A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter.
Flavocytochrome c-sulfide dehydrogenase (FCSD), an enzyme that catalyzes the reversible conversion of sulfide to elemental sulfur in vitro, is common to bacteria that utilize reduced sulfur compounds as electron donors in the process of carbon dioxide fixation. FCSD is a heterodimer containing two different cofactors, a flavin (FAD) and one or two heme c groups, located on the separate protein subunits. Efforts to produce the holoproteins of the soluble Allochromatium vinosum FCSD and the membrane-bound Ectothiorhodospira vacuolata protein in Escherichia coli using several expression systems were unsuccessful. Although all systems used were able to export the recombinant FCSDs to the periplasm, the proteins did not incorporate heme. In order to develop a new expression system involving photosynthetic hosts (Rhodobacter capsulatus, Rhodobacter sphaeroides and Ect. vacuolata), plasmid mobilisation from E. coli donors was studied. In the search for efficient promoters for such hosts, a system was developed combining the broad-host-range plasmid pGV910 and the promoter of the A. vinosum RuBisCo gene, rbcA. Conjugation was used to enable transfer from the expression plasmid of E. coli into Rba. capsulatus, Rba. sphaeroides strains and into Ect. vacuolata. Both Rhodobacter hosts were able to transcribe the genes coding for FCSD from the rbcA promoter and to produce detectable amounts of recombinant FCSD holoprotein. Western blots showed that the best production was obtained from cells grown photosynthetically on malate or acetate with sulfide. This system may prove to be of general use for the production of recombinant c-type cytochromes in homologous or related host systems.
De Smet
L
L
Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, Ledeganckstraat 35, 9000 Gent, Belgium.
Kostanjevecki
V
V
Guisez
Y
Y
Van Beeumen
J
J
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015153:Blotting, Western; D002841:Chromatiaceae / Q000201:enzymology* / Q000235:genetics*; D003227:Conjugation, Genetic; D003574:Cytochrome c Group / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D020587:Ectothiorhodospira / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D020035:Holoenzymes / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016992:Rhodobacter / Q000235:genetics* / Q000502:physiology; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004274:DNA, Recombinant; D020035:Holoenzymes; D011994:Recombinant Proteins; D010088:Oxidoreductases; C064500:flavocytochrome c sulfide dehydrogenase; D012273:Ribulose-Bisphosphate Carboxylase