The Presence and Role of Secondary Metabolites in Biofilm Forming Bacteria Isolated from Activated Sludge

Abstract

Biofilms are known as highly-structured microbial aggregates encased in a self-produced matrix composed of extracellular polymeric substances. Bacteria commonly grow in surface-associated (inert or organic) or suspended biofilms (bioflocs) in their natural environment. The role of secondary metabolites in regulation of bacterial biofilm formation has been frequently studied, however, there is still much to be learned regarding the role of secondary metabolites in biofilm communities given the diversity of secondary metabolites produced by bacteria. The thesis presented here explores the presence and role of secondary metabolites in biofilm forming bacteria, including an interplay between N-acetylglucosamine (NAG) and acylated homoserine lactone mediated gene expression. This thesis investigated the presence of quorum sensing (QS) and chitinase activities in chitin colonising bacteria isolated from activated sludge. The culture collection was dominated by Gammaproteobacteria, and QS, quorum quenching and chitinase activities are shown to be common among isolated bacteria. Further, a novel AHL-detection method was developed through modification of currently used AHL screening techniques. The novel method improved the AHL detection activity up to 1,000 fold in comparison to published reports. This thesis also investigated the impact of NAG on QS activities of LuxR, LasR and CviR regulated gene expression by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50% to 80% reduction at low millimolar concentrations (0.25 mM to 5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this thesis explored the role of Serratia secondary metabolite prodigiosin on biofilm formation of P. aeruginosa. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Overall, the results presented here described how secondary metabolites are prevalent in bacteria colonising on chitin surface, and the impact of NAG on bacterial secondary metabolism (quorum sensing). This study also described the inhibitory effect of prodigiosin on biofilm formation of P. aeruginosa.