Global control of bacterial translation by specific ribosome modification

Bacteria can interact with plants in a number of different ways. In addition to pathogenic bacteria that cause costly plant diseases, several species form mutually beneficial relationships with plants. These bacteria live off the organic molecules exuded by plant roots and in return they positively affect plant health and nutrition and suppress pathogenic fungal growth. My lab studies two plant-associated Pseudomonas species. These are the aggressive plant pathogen P. syringae, which causes economically destructive diseases including tomato speck, brown spot and bleeding canker, and the harmless, soil-dwelling species P. fluorescens. P. fluorescens colonises plant roots and displays effective biocontrol properties against pathogens, making it an attractive potential alternative to conventional chemical pesticides. The efficacy of P. syringae pathogenicity or P. fluorescens biocontrol is directly related to the ability of the bacteria to colonise their plant host. However, despite extensive research into plant infection, biocontrol and root colonisation, the internal bacterial signalling pathways that control these processes are only poorly understood. We seek to improve our understanding of these internal signals, with the eventual aims of fighting P. syringae infection, and modifying P. fluorescens to produce new, more effective biocontrol agents.
As part of our ongoing research into Pseudomonas signalling during plant interactions, we have investigated the RimK protein, which is predicted to interact with the protein production machinery of the bacterial cell. RimK appears to modify a small protein in the bacterial ribosome called RpsF. Subsequent experiments suggest that RimK activity towards RpsF affects ribosome stability, leading to altered ribosomal function and consequently to specific changes in the protein makeup of the cell. The rimK gene is part of an operon that also includes rimA, a gene encoding a cyclic-di-GMP (cdG) degrading enzyme. CdG is a bacterial signalling molecule that regulates diverse bacterial characteristics including motility and attachment to surfaces. The RimA and RimK proteins physically interact, consistent with a role for RimA (and possibly cdG) in RimK regulation. Deletion of the rimK gene led to decreased P. syringae virulence and reduced the efficiency of wheat root colonisation by P. fluorescens. Furthermore, the rimA and rimK genes were up-regulated during the later stages of P. fluorescens root colonisation, suggesting that RimK activity contributes to the adaptive response of Pseudomonas species to the plant environment.
With this proposal we will first determine how the RimK protein functions, and how it changes the stability and function of the ribosome. Next, we will examine the significance of RimA cdG metabolism, and how the Rim proteins interact with each other. We will also examine the total protein content of bacteria with the rimA gene deleted. Comparison of these results with those for a rimK deletion mutant will tell us whether or not the two genes function as part of the same signalling pathway. Finally, we will examine the function of RimK protein in P. syringae and in the P. fluorescens wheat root environment. To do this we will extract the total protein content of wild-type and rimK-mutant bacteria, either from P. syringae cultures or from P. fluorescens grown in model wheat root systems. We will then measure protein levels and use this data to determine how rimK deletion affects protein translation in different species and different environments. These data will allow us to determine both the effects of RimK activity, the protein changes that occur as a consequence of growth in the plant environment, and the importance of RimK activity for pathogenic and beneficial plant-microbe interactions.

Translational regulation plays a central role in controlling the environmental responses of bacterial species. We propose that the widespread glutamate ligase RimK functions as a global controller of bacterial mRNA translation. RimK modifies the ribosomal protein RpsF by the addition of C-terminal glutamate residues. Our preliminary data suggests that RimK activity changes both the stability and function of the ribosome, and hence the composition of the bacterial proteome. Deletion of rimK leads to significantly reduced virulence in the destructive plant pathogen Pseudomonas syringae and compromises wheat rhizosphere colonisation by the biocontrol bacterium Pseudomonas fluorescens. In both of these plant-associated Pseudomonads the rim operon also encodes RimA; a phosphodiesterase for the ubiquitous bacterial second messenger cyclic-di-GMP (cdG). RimA and RimK physically interact in vivo, and rimA deletion induces similar plant interaction phenotypes to delta-rimK, consistent with a role for RimA, and possibly cdG, in the regulation of RimK.
In this proposal we will first biochemically characterise RimK, and examine the effects of RpsF modification on ribosome structure and protein composition. Next, we will use Ribosomal Profiling to determine the mechanism for translational control by RimK. We will investigate interactions between the Rim proteins in vitro, and examine the role of cdG in the regulation of RimK with cdG binding assays and site-directed mutagenesis. We will examine the transcriptional regulation of the rim genes during growth in the plant environment using qRT-PCR and transcriptional reporter constructs. Finally, we will use quantitative proteomic analysis by iTRAQ tagging and LC-MS/MS to examine the physiological role of rimA, rimK deletion on virulence protein translation in P. syringae, and the in planta role of RimK during P. fluorescens rhizosphere colonisation.

WHO WILL BENEFIT FROM THIS RESEARCH, AND HOW?
Pseudomonas syringae is a ubiquitous and economically destructive plant pathogen that is responsible for reduced yields in a wide range of crops worldwide. [For example, New Zealand's kiwifruit industry recently came under pressure from a P. syringae outbreak that infected more than 40% of the nation's kiwifruit hectares.] This proposal will shed light on the mechanisms by which P. syringae controls the translation of proteins required for plant pathogenicity. In turn, this promises to uncover new avenues for research into phytopathogen control and crop protection.
Modern agricultural techniques rely on the extensive use of chemical pesticides to control plant pathogens. Such methods are both environmentally damaging and costly. Furthermore, they encourage the development of resistant pathogen populations that require treatment with ever-increasing pesticide concentrations. The development of plant growth promoting rhizobacteria (PGPR), soil microorganisms that stimulate plant growth and/or combat the spread of pathogens, represents an attractive potential alternative to conventional chemical pesticides. Pseudomonas fluorescens is one such PGPR, with P. fluorescens biocontrol agents currently commercially available, e.g. BlightBan A506 (NuFarm), a preventative treatment for use in the protection of fruit crops from Fire Blight (Erwinia amylovora, estimated to cost fruit growers in the U.S $100 million dollars/year). P. fluorescens is a well-studied PGPR species with significant potential for further commercial exploitation.
While the research we propose here is primarily of fundamental academic interest, by increasing our understanding of the molecular mechanisms that control P. syringae pathogenicity and P. fluorescens rhizosphere colonization, this work has clear applications for the control of phytopathogenic Pseudomonads, and for the development of commensal Pseudomonas sp. as biocontrol agents and crop growth promoters. For these reasons, the proposal aligns closely with the BBSRC strategic research priority area of Food Security.
In addition, the project presents the opportunity to acquire and develop a range of skills within a highly stimulating, multidisciplinary research environment. In this way the project will directly benefit the employed scientists, and will help to prepare them for their future research careers. Furthermore, these researchers will receive training in generic 'transferable' skills that are applicable to any area of employment. This will include the planning and organisation of a programme of research, the maintenance of accurate day-to-day records, the presentation of their research to a variety of different audiences and the preparation of scientific manuscripts.
WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH?
Academic research at the John Innes Centre (JIC) with commercial potential is patented through Plant Biosciences Ltd (PBL), the JIC-associated knowledge transfer company. PBL works to bring the results of research in microbial and plant sciences at the JIC into public use for public benefit through commercial exploitation. PBL meets all patent filing, marketing and licensing expenses in respect of technologies it develops for JIC. As stated elsewhere in this proposal, we will regularly assess our research findings with PBL for potential opportunities for commercial exploitation and/or intellectual property.