Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis.

Abstract

Catabolite repression of the Bacillus subtilis a-amylase gene (amyE) involves an operator sequence located just downstream of the promoter (amyR), overlapping the transcription start site. Oligonucleotide site-directed mutagenesis of this sequence identified bases required for catabolite repression. Two mutations increased both the 2-fold symmetry of the operator and the repression ratio. Although many mutations reduced the repression ratio 3-to 11-fold, some also caused a 2-fold or greater increase in amylase production. Others caused hyperproduction without affecting catabolite repression. Homologous sequences in other catabolite-repressed B. subtilis promoters suggest a common regulatory site may be involved in catabolite repression.Enzymes involved in the metabolism of complex carbon and energy sources are unnecessary under conditions of abundant, readily metabolized alternatives such as glucose. The repression of these enzymes by glucose has been termed catabolite repression (1). In Escherichia coli, catabolite repression is mediated through the cAMP receptor protein (CRP, also called CAP, catabolite gene activator protein), which in the presence of cAMP binds specific DNA sites near promoters and activates transcription (for reviews, see refs. 2-4). Specific contacts are made between CRP, which functions as a dimer, and the CRP-binding sites on the DNA. The binding sites are roughly homologous and have a partial 2-fold symmetry in their consensus sequence (5-7). Recent evidence suggests that transcriptional activation is mediated by protein-protein interaction between CRP and RNA polymerase (8).Like E. coli, Bacillus subtilis is subject to catabolite repression but by a different mechanism. cAMP cannot normally be found in Bacillus species (9,10), except under conditions of oxygen limitation (11) BRE (trpC2, recE4, amyE). The plasmids used were p5'aGR10 (24), pAMY10 (25), pARED, and pGEMR1F (23). Plasmid pARED is a derivative of pAMY10 from which the EcoRI fragment containing the 5' end ofthe amylase structural gene and the amyR region has been deleted. The E. coli phagemid pGEMR1F (Fig. 1) contains the EcoRI fragment deleted in the generation of pARED. Subcloning the EcoRI fragment containing operator mutations from pGEMR1F back into pARED reconstitutes the amylase gene and creates a plasmid identical to pAMY10 except for the mutations. In a similar way, pAR1GR10 was created by subcloning this EcoRI fragment from p5'aB10, which contains the gralO mutation. The operator mutant plasmids were named pGEMRlMx or pARlMx, where x equals the identification number of the mutant, as noted in Tables 1 and 2. All strains were grown as described (23). Antibiotic concentrations were 10 ,ug/ml for chloramphenicol and 50 ,g/ml for ampicillin.Enzymes, Reagents, and Amylase Assays. All chemicals and reagents were at least reagent grade and were purchased from Sigma. Restriction enzymes, EcoRI and Hpa I, and DNA ligase were purchased from Promega Biotec. Amylase assays were done as reported (24).Plasmid Isolation...

Transcription–repair coupling factor is involved in carbon catabolite repression of the Bacillus subtilis hut and gnt operons

“…To test this hypothesis, the regulation of the amyE and bglPH genes was examined in wild-type and mfd mutant cells. Expression of the amyE gene, which encodes ␣-amylase, is activated ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 27, 1031-1038 at the end of exponential growth in nutrient sporulation medium (NSM) and is subject to CCR (Weickert and Chambliss, 1990). The amyE cre overlaps the transcriptional start site of the amyE gene (Weickert and Chambliss, 1990).…”

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

“…l A , this region (nucleotides -48 to +8) was found to contain two tandem sequences (nucleotides -46 to -33, and -23 to -10) exhibiting considerable sequence similarity to a consensus sequence of cre (5'-TGWAANCGNTNWCA-3') (Weickert and Chambliss, 1990). These two sequences have been previously suggested to be cre-like sequences (Weickert and Chambliss, 1990). To determine whether or not these cre-like Weickert and Chambliss (1990), and '-35' and '-10 represent the -35 and -10 regions of the gnt promoter, respectively.…”

“…These two sequences have been previously suggested to be cre-like sequences (Weickert and Chambliss, 1990). To determine whether or not these cre-like Weickert and Chambliss (1990), and '-35' and '-10 represent the -35 and -10 regions of the gnt promoter, respectively. The gnt inserts of plasmids pGNT24M39T, pGNT24M34T, pGNT24M16T and pGNT24MIlT carry the -39G-T, -34'2-T, -16G-T and -11C-T substitutions, respectively.…”

“…The non-coding and coding strands of the cre sequence (nucleotides -60 to -21) are shown, the protected regions being boxed. TGWAANCGNTNWCA is a consensus sequence of cres (Weickert and Chambliss, 1990) sequences actually function as cres, we introduced the respective base substitutions of -39G and -34C, and -16G and -11C, with T into the upstream and downstream cre-like sequences in the gnt sequence of plasmid pGNT24 (Fig. IA); these G and C residues correspond to the second G and C (positions 8 and 13) of the consensus sequence (5'-TGWAANCGNTNWCA-3'), which are highly conserved bases among the various cres, respectively (Hueck et a/., 1994 plasmids pGNT24M39T, and pGNT24M34T, carrying the respective substitutions of -39G -T and -34C-T, is shown in Table 1.…”

“…Several research groups have recently presented evidence that Bacillus subtilis might have a common negative regulatory mechanism underlying its catabolite repression, which involves CcpA, a member of the GalR-Lac1 family of bacterial regulator proteins (Henkin etal., 1991;Henkin, 1996), and a catabolite-responsive element (we) (Weickert and Chambliss, 1990;Hueck et a/., 1994; for reviews, see Saier etal., 1996). This mechanism is completely different from the positive regulatory mechanism operative in enteric bacteria, which involves cyclic AMP (CAMP) and the CAMP receptor protein (CRP).…”