Plasmid manipulation of bacterial gene regulatory networks

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences


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 research 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. This project provides a conceptual step change in the way we think about HGT, beyond mobile elements as passive vehicles of genetic exchange to evolving entities capable of manipulating their bacterial hosts through reprogramming gene regulation. The key benefits deriving from this research will therefore be increased knowledge and understanding of bacterial evolution among the following groups:

1. 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. Our project examining plasmid dynamics in the phytosphere will help us to better understand the potential drivers of plasmid spread in natural environments.

2. 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. Our discoveries will advance understanding of the fundamental processes of bacterial evolution. Thus, we aim to add knowledge via curriculum changes and to change schoolchildren perceptions and knowledge (via our Pathways to Impact strategy). 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.

3. Undergraduate and postgraduate students will benefit from interactions with all the researchers, both through the underpinning of teaching with research (i.e. the new discoveries), but also through research seminars at both home and external institutions, a horizontal gene transfer module in a MOOC, and via summer and final year projects.

4. 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.


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publication icon
Billane K (2022) Why do plasmids manipulate the expression of bacterial phenotypes? in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

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Brockhurst MA (2022) Ecological and evolutionary solutions to the plasmid paradox. in Trends in microbiology

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/R014884/1 03/12/2018 30/05/2020 £441,024
BB/R014884/2 Transfer BB/R014884/1 31/05/2020 02/12/2022 £248,963
Description We have published a major review paper on the ecology and evolution of pangenomes focusing on the role of horizontal gene transfer by mobile genetic elements such as plasmids. Published in Current Biology. We have published a review paper explaining the mechanisms by which plasmids are retained in bacterial genomes. Published in Trends in Microbiology. We have published a review paper about the consequences of plasmid manipulation of bacterial gene regulation and phenotypes for the spread of plasmids and horizontal gene transfer. Published in Philosophical Transactions of the Royal Society B.

The publication of experimental data has been severely delayed by the COVID-19 pandemic but we are currently writing up two manuscripts for publication:
- The first characterises the molecular mechanisms and phenotypic effects of plasmid-chromosome regulatory crosstalk and their evolution. These data show that a plasmid encoded regulator modulates a chromosomal global regulatory system to alter key phenotypes of the bacterium including motility and attachment and the production of secreted products.
- The second explores the longterm dynamics of plasmids in the plant rhizosphere. These data show that plasmids persist in soil rhizosphere communities and that this stability is explained by the gain of compensatory mutations to ameliorate the cost of plasmid carriage. This is the first report of compensatory evolution to stabilise a plasmid in a natural plant-associated community. Moreover we show that the plasmid is capable of spreading into diverse members of the bacterial community by conjugation.
Exploitation Route Our results demonstrate the importance of plasmids in bacterial genome evolution and in the spread of adaptive traits among bacterial taxa in a natural environment. Moreover we demonstrate that plasmids are capable of manipulating bacterial phenotypes for their own benefit and in doing so promote their spread into bacterial communities. These findings will advance our understanding of the dynamics of horizontal gene transfer in bacterial communities which is a key process driving e.g. the spread of antimicrobial resistance.
Sectors Agriculture, Food and Drink,Environment,Healthcare

Description We have engaged with the general public to enhance their knowledge and understanding of the role for microbes in health (including soils, plants and animals). The research has fed into a Parliamentary Office of Science and Technology POSTnote. We have published a comic book based on our research into plasmid biology "Luna and Simon: Bizarre Bacteria and Peculiar Plasmids" written by Edward Ross. Available in print and online.
First Year Of Impact 2018
Sector Agriculture, Food and Drink,Creative Economy,Digital/Communication/Information Technologies (including Software),Education,Environment,Culture, Heritage, Museums and Collections
Impact Types Cultural,Societal,Policy & public services

Description Public engagement comic book 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Comic book "Luna and Simon: Bizarre Bacteria and Peculiar Plasmids" written by Edward Ross based on our research and accompanying website. Printed and online. Production featured workshops for 20 school children. Over 500 printed copies have been distributed and far more downloaded from the website.
Year(s) Of Engagement Activity 2020
Description Workshops for a public engagement comic book 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Workshop for a group of school age children to develop a comic book about microbes and their mobile genetic elements. Involved creative tasks and discussion around scientific topics depicted in the draft comic book. Sparked enthusiastic discussion and questions.

Comic book to be published in 2021.
Year(s) Of Engagement Activity 2019,2020