Obiettivo
It was the aim of this project to identify and to functionally characterize cis- and trans-acting elements involved in catabolite repression (CR) in several food-grade Gram-positive bacteria. We wanted to detect the mode of action and possible interactions of these elements (operator, repressor, corepressor) participating in the signalling pathway which leads from the uptake of a well metabolizable carbon source to reduced expression of CR-sensitive catabolic genes. The detailed understanding of CR in industrially important food-grade bacteria was expected to allow in a directed and predetermined way to construct strains resistant to CR and exhibiting improved production and fermentation properties.
At the beginning of this project it was generally believed that CR in Gram-positive bacteria does not follow a single mechanism, but that several independent mechanisms of CR exist. It was known for example that a repressor-like protein CcpA (catabolite control protein A) was involved in CR of the a-amylase encoding gene in B.subtilis and that an operator-like cis-acting element existed which was also involved in the regulation of expression of this gene. During the course of this project, the general involvement of CcpA in CR of Gram-positive bacteria was established. CcpA has been shown to exist in all investigated low GC Gram-positive bacteria. We cloned and sequenced the ccpA gene from Bacillus megaterium, Staphylococcus xylosus, Lactobacillus casei, Leuconostoc lactis, Lactobacillus pentosus and Lactococcus lactis. Mutations in ccpA were found to exhibit a pleiotropic relief from CR in all investigated Gram-positive bacteria.
We have shown that the cis-acting element cre (catabolite responsive element) occurs in the promoter region or within the first gene of numerous CR-sensitive operons of Gram-positive bacteria. The 14 bp long consensus sequence resembles the Escherichia coli lac and gal operator sites. Mutations affecting a certain cre led to the loss of CR of only the corresponding catabolic operon. It was thought that CR in Gram-positive bacteria is mediated by the binding of CcpA to the cre, thereby preventing the expression of CR-sensitive operons. However, attempts to demonstrate in vitro the interaction of CcpA with the cis-acting element cre remained unsuccessful for a long time.
It was an interesting finding within this project that CR in Gram-positive bacteria requires the ATP-dependent phosphorylation of Ser-46 of HPr, a phosphocarrier protein of the PEP:glycose phosphotranferase system (PTS). This post-translational modofication reaction is catalyzed by the metabolite-activated HPr kinase. Mutants in which Ser-46 of HPr was replaced by a non-phosphorylatable amino acid were insensitive to CR and exhibited a phenotype similar to ccpA mutants. An interaction of CcpA with P-Ser-HPr, but not unphosphorylated HPr, could be demonstrated in vitro. P-Ser-HPr was found to function as co-repressor. The complex of P-Ser-HPr and CcpA has been shown to bind to the Bacillus gnt and xyl cre in vitro. Mutations in the gnt cre prevented the interaction of the complex between repressor and co-repressor with the target DNA.
Although the CcpA/P-Ser-HPr-dependent mechanism seems to represent the main mechanism of CR in Gram-positive bacteria, a novel gene involved in CR has been cloned and sequenced from S.xylosus. This gene has been demonstrated to encode glucose kinase. At the present it is not clear whether glucose kinase interacts with CcpA or P-ser-HPr or whether it constitutes an independent mechanism of CR.
Numerous PTS and non-PTS carbohydrate uptake systems have been investigated in the course of this project. Several genes of carbohydrate uptake systems and of their regulatory components have been cloned and sequenced and the encoded proteins have been studied at the molecular level.
MAJOR SCIENTIFIQUE BREAKTROUGHS:
In addition to the cis-acting catabolite responsive element (cre), two trans-acting components have been demonstrated to be involved in the main mechanism of CR of Gram-positive bacteria: Seryl-phosphorylated HPr and CcpA. These two proteins form a complex, which binds to the cre of CR-sensitive operons. The existence of seryl-phosphorylated HPr and CcpA in most Gram-positive bacteria suggests that the CcpA/P-Ser-HPr-dependent mechanism represents the main mechanism of CR in Gram-positive bacteria. A novel protein, the enzyme glucose kinase, has been demonstrated to be involved in CR in Staphylococcus xylosus. The genes encoding the catabolite control protein CcpA and the general PTS proteins HPr and enzyme I as well as several sugar-specific PTS proteins have been cloned and sequenced from different bacteria.
Campo scientifico
- natural sciencesbiological sciencesmicrobiologybacteriology
- natural sciencesbiological sciencesgeneticsmutation
- natural sciencesbiological sciencesbiochemistrybiomoleculescarbohydrates
- natural scienceschemical sciencesorganic chemistryamines
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
Invito a presentare proposte
Data not availableMeccanismo di finanziamento
CSC - Cost-sharing contractsCoordinatore
69367 LYON
Francia