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Brucella BioR regulator defines a complex regulatory mechanism for bacterial biotin metabolism.
- Source :
-
Journal of bacteriology [J Bacteriol] 2013 Aug; Vol. 195 (15), pp. 3451-67. Date of Electronic Publication: 2013 May 31. - Publication Year :
- 2013
-
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.
- Subjects :
- Agrobacterium tumefaciens genetics
Amino Acid Sequence
Artificial Gene Fusion
Base Sequence
Binding Sites
DNA, Bacterial metabolism
Electrophoretic Mobility Shift Assay
Gene Expression
Gene Expression Profiling
Genes, Reporter
Genetic Complementation Test
Molecular Sequence Data
Promoter Regions, Genetic
Protein Binding
Protein Multimerization
Real-Time Polymerase Chain Reaction
Repressor Proteins chemistry
Repressor Proteins genetics
Repressor Proteins isolation & purification
Sequence Alignment
beta-Galactosidase analysis
beta-Galactosidase genetics
Biotin metabolism
Brucella melitensis genetics
Brucella melitensis metabolism
Gene Expression Regulation, Bacterial
Repressor Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1098-5530
- Volume :
- 195
- Issue :
- 15
- Database :
- MEDLINE
- Journal :
- Journal of bacteriology
- Publication Type :
- Academic Journal
- Accession number :
- 23729648
- Full Text :
- https://doi.org/10.1128/JB.00378-13