| Popis: |
Most bacteria grow in multicellular communities known as biofilms, which are generally physiologically different from their planktonic counterparts. At the end of the biofilm life-cycle, bacteria disperse as free living single cells in response to environmental stimuli such as nutrient limitation. Carbon starvation has been shown to induce dispersal of biofilms of the opportunistic pathogen Pseudomonas aeruginosa. It has been shown that cAMP plays a role in mediating biofilm dispersal during glucose starvation. However, the molecular pathway controlling dispersal is not well defined. This dissertation characterises the molecular pathway regulating dispersal of P. aeruginosa PAO1 biofilms during carbon starvation that involves cAMP signaling. The cAMP/dispersal pathway was investigated in part by studying the starvation dispersal response of different mutants of the cAMP signalling pathway; the adenylate cyclases cyaA, cyaB (which produce cAMP) and the cAMP receptor protein gene (CRP) (cbpA). The results suggest that starvation induced dispersal is partially mediated through a functional CyaA, while the global pool of cAMP, which is largely regulated by CyaB, does not mediate the dispersal response. Further, cbpA encodes a CRP and was not required for the starvation dispersal response. This suggests that cAMP works by means of another, currently unidentified CRP homologue. In addition, the cAMP-CRP regulon has been shown here to play an important role in stress survival of P. aeruginosa, including nutrient and oxidative stress. Carbon starvation impacted the cellular cAMP concentration and expression of two genes encoding for adenylate cyclase proteins; cyaA and cyaB, as well as the CRPs, vfr and cbpA. Carbon starvation supressed the expression of cAMP signaling pathway and effector proteins. The results showed a transient state of decrease in the intracellular cAMP concentration of both adenylate cyclases in P. aeruginosa upon induction of starvation dispersal followed by significant increase. Finally, the role of PA3327 in the starvation dispersal response was investigated. PA3327 encodes a putative non-ribosome peptide synthetase (NRPS). The PA3327 mutant biofilm failed to disperse in response to glucose starvation. In addition, the PA3327 mutant showed significant growth limitation under extreme iron limitation. |
