QscR-mediated transcriptional activation of serine cycle genes in Methylobacterium extorquens AM1.
QscR, a LysR-type regulator, is the major regulator of assimilatory C1 metabolism in Methylobacterium extorquens AM1. It has been shown to interact with the promoters of the two operons that encode the majority of the serine cycle enzymes (sga-hpr-mtdA-fch for the qsc1 operon and mtkA-mtkB-ppc-mclA for the qsc2 operon), as well as with the promoter of glyA and its own promoter. To obtain further insights into the mechanisms of this regulation, we mapped transcriptional start sites for the qsc1 and qsc2 operons and for glyA via primer extension analysis. We also identified the specific binding sites for QscR upstream of the qsc1 and qsc2 operons and glyA by DNase I footprinting. The QscR protected areas were located at nucleotides -216 to -165, nucleotides -59 to -26, and nucleotides -72 to -39 within the promoter-regulatory regions upstream of transcriptional starts of, respectively, qsc1, qsc2 and glyA. To examine the nature of the metabolic signal that may influence QscR-mediated regulation of the serine cycle genes, Pqsc1::xylE translational fusions were constructed and expression of XylE monitored in the wild-type strain, as well as in knockout mutants defective in a variety of methylotrophy functions. The data from these experiments pointed toward formyl-H4F being a coinducer of QscR and possibly the major signal in the regulation of the serine cycle in M. extorquens AM1. The ability of formyl-H4F to enhance the binding of QscR to a specific region upstream of one of the serine cycle operons was demonstrated in gel retardation experiments.
Kalyuzhnaya
Marina G
MG
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-1750, USA. mkalyuzh@u.washington.edu
Lidstrom
Mary E
ME
D024506:5' Flanking Region / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D005575:Formyltetrahydrofolates; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D020601:Methylobacterium extorquens / Q000235:genetics*; D008969:Molecular Sequence Data; D009876:Operon; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
D005575:Formyltetrahydrofolates; D014157:Transcription Factors; D012694:Serine