Genetic and biochemical characterization of protein complexes that are essential for bacterial chromosome segregation and maintenance

Lead Research Organisation: University of East Anglia
Department Name: Graduate Office

Abstract

Protein-protein interactions (molecular "handshakes") are crucial for the operation and function of all living organisms. These interaction interface are often formed by a subset of key amino acid residues from each protein partner. Interfacial residues often vary between orthologs, indicating some degree of mutability or degeneracy. But it is unclear how plastic the interface is and how the combination of key amino acid residues at the interface supports a robust and specific protein-protein interaction. This DTP PhD project aims to comprehensively characterise the sequence space around an ancestral protein ParB that is crucial for bacterial chromosome segregation. The student will:

(i) characterise the effect on functions of all possible mutations and combination of mutations at a subset of amino acids that are crucial for ParB-ParB interaction by saturated mutagenesis and deep sequencing.

(ii) validate the functionality of the identified ParB variants by a combination of in vitro and in vivo techniques.

(ii) project the mutational sequence space on to the three-dimensional structure of ParB to fully characterise the sequence-structure-function relationship.

We use the Caulobacter crescentus chromosome partitioning protein B (ParB) as an ideal model system for this project.

Publications

10 25 50
 
Description DNA damage response in bacteria has been widely studied in species such as Escherichia coli, but poorly investigated in other bacterial species. We aimed to study the DNA-damage response in a different class of alphaproteobacteria with a different chromosome organization and segregation pattern. In this study, we report the discovery of a novel 37-amino-acid protein with an important effect on maintaining cell fitness under DNA-damaging conditions in C. crescentus. CalP is a small amphipathic transmembrane protein with Nout-Cin topology that polymerises generating a homooligomer in the inner cell membrane. Sensitivity assays confirm the deficient phenotype of ?calP when cells were exposed to antibiotics such as mitomycin C (MMC) norfloxacin, ciprofloxacin or MMS. Complementation analysis confirms the restoration of the WT phenotype when cells were challenged with MMC or norfloxacin. The induction of DNA damage with MMC causes downregulation of calP in a recA mutant strain unlike the expression of calP in WT, which is upregulated upon induction of DNA damage. Immunoblot assays show that CalP production increases following DNA damage with MMC, norfloxacin, or MMS. Nevertheless, CalP production did not increase in ?recA despite the DNA damage caused by MMC. ?calP mutants are more permeable to MMC-TRC (MMC linked to the Texas Red Cadaverine fluorophore) than WT, so CalP may have an efflux pump-like function. Overall, our results suggest that CalP could be involved in DNA damage tolerance/response in C. crescentus. However, the elucidation of the role and molecular mechanisms of CalP requires further investigation.
Exploitation Route Outcomes from our study will be relevant to understand the mode of actions of DNA-damaging antibiotics/anticancer drugs such as Norfloxacin or Mitomycin C. Outcomes from our research is also relavant to understanding how certain bacterial sepecies are resistant to these antibiotics.
Sectors Healthcare