title: Brucella BioR regulator defines a complex regulatory mechanism for bacterial biotin metabolism.
abstract: The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of expression of bioY and bio operons that separately function in biotin transport and the biosynthesis pathway.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana, Illinois, USA. fyj999@gmail.com
authors.forename: Youjun
authors.initials: Y
authors.lastname: Feng
authors.forename: Jie
authors.initials: J
authors.lastname: Xu
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.forename: Zeliang
authors.initials: Z
authors.lastname: Chen
authors.forename: Swaminath
authors.initials: S
authors.lastname: Srinivas
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism*; D017347:Brucella melitensis / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D055503:Protein Multimerization; D060888:Real-Time Polymerase Chain Reaction; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D012097:Repressor Proteins; D001710:Biotin; D001616:beta-Galactosidase