Conformational and thermodynamic changes of the repressor/DNA operator complex upon monomerization shed new light on regulation mechanisms of bacterial resistance against beta-lactam antibiotics

In absence of β-lactam antibiotics, BlaI and MecI homodimeric repressors negatively control the expression of genes involved in β-lactam resistance in Bacillus licheniformis and in Staphylococcus aureus. Subsequently to β-lactam presence, BlaI/MecI is inactivated by a single-point proteolysis that separates its N-terminal DNA-binding domain to its C-terminal domain responsible for its dimerization. Concomitantly to this proteolysis, the truncated repressor acquires a low affinity for its DNA target that explains the expression of the structural gene for resistance. To understand the loss of the high DNA affinity of the truncated repressor, we have determined the different dissociation constants of the system and solved the solution structure of the B. licheniformis monomeric repressor complexed to the semi-operating sequence OP₁ of blaP (1/2OP₁blaP) by using a de novo docking approach based on inter-molecular nuclear Overhauser effects and chemical-shift differences measured on each macromolecular partner. Although the N-terminal domain of the repressor is not subject to internal structural rearrangements upon DNA binding, the molecules adopt a tertiary conformation different from the crystallographic operator–repressor dimer complex, leading to a 30° rotation of the monomer with respect to a central axis extended across the DNA.

These results open new insights for the repression and induction mechanisms of bacterial resistance to β-lactams.

     

Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix

Lambda repressors bind cooperatively to adjacent pairs of operator sites. Here we show that repressors bind cooperatively to pairs of operator sites whose centers have been separated by five or six turns of the helix. No cooperativity is observed when the centers of these sites are on opposite sides of the DNA helix. Cooperativity depends upon the same part of the protein (the carboxyl domain) that mediates cooperativity when the sites are adjacent. As the repressors bind, the DNA between the sites becomes alternately sensitive and resistant to DNAase I cleavage at half turn intervals. We suggest that when repressors bind cooperatively to separated sites, the DNA forms a loop, thus allowing the two repressors to touch.     

The CTXphi repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes

CTX is a filamentous bacteriophage that encodes cholera toxin and integrates into the Vibrio cholerae genome to form stable lysogens. In CTX lysogens, gene expression originating from the rstA phage promoter is repressed by the phage-encoded repressor RstR. The N-terminal region of RstR contains a helix-turn-helix DNA-binding element similar to the helix-turn-helix of the cI/Cro family of phage repressors, whereas the short C-terminal region is unrelated to the oligomerization domain of cI repressor. Purified His-tagged RstR bound to three extended 50-bp operator sites in the rstA promoter region. Each of the RstR footprints exhibited a characteristic staggered pattern of DNase I-accessible regions that suggested RstR binds DNA as a dimer-of-dimers. In gel permeation chromatography and cross-linking experiments, RstR oligomerized to form dimers and tetramers. RstR was shown to be tetrameric when bound to operator DNA by performing mobility shift experiments with mixtures of RstR and a lengthened active variant of RstR. Binding of RstR to the high affinity O1 site could be fit to a cooperative model of operator binding in which two RstR dimers associate to form tetrameric RstR-operator complexes. The binding of RstR dimers to the left or right halves of O1 operator DNA was not observed in mobility shift assays. These observations support a model in which protein-protein contacts between neighboring RstR dimers contribute to strong operator binding.     

Studies of the repressor (BlaI) of β-lactamase synthesis in Staphylococcus aureus

Purified BlaI, the putative repressor of the β-lactamase operon in Staphylococcus aureus , binds specifically to two regions of dyad symmetry (operators) located in the blaZ–blaR1 intergenic region. BlaI binds with similar affinity to the two regions and to the related sequence upstream of the mec gene found in methicillin-resistant strains of S. aureus , providing physical evidence for the cross-talk previously observed between these systems. A change from a lysine in the N-terminus of BlaI to an alanine or deletion of the C-terminal 23 amino acids severely reduces its DNA-binding ability, demonstrating the functional importance of both the N- and C-termini. An operator DNA–protein complex observed with crude cell lysates from repressed cells, indistinguishable from that observed with purified BlaI, was eliminated by induction of the β-lactamase operon. Furthermore, BlaI is proteolytically cleaved in response to the addition of inducer in a blaR1 -dependent manner, providing primary evidence for the molecular basis of induction. Thus, BlaI is shown to be the repressor of the β-lactamase system.     

Lambda repressor mutations that increase the affinity and specificity of operator binding

Intragenic, second-site reversion has been used to identify amino acid substitutions that increase the affinity and specificity of the binding of lambda repressor to its operator sites. Purified repressors bearing the second-site substitutions bind operator DNA from 3 to 600 fold more strongly than wild type; these affinity changes result from both increased rates of operator association and decreased rates of operator dissociation. Three of the revertant substitutions occur in the alpha 2 and alpha 3 DNA binding helices of repressor and seem to increase affinity by introducing new salt-bridges or hydrogen bonds with the sugar-phosphate backbone of the operator site. The fourth substitution alters the alpha 5 dimerization helix of repressor and appears to increase operator affinity indirectly.     

P22 repressor mutants deficient in co-operative binding and DNA loop formation.

We show here, both in vivo and in vitro, that P22 repressor binds co-operatively to operator sites separated by an integral number of turns of the DNA helix. We measure this co-operativity in vivo using an assay in which repression of a promoter requires co-operative binding of P22 repressors to two separated (non-adjacent) operator sites. We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co-operative binding. Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated. We purified the two mutants most deficient in co-operative binding, and found that they bind non-co-operatively in vitro to adjacent as well as to non-adjacent pairs of operator sites.     

Degradation of the DNA-binding domain of wild-type and i-d lac repressors in Escherichia coli.

It has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in vivo and in vitro, but still bind inducer and are able to form tetramers (i-d repressors), could be divided into two groups by their capacity or incapacity to bind non-specifically to the phosphate groups of the DNA backbone. All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal of residue 52 showed uneffected non-specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal of residue 53 did not bind to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene were endogenously degraded. The degradation in vivo only affects the amino-terminal 50-60 residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of binding inducer in vivo and in vitro. The proteolytic attack presumably takes place during synthesis of the i-d repressors, since the resulting fragments are stable, both in vivo (as shown by a pulse-chase experiment) and in vitro. The proteolysis in vivo depends on the growth conditions of the bacteria and is higher in cells grown in minimal media than in rich media. Wild-type repressor is only susceptible to limited proteolysis in cells grown in minimal media but not in cells grown in rich media. The results suggest that the majority of the sequence alterations before residue 53 in missense mutant i-d lac repressor proteins affect the three-dimensional structure of the amino-terminal DNA-binding domain of the repressor protein, making it susceptible to proteolytic attack by one or several intracellular proteases.     

Purification and DNA binding properties of the blaI gene product, repressor for the beta-lactamase gene, blaP, of Bacillus licheniformis.

The location of the repressor gene, blaI, for the beta-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blaI open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blaI, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074-1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of BlaI as 5-10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of BlaI to DNA was detected by the slower migration of protein DNA complexes during polyacrylamide gel electrophoresis. BlaI was shown to selectively bind DNA fragments carrying the promoter regions of blaI and blaP.     

A Peptidoglycan Fragment Triggers β-lactam Resistance in Bacillus licheniformis

To resist to β-lactam antibiotics Eubacteria either constitutively synthesize a β-lactamase or a low affinity penicillin-binding protein target, or induce its synthesis in response to the presence of antibiotic outside the cell. In Bacillus licheniformis and Staphylococcus aureus, a membrane-bound penicillin receptor (BlaR/MecR) detects the presence of β-lactam and launches a cytoplasmic signal leading to the inactivation of BlaI/MecI repressor, and the synthesis of a β-lactamase or a low affinity target. We identified a dipeptide, resulting from the peptidoglycan turnover and present in bacterial cytoplasm, which is able to directly bind to the BlaI/MecI repressor and to destabilize the BlaI/MecI-DNA complex. We propose a general model, in which the acylation of BlaR/MecR receptor and the cellular stress induced by the antibiotic, are both necessary to generate a cell wall-derived coactivator responsible for the expression of an inducible β-lactam-resistance factor. The new model proposed confirms and emphasizes the role of peptidoglycan degradation fragments in bacterial cell regulation.     

Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors

Members of the IclR family of regulators are proteins with around 250 residues. The IclR family is best defined by a profile covering the effector binding domain. This is supported by structural data and by a number of mutants showing that effector specificity lies within a pocket in the C-terminal domain. These regulators have a helix-turn-helix DNA binding motif in the N-terminal domain and bind target promoters as dimers or as a dimer of dimers. This family comprises regulators acting as repressors, activators and proteins with a dual role. Members of the IclR family control genes whose products are involved in the glyoxylate shunt in Enterobacteriaceae , multidrug resistance, degradation of aromatics, inactivation of quorum-sensing signals, determinants of plant pathogenicity and sporulation. No clear consensus exists on the architecture of DNA binding sites for IclR activators: the MhpR binding site is formed by a 15-bp palindrome, but the binding sites of PcaU and PobR are three perfect 10-bp sequence repetitions forming an inverted and a direct repeat. IclR-type positive regulators bind their promoter DNA in the absence of effector. The mechanism of repression differs among IclR-type regulators. In most of them the binding sites of RNA polymerase and the repressor overlap, so that the repressor occludes RNA polymerase binding. In other cases the repressor binding site is distal to the RNA polymerase, so that the repressor destabilizes the open complex.