Regulator Of Carbon Catabolite Repression Research Articles

Identification of a co-repressor binding site in catabolite control protein CcpA.

The catabolite control protein CcpA is the central regulator of carbon catabolite repression in Bacilli and other Gram-positive bacteria. A comparison of 12 CcpA-like sequences with regulators from the LacI/GalR family defines a CcpA subfamily based on extensive similarities found among CcpAs and not in 32 other members of the family. These amino acids are clustered in three blocks in the CcpA sequence. Their interpretation, assuming a PurR-like fold, reveals that almost all of them are surface exposed and form a continuous patch on the N-terminal subdomain of the protein core extending into the DNA reading head. We introduced nine single amino acid exchanges in the subfamily specific residues of CcpA from Bacillus megaterium. Six mutants, namely CcpA47RS, 79AE, 89YE, 295YR, 299YE and 303RD, are inactive or severely impaired in catabolite repression, underlining their relevance for CcpA function. They are negatively transdominant over wild-type CcpA demonstrating their ability to correctly fold for dimerization. Five of them are unable or impaired in binding HPr-Ser-46-P in vitro, establishing a correlation between catabolite repression efficiency and HPr-Ser-46-P binding. These results support the hypothesis that the conserved region in CcpA is the HPr-Ser-46-P binding site.

CcrA1: a mutation in Streptomyces coelicolor that affects the control of catabolite repression.

The regulation of carbon utilization is of central importance in the gene expression pathways for both morphological development and antibiotic production in Streptomyces species. We report the identification and characterization of a mutation in Streptomyces coelicolor, ccrA1, that affects the expression of several catabolite-controlled promoters. ccrA1 mutants are altered in expression of galP1, the glucose-sensitive, galactose-dependent promoter of the galactose utilization operon; in expression of the glycerol utilization operon, which is glucose sensitive and glycerol dependent; and in expression of chi63, the glucose-sensitive chitin-dependent promoter of a gene involved in chitin utilization. ccrA1 has no effect on the expression of galP2, a promoter that directs constitutive transcription of the galE and galK genes. ccrA1 maps to a region of the S. coelicolor genome which distinguishes it from other mutations known to be involved in catabolite control. We suggest that ccrA1 identifies a gene whose product may be involved in the general regulation of carbon catabolite repression in this complex bacterium.

Sequence analysis of the Aspergillus nidulans pectate lyase pelA gene and evidence for binding of promoter regions to CREA, a regulator of carbon catabolite repression.

The nucleic acid and deduced amino-acid sequences of the pectate lyase gene (pelA) from Aspergillus nidulans are presented. The pelA gene contains two short introns, 68 and 49 bp in length, and encodes a peptide of 326 amino acids. Five transcriptional start sites are clustered between 65 and 79 bp upstream of the start codon as determined by primer extension. Comparison of the amino-acid sequences of pectate or pectin lyases from bacteria, fungi and plants revealed less than 30% overall identity. However, five regions within these enzymes, in particular domains associated with the active site, are highly conserved with amino-acid similarities greater than 50%. Phylogenetic analysis using the principle of parsimony (PAUP 3.1.1) showed that pelA is most closely related to pectate lyases from plants rather than pectin lyases from other fungi. Previously, pelA was shown to be induced by polygalacturonic acid and repressed in the presence of preferred carbon sources, such as glucose. Gel mobility shift analysis indicates that a PstI-SphI fragment from the pelA promoter binds to a fusion protein composed of the N-terminal part of CREA, a protein involved in carbon catabolite repression, and glutathione-S-transferase. This result suggests CREA may contribute to the regulation of pelA expression.

Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans.

The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented. The gene contains no introns. The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein. The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein. A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition. Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation. This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.

Analysis of the creA Gene, a Regulator of Carbon Catabolite Repression in Aspergillus nidulans

The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented. The gene contains no introns. The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein. The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein. A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition. Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation. This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.