data that can be used by the stakeholders to benchmark biophysical assessments for management of marine-protected areas in Liloan. NEW INFORMATION: This data paper provides baseline information on the health of the coral reefs of the MPAs in Liloan, Cebu. Datasets covering physico-chemical and biological parameters inclusive of water quality, coral reef cover, fish and plankton occurrence and abundance were determined using the standard protocols for surveying tropical marine resources. The results will serve as a benchmark in formulating guidelines and implementing relevant policies for the effective management and protection of the MPAs in Liloan, Cebu, Philippines.