Hydrogen-Sulfur Autotrophy in the Hyperthermophilic Archaebacterium,Pyrodictium brockii

Abstract

Research Summary#.,Our objective in this project was to investigate the bioenergetics and physiology of high temperature bacteria. The initial focus was on a hyperthermophilic chemolithotroph, Pyrodictitim brockii (Stetter et al., 1983;Parameswaran et al., 1988; Pihl et al., 1989). This bacterium was chosen for several reasons: it has the reported highest optimum growth temperature (105 0 C) for any organism and it clearly reduces sulfur to sulfide to fulfill some or all of its energetic requirements. Although small amounts of yeast extract stimulate growth, growth proceeds mainly through hydrogen/sulfur autotrophy apparently centered around a series of electron transfer reactions in the cell membrane (Pihl et al., 1989; Pihl et al., 1990). Initially, based on the work on less thermophilic archaebacteria (Danson, 1988), we thought that the absence of significant heterotrophy would simplify the analysis of energetics and allow us to follow growth stoichiometry through gas analysis (i.e., by following the patterns of utilization of CO 2 , H2 and polysulfides, and production of H 2 S). An additional objective was to correlate membrane permeability with changes in energetic efficiency at different temperatures for the bacterium; the hypothesis is that the membranes of high temperature bacteria might be relatively permeable creating an energetic burden (McKay et al., 1982).Continuous culture techniques, developed for P. furiosus (Brown and Kelly, 1989), were modified to grow P. brockii under sulfur-or hydrogen-limiting conditions. Methods for feeding suspensions of colloidal sulfur or polysulfides were developed and a technique to measure cell membrane permeability was adapted to high temperature. Considerable effort was directed at determining the stoichiometry of hydrogen/sulfur autotrophy . However, after much effort, it was found that difficulties in obtaining sufficiently quantitative data for determining maximal yields and maintenance requirements on hydrogen and sulfur made pursuing the planned study infeasible. Also, extremely low biomass yields (2E7 cells/ml corresponding to approximately 5 mg/l cell dry weight) available for proton motive force and membrane permeability measurements and determination of important enzyme activities caused us to re-assess our choice of bacterium for study.As an alternative, we switched to Pyrococcus furiosus, an organism with which we had considerable experience. In many ways, this has proved to be a much better system for study, particularly in view of reproducibility of growth experiments and biomass yields. Also, some details of P. furiosus's physiology have been worked out and a growing list of enzymes from this bacterium have been purified and studied (e.g., Adams, 1990;Aono et al., 1989;Blumentals et al., 1990;Brown et al., 1990; Brown and Kelly, in preparation;Bryant and Adams, 1989;Connaris et al., 1991;Costantino et al., 1990; Blumentals et al., in preparation;Eggen et al., 1990;Koch et al., 1990; Klingeberg et al., 1991). We have been able to use many of the techniq...