Microbial metal reduction contributes to biogeochemical cycling, and reductive precipitation provides the basis for bioremediation strategies designed to immobilize radionuclide contaminants present in the subsurface. Facultatively anaerobic ×-proteobacteria of the genus Shewanella are present in many aquatic and terrestrial environments and are capable of respiration on a wide range of compounds as terminal electron acceptor including transition metals, uranium and transuranics. S. putrefaciens is readily cultivated in the laboratory and a genetic system was recently developed to study U(VI) reduction in this organism. U(VI) reduction-deficient S. putrefaciens point mutant Urr14 (hereafter referred to as CCMB1) was found to retain the ability to respire several alternate electron acceptors. In the present study, CCMB1 was tested on a suite of electron acceptors and found to retain growth on electron acceptors with high reduction potential (E¡¬0) [O2, Fe(III)-citrate, Mn(IV), Mn(III)-pyrophosphate, NO3-] but was impaired for anaerobic growth on electron acceptors with low E¡¬0 [NO2-, U(VI), dimethyl sulfoxide, trimethylamine N-oxide, fumarate, ×-FeOOH, SO32-, S2O32-]. Genetic complementation and sequencing analysis revealed that CCMB1 contained a point mutation (H108Y) in a CcmB homolog, an ABC transporter permease subunit required for c-type cytochrome maturation in E. coli. The periplasmic space of CCMB1 contained low levels of cytochrome c and elevated levels of free thiol equivalents (-SH), an indication that redox homeostasis was disrupted. Anaerobic growth ability, but not cytochrome c maturation activity, was restored to CCMB1 by adding exogenous disulfide bond-containing compounds (e.g., cystine) to the growth medium. To test the possibility that CcmB transports heme from the cytoplasm to the periplasm in S. putrefaciens, H108 was replaced with alanine, leucine, methionine and lysine residues via site-directed mutagenesis. Anaerobic growth, cytochrome c biosynthesis or redox homeostasis was disrupted in each of the site-directed mutants except H108M. The results of this study demonstrate, for the first time, that S. putrefaciens requires CcmB to produce c-type cytochromes under U(VI)-reducing conditions and maintain redox homeostasis during growth on electron acceptors with low E¡¬0. The present study is the first to examine CcmB activity during growth on electron acceptors with widely-ranging E¡¬0, and the results suggest that cytochrome c or free heme maintains periplasmic redox poise during growth on electron acceptors with E¡¬0 < 0.36V such as in the subsurface engineered for rapid U(VI) reduction or anoxic environments dominated by sulfate-reducing bacteria. A mechanism for CcmB heme translocation across the S. putrefaciens cytoplasmic membrane via heme coordination by H108 is proposed.
| Identifer | oai:union.ndltd.org:GATECH/oai:smartech.gatech.edu:1853/19846 |
| Date | 11 October 2007 |
| Creators | Dale, Jason Robert |
| Publisher | Georgia Institute of Technology |
| Source Sets | Georgia Tech Electronic Thesis and Dissertation Archive |
| Detected Language | English |
| Type | Dissertation |
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