Application of crossover-PCR-mediated deletion-insertion mutagenesis to analysis of the bdhA-xdhA2-xdhB2 mixed-function operon of Sinorhizobium meliloti.
The bdhA-xdhA2-xdhB2 mixed-function operon was used to demonstrate the application of crossover PCR to the construction of in-frame, non-polar deletion-insertion mutations in Sinorhizobium meliloti. Replacement of a 474-bp internal portion of the 774-bp coding sequence of bdhA with a 21-bp in-frame synthetic sequence resulted in loss of the bdhA-encoded d-3-hydroxybutyrate dehydrogenase activity. Such mutants retained the xanthine oxidase activity encoded by xdhA2-xdhB2, thus illustrating the non-polar nature of the mutation. This method of constructing unmarked, in-frame deletions should be generally applicable to functional genomics studies in S. meliloti and other alpha-Proteobacteria.
Sukdeo
Nicole
N
Department of Biology, University of Waterloo, 200 University Avenue West, N2L 3G1, Waterloo, ON, Canada.
Charles
Trevor C
TC
D020155:3-Hydroxybutyric Acid / Q000378:metabolism; D017353:Gene Deletion; D016680:Genome, Bacterial; D006884:Hydroxybutyrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009876:Operon / Q000502:physiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016962:Sinorhizobium meliloti / Q000201:enzymology / Q000235:genetics*; D014969:Xanthine Oxidase / Q000378:metabolism
D006884:Hydroxybutyrate Dehydrogenase; D014969:Xanthine Oxidase; D020155:3-Hydroxybutyric Acid