Modeling of inducer exclusion and catabolite repression based on a PTS-dependent sucrose and non-PTS-dependent glycerol transport systems in Escherichia coli K-12 and its experimental verification.

We used genetically engineered sucrose positive Escherichia coli K-12 derivatives as a model system for the modeling and experimental verification of regulatory processes in bacteria. These cells take up and metabolize sucrose by the phosphoenolpyruvate (PEP)-dependent sucrose phosphotransferase system (Scr-PTS). Expression of the scr genes, which cluster in two different operons (scrYAB and scrK), is negatively controlled by the ScrR repressor. Additionally, expression of the scrYAB operon, but not of the scrK operon is positively controlled by the cAMP-CRP complex. Modeling of sucrose transport and metabolism through the Scr-system and of the scr gene expression has been performed using a modular and object-orientated new approach. To verify the model and identify important model parameters we measured in a first set of experiments induction kinetics of the scr genes after growth on glycerol using strains with single copy lacZ operon fusions in the scrK or scrY genes, respectively. In a second set of experiments an additional copy of the complete scr-regulon was integrated into the chromosome to construct diplogenotic strains. Differences were observed in the induction kinetics of the cAMP-CRP-dependent scrY operon compared to the cAMP-CRP independent scrK operon as well as between the single copy and the corresponding diplogenotic strains.     

Molecular analysis of sucrose metabolism of Erwinia amylovora and influence on bacterial virulence

Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scr regulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genes scrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scr regulons from Klebsiella pneumoniae and plasmid pUR400. scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) of E. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a K(m) of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYAB promoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora.     

Carbon catabolite repression of sucrose utilization in Staphylococcus xylosus: catabolite control protein CcpA ensures glucose preference and autoregulatory limitation of sucrose utilization

Sucrose utilization in Staphylococcus xylosus is dependent on two genes, scrA and scrB; encoding a PTS permease and a sucrose phosphate hydrolase, respectively. The genes are encoded on separate loci and are transcribed from two promoters, P(scrA) and P(scrB), both of which are controlled by the repressor ScrR by binding to the operator sequences O(A) and O(B). In the scrA promoter region, a catabolite-responsive element (cre), operator for the global catabolite control protein CcpA, is also present, but its contribution to scrA regulation has not been determined. Using an integrative promoter probe plasmid, the activities of the promoters P(scrA) and P(scrB) were determined under different growth conditions. Both promoters are induced by sucrose and induction is prevented when glucose is also present. Without a functional CcpA, glucose-mediated prevention of induction is lost, clearly demonstrating that CcpA ensures hierarchical sugar utilization with glucose as preferred substrate. Measurements of promoter activities in the absence of a functional ScrR repressor indicated that CcpA also acts upon the operators O(A) and O(B), albeit not as efficiently as on the genuine cre in P(srcA). Besides determining the choice of the carbon source, CcpA has a second effect on sucrose gene expression. When sucrose is the sole carbon source, sucrose catabolism activates carbon catabolite repression and CcpA prevents full induction of the sucrose utilization genes by partially repressing the scrA promoter. Thus, CcpA-dependent regulation serves as a built-in autoregulatory device to restrict sucrose uptake.