Prokaryotic transcription: a new direction?

Project Outline

In all lifeforms DNA is transcribed to create RNA templates for protein synthesis. This is known as the central dogma of molecular biology. Transcription is stimulated by regions of DNA called promoters. It has long been assumed that promoters drive transcription in one direction only. It seems illogical that promoters might simultaneously drive transcription “in reverse”. However, this phenomenon was reported for mammalian cells in 2008. Using genome-scale approaches, these studies concluded that mammalian promoters are inherently bi-directional; antisense and mRNA typically originate from the same region of DNA. This remarkable observation was met with some scepticism and any role remains a puzzle. In the intervening years, it has been assumed that the same does not happen in prokaryotic cells. However, in our recent work we have shown that bi-directional promoters are common in all prokaryotes (1). In this project, you will work on understanding what the role of bi-directional promoters is, how they work, and how they are regulated. Your research will involve the use of genetic, biochemical, genomic and bioinformatics tools.

References

Warman EA, Forrest D, Guest T, Haycocks JJRJ, Wade JT, Grainger DC (2021) Widespread divergent transcription from bacterial and archaeal promoters is a consequence of DNA-sequence symmetry. Nat Microbiol. 6(6):746-756

Techniques

Protein purification, Chromatin Immunoprecipitation, Illuminia Sequencing and associated bioinformatics, PCR, Radioisotopes, Microscopy, In vitro DNA binding assays, Reporter assays, Microbial cell culture, mutagenesis, lab based evolution

Understanding genome dynamics in the multiple antibiotic resistant ESKAPE pathogen Acinetobacter baumannii

Project Outline

The “ESKAPE” pathogens, which evade most antibiotics, are a small group of bacteria, identified by the WHO in 2017, as presenting the greatest risk to global human health. Amongst these organisms, Acinetobacter baumannii, has been identified as the number one priority. The microbe thrives in hospitals, resists most antibiotics, and frequently rearranges its genome. In the latter respect, mobile DNA elements (particularly transposons) are a key driver of genome plasticity. How this relates to the organism’s success is unknown. We have developed a new genomic method to track genome rearrangements across entire A. baumannii populations and we are using this to understand how chromosomal changes give the organism an advantage in hospital settings (e.g. by better resisting antibiotics or causing more infections).

You will use a combination of genomic, genetic, molecular and biochemical tools. These will be applied to our current laboratory isolates of A. baumannii, and new isolates that we obtain from infected patients at the nearby Queen Elizabeth hospital. The ultimate goal is to understand the mechanisms by which genome rearrangements allow the bacterium to better infect the human host and persist in hospital environments.

References

Mea HJ, Yong PVC, Wong EH (2021) An overview of Acinetobacter baumannii pathogenesis: Motility, adherence and biofilm formation. Microbiol Res. 247:126722

Techniques

Protein purification, Chromatin Immunoprecipitation, Illuminia Sequencing and associated bioinformatics, PCR, Radioisotopes, Microscopy, In vitro DNA binding assays, Reporter assays, Microbial cell culture, mutagenesis, drug uptake assays, antibiotic sensitivity assays, chemical genomics

Understanding how pandemic microbes evolve

Project Outline

Understanding the rules of species evolution is a fundamental question in biology. The emergence, spread and demise of harmful organisms, which can cause pandemics, is particularly important. You will focus on the bacterial species Vibrio cholerae. Recent advances in phylogenetics, model systems, and molecular tools, provide a timely opportunity to substantially progress our understanding. Your goal is to determine how evolution drives species development and pandemic potential. You will take a multi-level approach encompassing global species genetics, model ecosystems, and molecular mechanisms.

Currently, we cannot explain how the strain causing the current cholera pandemic arose, let alone predict why, where or when the next one might arise. The “success” of V. cholerae depends on its ability to adapt to different surroundings. Normally, the bacterium resides in aquatic environments and persists by forming biofilms on the chitinous surfaces of plankton and shellfish. These biofilms are rapidly disassembled on ingestion by a human or aquatic host. Following host colonisation, disease is caused by the extrusion of toxins (1). Hence, to understand what is special about the “pandemic” strain, it is essential to understand how the bacterium is able to switch between lifestyles. Recent work has generated a wealth of V. cholerae genome sequences (>10,000) from decades of outbreaks associated with the current worldwide pandemic (2,3). Our laboratory is using these data to try and understand key genetic changes that influence lifestyle choice and pandemic potential. Our work involves the application of bacterial genomics, molecular biology, and host organism colonisation models, to study lifestyle changes (4,5).

References

1. Nelson, EJ, Harris, JB, Morris, JG, Calderwood, SB, Camilli, A. (2009) Cholera transmission: the host, pathogen and bacteriophage dynamic. Nature Reviews Microbiology 7, 693-702.
2. Domman D, Quilici ML, Dorman MJ, Njamkepo E, Mutreja A, Mather AE et al. (2017) Science. Integrated view of Vibrio cholerae in the Americas. 358:789-793.
3. Weill FX, Domman D, Njamkepo E, Tarr C, Rauzier J, Fawal N et al. (2017) Genomic history of the seventh pandemic of cholera in Africa. Science. 358:785-789.
4. Manneh-Roussel J, Haycocks JRJ, Magán A, Perez-Soto N, Voelz K, Camilli A, Krachler AM, Grainger DC. (2018) cAMP Receptor Protein Controls Vibrio cholerae Gene Expression in Response to Host Colonization. mBio. 9:e00966-18.
5. Haycocks, JRJ, Warren, GZL, Walker, LM, Chlebek, JL, Dalia, TN, Dalia, AB, Grainger, D (2019) The quorum sensing transcription factor AphA directly regulates natural competence in Vibrio cholerae. PLoS Genet. 15:e1008362

Techniques

Protein purification, Chromatin Immunoprecipitation, Illuminia Sequencing and associated bioinformatics, PCR, Radioisotopes, Microscopy, In vitro DNA binding assays, Reporter assays, Microbial cell culture, mutagenesis

Understanding Multiple Antibiotic Resistance in Gram-negative Bacteria

Project Outline

Antibiotics have transformed medical practice, increased life expectancy and, together with vaccination, led to the near eradication of many bacterial diseases. However, overuse, including in animals, has potentiated the emergence of resistant bacteria. Such bacteria now cause millions of infections annually, with thousands of lives lost at a societal cost of billions of dollars. Moreover, the occurrence of untreatable infections is increasingly common. This disturbing trend is an immediate threat to health in every region of the world; antibiotic-resistant bacteria have the potential to affect anyone, of any age, in any country. The multiple antibiotic resistance (mar) regulon, and homologous systems, are continually implicated. Briefly, the mar operon encodes a global gene regulatory system that controls expression of genetic determinants which confer antibiotic resistance. We hypothesise that the majority of targets for MarA, and the underlying molecular mechanisms of antibiotic resistance, are undefined. This project will combine genomic tools with focused molecular biology to identify new mechanisms of antibiotic resistance and new drug targets.

References

Sharma P, Haycocks JRJ, Middlemiss AD, Kettles RA, Sellars LE, Ricci V, Piddock LJV, Grainger DC. (2017) The multiple antibiotic resistance operon of enteric bacteria controls DNA repair and outer membrane integrity. Nat Commun. 8:1444.

Techniques

Protein purification, Chromatin Immunoprecipitation, Illuminia Sequencing and associated bioinformatics, PCR, Radioisotopes, Microscopy, In vitro DNA binding assays, Reporter assays, Microbial cell culture, mutagenesis, drug uptake assays, antibiotic sensitivity assays.