xylB Genes Research Articles

Cloning and characterization of the xyl genes from Escherichia coli.

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.

Molecular cloning of TOL genes xylB and xylE in Escherichia coli

The xylB and xylE genes in the TOL plasmid of Pseudomonas putida mt-2, which code for benzyl alcohol dehydrogenase and catechol 2,3-oxygenase, respectively, were cloned onto plasmid pBR322 in Escherichia coli for detailed mapping. The xylB gene was mapped in a 2.9-kilobase region within the BamHI BC fragment of pTN2, an in vivo RP4-TOL recombinant, whereas the xylE gene was mapped in a 1.8-kilobase region within the BamHI BD fragment. The directions of transcription of these genes were deduced from the expression of the cloned genes which had been ligated in orientations opposite pBR322 at its BamHI site within the tetracycline resistance gene. The xylB and xylE genes are inducible by a specific inducer of the TOL pathway genes in the RP4-TOL recombinant, whereas they are not inducible in the pBR322-TOL hybrids. The regulatory regions involved in expression of the xylB and xylE genes do not appear to be located in the vicinity of the structural genes. Catechol 2,3-oxygenase formed in E. coli carrying an xylE-containing plasmid is similar, or identical, to that formed in P. putida carrying the TOL plasmid.