Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.     

Glycerol Utilization Gene Cluster in Streptomyces clavuligerus

The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.Streptomyces clavuligerus ATCC 27064 produces the β-lactamase inhibitor clavulanic acid (CA). This compound is formed from arginine (17) and the three-carbon molecule glyceraldehyde-3-phosphate (6) which are condensed by the carboxyethylarginine synthase, the first enzyme of the pathway, encoded by ceaS2. Mutants disrupted in this gene do not produce CA in tryptic soy broth or starch-asparagine (SA) medium but form moderate amounts of CA in glycerol-supplemented media, probably due to glycerol utilization through the duplicated CeaS1 carboxyethylarginine synthase (10).The role of d-glyceraldehyde-3-phosphate as a CA precursor was further supported by the construction of a glyceraldehyde-3-phosphate dehydrogenase (gap1) mutant of S. clavuligerus, which produces 180 to 210% CA in comparison to the wild-type strain due to higher availability of the glyceraldehyde-3-phosphate precursor (9).Genes for glycerol utilization in Streptomyces coelicolor form an operon, gylCABX (15, 16), containing a gene for a putative glycerol transporter, a glycerol kinase, a glycerol-3-phosphate dehydrogenase, and a gene of unknown function. They are preceded by gylR (5), which encodes a glycerol-inducible repressor controlling both gylR and the gylCABX operon. Glycerol induction and glucose catabolite repression of the glp genes are thought to be directly related to the GylR protein in S. coelicolor (5).Due to the importance of the glycerol flow for CA production, we decided to analyze the glycerol-utilizing gene cluster of S. clavuligerus.     

Characterization of glucose-repression-resistant mutants of Bacillus subtilis: identification of the glcR gene

In Bacillus subtilis, carbon catabolite repression (CCR) is mediated by the pleiotropic repressor CcpA and by ATP-dependent phosphorylation of the HPr protein of the phosphotransferase system (PTS). In this study, we attempted to identify novel genes that are involved in the signal transduction pathway that ultimately results in CCR in the presence of repressing carbon sources such as glucose. Seven mutants resistant to glucose repression of the levanase operon were isolated and characterized. All mutations were trans-acting and pleiotropic as determined by analyzing CCR of beta-xylosidase and of the sacPA and bglPH operon. Moreover, all mutations specifically affected repression exerted by glucose but not by other sugars. The mutations were mapped to three different loci on the genetic map, ptsG, glcR, and pgi. These three genes encode proteins involved in glucose metabolism. A novel repressor gene, glcR (ywpI), defined by two mutations, was studied in more detail. The glcR mutants exhibit loss of glucose repression of catabolic operons, a deficiency in glucose transport, and absence of expression of the ptsG gene. The mutant GlcR proteins act as super-repressors of ptsG expression.     

Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phos-phorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (cre_down), this operon contains another ere located in the promoter region (cre_up). A translational gntR-lacZ fusion expressed under the control of various combinations of wild-type and mutant cre_down and cre_up was integrated into the chromosomal amyE locus, and then catabolite repression of p-galac-tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by cre_up was probably independent of catabolite repression exerted by cre_down; both creup and cre_down catabolite repression involved CcpA. Catabolite repression exerted by cre_up was independent of P-ser-HPr, and catabolite repression exerted by cre_down was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize cre_up, in contrast to its specific recognition of cre_down. However, CcpA complexed with glucose-6-phosphate specifically recognized cre_up as well as cre_down, but the physiological significance of this complexing is unknown.     

Antisense Silencing of the creA Gene in Aspergillus nidulans

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.     

D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC

d-Fucose, a nonmetabolizable analogue of l-arabinose, prevents growth of Escherichia coli B/r on a mineral salts medium plus l-arabinose by inhibiting induction of the l-arabinose operon. Mutations giving rise to d-fucose resistance map in gene araC and result in constitutive expression of the l-arabinose operon. Most of these mutations also permit d-fucose to serve as a gratuitous inducer. It is concluded that d-fucose-resistant mutants produce an araC gene product with an altered inducer specificity. Addition of l-arabinose to cells induced with the gratuitous inducer, d-fucose, resulted in severe transient repression of operon expression followed by permanent catabolite repression. Transient repression but no permanent catabolite repression was obtained when cells unable to metabolize l-arabinose were employed. It is concluded that transport of l-arabinose alone is sufficient to achieve transient repression of its own operon, but that metabolism of l-arabinose must occur to achieve permanent catabolite repression of the l-arabinose operon. This general effect has been termed "self-catabolite repression."     

Catabolite repression of the lac operon. Separate repression of two enzymes

1. Catabolite repression of β-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol–minimal medium and in glucose–minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for β-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for β-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of β-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of β-galactosidase and thiogalactoside transacetylase is separate but equal.