Identification of an upstream regulatory sequence that mediates the transcription of mox genes in Methylobacterium extorquens AM1.
A multiple A-tract sequence has been identified in the promoter regions for the mxaF, pqqA, mxaW, mxbD and mxcQ genes involved in methanol oxidation in Methylobacterium extorquens AM1, a facultative methylotroph. Site-directed mutagenesis was exploited to delete or change this conserved sequence. Promoter-xylE transcriptional fusions were used to assess promoter activity in these mutants. A fiftyfold drop in the XylE activity was observed for the mxaF and pqqA promoters without this sequence, and a five- to sixfold drop in the XylE activity was observed for the mxbD and mxcQ promoters without this sequence. Mutants were generated in the chromosomal copies in which this sequence was either deleted or altered, and these mutants were unable to grow on methanol. When one of these sequences was added to Plac of Escherichia coli, which is a weak constitutive promoter in M. extorquens AM1, the activity increased two- to threefold. These results suggest that this sequence is essential for normal expression of these genes in M. extorquens AM1, and may serve as a general enhancer element for genetic constructs in this bacterium.
Zhang
Meng
M
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2125, USA.
FitzGerald
Kelly A
KA
Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2125, USA.
Lidstrom
Mary E
ME
Department of Microbiology, University of Washington, Seattle, WA 98195-2125, USA.|Department of Chemical Engineering, University of Washington, Seattle, WA 98195-2125, USA.
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D000432:Methanol / Q000378:metabolism*; D020601:Methylobacterium extorquens / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D000432:Methanol