Regulatory mechanisms of the Sin Quorum Sensing System and its impact on survival of the soil-dwelling bacterium Sinorhizobium meliloti

The Sin Quorum Sensing (QS) system of the soil bacterium Sinorhizobium meliloti controls genes involved in a variety of cellular processes such as exopolysaccharide (EPS) production, motility, nitrogen fixation, and transport of metals and small molecules. The system consists of SinI, an N-acylhomoserine lactone (AHL) synthase, SinR, the LuxR-type transcriptional regulator of sinI, and ExpR, the LuxR-type master transcriptional regulator. The aims of this study are to understand the mechanisms and functions of the Sin QS, as well as its importance to survival of S. meliloti. Some of the regulatory target genes of the Sin QS have been previously shown to contain a promoter sequence that binds specifically to AHL-activated ExpR. In the first part of this study, the mechanisms of the ExpR transcriptional regulatory network were explored. The results confirmed 7 previously detected ExpR-DNA binding sites and added 26 novel sites, some of which regulate genes previously unknown to be members of the ExpR regulon. ExpR regulates the expression of the target genes in an AHL dependent manner. The data indicate that the location of the ExpR-binding site with respect to the relevant transcription start determines whether ExpR/AHL activates or represses promoter activity. Furthermore, the strength of the response is dependent upon the concentration of AHLs. This suggests a type of temporal gene expression program whereby the activity of each promoter is subjected to a specific range of AHL concentration since AHL accumulation and concentration varies with the age of the culture. In the second part of this study, the regulation of the Sin QS itself was further investigated. Until recently, all LuxR-type proteins were thought to bind to AHLs as the inducer. Unexpectedly, the results confirm that, in contrast to ExpR, the activity of SinR on sinI expression is independent of AHLs. The results also indicate that RNase E, an endoribonuclease that is essential for cell viability, regulates sinI expression by specifically targeting the 5’-UTR of sinI mRNA. Overexpression of rne resulted in a shorter half-life of sinI mRNA and a strong reduction of AHL accumulation. The results suggest that RNase E-dependent degradation of sinI mRNA from the 5’ end is one of the steps mediating a high turnover of sinI mRNA, which allows the Sin QS system to respond rapidly to changes in transcriptional control of AHL production. This is the first report of a specific regulatory interaction between QS and an essential component of cell viability in S. meliloti. The last part of this study involves the impact of the Sin QS on fitness of S. meliloti. Cultivation under standard laboratory conditions demonstrated aggressive invasions of QS-deficient expR mutants in the QS-efficient wild type population. Various mutants were tested in a series of competition assays. The results suggest that ExpR has a negative effect on bacterial fitness under standard laboratory conditions and that this effect is dependent upon EPS and flagellum production. However, when bacteria were exposed to severe stress, i.e. desiccation, survival was mostly dependent upon ExpR. In contrast, symbiotic potential was not enhanced by ExpR. Altogether, the results reveal that QS can have either positive or negative impact on fitness, depending on the context. It is, on one hand, a beneficial trait that helps bacteria to survive from severe stress but, on the other hand, tends to be eliminated under low stress and nutrient rich conditions