A novel plant pathogenesis regulatory system in Erwinia: functional analysis of a new post-transcriptional input to bacterial quorum sensing control.

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

This proposed research involves the study of a new gene in a bacterial pathogen of plants. The bacterial pathogen (Erwinia) causes rotting diseases of potato and other plants and is important in potato crop production. The bacteria attack the plant by producing a spectrum of enzymes that can degrade the cell walls of the plant leading to the commercially-significant rotting disease symptoms. The ability of the bacteria to make these plant cell wall degrading enzymes is affected by the cell population density of the bacteria; the enzymes are only made in abundance when the bacterial density if high. The bacteria make a small diffusible chemical signal called OHHL and the concentration of this molecule is a direct reflection of bacterial density. In this process (quorum sensing) the bacteria use the chemical signal to communicate with each other and thereby link cell density to the ability to aggressively rot the plant. If the ability to enact the process of quorum sensing is blocked then the bacteria are no longer capable of causing disease. Consequently, a deeper understanding of the mode of action of quorum sensing in this potato pathogen is important for our fundamental appreciation of how bacterial pathogens communicate with each other and, if we can understand the process, how we might intervene in the disease. There are no chemical control systems available for potato diseases caused by Erwinia and so fundamental understanding of how it causes infection is the only route to an eventual rational therapy. In this proposal we will investigate the role of a new gene (ECA2020) that we have shown recently to be involved in the process of quorum sensing. The product of this gene affects the expression/functionality of one of the key proteins (VirR) involved in quorum sensing. We will study the way ECA2020 operates by investigating which other proteins it interacts with and we will test the hypothesis that it may have some functions similar to proteins from higher organisms that are involved in messenger RNA processing. We will look for bacterial mutants that can bypass their dependence on the ECA2020 system and we will study the precise impact on production of the fully active VirR protein. We will also test the possibility that the contiguous gene (ECA2019) might be involved in a related process and we will investigate how the ECA2020 gene is itself regulated. The overall aim is to try to work out how this new gene modulates the function of the quorum sensing system during plant infection and disease initiation because this might be a target in the longer term for control of potato rotting diseases caused by Erwinia.

Technical Summary

Erwinia carotovora subspecies atroseptica (Eca) causes potato blackleg disease. Eca produces >20 plant cell wall degrading enzymes and multiple additional determinants that affect virulence, including secondary metabolite toxins, Type VI targeting systems and proteins of unknown function. All known virulence factors are regulated by quorum sensing (QS) during plant infection. In QS the expI/carI gene encodes a LuxI-type enzyme that makes OHHL (the N-acyl homoserine lactone intercellular chemical communication signal for Eca). At high cell density the concentration of OHHL reaches the threshold level that switches on virulence factor elaboration. The main regulator of virulence in Eca is VirR. VirR is a LuxR-type regulator and the central repressor of virulence gene expression. The current model is that OHHL binds to the VirR repressor leading to de-repression of virulence genes, although probably indirectly rather than by direct action on virulence genes. We wanted to identify new inputs to virR regulation. Using a virR::lacZ fusion screen we identified a novel regulatory input to the QS process in Eca. Mutation of the ECA2020 gene led to down-regulation of virR expression. The ECA2020 protein shows some sequence relatedness with eukaryotic adenosine deaminases (ADARs) that control gene expression via edited base substitutions in target mRNA, affecting function. Preliminary evidence suggests that virR mRNA may be processed in the ECA2020 mutant leading to truncated VirR production and loss of functionality. We will test the hypothesis directly that ECA2020 acts in this post-transcriptional way; a mechanism that would be unique in a eubacterium. We will assess the impact of mutation of the ECA2020 residues conserved in ADARs, search for ECA2020-interacting proteins that may be partners in the regulation, and determine if ECA2019 has any related role in virR expression. We will study regulation of ECA2020 by bypass mutagenesis to define hierarchical controls.

Planned Impact

Who will benefit from this research? Potential beneficiaries include potato growers and distributers and, ultimately, the consumer. Furthermore, the basic principles of QS control of pathogenesis radiates across plant, animal and human pathogens. Knowledge gleaned from our studies might have relevance and wider applications in plant pathology, and in control/therapy of some animal/human diseases due to Gram-negative bacteria that use a QS mode of virulence regulation. Thus the study has longer term implications for agricultural productivity and, potentially, for medicine. QS systems could also have potential applications in industrial fermentations as easily manipulated gene induction and repression control systems in the manufacture of biotechnologically useful products. Such QS systems are also potentially useful in chemical biology and synthetic biology arenas where simple, modular gene regulation cassettes might find applications where there is a need to engineer metabolic process controls or report on physiological events in bacteria. Thus, our studies in the longer term could have applicability in far wider areas than potato crop disease. In effect a wide spectrum of possibilities might emerge from a basic understanding of how Erwinia uses intercellular chemical communication to regulate disease of a crop plant. How will they benefit from this research? Crop losses due to plant disease and biodeterioration are significant, on a global scale. Indeed, it is estimated that the latter crop losses may account for 10-30% of bulk food production, globally; this is obviously a major issue. If our studies lead eventually to new potato disease control procedures (for blackleg in the field and soft rot in storage) this could diminish crop losses and enhance profitability in agriculture and aspects of the food production and distribution chain in the UK and globally. Decreasing potato crop losses could also enhance our domestic (UK) food security and so, even marginal impacts on our ability to control potato crop pathogenesis has value. On a global scale, improvement in potato crop productivity through decreased deterioration could have impacts on food availability and health in poorer countries through knowledge transfer. The potential biotechnological applications of QS research would require agricultural and industrial sector participation and exploitation with investment over a longer time scale than envisaged in this current proposal. The PDRA employed on this project will acquire diverse skills in molecular microbiology, cutting edge 'omics technologies, bioinformatics, and experience in teaching and science communication. What will be done to ensure that they have the opportunity to benefit from this research? The research outcomes will be disseminated to scientists (from universities, institutes and commercial organisations) via peer reviewed international publications and lectures and posters presented at domestic and international symposia. When publishing in open access journals, there will be a global accessibility of the knowledge generated from this work. We have a track record over the past 20 years of BBSRC-CASE studentships (two currently running with UK companies in other projects) and collaboration with UK research institutes. We will try to further extend such associations as a way of leveraging additional funding for our research programmes and to generate added value through synergy of research interests. Cambridge university takes a very pragmatic and enlightened industry-friendly stance that encourages commercial collaborations and the generation of spin-out companies, after the establishment of solid IP positions. We have a track record of lodging patent applications on carbapenem antibiotics and on cryptic gene regulation e.g. Salmond et al (US Patent 5821077 - issued 1998); Salmond et al (WO/1995/032294) and have recently filed a patent application on phage abortive infection in late 2008.

Publications

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Mansfield J (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. in Molecular plant pathology

 
Description The bacterium, Erwinia (Pectobacterium) is a significant agricultural problem in the UK potato industry. It causes soft rot and blackleg disease, leading to commercial losses. There is no effective chemical control method for Erwinia infection. There are no fully resistant potato cultivars available commercially and the genetic manipulation of the crop for consumption is currently considered to be unacceptable in the UK. Consequently, there is a need to understand how the pathogen is transmitted and how it kills potato plants. The main (but not sole) factors in the disease is the production of a battery of plant cell wall degrading enzymes (PCWDEs) by the bacterial pathogen. The production of these enzymes is very tightly regulated by the bacterium. There are many environmental factors that impact on PCWDE production, including temperature, pH, humidity, plant stress and the size of the bacterial population. The bacteria have evolved a mechanism for sensing the numbers of cells in the local population - this is called quorum sensing (QS). In QS the bacteria make a small, freely diffusible molecule that acts as a chemical signal that allows the bacterial pathogen to monitor cell density. At low density the PCWDEs are repressed but at higher cell density the relative concentration of the signal increases and activates the simultaneous production and release of the PCWDEs by all the bacteria, leading to aggressive rotting of the potato plant. Therefore we need to understand all regulatory mechanisms used by the bacteria to control the production of their PCWDEs and that includes a complete understanding of the QS control system. This grant covered research into how QS is modulated to control PCWDE production - and virulence in potato. We investigated a bacterial gene called ECA2020, mutation of which affected the expression of the virulence regulator, VirR. Microarray experiments (allowing us to look at expression of all genes in the pathogen) allowed us to compare expression in a virR, expI and virR/expI mutant to wild type Erwinia (Pectobacterium). These showed that a mutation in the virR gene bypassed the majority of transcriptional changes found in an expI mutant (affected in QS). A technique called Chromatin Immunoprecipitation microarray analysis was performed with tagged VirR to identify its binding sites across the Erwinia genome. Using purified VirR, we determined the minimum DNA site required for VirR binding. Other post-transcriptional investigations were done involving a protein called Hfq and this had a strong impact on virulence in Erwinia. Overall, our work has added new insight into the events that happen after the VirR regulator gene is transcribed and how this might impact on virulence in potato.
Exploitation Route Research outcomes from this project that could have relevance to plant disease control, because the main theme of the project is the regulation of the virulence factors elaborated by this bacterial phytopathogen. Increasing understanding of Pectobacterium, a potato pathogen, infections is of increasing interest given the implications for UK food security and the economic impacts this pathogen has on seed potato exports. This study identified new regulatory inputs involved in Pectobacterium virulence, which can be further exploited for agricultural and biocontrol purposes.
Sectors Agriculture, Food and Drink,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description A Postdoctoral Research Associate who worked on this grant moved to a position in the UK healthcare industry, thereby retaining her acquired skills within UK Plc. As a part of this research programme, the central regulator of virulence, virR, in this plant pathogen, was defined. The definition of this new regulator along with its cognate quorum sensing LuxI-type protein, could define a new target for therapeutic intervention in phytopathogenesis.
First Year Of Impact 2014
Sector Agriculture, Food and Drink,Education,Environment,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

 
Description Scottish Science Advisory Council
Geographic Reach National 
Policy Influence Type Citation in other policy documents
 
Title Development of plasposon mutagenesis system for generic use in Gram-negative bacteria 
Description Development of an engineered transposon mutagenesis system that allows mutagenesis of diverse bacteria for facile mutagenesis, cloning of insertion sites and sequencing. 
Type Of Material Biological samples 
Year Produced 2016 
Provided To Others? Yes  
Impact The system makes it easier to mutate genes, clone insertion sites and sequence them 
 
Description Colworth Prize Lecture, Society for General Microbiology Meeting, Harrogate, UK (2011) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presentation of the Colworth Prize Lecture, Society for General Microbiology Meeting, Harrogate, UK (2011)

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited Member, Reception, House of Lords, London, Role of Microbes in Food Security (2011) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited member at a Lunchtime Reception hosted by Lord Soulsby of Swaffham Prior, Cholmondley Room, House of Lords, London, to highlight the Role Played by Microbes in Food Security and Safety (November 2011).

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Invited Talk at the UK-Japan Systems & Synthetic Biology Workshop, 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at the UK-Japan Systems & Synthetic Biology Workshop, BBSRC-funded, University of Surrey, UK (2010)

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Invited lecture to the Danish Society for Biochemistry and Molecular Biology Symposium, Copenhagen, 2010 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited Talk at the Danish Society for Biochemistry and Molecular Biology. Symposium on "Small Talk: Chemical Communication in Bacteria". Copenhagen, Denmark. 2010

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Invited member at "Science on a Plate", The Scottish Parliament, Edinburgh, 2012 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited member at Science on a Plate - A series of presentations of Food Security and related topics, presenting the practical outputs and translational products from Scottish Research Institutes, The Scottish Parliament, Edinburgh (February 2012). Outreach.

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Invited talk at the Japan-UK Microbial Systems Biology Conference, Institute for Advanced Biosciences, University of Keio, Tsuruoka, Japan (2011) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited talk at the Japan-UK Microbial Systems Biology Conference, Institute for Advanced Biosciences, University of Keio, Tsuruoka, Japan (2011). Invitation to George Salmond, but the talk was given by Postdoctoral Research Assistant (Dr Rita Monson - BBSRC-funded PDRA) due to family commitments.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Open Days 2009, 2010, 2011 and 2012 - assorted 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact The PDRA hosted at least 2 open days every year from 2009 till present as a part of the University's Open Days (in September every year, and one in April). These open day sessions have involved presentations on various topics e.g. a recent one was entitled 'How can we chemically and biologically describe the composition of the human body?'.

The PDRA also attended an open day at City and Islington College to discuss Careers in Science in April 2012.
Year(s) Of Engagement Activity 2009,2010,2011,2012
 
Description Open days 2013 -assorted 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact April 11 - - The PDRA gave a lecture as part of the Cambridge Admissions Office open day to students interested in medicine, biological natural sciences and veterinary sciences.

July 4th, 5th, September 20th - - The PDRA hosted a lunch each day for students interested in attending the University as part of Open Days.
Year(s) Of Engagement Activity 2013
 
Description Sixth Form College talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact A talk to a group of students and staff in Hills Road 6th Form College (Cambridge) on microbes, using our own research as examples of the importance of microbes in, among others, disease, antibiotics, chemical signaling (quorum sensing), and bioluminescence. Followed by a Q&A session.
Year(s) Of Engagement Activity 2013
 
Description Talk at Cold Spring Harbor Meeting on Molecular Genetics of Bacteria and Phages, Madison, Wisconsin, USA 2011 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Talk at the Cold Spring Harbor Meeting on Molecular Genetics of Bacteria and Phages, Madison, Wisconsin, USA 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Talk at the American Society for Microbiology, Conference on Bacterial Cell-Cell Communication, Miami, Florida, USA, 2011 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach international
Primary Audience Participants in your research or patient groups
Results and Impact Talk at the American Society for Microbiology, Conference on Bacterial Cell-Cell Communication, Miami, Florida, USA , 2011

no actual impacts realised to date
Year(s) Of Engagement Activity 2011