Cloning and expression of Chlorobium vibrioforme uroporphyrinogen decarboxylase gene in a Salmonella heme auxotroph.
To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase ( hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant ( hemA(-) hemE(-)) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert.
Xu
Kaiping
K
Department of Biology, Norfolk State University, VA 23504, USA.
Ndivo
Victoria
V
Majumdar
Debabrata
D
D019414:Chlorobi / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003305:Coproporphyrinogens / Q000378:metabolism; D003470:Culture Media; D006418:Heme / Q000378:metabolism*; D012475:Salmonella / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014575:Uroporphyrinogen Decarboxylase / Q000235:genetics* / Q000378:metabolism*; D014577:Uroporphyrinogens / Q000378:metabolism
D003305:Coproporphyrinogens; D003470:Culture Media; D014577:Uroporphyrinogens; D006418:Heme; D014575:Uroporphyrinogen Decarboxylase