Sulfur reduction in the hyperthermophilic bacterium {\it Thermotoga neapolitana\/}
Date of Completion
January 1997
Keywords
Biology, Molecular|Biology, Microbiology|Chemistry, Biochemistry
Degree
Ph.D.
Abstract
The primary goal of this research is to determine the importance of sulfur reduction (sulfidogenesis) in the central metabolism of the hyperthermophilic bacterium Thermotoga neapolitana. Growth medium was optimized to maximize cell yields for purification of the sulfur reductase. Supplementing the medium with cystine, dimethyl disulfide or tetrathionate allowed cells to grow under otherwise inhibitory concentrations of hydrogen whereas thiosulfate did not. An assay was developed to monitor enzymatic reduction of sulfur, or more accurately polysulfides. In addition to the known polysulfide reducing activity of the hydrogenase, a second cytoplasmic enzyme called sulfide dehydrogenase was found in cell extracts. Sulfide dehydrogenase specific activity was up to 48-fold higher than the polysulfide reducing activity of the hydrogenase indicating that sulfide dehydrogenase is more physiologically relevant. Polysulfide (K$\rm\sb{m}$ 0.15 mM) is preferred over sulfur as an electron acceptor and the enzyme is more active with NADH (K$\rm\sb{m}$ 0.03 mM) than NADPH (K$\rm\sb{m}$ 0.41 mM). Sulfide dehydrogenase was purified from cell extracts and consists of one or more 45 kDa subunits. Further characterization of this enzyme was not complete due to a low yield during purification. An N-terminal amino acid sequence was obtained and showed similarity to dihydrolipoamide dehydrogenases and other pyridine nucleotide-dependent disulfide dehydrogenases. Cloning of the sulfide dehydrogenase gene was done using degenerate primers designed from the N-terminal sequence data. The gene was amplified from chromosomal DNA by PCR. A PCR product of 578 bp was cloned, sequenced and found to contain 487 bp of an estimated 1.1 kb gene encoding the sulfide dehydrogenase. A database search with this fragment showed the highest similarity to a putative NADH oxidase from Methanococcus jannaschii.^ Investigations of other dehydrogenase activities in cells extracts revealed two other NADH-dependent activities in addition to a glycerol dehydrogenase. An aerobic NADH oxidizing activity, presumably an NADH oxidase, and an NADH:methyl viologen oxidoreductase (NMOR), presumably an NADH:ferredoxin oxidoreductase could be resolved from the sulfide dehydrogenase by chromatography. NMOR was found to be membrane associated and experiments were begun to discern if this enzyme is involved in energy conservation. ^
Recommended Citation
Childers, Susan Elizabeth, "Sulfur reduction in the hyperthermophilic bacterium {\it Thermotoga neapolitana\/}" (1997). Doctoral Dissertations. AAI9737399.
https://digitalcommons.lib.uconn.edu/dissertations/AAI9737399