Different residues in periplasmic domains of the CcmC inner membrane protein of Pseudomonas fluorescens ATCC 17400 are critical for cytochrome c biogenesis and pyoverdine-mediated iron uptake

Authors

  • Ahmed Gaballa,

    1. Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussels, Belgium.,
    2. Laboratory of Microbial Interactions, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium.,
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  • Christine Baysse,

    1. Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussels, Belgium.,
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  • Nico Koedam,

    1. Laboratory of Microbial Interactions, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium.,
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  • Serge Muyldermans,

    1. Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussels, Belgium.,
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  • Pierre Cornelis

    1. Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussels, Belgium.,
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Pierre Cornelis. E-mail PCORNEL@vub.ac.be; Tel. (2) 359 02 21; Fax (2) 359 03 99.

Abstract

The inner membrane protein CcmC (CytA) of Pseudomonas fluorescens ATCC17400, which has homologues in several bacteria and plant mitochondria, is needed for the biogenesis of cytochrome c. A CcmC-deficient mutant is also compromised in the production and utilization of pyoverdine, the high-affinity fluorescent siderophore. A topological model for CcmC, based on the analysis of alkaline phosphatase fusions, predicts six membrane-spanning regions with three periplasmic loops. Site-directed mutagenesis was used in order to assess the importance of some periplasm-exposed residues, conserved in all CcmC homologues, for cytochrome c biogenesis, and pyoverdine production/utilization. Despite the conservation of the residues His-61, Val-62 and Pro-63 in the first periplasmic loop, and Leu-184, His-185 and Gln-186 in the third periplasmic loop, their simultaneous replacement with Ala only partially affected cytochrome c biogenesis and pyoverdine production/utilization. Simultaneous replacements of residues Trp-115 and Gly-116 in the second periplasmic loop substantially affected pyoverdine production/utilization but not cytochrome c production. An Ala substitution of Asp-127, in the second periplasmic loop, resulted in decreased production of cytochrome c, slower growth in conditions of anaerobiosis and reduced pyoverdine production. On the other hand, a mutation in Trp-126, also in the second periplasmic loop, totally suppressed the production of cytochrome c, whereas it had no effect on the production and utilization of pyoverdine. These results show a differential involvement of amino acid residues in periplasmic domains of CcmC in cytochrome c biogenesis and pyoverdine production/utilization.

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