The Desulfuromonas acetoxidans triheme cytochrome c7 produced in Desulfovibrio desulfuricans retains its metal reductase activity.
Multiheme cytochrome c proteins that belong to class III have been recently shown to exhibit a metal reductase activity, which could be of great environmental interest, especially in metal bioremediation. To get a better understanding of these activities, the gene encoding cytochrome c7 from the sulfur-reducing bacterium Desulfuromonas acetoxidans was cloned from genomic DNA by PCR and expressed in Desulfovibrio desulfuricans G201. The expression system was based on the cyc transcription unit from Desulfovibrio vulgaris Hildenborough and led to the synthesis of holocytochrome c7 when transferred by electrotransformation into the sulfate reducer Desulfovibrio desulfuricans G201. The produced cytochrome was indistinguishable from the protein purified from Desulfuromonas acetoxidans cells with respect to several biochemical and biophysical criteria and exhibited the same metal reductase activities as determined from electrochemical experiments. This suggests that the molecule was correctly folded in the host organism. Desulfovibrio desulfuricans produces functional multiheme c-type cytochromes from bacteria belonging to a different genus and may be considered a suitable host for the heterologous biogenesis of multiheme c-type cytochromes for either structural or engineering studies. This report, which presents the first example of the transformation of a Desulfovibrio desulfuricans strain by electrotransformation, describes work that is the first necessary step of a protein engineering program that aims to specify the structural features that are responsible for the metal reductase activities of multiheme cytochrome c7.
Aubert
C
C
Unité de Bioénergétique et Ingéniérie des Protéines, Centre National de la Recherche Scientifique, Marseilles, France.
Lojou
E
E
Bianco
P
P
Rousset
M
M
Durand
M C
MC
Bruschi
M
M
Dolla
A
A
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D003901:Desulfovibrio / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016999:Sulfur-Reducing Bacteria / Q000235:genetics* / Q000378:metabolism*
D003574:Cytochrome c Group; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D008670:Metals; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010088:Oxidoreductases