Download files
Abstract
Bacteria in the environment live predominantly as surface-attached communities, called biofilms, and are different to their free-living planktonic counterparts [1-3]. Biofilms are responsible for 65% of infections in humans [4] and cause problems in industrial settings. Carbon starvation has been shown to induce dispersal of biofilms of the opportunistic pathogen, Pseudomonas aeruginosa [5-6]; however, the molecular pathway controlling dispersal is unknown. This dissertation aimed to characterise the molecular pathway regulating dispersal of P. aeruginosa PAO1 biofilms during glucose starvation.
An improved online biofilm monitoring system was used to continuously quantify biofilm formation and dispersal. Glucose starvation was applied to 4 day-old biofilms, resulting in dispersal. Biofilms of strains mutated in genes that are involved in biofilm formation and/or dispersal (i.e. nirS, vfr, bdlA, rpoS, lasRrhlR and Pf4 bacteriophage) behaved similarly to the wild type strain when starved, suggesting that these genes are not associated with glucose starvation-induced dispersal. Thus, the molecular pathway regulating starvation-induced dispersal may be distinct from dispersal or biofilm development mediated by those regulators (e.g. nitric oxide mediated dispersal). In contrast, cAMP was found to be important as biofilms of the cyaA mutant strain were unable to disperse.
Results showed that biofilms treated with a proton ionophore were defective for dispersal, suggesting that energy derived from oxidative phosphorylation is essential for dispersal. In contrast, induction of the stringent response or translation inhibition did not inhibit dispersal of glucose-starved biofilms, indicating that neither pathway was required. Proteomic analysis revealed that more than 100 proteins were differentially expressed in starved vs. unstarved cells of both biofilm and planktonic cells. These proteins belonged to various functional classes including motility, energy and carbon metabolism. Further, a non-dispersing mutant was identified that carried a transposon insertion in a probable non-ribosomal synthase, but the product of this gene is still being characterised.
Overall, the results showed that carbon starvation-induced dispersal of P. aeruginosa biofilms is cAMP and energy dependent and may also require flagellar motility. The online biofilm monitoring system is an advanced, reliable, inexpensive biofilm growth system that continuously quantifies biomass accumulation biofilms or dispersal cells in the effluent.