AbstractThe Gram-negative bacterium, Serratia marcescens, is an important opportunistic human pathogen capable of causing serious infections, particularly in immunocompromised patients. S. marcescens Db10 is an insect pathogen that is closely related to S. marcescens strains which cause disease in humans. The genome sequence of S. marcescens Db10 is available and tools exist which allow the genetic manipulation of this bacterium, making it an ideal model organism. In this work, contact dependent and independent antibacterial strategies employed by S. marcescens Db10 are investigated at the molecular level.
Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are widespread in bacteria and fungi and are responsible for the biosynthesis of many metabolites with medically relevant properties, including antibiotics. The activity of NRPS and PKS enzymes is dependent on their modification by a phosphopantetheinyl transferase (PPTase) enzyme. In this work we identify the biosynthetic genes, alb2-6, which are required for the production of the NRPS-PKS antibiotic, althiomycin, in S. marcescens Db10. The role of the Major Facilitator Superfamily protein, Alb1, in the export of and resistance to althiomycin is also defined. Using a combination of bioinformatic, biochemical and genetic approaches we demonstrate that a PPTase enzyme, PswP, is essential for althiomycin production. We further show that PswP is required for the biosynthesis of the surfactant, serrawettin W2, and an enterobactin-like siderophore. These PswP dependent metabolites are all shown to possess antimicrobial activity against the Gram-positive bacterium, Staphylococcus aureus.
The Type VI Secretion System (T6SS) is the most recently identified Gramnegative bacterial secretion system and has been demonstrated to play important roles in mediating contact dependent interactions with neighbouring bacterial and eukaryotic cells. S. marcescens Db10 is known to encode an anti-bacterial T6SS. In this study, S. marcescens Db10 strains are constructed which harbour fusions of T6SS components to fluorescent proteins. The functionality of these fusion proteins and their localisation profiles are analysed by fluorescence microscopy. We demonstrate that different sub-complexes of the T6SS show distinct patterns of localisation. We additionally show that the T6SS assembles within many cells within a population of S. marcescens Db10 and that assembly is not dependent on the presence of neighbouring cells. Strains constructed in this study will comprise a toolbox for further investigations into the mechanisms of assembly and dynamics of the T6SS.
|Date of Award
|Nicola Stanley-Wall (Supervisor) & Sarah Coulthurst (Supervisor)