Dissimilatory Fe(III) and Mn(IV) Reduction by Shewanella putrefaciens Requires ferE a Homolog of the pulE (gspE) Type II Protein Secretion Gene

摘要

Shewanella putrefaciens strain 200 respires anaerobically on a wide range of compounds as the sole terminal electron acceptor, including ferric iron [Fe(III)] and manganese oxide [Mn(IV)]. Previous studies demonstrated that a 23.3-kb S. putrefaciens wild-type DNA fragment conferred metal reduction capability to a set of respiratory mutants with impaired Fe(III) and Mn(IV) reduction activities (T. DiChristina and E. DeLong, J. Bacteriol. 176:1468–1474, 1994). In the present study, the smallest complementing fragment was found to contain one open reading frame (ORF) (ferE) whose translated product displayed 87% sequence similarity to Aeromonas hydrophila ExeE, a member of the PulE (GspE) family of proteins found in type II protein secretion systems. Insertional mutants E726 and E912, constructed by targeted replacement of wild-type ferE with an insertionally inactivated ferE construct, were unable to respire anaerobically on Fe(III) or Mn(IV) yet retained the ability to grow on all other terminal electron acceptors. Nucleotide sequence analysis of regions flanking ferE revealed the presence of one partial and two complete ORFs whose translated products displayed 55 to 70% sequence similarity to the PulD, -F, and -G homologs of type II secretion systems. A contiguous cluster of 12 type II secretion genes (pulC to -N homologs) was found in the unannotated genome sequence of Shewanella oneidensis (formerly S. putrefaciens) MR-1. A 91-kDa heme-containing protein involved in Fe(III) reduction was present in the peripheral proteins loosely attached to the outside face of the outer membrane of the wild-type and complemented (Fer⁺) B31 transconjugates yet was missing from this location in Fer mutants E912 and B31 and in uncomplemented (Fer⁻) B31 transconjugates. Membrane fractionation studies with the wild-type strain supported this finding: the 91-kDa heme-containing protein was detected with the outer membrane fraction and not with the inner membrane or soluble fraction. These findings provide the first genetic evidence linking dissimilatory metal reduction to type II protein secretion and provide additional biochemical evidence supporting outer membrane localization of S. putrefaciens proteins involved in anaerobic respiration on Fe(III) and Mn(IV).