ROLE OF GENES BELONGING TO METABOLIC PATHWAYS (SULFATE ASSIMILATION AND PYRIMIDINE BIOSYNTHESIS) IN THE PRODUCTION OF EXTRACELLULAR STRUCTURES IN ESCHERICHIA COLI

Most bacteria are able to grow as single cells or organized in microbial communities attached to solid surfaces, known as biofilms. Growth as a biofilm confers bacterial cells different advantages, such as increased resistance to environmental stresses, antimicrobial agents and it also protects the bacteria from host immune system. During the transition from planktonic cells to biofilm bacteria undergo a genome-wide reprogramming of gene expression that leads to the production of specific biofilm determinants and to deep physiological changes. This process requires the consumption of a large amount of energy and therefore the metabolic state of the cell plays a central role in regulating the biosynthesis of adhesion structures. Indeed, several metabolic pathways and metabolites have been shown to directly control the production of biofilm determinants. During my Ph.D, I have tackled the problem of how different metabolic pathways can regulate the production of extracellular structures involved in biofilm formation in Escherichia coli. In particular, we showed that curli and cellulose, the two main adhesion factors in E. coli, are affected by nucleotide biosynthetic pathways: transcription of curli encoding operons responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. Furthermore, we showed that curli production is also hindered by the reduction of purine nucleotides. Since curli and cellulose are pivotal for biofilm formation, nucleotide biosynthesis could be a good candidate for drugs endowed with anti-biofilm activity. Indeed, we showed that the drug azathioprine, which inhibits nucleotides biosynthesis, could prevent biofilm formation in clinical isolates of E. coli through the reduction of the nucleotide pools available for the synthesis of the second messenger c-di-GMP. Nucleotide biosynthesis is not the only metabolic process that influences the production of biofilm determinants in E. coli. We showed that curli fibres production is also affected by yet another biosynthetic pathway, namely, sulphate reduction for cysteine/methionine biosynthesis. Our data suggest that accumulation of pathway intermediate phosphoadenosine 5’-phosphosulfate (PAPS) affect the production of curli fibres and of other extracellular structures. Therefore my results strongly support the idea that metabolic fluxes of different essential elements play a crucial role in controlling cell surface reorganization. Accumulation of intermediate metabolites is instrumental in relaying to the bacterial cell conditions of lack or abundance of a given element, triggering the adequate responses. The molecular mechanisms involved in this process seem to be extremely complex and probably involve gene regulation control at transcription initiation and RNA and protein stability. Extracellular structures regulation by metabolic cues.