Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding

We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis. By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA. We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. coli. We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E. coli genome that has a very low binding efficiency compared with the bio operator region.     

Isolation and characterization of Escherichia coli birA intragenic suppressors

Summary The biotin (bio) operon in Escherichia coli is negatively regulated by BirA, a bifunctional protein with both repressor and biotin-activating functions. Twenty-five heatresistant revertants of three temperature-sensitive birA alleles (birA 85, bir A 104 and bir A 879) were isolated and categorized into five growth and six repression classes. The revertants appear to increase biotin activation by raising the specific activity of BirA and/or, increasing the number of enzyme molecules. The 19 bir A 85 revertants displayed a broad range of activity for both enzyme and repressor functions, and may represent intragenic second-site suppressor mutations. The bir A 85 revertants included a novel class of bio superrepressor mutations. Repressor titration experiments suggested that many of the bir A 85 revertants increase BirA concentrations above wild-type levels because the repressors were not competed from the chromosomal bio operator by multicopy bio operator plasmids. The majority of the bir A 104 revertants resulted in both wild-type repressor and enzyme activity; they are possibly true revertants in which the amino acid residue altered by the bir A 104 mutation has been substituted by the wild-type or a chemically similar amino acid.     

Co-repressor induced order and biotin repressor dimerization: a case for divergent followed by convergent evolution

BirA catalyzes the adenylation and subsequent covalent attachment of biotin to the biotin carboxyl carrier protein (BCCP). In the absence of apo-BCCP, biotin-5'-AMP acts as a co-repressor that induces BirA dimerization and binding to the bio operator to repress biotin biosynthesis. The crystal structures of apo-BirA, and BirA in complex with biotin have been reported. We here describe the 2.8A resolution crystal structure of BirA in complex with the co-repressor analog biotinol-5'-AMP. It was previously shown that the structure of apo-BirA is monomeric and that binding of biotin weakly induces a dimeric structure in which three disordered surface loops become organized to form the dimer interface. The structure of the co-repressor complex is also a dimer, clearly related to the BirA.biotin structure, but with several significant conformational changes. A hitherto disordered "adenylate binding loop" forms a well-defined structure covering the co-repressor. The co-repressor buttresses the dimer interface, resulting in improved packing and a 12 degrees change in the hinge-bending angle along the dimer interface relative to the BirA.biotin structure. This helps explain why the binding of the co-repressor is necessary to optimize the binding of BirA to the bioO operator. The structure reveals an unexpected use of the nucleotide-binding motif GXGXXG in binding adenylate and controlling the repressor function. Finally, based on structural analysis we propose that the class of adenylating enzymes represented by BirA, lipoate protein ligase and class II tRNA synthetases diverged early and were selected based on their ability to sequester co-factors or amino acid residues, and adenylation activity arose independently through functional convergence.     

Overproduction and rapid purification of the biotin operon repressor from Escherichia coli

The Escherichia coli biotin operon repressor protein (BirA) has been overexpressed at the level of 0.5-1% of the total cellular protein from the plasmid pMBR10. Four lines of evidence demonstrated that authentic BirA protein was produced. First, birA plasmids complemented birA mutants for both the repressor and biotin holoenzyme synthetase activities of BirA. Second, biotin holoenzyme synthase activity was increased in strains containing the overproducing plasmids. Third, deletion of sequences flanking the birA gene did not alter production of the 35-kDa BirA protein, but insertion of oligonucleotide linkers within the birA coding region abolished it. Fourth, the 35-kDa protein copurified with the biotin binding activity normally associated with BirA. The birA protein has been purified to homogeneity in a three-step process involving chromatography on phosphocellulose and hydroxyapatite columns.     

A new dehydratase conferring innate resistance to thiacetazone and intra‐amoebal survival of Mycobacterium smegmatis

Nontuberculous mycobacteria are innately resistant to most antibiotics, although the mechanisms responsible for their drug resistance remain poorly understood. They are particularly refractory to thiacetazone (TAC), a second‐line antitubercular drug. Herein, we identified MSMEG_6754 as essential for the innate resistance of Mycobacterium smegmatis to TAC. Transposon‐mediated and targeted disruption of MSMEG_6754 resulted in hypersusceptibility to TAC. Conversely, introduction of MSMEG_6754 into Mycobacterium tuberculosis increased resistance 100‐fold. Resolution of the crystal structure of MSMEG_6754 revealed a homodimer in which each monomer comprises two hot‐dog domains characteristic of dehydratase‐like proteins and very similar to the HadAB complex involved in mycolic acid biosynthesis. Gene inactivation of the essential hadB dehydratase could be achieved in M. smegmatis and M. tuberculosis only when the strains carried an integrated copy of MSMEG_6754, supporting the idea that MSMEG_6754 and HadB share redundant dehydratase activity. Using M. smegmatis‐Acanthamoeba co‐cultures, we found that intra‐amoebal growth of the MSMEG_6754 deleted strain was significantly reduced compared with the parental strain. This in vivo growth defect was fully restored upon complementation with catalytically active MSMEG_6754 or HadABC, indicating that MSMEG_6754 plays a critical role in the survival of M. smegmatis within the environmental host.     

Ligand-linked structural changes in the Escherichia coli biotin repressor: the significance of surface loops for binding and allostery.

The Escherichia coli repressor of biotin biosynthesis (BirA) is an allosteric site-specific DNA-binding protein. BirA catalyzes synthesis of biotinyl-5'-AMP from substrates biotin and ATP and the adenylate serves as the positive allosteric effector in binding of the repressor to the biotin operator sequence. Although a three-dimensional structure of the apo-repressor has been determined by X-ray crystallographic techniques, no structures of any ligand-bound forms of the repressor are yet available. Results of previously published solution studies are consistent with the occurrence of conformational changes in the protein concomitant with ligand binding. In this work the hydroxyl radical footprinting technique has been used to probe changes in reactivity of the peptide backbone of BirA that accompany ligand binding. Results of these studies indicate that binding of biotin to the protein results in protection of regions of the central domain in the vicinity of the active site and the C-terminal domain from chemical cleavage. Biotin-linked changes in reactivity constitute a subset of those linked to adenylate binding. Binding of both bio-5'-AMP and biotin operator DNA suppresses cleavage at additional sites in the amino and carboxy-terminal domains of the protein. Varying degrees of protection of the five surface loops on BirA from hydroxyl radical-mediated cleavage are observed in all complexes. These results implicate the C-terminal domain of BirA, for which no function has previously been known, in small ligand and site-specific DNA binding and highlight the significance of surface loops, some of which are disordered in the apoBirA structure, for ligand binding and transmission of allosteric information in the protein.     

Regulation of amino sugar utilization in Bacillus subtilis by the GntR family regulators,NagR and GamR

In Bacillus subtilis separate sets of genes are implicated in the transport and metabolism of the amino sugars, glucosamine and N‐acetylglucosamine. The genes for use of N‐acetylglucosamine (nagAB and nagP) are found in most firmicutes and are controlled by a GntR family repressor NagR (YvoA). The genes for use of glucosamine (gamAP) are repressed by another GntR family repressor GamR (YbgA). The gamR‐gamAP synton is only found in B. subtilis and a few very close relatives. Although NagR and GamR are close phylogenetically, there is no cross regulation between their operons. GlcN6P prevents all binding of GamR to its targets. NagR binds specifically to targets containing the previously identified dre palindrome but its binding is not inhibited by GlcN6P or GlcNAc6P. GamR‐like binding sites were also found in some other Bacilli associated with genes for use of chitin, the polymer of N‐acetylglucosamine, and with a gene for another GamR homologue (yurK). We show that GamR can bind to two regions in the chi operon of B. licheniformis and that GamR and YurK are capable of heterologous regulation. GamR can repress the B. licheniformis licH‐yurK genes and YurK can repress B. subtilis gamA.     

Cloning of Schizosaccharomyces pombe bio2 by Heterologous Complementation of a Saccharomyces cerevisiae Mutant

A Saccharomyces cerevisiae mutant affected in the last step of the biotin biosynthesis pathway was isolated by using a transposon mutagenesis method. The gene BIO2, encoding a biotin synthase, is shown to be interrupted in this mutant. Heterologous complementation experiment allowed the cloning and the characterization of a novel bio gene: bio2, encoding biotin synthase from Schizosaccharomyces pombe. Received: 4 June 1999 / Accepted: 12 July 1999     

Interconnection of the mycobacterial heparin-binding hemagglutinin with cholesterol degradation and heme/iron pathways identified by proximity-dependent biotin identification in Mycobacterium smegmatis

Deciphering protein–protein interactions is a critical step in the identification and the understanding of biological mechanisms deployed by pathogenic bacteria. The development of in vivo technologies to characterize these interactions is still in its infancy, especially for bacteria whose subcellular organization is particularly complex, such as mycobacteria. In this work, we used the proximity-dependent biotin identification (BioID) to define the mycobacterial heparin-binding hemagglutinin (HbhA) interactome in the saprophytic bacterium Mycobacterium smegmatis. M. smegmatis is a commonly used model to study and characterize the physiology of pathogenic mycobacteria, such as Mycobacterium tuberculosis. Here, we adapted the BioID technology to in vivo protein–protein interactions studies in M. smegmatis, which presents several advantages, such as maintaining the complex organization of the mycomembrane, offering the possibility to study membrane or cell wall-associated proteins, including HbhA, in the presence of cofactors and post-translational modifications, such as the complex methylation pattern of HbhA. Using this technology, we found that HbhA is interconnected with cholesterol degradation and heme/iron pathways. These results are in line with previous studies showing the dual localization of HbhA, associated with the cell wall and intracytoplasmic lipid inclusions, and its induction under high iron growth conditions.