A prototype of a bacterial mitotic spindle: moving DNA molecules apart through a polymerization-based engine

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Bacterial multidrug resistance is a global problem that needs urgent action as it poses a serious threat to human health worldwide. The fraction of patients carrying resistant strains has escalated to levels that put the control of bacterial infections in jeopardy. Antimicrobial resistance constitutes a heavy burden on health care resources: it has been estimated that these types of infections cost the NHS around #1 billion a year. In the case of tuberculosis, the treatment of a patient carrying a multidrug resistant strain ranges from $500 to $6,000. The emergence of multidrug resistant pathogens among bacterial populations results either from chromosomal mutations or from the horizontal transfer of resistance genes often present on mobile genetic elements such as plasmids, transposons and pathogenicity islands. Multidrug resistance plasmids harbour their own survival system, a partition cassette, which ensures an equitable segregation of the plasmids from one generation to the next at cell division. When this system malfunctions, the plasmid is not stably inherited and after some generations is ultimately lost. Shedding light on the molecular mechanisms responsible for the stable inheritance and maintenance of multidrug resistance plasmids could lead to the discovery of targets, which may be exploited to develop new therapeutic agents. The partition cassette of the multidrug resistance plasmid TP228 consists of the parFG genes and upstream noncoding sequence which harbours a series of direct and invert repeats. ParF is an ATPase that polymerizes into multistranded filaments; ParG is a DNA-binding protein that contacts the repeats in the noncoding region. The aim of this project is to dissect the ParFG partition system by using complementary approaches. The research objectives include: 1) in vivo localization of ParF protein by fluorescence or immunofluorescence microscopy; 2) analysis of the dynamics of the ParF polymerization/depolymerization cycle by Dynamic Light Scattering; 3) investigation of the role of ParG in ParF filament bundling by using N-terminal truncated mutants of ParG in sedimentation assays and electron microscopy experiments; 4) study of the mechanism whereby ParG stimulates ParF ATPase activity by testing various ParG mutants in ATPase assays; 5) analysis of the potential interplay between ParF and the MinD site-selection cell division protein by the use of genetic, biochemical and cellular biology approaches; and 6) determination of the structure of the ParF protein by NMR or X-ray crystallography. The proposed studies will make important contributions in unravelling the molecular details of the segregation process of multidrug resistance plasmids.