Global control of bacterial translation by specific ribosome modification

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences


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.

Technical Summary

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.

Planned Impact

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.
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.
Description We have identified and characterised a pathway used by many pathogenic and beneficial bacteria to control their behaviour by modifying their protein production machinery. This allows the bacteria to adapt to their surroundings, either attaching to surfaces and scavenging for nutrients, or swimming around and colonising their environment/their hosts.
If we disrupt this system in the important pathogenic bacterium Pseudomonas aeruginosa, it can no longer infect plant tissue or damage red blood cells, pointing to a possible use for this research in the development of novel anti-infective treatments.
Exploitation Route This work revises the current models for control of microbial protein production in a large number of different bacterial species. This may in turn have far reaching implications for many different areas of microbial science. As stated above, this research identifies a possible new target for anti-infective drug design. Much of the research was carried out in plant-associated bacteria, and there is also the possibility that this could be exploited in the development of plant protection products. In carrying out this work, we have developed several new advanced methods for studying gene regulation, which refine and expand on existing techniques to achieve much deeper levels of resolution. These tools are likely to be useful for other scientists studying complex regulatory pathways in bacteria.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology

Description Plasmid manipulation of bacterial gene regulatory networks
Amount £485,682 (GBP)
Funding ID BB/R018154/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 12/2018 
End 09/2021
Title Integrated analysis of global genetic datasets 
Description Reductive analysis of individual signalling pathways is inherently limited in its ability to fully explain the implications of e.g. environmental change on the global regulatory network of microorganisms. In order to enable a deeper understanding of these complex signalling networks we have developed a bioinformatic pipeline that enables the simultaneous consideration of several regulatory layers, at the whole-cell scale. Using the Hfq transcriptional/translational regulatory network in the model bacterium Pseudomonas fluorescens as a test case, we used extensive 'omic-analyses to assess how hfq deletion affects mRNA abundance, translation and protein abundance. The subsequent, multi-level integration of these datasets enabled us to highlight discrete contributions by Hfq to gene regulation at different regulatory levels. This integrative approach to global signalling may be used to dissect individual signalling networks, or to understand how bacterial cells adapt to changes in their environments to a far greater resolution than is available using conventional molecular microbiology or individual 'omic analyses. 
Type Of Material Improvements to research infrastructure 
Year Produced 2017 
Provided To Others? Yes  
Impact This analytical approach has been published (Analyzing the Complex Regulatory Landscape of Hfq-an Integrative, Multi-Omics Approach, L Grenga, G Chandra, G Saalbach, CV Galmozzi, G Kramer, JG Malone, Frontiers in microbiology 8, 1784), and is currently being adapted to examine the RimABK pathway, which forms the main focus of this project. This follow-on work will be published in 2018. 
Description Establishment of Ribo-seq in the Malone Lab 
Organisation Heidelberg University
Country Germany 
Sector Academic/University 
PI Contribution Dr Lucia Grenga undertook a research visit to Univ. Heidelberg (Germany) for 4 weeks in 2015 to learn how to conduct Ribosomal profiling experiments. She then transferred this knowledge back to my group in the form of detailed protocols and materials
Collaborator Contribution Dr Gunter Kramer's lab in Heidelberg shared their skills and know-how with us during the 2015 research visit.
Impact Direct outputs include the 2017 Frontiers in Microbiology paper listed in the publication section (with Heidelberg co-authors).
Start Year 2015
Description A view from the lab - blog interview 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview for a science blog - A View From The Lab.
Year(s) Of Engagement Activity 2018
Description Educational film (UEA) 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Contributed to an educational film showing A-level students how mathematics is used in the world of science.
Year(s) Of Engagement Activity 2016
Description JIC50 Molecular Microbiology presentation 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Contributed to the design and running of the molecular microbiology department stall at the JIC 50 year open day.
Year(s) Of Engagement Activity 2017
Description Norwich Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Lecture to the public as part of Norwich Science festival, explaining the science and relevance of plant-microbial interactions in the soil. Positive audience feedback including two people who expressed interest in the JIC as a potential future workplace.
Year(s) Of Engagement Activity 2017