title: PdhR, the pyruvate dehydrogenase repressor, does not regulate lipoic acid synthesis.
abstract: Lipoic acid is a covalently-bound enzyme cofactor required for central metabolism all three domains of life. In the last 20 years the pathway of lipoic acid synthesis and metabolism has been established in Escherichia coli. Expression of the genes of the lipoic acid biosynthesis pathway was believed to be constitutive. However, in 2010 Kaleta and coworkers (BMC Syst. Biol. 4:116) predicted a binding site for the pyruvate dehydrogenase operon repressor, PdhR (referred to lipA site 1) upstream of lipA, the gene encoding lipoic acid synthase and concluded that PdhR regulates lipA transcription. We report in vivo and in vitro evidence that lipA is not controlled by PdhR and that the putative regulatory site deduced by the prior workers is nonfunctional and physiologically irrelevant. E. coli PdhR was purified to homogeneity and used for electrophoretic mobility shift assays. The lipA site 1 of Kaleta and coworkers failed to bind PdhR. The binding detected by these workers is due to another site (lipA site 3) located far upstream of the lipA promoter. Relative to the canonical PdhR binding site lipA site 3 is a half-palindrome and as expected had only weak PdhR binding ability. Manipulation of lipA site 3 to construct a palindrome gave significantly enhanced PdhR binding affinity. The native lipA promoter and the version carrying the artificial lipA3 palindrome were transcriptionally fused to a LacZ reporter gene to directly assay lipA expression. Deletion of pdhR gave no significant change in lipA promoter-driven β-galactosidase activity with either the native or constructed palindrome upstream sequences, indicating that PdhR plays no physiological role in regulation of lipA expression.
authors.affiliation: Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases & State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, PR China; Department of Medical Microbiology and Parasitology, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, PR China; Department of Microbiology, University of Illinois at Urbana-Champaign, IL 61801, USA.
authors.forename: Youjun
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign, IL 61801, USA; Department of Biochemistry, University of Illinois at Urbana-Champaign, IL 61801, USA. Electronic address: j-cronan@life.uiuc.edu.
authors.forename: John E
authors.initials: JE
authors.lastname: Cronan
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D011485:Protein Binding; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008063:Thioctic Acid / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C079999:LipA protein, Bacteria; C084955:PdhR protein, E coli; D012097:Repressor Proteins; D008063:Thioctic Acid
keywords: 2-Oxoglutarate dehydrogenase; Electrophoretic mobility shift assays (EMSA); LipA. lipoic acid; PdhR; Pyruvate dehydrogenase