Quorum Sensing in Sinorhizobium meliloti

Abstract

Patankar, Arati V., Quorum Sensing in Sinorhizobium meliloti. Doctor of Philosophy (Microbiology and Immunology), December 2008, 170 pp., 14 tables, 23 illustrations, bibliography, 212 titles. The overall goal of this study was to elucidate the role of a series of transcriptional regulators and potential signal molecules in the coordination of gene regulation in Sinorhizobium meliloti. The agriculturally important gram-negative soil bacterium S. meliloti, forms a symbiotic association with its host legume, Medicago sativa (alfalfa); thereby serving as a good model for studying host-bacterial interactions. Often, bacteria associated with eukaryotic hosts utilize global gene regulatory systems to coordinate their behavior in order to establish pathogenic or symbiotic associations. Quorum sensing is one such form of bacterial gene regulation which is mediated by signaling molecules and regulatory proteins in a population density dependent manner. In S. meliloti, the process of quorum sensing has been shown to play an important role in the relationship with its host plant. Control of essential processes such as plant nodulation and exopolysaccharide production has been attributed to the Sin/ExpR quorum-sensing system of S. meliloti. Interestingly, S. meliloti contains four additional (SMc04032, SMc00658, Smc00878 and SMc00877) putative quorum-sensing response regulators whose regulatory network was not known. The predicted protein sequences of these genes contain features typical of the LuxR family of proteins i.e., an N-terminal signal binding domain and C-terminal helix-turn-helix DNA biding domain. In order to identify their regulatory role, mutants of the response regulators were constructed and their expression profile was determined by employing genome-wide microarray and real-time PCR expression analysis. Through these analyses, it was determined that the SMc004032 locus controls expression of genes involved in the active methyl cycle, while the SMc00658, SMc00878 and SMc00877 loci control expression of genes from the denitrification of pathway of S. meliloti. Further, through phenotypic studies it was established that SMc04032 impacts stress response adaptation, and effective competition for plant nodulation. This suggests that SMc04032 could play a role in bacterial survival in the soil as well as within the host. The ability to denitrify is highly variable in different strains of S. meliloti. Through growth and enzymatic assays, it was established that the wild-type strain of this study, S. meliloti Rm8530, is a partial dentrifier in which, the capacity to metabolize nitrate is impaired. It was further determined that SMc00658, SMc00878 and SMc0877 modulated nitrite reductase activity under aerobic conditions, implying that these genes are involved in aerobic denitrification and therefor probably play a role in detoxification in S. meliloti. Based on the sequenced-genome analysis, S. meliloti possess homologs of other mediators of quorum sensing, that might be responsible for the synthesis of novel signal molecules. Bioreporter strains and mass spectrometry analysis were employed to identify production of cyclic dipeptides in S. meliloti. These compounds have been previously reported as quorum-sensing signal molecules in several bacteria. The results presented in this study provide a better understanding of S. meliloti’s metabolic and physiological properties and will be fundamental in future studies of bacterial interaction with its host and survival within its ecological niche.