Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene

Weickert, Michael J.; Chambliss, Glenn H.

doi:10.1128/jb.171.7.3656-3666.1989

Cited by 29 publications

(23 citation statements)

References 49 publications

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“…The mechanism of catabolite repression in grampositive bacteria, which is unlikely to involve cyclic AMP, is largely unknown. Interestingly, catabolite repression of amylase production and aconitase synthesis in B. subtilis are postulated to involve negative regulatory protein(s) as well (11,23,36). However, in the amylase system, the binding site for the catabolite repressor is downstream of the transcription start site, not within the promoter region.…”

Section: Discussionmentioning

confidence: 99%

Repression and catabolite repression of the lactose operon of Staphylococcus aureus

Oskouian

Stewart

1990

J Bacteriol

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The lacR gene encodes the repressor of the lactose operon of S. aureus. The nucleotide sequence of this gene and the promoter-operator region of the operon are reported. The lacR gene encodes a protein with a molecular weight of 28,534. This protein was found to share sequence homology with the DeoR protein, the repressor of the E. coli deoxyribonucleotide operon. Directly and invertedly repeated sequences were found associated with the promoter for the structural genes of the operon. These sequences were examined by site-directed mutagenesis and found to be important in repressor binding and in the binding of a catabolite repressor. Evidence is presented in support of a model for catabolite repression of the operon which involves a negative-acting transcriptional regulator which binds to the promoter region of the operon and prevents transcription.The uptake of lactose by Staphylococcus aureus takes place via a phosphoenol pyruvate-dependent phosphotransferase system (reviewed in references 25 and 28). Lactose is phosphorylated upon entry into the cell by the joint action of two sugar-specific components, enzyme 11Lac (EIILaC) and enzyme I11Lac (ElIlLac). The intracellular phosphorylated lactose is then cleaved into glucose and galactose-6-phosphate by phospho-fi-galactosidase (14). The galactose-6-phosphate generated is further metabolized via a tagatosephosphate pathway (2).The lactose (lac) genes of S. aureus have been shown to be inducible with the addition of either lactose or galactose to the culture medium. Galactose-6-phosphate was found to be the actual intracellular inducer (22). Additionally, the lac genes are subject to catabolite repression (20). The significance of the glucose effect lies in the fact that cyclic AMP, which plays a critical role in catabolite repression in Escherichia coli, is not found, at least in physiologically significant concentrations, in S. aureus (3; unpublished data). The mechanism of catabolite repression in gram-positive bacteria is unknown.Our laboratory has reported the cloning of the structural genes of the staphylococcal lac operon (5-7, 29). These genes are arranged as a heptacistronic operon, according to data obtained by nucleotide sequence analysis. The organization of the operon is presented in Fig. 1. The terminal three genes in the operon, lacFEG, encode EIITLaC, EIILaC, and phospho-p-galactosidase, respectively (5, 7). The functions of the lacA-D determinants are currently under investigation (29). We previously reported the cloning of the gene encoding the repressor of the lac operon (lacR), which was found to lie immediately upstream of the promoter-proximal end of the lacA-G genes (24).In this report, the nucleotide and deduced amino acid sequences of lacR are presented, as are experiments to define the repressor target site, the lac operator. In addition, the promoter of the operon has been identified through deletion and gene fusion analysis. We will also present data pertaining to the mechanism of catabolite repression of this * Corresponding author. o...

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Section: Discussionmentioning

confidence: 99%

Repression and catabolite repression of the lactose operon of Staphylococcus aureus

Oskouian

Stewart

1990

J Bacteriol

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“…All of the PCR mutants were confirmed by sequencing using double-stranded plasmid DNA templates (29).…”

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Identification of the staphylococcal enterotoxin A superantigen binding site in the beta 1 domain of the human histocompatibility antigen HLA-DR.

Herman

Labrecque

Thibodeau

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

102

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The staphylococcal enterotoxin A (SEA) is a superantigen that must bind to class II molecules of the major histocompatibility complex to be recognized by T cells. In humans, most HLA-DR class II allelic and isotypic forms, such as DR1, bind SEA well. DRw53 is an exception, binding SEA very poorly. We have localized this difference to a single residue (amino acid 81) in the fl1 domain. A highly conserved histidine at residue 81 allows SEA binding, but a tyrosine does not. Residue 81 is predicted to lie in an a-helix on the surface of the molecule, with its side chain pointing up out of the pocket associated with binding of conventional peptide antigens. This rinding supports the hypothesis that superantigens and conventional antigens bind to different sites on the class II molecule.Recently, a class of antigens has been characterized that stimulates T cells predominantly through the 13chain variable region element of the T-cell receptor (1-3). They differ from conventional peptide antigens, which require specific interaction with several variable elements of the T-cell receptor (4, 5). These molecules have been termed superantigens, since they stimulate large numbers of T cells (2). The superantigen family includes mouse self superantigens encoded by mouse mammary tumor viruses (6-8) and a number of microbial exotoxins implicated in severe shock-like disorders. Among the best characterized superantigens are the staphylococcal exotoxins, a family of basic secretory proteins of 22-30 kDa such as the staphylococcal enterotoxins (SEs) SEA, SEB, SEC1-3, SED, and SEE and the toxic shock syndrome toxin (9-11).Superantigen binding to major histocompatibility complex (MHC) class II molecules is a prerequisite for T-cell stimulation (2,(12)(13)(14) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…This mutation is in an operator-like region having 2-fold symmetry and partial homology to the lac and gal operators (18). Deletion analysis eliminated the possibility that any other sequence in the promoter region plays a critical role in catabolite repression (23).…”

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“…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.…”

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

Weickert

Chambliss

1990

Proc. Natl. Acad. Sci. U.S.A.

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271

268

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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...

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Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene

Cited by 29 publications

References 49 publications

Repression and catabolite repression of the lactose operon of Staphylococcus aureus

Repression and catabolite repression of the lactose operon of Staphylococcus aureus

Identification of the staphylococcal enterotoxin A superantigen binding site in the beta 1 domain of the human histocompatibility antigen HLA-DR.

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

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