Comparative analysis of regulatory mechanisms related to the second messenger c-di-GMP in Pseudomonas and Xanthomonas

Abstract

Bacteria were once considered as unicellular independent organisms, but in the last decades it has become clear that they can communicate with each other (both members of the same species and different species) and to their hosts and that they can alternate lifestyles characterized by planktonic motile cells or surface-attached cells living in biofilms. In the latter, bacteria become more resistant to harsh conditions, not only because they are protected by an extracellular matrix, but also because they adopt specific physiological state, which results from processes with significant parallels to cell development and differentiation observed in eukaryotes. The molecular mechanisms responsible for the decision between planktonic and biofilm lifestyles have been subject for academic research but are also relevant to the medical, agricultural and biotechnological methods employed to combat bacterial biofilms. Besides quorum sensing, in which individuals of a population respond to a threshold concentration of auto-inductor molecules in the medium, the second messenger cyclic bis-(3',5')-di-guanosine monophosphate (c-di-GMP) is of utmost importance in the decision between motile and sessile lifestyles. High intracellular levels of c-di-GMP favour sessility and the production of exopolysaccharides, whereas lower levels lead to increased motility and biofilm dispersion.Genes coding for proteins that function as diguanilate cyclases, c-di-GMP phosphodiesterases and c-di-GMP receptors are present in large numbers in most bacterial genomes, indicating that various pathways are used for the fine regulation of the processes discussed above. C-di-GMP may act in different levels, binding to proteins and modulating their catalytic or regulatory functions, and also to riboswitches present in some recently identified mRNA 5´UTRs. Binding of c-di-GMP to these macromolecules can have positive or negative effects, depending on the particular nature of the target. This project intends to analyze how c-di-GMP altered levels affect the protein profiles of the phytopatogen Xanthomonas campestris pv. citri 306 (XAC) and Pseudomonas aeruginosa PA14, an opportunist pathogen, in a search for novel regulatory mechanisms. Strains carrying mutations or overexpressing genes related to c-di-GMP metabolism will be generated and their protein profiles evaluated by 2D electrophoresis. Differentially expressed proteins will be identified by mass spectrometry. Common patterns can lead to the disclosure of novel conserved regulatory pathways affected by the intracellular concentration of the second messenger c-di-GMP in protein levels and/or post-translational modifications. (AU)