Noisy Strep

Lead Research Organisation: Newcastle University
Department Name: Inst for Cell and Molecular Biosciences


Summary. Streptococcus pneumoniae is a major pathogen causing invasive (pneumonia, meningitis, bacteraemia) and non-invasive (acute otitis media, sinusitis) disease in children and in elderly and/or immuno-compromised adults. The last decades have seen the emergence and spread of pneumococcal strains with multiple antibiotic resistance posing a serious threat to human health. The strategy of the pathogen development is regulated by so called bistability, and may lead to formation of drug resistant forms of bacteria, such as biofilms. Transcriptional noise is the main determinant of switching of bistable systems. Therefore, understanding of molecular mechanisms giving birth to the noise in transcription in S. pneumoniae is highly medically relevant. By using cutting-edge techniques in biochemistry and microbiology as well as mathematical modelling we will address the problem of origins of transcriptional noise in S. pneumonia. This will result in a high resolution systems-level understanding of the role of transcriptional noise in gene regulation of a human pathogen. The results will potentially provide a new target for intelligent drug design to manipulate differentiation and life-strategy decisions of pathogenic bacteria.

Technical Summary

Technical Summary. Bistable switches are the key elements of the regulatory networks governing cell development, differentiation and life-strategy decisions. Transcriptional noise is a main determinant that causes switching between different states in bistable systems. By using the human pathogen Streptococcus pneumoniae as a model bacterium, we will investigate how transcriptional fidelity and processivity influence (noisy) gene expression and participate in bistability. To study this question, we will use both natural and synthetic S. pneumoniae bistable switches as a highly sensitive probe for transcriptional noise. We will screen for mutations of the transcriptional apparatus that display altered bistability. A pure in vitro transcription system for S. pneumoniae will be set up and used to quantitatively characterize effects of these mutations on transcription. Detailed single-cell analysis using time-lapse microscopy will yield quantifiable data on the effects of the mutations on switching times and probabilities. Mathematical models that take transcription fidelity and processivity into account will be used to pinpoint parameters which most strongly affect the switching probabilities of our bistable networks. A global model encompassing all our in vivo and in vitro data will yield a high resolution systems-level understanding of the role of transcriptional noise in gene regulation of a human pathogen. Genetic and biochemical characterization of mutant RNAPs and/or accessory factors will yield molecular insights into the fundamental mechanisms of transcription. Furthermore, our results might lead to novel drug discovery projects specifically aimed to reduce or increase transcriptional noise to prevent unwanted development of pathogenic bacteria such as S. pneumoniae.

Planned Impact

Impact Summary Beneficiaries within the 'commercial private sector', are UK and Holland based pharmaceutical companies, named in the Impact Plan, (letters of support are available upon request). Both immediate and long term beneficiaries are in the sphere of investigation of infectious disease caused by bacteria and drug design. As a result, potential long term beneficiaries will be health organisations and consequently the wider public. Additional potential benefits for the 'wider public' will be in publicising the research via press releases, interviews, etc. S. pneumoniae is a major pathogen causing invasive (pneumonia, meningitis, bacteraemia) and non-invasive (acute otitis media, sinusitis) disease in children and in elderly and/or immuno-compromised adults. The last decades have seen the emergence and spread of pneumococcal strains with multiple antibiotic resistances posing a serious threat to human health. Research may lead to novel antimicrobial targets. It is foreseen that novel targets to tune noise and/or prevent unwanted differentiation processes such as competence development and biofilm formation, will be investigated for patent possibilities. Drugs that prevent S. pneumoniae from forming highly resistant biofilms for instance, will render the bacterium more sensitive to common antibiotics. Dual treatment with standard antibiotics and a compound that prevents initiation of competence development might result in reduced occurrence of multidrug resistant bacteria. Therefore, a potential benefit for the mentioned parties will be in novel targets for drug design and pathogen manipulation that will be investigated in our study. The drug design companies (mentioned in the Impact Plan) expressed considerable interest in our work. We agreed for a meeting half-way into the project for discussions of possible patenting and exploitation of our finding. Moreover, one of the companies is based in the same University as one of the Partners of the consortium, allowing intensive communication of our group with the researches of this company. The leading European laboratory that models Streptococcus infection in animal systems (see Impact Plan) is keen to test the mutant bacteria that will be obtained in our work in their experimental systems. This will be the next step of exploitation of our results bringing them to a more medical sphere, which may also lead to further integration with the 'commercial private sector'. The Impact activities will be managed by the members of the consortium and supported by the respective institutions. In the Newcastle University it will be supported by NU commercial development team and press office that are partly funded via this grant.
Description We set up in vitro transcription system for S. pneumoniae RNAP and its factors -We performed deep sequencing of S. pneumoniae transcriptome and its mutant lacking Gre factor -We discovered that transcription elongation can be a rate liming step of transcription cycle on highly expressed genes -We discovered that the pausing of transcription can cause queuing of RNAPs, which blocks expression of the gene and is detrimental to cell viability -We discovered that the function of Gre in vivo is to resolve queues of RNAPs -We showed that Gre factor acts as a subunit of RNAP without dissociation from elongation complex after cleavage reaction. -We found that the error rate of transcription in vivo is much higher than was proposed earlier. -We showed that Gre has relatively little effect on transcription fidelity, as was proposed earlier, suggesting that accuracy of RNA synthesis is mainly achieved by intrinsic properties of RNAP.
Exploitation Route We have found that transcription factor Gre is crucial for viability of Streptococcus pneumonia cells. Moreover, strain with Gre deletion, which is very sick and prone to lyse quickly, did not develop any suppressor mutations to overcome negative effects of deletion. This means that Gre factor is attractive target for development of new antibiotics to fight Streptococcus pneumonia infections.
Sectors Healthcare

Title The recources generated 
Description Our consortium generated around ~100 new plasmids and ~100 new S. pneumoniae strains. We established in vitro transcription system with S. pneumoniae RNAP and its factors. We developed a number of promoters and templates with different strengths and properties that can be used by other researchers. We developed methods for analysis of S. pneumoniae RNAP pausing and rate of transcription in vivo. We have highly improved the technique of assembled elongation complexes, by in depth analysis of the properties of few dozens of various complexes. The properties of the complexes are published and can be used in crystallography, Cryo-EM and single-molecule techniques. We obtained first deep transcriptome analysis of S. pneumoniae, which is deposited in data base of the SysMo (below), and which can be used for analysis of start/termination sites, operons, etc. Our consortium partners developed new bioinformatics tools for analysis of S. pneumoniae transcriptome sequencing data. 
Type Of Material Cell line 
Provided To Others? No  
Title RNA-Seq Dataset 
Description We have performed RNA-Seq on wild type S. pneumoniae and on the ?greA mutant. Two large RNA-Seq data sets are available via Sysmo-Seek ( presented as llumina fastq format (S. pneumoniae wild type D39, and its ?greASpn derivative). 
Type Of Material Database/Collection of data 
Year Produced 2011 
Provided To Others? No  
Impact No actual impacts realised to date 
Description Collaboration with Dr Stefan Klumpp on developing stochastic model of transcription elongation 
Organisation Max Planck Society
Department Max Planck Institute of Colloids and Interfaces
Country Germany 
Sector Charity/Non Profit 
PI Contribution To gain deeper insights into the molecular mechanism of transcription factor GreA, we developed a stochastic model of transcription in collaboration with the MPI in Potsdam. This model was build upon a framework of previously established model for rRNA transcription in E. coli (Klumpp and Hwa, 2008). We have found that simulations of the model are in high agreement with our experimental results. Model also predicts that transcription elongation speed per se in unaffected by greA deletion. Indeed, this prediction was experimentally confirmed by direct measurement of transcription elongation rate in vivo. Instead, transcription is reduced because of the occurrence of transcription traffic jams, which are otherwise efficiently resolved by GreA. This is a great example whereby modelling helped us in explaining the in vivo data and generated new testable hypothesis. This work is now close to submission to a top journal.
Start Year 2012