A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter

Citation
L. De Smet et al., A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter, ARCH MICROB, 176(1-2), 2001, pp. 19-28
Citations number
67
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
ARCHIVES OF MICROBIOLOGY
ISSN journal
0302-8933 → ACNP
Volume
176
Issue
1-2
Year of publication
2001
Pages
19 - 28
Database
ISI
SICI code
0302-8933(200107)176:1-2<19:ANSFHE>2.0.ZU;2-C
Abstract
Flavocytochrome c-sulfide dehydrogenase (FCSD), an enzyme that catalyzes th e 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 d ifferent 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 Ectothiorhodospi ra vacuolata protein in Escherichia coli using several expression systems w ere unsuccessful. Although all systems used were able to export the recombi nant FCSDs to the periplasm, the proteins did not incorporate heme. In orde r to develop a new expression system involving photosynthetic hosts (Rhodob acter capsulatus, Rhodobacter sphaeroides and Ect. vacuolata), plasmid mobi lisation. from E. coli donors was studied. In the search for efficient prom oters for such hosts, a system was developed combining the broad-host-range plasmid pGV910 and the promoter of the A. vinosum RuBisCo gene, rbcA. Conj ugation was used to enable transfer from the expression plasmid of E. coh i nto Rba. capsulatus, Rba. sphaeroides strains and into Ect. vacuolata. Both Rhodobacter hosts were able to transcribe the genes coding for FCSD from t he rbcA promoter and to produce detectable amounts of recombinant FCSD holo protein. Western blots showed that the best production was obtained from ce lls grown photosynthetically on malate or acetate with sulfide. This system may prove to be of general use for the production of recombinant c-type cy tochromes in homologous or related host systems.