Impacto de la acetilacion de CRP sobre el metabolismo de Escherichia coli

Abstract

Escherichia coli is a bacterium that has been established as a prokaryotic model in the area of Biotechnology and for industrial applications. Therefore, discovering the mechanisms involved in the regulation of the E. coli metabolism will contribute to the use of this microorganism as an on demand cellular factory. The prokaryotes metabolism can be regulated by post-translational modifications. Thus, many investigations have focused on acetylation as one of the most important post-translational modifications, which have been found is widely involved in several cellular processes. The catabolite regulator protein (CRP) is a global transcription factor of E. coli involved in controlling the "catabolic repression by carbon source" (RCC). CRP needs to bind cAMP to carry out activation of genic expression. Genes controlled by CRP can be classified as "CRP-dependent promoters of type I, type II or type III". Additionally, CRP is modified by acetylation in several lysines, although this thesis has focused on studying K152, K166 and K100. Particularly, K100 is the most interesting because it is involved in the direct interaction with RNA Polymerase through the Activating Region 2 in the transcription of type II promoters. The main objective of this thesis was to elucidate the role of CRP acetylation and ascertain how this post-translational modification affects the physiology of E. coli and, more specifically, the genes regulated by CRP. To achieve this goal Molecular Biology techniques, protein purifications, protein characterization, gene expression analysis by microarrays and RT-qPCR, study of the protein-protein interaction through electrophoretic mobility shift assay (EMSA) and in vitro transcription to, it has been used. Study of the E. coli physiology studies in different carbon sources showed differences in lysines acetylation. Thus, the acetylation of lysines 152 and 166 only seems to be evident when acetate is used as the carbon source, while the acetylation of lysine 100 depended on the carbon source considered. For this reason, the major impact of lysine 100 on the metabolism of E. coli was demonstrated. Proteins simulating acetylated (CRP K100Q) and deacetylated (CRP K100R) states were purified and characterized, and both of them showed similar thermodynamic parameters, leading to conclude that the acetylation of lysine 100 did not alter their stability. Global transcriptome analysis of E. coli K100 mutants growing in glucose or acetate as carbon source, demonstrated that the acetylation of lysine 100 is detrimental to gene expression, while the deacetylation state seems to be the most beneficial for the transcription of type II promoters. In vitro transcription analysis using fepA and gatY promoters showed a worse interaction between CRP and RNA Polymerase, which is caused by the acetylation of lysine 100 in CRP. This way, the damage produced in the transcriptional rate by the acetylation of the lysine 100 of CRP was corroborated. In addition, the importance of the distance between the location of CRP and RNA Polymerase in DNA was studied. The results showed that the transcription did not occur in cases where the distance between CRP and RNA Polymerase was smaller due to steric hindrance, while the optimal distance between CRP and RNA Polymerase is provided by the semisynthetic promoter CC(-41,5)D4. The main conclusions of this thesis are: the E. coli acetylation state is totally dependent on the carbon source and culture stage, which is reflected in the number and diversity of lysines acetylated in CRP in the considered conditions. Also, the acetylation of lysine 100 seems to have important repercussions for bacterial physiology and gene expression. The E. coli transcriptome analysis under the influence of CRP acetylation enabled us to understand the role of acetylation in lysine 100 of CRP, which diminishes gene expression levels of type II promoters. The gene expression differences observed, as a function of the acetylation state of CRP, are due to the worse CRP-RNA Polymerase interaction during the transcription, as a consequence of the damage caused by eliminating the positive charge provided by lysine 100 when acetylation occurs.