Isolation and sequence of omcA, a gene encoding a decaheme outer membrane cytochrome c of Shewanella putrefaciens MR-1, and detection of omcA homologs in other strains of S. putrefaciens.
The sequence of the omcA gene, which encodes a decaheme cytochrome c that is localized to the outer membrane (OM) of Shewanella putrefaciens MR-1, was determined. The 2202 bp nucleotide sequence of omcA encodes for 734 amino acids with a predicted molecular protein mass of 78.6 kDa. Comparison with the amino-terminal sequence of the mature protein suggests the presence of a hydrophobic leader sequence which is cleaved during translocation of the protein to the OM. This leader sequence has a lipoprotein consensus sequence for signal peptidase II at the cleavage site. The predicted mature protein is comprised of 708 amino acids with a predicted molecular mass of 75.8 kDa, but the addition of ten covalently attached heme c groups and covalent lipid modification to the amino-terminal cysteine increases the predicted mass to 82.7 kDa. This is consistent with its apparent mass of 83 kDa in SDS-PAGE gels. The predicted amino acid sequence for the OmcA protein shows no significant homology to known proteins. A RNA of approx. 2300 bases that hybridizes to the omcA gene was detected in anaerobically grown MR-1 cells. The size of this transcript is similar to the coding region of the omcA gene, suggesting that it is not part of a multicistronic operon. Similar to MR-1, four other strains of S. putrefaciens were all found to localize a majority of their membrane-bound cytochromes to the OM when grown under anaerobic conditions, and all contained an OM cytochrome of similar size to OmcA. In two of these strains, MR-4 and MR-8, a homolog of omcA was identified by RT-PCR and Southern blotting using primers and probes specific for omcA of MR-1. Western blot analysis using a polyclonal antibody to OmcA was similarly positive in strains MR-4 and MR-8. Partial nucleotide sequence analysis of these homologs demonstrated 74-77% predicted amino acid homology with OmcA of MR-1. In contrast, strains MR-30 and MR-42 tested negative for omcA homologs by Southern and Northern blots, RT-PCR, and Western blots.
Myers
J M
JM
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Myers
C R
CR
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D001483:Base Sequence; D003574:Cytochrome c Group / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics; D006092:Gram-Negative Facultatively Anaerobic Rods / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004269:DNA, Bacterial; C114590:omc A protein, Shewanella putrefaciens