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Microbiology 145 (1999), 3047-3057
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Microbiology (1999), 145, 3047-3057.
© 1999 Society for General Microbiology

Genetics and Molecular Biology

Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB

M. Dudley Pagea,1 and Stuart J. Ferguson1

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK, and Oxford Centre for Molecular Sciences, New Chemistry Building, South Parks Road, Oxford, OX1 3QT, UK1

Author for correspondence: M. Dudley Page. Tel: +31 20 4447183. Fax: +31 20 4447229. e-mail: mpage{at}bio.vu.nl

Each of the Paracoccus denitrificans genes in the c-type cytochrome biogenesis gene cluster ccmABCDG, plus the two flanking genes ORF117 and hisH, were individually disrupted by {Omega} insertion. Resultant phenotypes were restored to the wild-type by complementation from a set of plasmids. All of the ccm genes, but neither ORF117 nor hisH, were required for c-type cytochrome biogenesis; only ccmG was also implicated in the biosynthesis of cytochrome aa3. Disruption of ccmC or ccmG resulted in failure to grow on rich media, but disruption of ccmA, ccmB or ccmD did not. The ccmC mutant, but not the ccmA, ccmB or ccmD mutants, also exhibited the increased sensitivity to growth inhibition by oxidized thiol compounds previously observed for the ccmG mutant. In contrast to the ccmG mutant, however, growth of the ccmC mutant on rich media could not be restored by DTT. Siderophore biosynthesis and/or secretion by P. denitrificans was also attenuated by disruption of ccmC and ccmG but not of ccmA, ccmB or ccmD. These results indicate that CcmC can function independently of CcmA, CcmB and CcmD despite other evidence that these gene products form an ATP-binding cassette (ABC)-type-transporter with the subunit composition (CcmA)2-CcmB-CcmC or (CcmA)2-CcmB-CcmC-CcmD, and also suggest a possible link between the functions of CcmC and CcmG.

Keywords: Paracoccus denitrificans, c-type cytochrome biogenesis, putative ABC-transporter, siderophores

Abbreviations: ABC, ATP-binding cassette; CAS, chrome azurol sulphonate; HEDS, 2-hydroxyethyldisulphide; TMPD, tetramethylphenylenediamine

The GenBank accession number for the sequence determined in this work is Z71971.

a Present address: Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.




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