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PMID: 2158969 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Paracoccus denitrificans cytochrome c1 gene replacement mutants.

Journal of bacteriology ·Vol. 172 ·No. 5 ·1990-05-00 ·Pages 2392-400

Gerhus E, Steinrücke P, Ludwig B

Abstract

We describe the construction and characterization of gene replacement mutants for the respiratory chain component cytochrome c1 in the bacterium Paracoccus denitrificans. Its structural gene (fbcC) was inactivated by insertion of the kanamycin resistance gene, introduced into a suicide vector, and conjugated into Paracoccus; chromosomal mutants obtained by homologous recombination were selected by antibiotic resistance screening and further characterized biochemically. They showed the complete spectral, enzymatic, and immunological loss of the fbcC gene product together with a serious defect in the assembly of the two other gene products of the fbc operon, cytochrome b and the FeS protein. A possible role of the cytochrome c1 in the assembly process for the enzyme complex is discussed. A functional restoration to wild-type phenotype was achieved by complementing in trans with a newly constructed broad-host-range vector carrying the fbcC gene cassette. When the complete fbc operon was present on this vector, overexpression of complex III subunits was observed. Apart from their physiological significance, such mutants are a prerequisite for probing structure-function relationships by site-directed mutagenesis in order to understand molecular details of electron transport and energy transduction processes of this respiratory enzyme in bacteria and in mitochondria.

MeSH Terms
Blotting, Southern Cytochrome c Group Cytochromes c1/genetics Escherichia coli/genetics Genes, Bacterial Genetic Complementation Test Genotype Mutation Paracoccus denitrificans/enzymology,genetics Plasmids Recombination, Genetic Restriction Mapping
Chemicals
Cytochrome c Group Cytochromes c1
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Gerhus E
Institute of Biochemistry, Medical University of Lübeck, Federal Republic of Germany.
Steinrücke P
Ludwig B
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/jb.172.5.2392-2400.1990
Published
1990-05-00
Pages
2392-400
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC208874
Subset
IM
External Links
PubMed source DOI: 10.1128/jb.172.5.2392-2400.1990 PMC Full Text View journal details
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