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.
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.
Gaballa
A
A
Department of Immunology, Parasitology, and Ultrastructure, Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussels, Belgium.
Baysse
C
C
Koedam
N
N
Muyldermans
S
S
Cornelis
P
P
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D003574:Cytochrome c Group / Q000096:biosynthesis*; D007501:Iron / Q000493:pharmacokinetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides; D019897:Periplasm / Q000378:metabolism*; D010860:Pigments, Biological / Q000235:genetics / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000235:genetics* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C116246:CcmC protein, bacteria; D003574:Cytochrome c Group; D008565:Membrane Proteins; D009842:Oligopeptides; D010860:Pigments, Biological; C042453:pyoverdin; D007501:Iron