Papel de las proteinas reguladoras de la familia RSM en la señalización mediada por diguanilato cíclico y el modo de vida multicelular de pseudomonas putida kt2440

Abstract

In the environment, most bacteria form multicellular communities associated to surfaces, called biofilms (Wingender et al., 1999). In a biofilm, bacterial cells anchored to biotic or abiotic solid surfaces are surrounded by a complex self-produced matrix consisting on exopolysaccharides, proteins, lipids and extracellular DNA(Hall-Stoodley et al., 2004). The structural components of the biofilm matrix give rise to a robust structure that aids in the protection of enclosed bacterial populations from adverse conditions. Biofilm formation is a highly regulated process which responds to different environmental signals (Costerton et al., 1999; Flemming and Wingender, 2010; Römling and Balsalobre, 2012). The regulatory network that connects these stimuli with changes in gene expression is complex and not totally understood. In this network, the intracellular second messenger cyclic diguanilate (c-di-GMP) is a key element that modulates the transition from motile, planktonic life to the biofilm lifestyle (Zhang and Dong, 2004). This Doctoral Thesis has been focused on the role of the Rsm family of global post-transcriptional regulators on bacterial biofilm formation and c-di-GMP signaling, using as a model system Pseudomonas putida KT2440, a bacterium of agronomic interest for its properties as PGPR. Earlier work in the research group had established that the two-component regulatory system GacS/GacA controls expression of adhesins required for biofilm formation in this bacterium. Given that in other microorganisms this two-component system is part of a regulatory cascade mediated by Rsm family proteins, the objectives of this Thesis have included the characterization of these proteins in P. putida KT2440, analyzing their involvement in biofilm formation and c-di-GMP signaling, and to begin studying their role as global regulators. Chapter 1, “Self-Regulation and Interplay of Rsm Family Proteins Modulate the Lifestyle of Pseudomonas putida”, describes the role of the three proteins of the CsrA/RsmA family of post-transcriptional regulators present in P. putida KT2440 (RsmA, RsmE and RsmI) in the lifestyle of this bacterium. For that purpose, single, double and a triple mutant were constructed and characterized, and the three genes overexpressed under the control of a constitutive promoter. The triple mutant showed reduced swimming and swarming motility, and altered dynamics of biofilm formation. The biomass attached to abiotic surfaces is higher than in the wild type, but the biofilm disperses earlier, although the phenotype varies on different surfaces. On the other hand, overexpression of RsmE or RsmI causes reduced biofilm formation. Expression analysis of the different adhesins and exopolysaccharides revealed that these alterations could be due to changes in the extracellular matrix and in the timing of synthesis of its elements (Huertas-Rosales et al., 2016) In chapter 2, “The Pseudomonas putida CsrA/RsmA homologues negatively affect c-di-GMP pools and biofilm formation through the GGDEF/EAL response regulator CfcR”, we expanded the study of the regulation of cfcR expression. This gene encodes the unique response regulator with GGDEF/EAL domains in P. putida KT2440. Expression of cfcR is regulated at the transcriptional level by RpoS, ANR and FleQ, and its diguanylate cyclase activity requires the multisensor hybrid histidine kinase CfcA. In this chapter, an additional level of regulation is described, operating at the post-transcriptional level through RsmA, RsmE and RsmI. Direct binding of these proteins to a specific motif overlapping the initiation codon of cfcR (5´-CAUGGAUG-3´) is demonstrated. The lack of Rsm proteins causes de-repression of cfcR. We also show that in stationary phase the c-di-GMP pool in KT2440 is mostly dependent on the activity of CfcR, and that in a triple rsm mutant the levels of second messenger are not only higher but increase earlier during growth. Results from this chapter allow us to establish that the Gac/Rsm/c-di-GMP signaling cascade operates in P. putida through CfcR. In the third charper “Global analysis of the Rsm regulon in Pseudomonas putida KT2440”, a RIP-seq study was performed to elucidate all possible genes that are directly regulated by Rsm proteins in P. putida. Over 400 genes, intergenic regions and potential sRNA are targets for these proteins, some being specific for each of them and others being shared by two (95) or the three (38) Rsm proteins. We have confirmed that cfcR and elements involved in its regulation are among the identified targets, as expected from the results in chapter 2. Other biofilm related genes have also been found in this study. Although the analysis of targets is still preliminary, thanks to this work we have a long list of genes and new sRNA that will allow us to complete the regulatory map associated to Rsm proteins in P. putida KT2440.