Metabolic regulation of denitrification in the opportunistic pathogen, Pseudomonas aeruginosa
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
Theme: Industrial Biotechnology and Bioenergy
Pseudomonas aeruginosa is an opportunistic human pathogen commonly associated with hospital ICUs and chronic airway infections. Although less well-known among the general public than other "headline-grabbing" bacterial pathogens such as MRSA or E. coli, P. aeruginosa infections are particularly dreaded by clinicians. This is due to the intrinsically high antibiotic resistance of the bacterium, making it a fixture in most "top ten" lists pertaining to AMR (a cross-council priority). Indeed, P. aeruginosa is one of the six "ESKAPE" pathogens that are the cause of most gram-negative nosocomial infections.
As part of BBSRC grant BB/M019411/1, we recently found that during growth on acetate as a sole carbon source, the denitrification pathway of P. aeruginosa is up-regulated. Indeed, RNA-Seq analyses revealed that some 20 gene products comprising the Nar/Nir/Nos/Nor denitrification pathways are the most highly up-regulated transcripts in the cell. This was a surprising observation because transcripts encoding the known regulators of denitrification (Anr and Dnr) were not modulated at all, suggesting that in these conditions, denitrification is controlled by some other mechanism(s). The practical relevance of this is that (i) fatty acids and other acetate-producing substrates are now known to be the preferred energy source for P. aeruginosa during many infection scenarios, and (ii) denitrification is critically-important in industry for microaerobic bacterial growth. Therefore, the key question and the main aim of this project, is to understand why P. aeruginosa turns on the denitrification pathways during growth on acetate, and what regulates this.
The project is industrially-relevant. It has been known for many years that the denitrification pathways are strongly turned on in activated sludge by growth on acetate and/or ethanol, yet remarkably, the biochemistry underpinning this important observation has not been pursued further. It is also worth noting that the UK skillset in basic microbial metabolism is dwindling, yet at the same time, the genomic revolution is creating opportunities that absolutely require expertise in this area.
We aim to use the tools of molecular microbiology (molecular biology, transposon mutagenesis etc) to identify genetic regulators of acetate-induced denitrification. In parallel, we will also use a systems biology approach. To do this, we will interrogate our RNA-Seq datasets to identify putative regulators of denitrification. Mutants containing Tn insertions in these will be extracted from our comprehensive Tn mutant library (an arrayed mutant library obtained from the UWGCG, and containing Tn insertions in every non-essential gene). Reporter gene constructs and western analyses will be used to confirm dysregulated denitrification gene expression, and the mutants will be phenotyped and biochemically characterized in considerable detail. This is a training-rich project with built-in fail-safes, and should yield a highly-trained student with a research profile attractive to both academia and industry.
Pseudomonas aeruginosa is an opportunistic human pathogen commonly associated with hospital ICUs and chronic airway infections. Although less well-known among the general public than other "headline-grabbing" bacterial pathogens such as MRSA or E. coli, P. aeruginosa infections are particularly dreaded by clinicians. This is due to the intrinsically high antibiotic resistance of the bacterium, making it a fixture in most "top ten" lists pertaining to AMR (a cross-council priority). Indeed, P. aeruginosa is one of the six "ESKAPE" pathogens that are the cause of most gram-negative nosocomial infections.
As part of BBSRC grant BB/M019411/1, we recently found that during growth on acetate as a sole carbon source, the denitrification pathway of P. aeruginosa is up-regulated. Indeed, RNA-Seq analyses revealed that some 20 gene products comprising the Nar/Nir/Nos/Nor denitrification pathways are the most highly up-regulated transcripts in the cell. This was a surprising observation because transcripts encoding the known regulators of denitrification (Anr and Dnr) were not modulated at all, suggesting that in these conditions, denitrification is controlled by some other mechanism(s). The practical relevance of this is that (i) fatty acids and other acetate-producing substrates are now known to be the preferred energy source for P. aeruginosa during many infection scenarios, and (ii) denitrification is critically-important in industry for microaerobic bacterial growth. Therefore, the key question and the main aim of this project, is to understand why P. aeruginosa turns on the denitrification pathways during growth on acetate, and what regulates this.
The project is industrially-relevant. It has been known for many years that the denitrification pathways are strongly turned on in activated sludge by growth on acetate and/or ethanol, yet remarkably, the biochemistry underpinning this important observation has not been pursued further. It is also worth noting that the UK skillset in basic microbial metabolism is dwindling, yet at the same time, the genomic revolution is creating opportunities that absolutely require expertise in this area.
We aim to use the tools of molecular microbiology (molecular biology, transposon mutagenesis etc) to identify genetic regulators of acetate-induced denitrification. In parallel, we will also use a systems biology approach. To do this, we will interrogate our RNA-Seq datasets to identify putative regulators of denitrification. Mutants containing Tn insertions in these will be extracted from our comprehensive Tn mutant library (an arrayed mutant library obtained from the UWGCG, and containing Tn insertions in every non-essential gene). Reporter gene constructs and western analyses will be used to confirm dysregulated denitrification gene expression, and the mutants will be phenotyped and biochemically characterized in considerable detail. This is a training-rich project with built-in fail-safes, and should yield a highly-trained student with a research profile attractive to both academia and industry.
Organisations
People |
ORCID iD |
| Martin Welch (Primary Supervisor) | |
| Stephen Trigg (Student) |
