In this work we present cloning and overexpression of lactococcal CcpA protein in Escherichia coli Xl1blue strain as a fusion with 6 x His tag. A high yield of the CcpA protein was obtained when the cells were cultured in liquid medium LB with 100 microg/ml ampicillin at 37 degrees C and subsequently for 4 h after induction by IPTG. The procedure let us obtain 5 mg of homogenous CcpA protein. G...

In low-GC Gram-positive bacteria, carbon catabolite repression (CCR) is exerted by transcriptional regulation through a protein complex consisting of catabolite control protein CcpA and serine phosphorylated phosphocarrier protein HPr (HPr-ser-P). We investigated the interaction between these components of Lactobacillus casei and Bacillus megaterium. CcpA of L. casei could not complement a B. m...

Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation o...

This study examines the anticonvulsant profile of interactions between 2-chloro-N6-cyclopentyladenosine (CCPA, a selective adenosine A1 receptor agonist) and four conventional antiepileptic drugs (AEDs: carbamazepine--CBZ, phenobarbital, phenytoin and valproate) in the mouse maximal electroshock seizure (MES) model. Acute adverse effects produced by AEDs in combination with CCPA were determined...

The sigma(B)-dependent promoter in front of the rsbV gene of Bacillus subtilis is induced approximately 5-fold in response to (1) the addition of 4% ethanol, (2) carbon starvation, and (3) phosphorous starvation. Binding sites for the global carbon and nitrogen regulators, CcpA and TnrA, were mutated, and the consequences of their loss and that of CcpA or TnrA were studied using rsbV-lacZ fusio...

CcpA was purified from Escherichia coli BL21 (lambda DE3)/pLysS carrying plasmid pTSC5, which was constructed by inserting the ccpA gene into the polycloning site of pGEM4. The purified protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent mass of 38 kDa but was eluted from a calibrated Bio-Gel P-100 column with an apparent mass of 75 kDa. Western blot (...

During this study His-tagged CcpA protein purified under native conditions to obtain a biologically active protein was used for molecular analysis of CcpA-dependent regulation. Using electrophoretic mobility shift assays it was demonstrated that CcpA of L. lactis can bind DNA in the absence of the HPr-Ser-P corepressor and exhibits DNA-binding affinity for nucleotide sequences lacking cre sites...

Carbon catabolite protein A (CcpA) is known to function as a major regulator of gene expression in different gram-positive organisms. Deletion of the ccpA homologue (saCOL1786) in Staphylococcus aureus was found to affect growth, glucose metabolization, and transcription of selected virulence determinants. In liquid culture, deletion of CcpA decreased the growth rate and yield; however, the eff...

The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low l...

Previous studies have suggested that the transcription factor CcpA, as well as the coeffectors HPr and Crh, both phosphorylated by the HprK kinase/phosphorylase, are primary mediators of catabolite repression and catabolite activation in Bacillus subtilis. We here report whole transcriptome analyses that characterize glucose-dependent gene expression in wild-type cells and in isogenic mutants l...