Plasmid manipulation of bacterial gene regulatory networks

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

Abstract

Genome sequencing has revealed that bacteria frequently exchange genes between species, speeding-up evolution by allowing it to proceed by big leaps rather than just the gradual accumulation of small changes. Genes often pass from one bacterial cell to another on circular pieces of DNA called plasmids. Acquiring a plasmid is typically costly to a bacterial cell. However, little is currently known about the causes of these fitness costs. One possible cause of fitness costs is that plasmids often carry genes that interfere with the switching on / off of bacterial genes causing these genes to be expressed at the wrong times. In the proposal, we investigate a common plant-associated bacterium, Pseudomonas fluorescens, where gaining a plasmid switches on around 20% of bacterial genes. Our preliminary data links this gene disruption to a widespread global regulatory pathway, Gac/Rsm, which controls the switch between chronic, biofilm-forming lifestyles and acute virulent states in many different bacterial species. This raises the intriguing possibility that plasmid-acquisition may reprogram the Gac/Rsm pathway, impacting key virulence- and competition-related behaviours in diverse bacterial species, including important human and plant pathogens. In this project, we will first unravel the molecular mechanism by which plasmids disrupt bacterial gene expression. Informed by the results of these experiments, we will examine the fitness costs and potential benefits of plasmid acquisition in complex plant-associated environments, and test whether the plasmid benefits from disrupting the Gac/Rsm pathway by boosting its rate of spread in bacterial populations growing on plants. These experiments will shed new light on a fundamental process in bacterial evolution, with relevance for understanding the sharing of genes encoding important functional traits (including antibiotic resistance) in natural communities.

Technical Summary

Our previous work showed evidence of large-scale dysregulation of bacterial gene expression following plasmid acquisition. We will define the mechanistic basis of the bacteria-plasmid regulatory cross-talk that causes this dysregulation, and determine its effects on bacterial phenotype and plasmid fitness. We will test the hypotheses (A) that plasmid regulatory proteins directly interfere with bacterial gene regulation, and (B) that plasmid manipulation of bacterial gene expression is an evolutionary adaptation increasing plasmid fitness.

We will combine bacterial genetics, biochemistry and omics technologies with experimental evolution, building on our previous work using the plant-associated bacterium Pseudomonas fluorescens SBW25 and its naturally co-occurring mercury resistance plasmid pQBR103. Our preliminary data strongly suggests that the pQBR103 plasmid uses a plasmid-borne ortholog of the global posttranscriptional regulator Rsm to manipulate the bacterial Gac/Rsm regulatory system, which controls a wide range of virulence- and competition-associated traits in Pseudomonas.

We will first determine how the plasmid-Rsm interacts with host proteins by co-immunoprecipitation, and mRNA by CLIP-seq analysis. Next, we will determine the effects of the plasmid-Rsm on gene regulation using integrated RNA-seq, Ribo-seq and iTRAQ proteomics analyses, and second-messenger signaling by mass spectrometry. In parallel, we will determine the effect of plasmid-Rsm on expression of Gac/Rsm regulated phenotypes such as motility, biofilm formation, extracellular secretion, metabolic phenotypes, and plant colonization using well-established assays. We will determine how the plasmid-Rsm affects plasmid dynamics on sugar beet over the growing-season in greenhouse experiments, and track the spread of the plasmid into the bacterial community using epic-PCR. Finally, whole genome sequencing of evolved clones will allow us to identify compensatory evolution in the phytosphere.

Planned Impact

Who will benefit from this research and how?

This is basic blue-skies research that will advance fundamental understanding of evolutionary processes and dynamics in bacterial communities. Nevertheless, bacterial evolution has a broad range of important impacts upon society, for example through the effects of rapid evolutionary change on the prognosis of clinical infections, the evolutionary emergence of antibiotic resistance, and evolutionary responses of microbial communities underpinning the functioning of ecosystems to environmental change. Despite the widespread and fundamental impact of rapid microbial evolution in general and horizontal gene transfer (HGT) in particular upon society, these evolutionary processes remain very poorly understood by the general public and policy-makers. The key benefits deriving from this research will therefore be increased knowledge and understanding of bacterial evolution among the following groups:

Secondary school age children: Teaching of evolution in Key Stages 2 and 3 of the National Curriculum is mainly theoretical and lacking in engaging practical classes. We will take experimental evolution into the school classroom allowing pupils to experience evolution in action themselves in real time, generating excitement about microbes and evolution and offering deeper experiential learning.

General public: Bacterial evolution is high on the news agenda due to the crisis in antimicrobial resistance (AMR), however few non-scientists realise that this societal problem is exacerbated by HGT-mediated evolution. Public engagement activities will enhance public understanding of HGT and put this into the context of AMR to show what we can all do to reduce the risks of AMR.

Policy makers in healthcare and agri-food sectors: HGT impacts the evolutionary emergence of AMR in the clinic and the spread of functional traits in soil bacterial communities. Designing policies and interventions that aim to e.g. limit the spread of AMR or conserve the functional diversity of soil bacterial communities, requires sharing knowledge and understanding of the dynamics of HGT and how these are shaped by the ecology of microbial communities and their environments arising from this research with stakeholders and policymakers in these sectors. We will engage with healthcare stakeholders via an established clinical network (PARC; PI Brockhurst is a member) and agri-food stakeholders via the N8 AgriFood Partnership facilitated by the N8 AgriFood Knowledge Exchange Fellows.

Publications

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Thompson CM (2020) Nucleotide second messengers in bacterial decision making. in Current opinion in microbiology

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/R018154/1 01/12/2018 30/06/2021 £485,682
BB/R018154/2 Transfer BB/R018154/1 01/07/2021 30/11/2021 £82,567
 
Title Comic book illustrating how plasmids work 
Description As part of the outreach element of our pathways to impact plan, we commissioned an artist to produce a comic book illustrating the scientific concepts in our project. 
Type Of Art Artwork 
Year Produced 2021 
Impact This comic book is currently in production, with an initial print run of a couple of thousand. 
 
Description We have discovered that small regulatory proteins control the fitness of plasmids (replicative circles of DNA, that may contain antibiotic resistance or other environmental/ medically relevant features) in bacteria in the soil. More recently, we have identified the genes that control different aspects of the relationship between the plasmid and the host genome. We are now in the process of working out how these genes do this, and the structures and functions of the proteins they encode.
Exploitation Route This work may have relevance for agricultural advice, to restrict the spread of antibiotic resistance in soils. It will provide fundamental insights into the control of important bacterial behaviours such as virulence, persistence of infection or antibiotic resistance.
Sectors Agriculture, Food and Drink,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Plasmid manipulation of translational regulation in bacteria 
Organisation University of Reading
Department School of Biological Sciences Reading
Country United Kingdom 
Sector Academic/University 
PI Contribution In addition to intellectual input, my lab provides specialist resources and research skills in molecular microbiology, protein biochemistry and plant-microbe interaction to this collaboration.
Collaborator Contribution Sheffield University (Brockhurst lab) are providing skills in evolutionary microbiology, soil microbiology and mathematical/statistical modelling. Reading University (Jackson lab) are providing intellectual input and know-how relating to bacterial genetics and microbiology. Both partners are funded by a companion grant to this one.
Impact None so far
Start Year 2018
 
Description Plasmid manipulation of translational regulation in bacteria 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution In addition to intellectual input, my lab provides specialist resources and research skills in molecular microbiology, protein biochemistry and plant-microbe interaction to this collaboration.
Collaborator Contribution Sheffield University (Brockhurst lab) are providing skills in evolutionary microbiology, soil microbiology and mathematical/statistical modelling. Reading University (Jackson lab) are providing intellectual input and know-how relating to bacterial genetics and microbiology. Both partners are funded by a companion grant to this one.
Impact None so far
Start Year 2018