Genetic suppression of the RNA regulator system controlling virulence and antibiotic biosynthesis in the phytopathogen Erwinia carotovora

This project involves a study of the control of multiple virulence factors in the bacterial plant pathogen, Erwinia carotovora. This bacterium causes soft rot and blackleg diseases of potato and it is commercially important. The bacteria make and secrete a collection of enzymes that degrade plant cell walls. This is the main, but not sole, mechanism of plant attack. The bacteria can 'talk' to each by chemical signalling in a process called 'quorum sensing'. By this mechanism the bacteria keep production of the plant degrading enzymes at a low level until the bacterial population reaches a high density when the bacteria secrete a lot of the enzyme in a burst of expression. This gives the plant no time to resist the lethal effects of the infection and leads to a very productive attack of the plant by the bacterial pathogen. A key control mechanism for the plant degrading enzymes is a system called the RsmAB system. The two factors that act in this RsmAB system are RsmA (a small protein) and rsmB, which is a small RNA molecule. The RsmA protein acts by degrading the products of the plant cell wall degrading enzyme genes. However, the rsmB molecules sequesters the RsmA protein to prevent it from assisting the degradation of the messenger molecules that make the enzymes. Bacterial mutants that cannot make the rsmB molecule have an excess of RsmA and this leads to excessive degradation of the messages that make the plant cell wall degrading enzymes. The rsmB mutants are not virulent because they make very little enzymes. We have exploited this fact to identify a totally new gene that, when mutated, can bypass (suppress) the effects of the rsmB mutation. This new suppressor gene is called rsmS and it nullifies the effect of the rsmB mutation. Our aims are to study the nature and the effects of the new rsmS gene, We intend to investigate the impacts of rsmS by the use of genetics, DNA microarray technology (transcriptomics) and advanced proteomics. We hope to understand how the new rsmS suppressor works to impact on the control of plant cell wall degrading enzymes in Erwinia. This information could lead eventually to a deeper understanding of regulation of pathogenicity in the plant pathogen. Ultimately, this type of information might be exploited as a route to intervening rationally in the plant disease.

The regulation of virulence in the plant pathogen, Erwinia carotovora is complex and is highly responsive to many environmental and physiological cues. Multiple virulence factors have been discovered, but the main ones are plant cell wall degrading enzymes (PCWDEs). Synthesis of the PCWDEs is co-ordinated and is extremely sensitive to bacterial cell population density in the process called 'quorum sensing'(QS). A major input to QS is the RsmAB regulatory system. The RsmA protein targets specific mRNAs for degradation in a post-transcriptional control mode. The rsmB small, regulatory RNA sequesters RsmA and prevents it acting in mRNA degradation. The stoichiometry of RsmA to rsmB is crucial. Mutants defective in the small RNA (rsmB) show reduced PCWDEs and reduced secondary metabolite (antibiotic) production. We have dicovered a novel suppressor mutation that allows the bacterium to bypass the pleiotropic impacts of the rsmB mutation. This new gene (rsmS) has homologues in other bacteria, but has no known function (until now). The main objectives of this research are to understand the nature and mechanism of action of rsmS and how it functions in the suppression of the rsmB phenotype to control virulence factor elaboration in the plant pathogen. The particular aims are: 1. To determine if rsmS acts as a protein or as a small, regulatory RNA. 2. To use transcriptomics and proteomics to determine the breadth of the physiological impacts of rsmS (and rsmB) in Erwinia. 3. To assess whether the rsmS suppressor acts via the RsmAB system or independently of this system. 4.To determine how rsmS is itself regulated Given the fact that homologues of rsmS exist in other pathogens, this study should have implications for pathogenesis and metabolism in other bacteria.