Cuhel Russell Lee

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Russell Lee

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  • Thesis
    Assimilatory sulfur metabolism in marine microorganisms
    (Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 1980-12) Cuhel, Russell Lee
    The reductive assimilation of sulfate into cellular organic sulfur compounds was studied in aerobic marine bacteria, with emphasis on the relationship between sulfur metabolism and protein synthesis. The goal of the study was to develop and apply a method for the quantitative assay of total bacterial protein synthesis in aerobic ocean waters. The study consisted of four parts: (1) The sulfate transport systems of two nutritionally different marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus, were characterized to provide information on environmental regulation of sulfate transport capacity. In common with terrestrial bacteria, the transport systems of both marine bacteria exhibit (a) size-selective competitive inhibition of sulfate uptake by sulfate analogs, (b) requirements for energy coupling,and (c) derepression of transport capacity as a result of sulfur starvation. Features which are unique to the marine bacteria include (a) a ten-fold lower affinity for sulfate (half-saturation constant ~200 μm), (b) derepression of transport capacity when grown with methionine as the sole source of sulfur, and (c) an inability to accumulate inorganic sulfate in excess of growth requirements. The different characteristics of the sulfate transport systems of the marine bacteria relative to terrestrial microorganisms are consistent with the saturating concentration of sulfate that is always present in their environment. Substantial differences also exist between the two marine bacteria, notably in the effect of thiosulfate on sulfate uptake. P. halodurans transports thiosulfate with a ten-fold higher affinity than sulfate. Sulfate and thiosulfate are mutually competitive inhibitors of transport, and the half-saturating concentration of thiosulfate for uptake also produces half-maximal inhibition of sulfate transport. Sulfate and thiosulfate transport systems both respond similarly to all inibitors. These facts implicate a common carrier for the two compounds. In contrast, sulfate transport in A. luteo-violaceus is relatively insensitive to thiosulfate. The effect of the suIfhydryl reagent pHMB is similarly much less pronounced than in P. halodurans. These and other differences indicate that the sulfate transport system of A. luteo-violaceus is unique among microorganisms. (2) Growth experiments with P. halodurans and A. luteo-violaceus were carried out over a range of nutritional regimes. Biomass parameters (cell counts, bulk protein, particulate carbon and nitrogen), total uptake of radioactive sulfate, and the distribution of sulfur in major biochemical components (low molecular weight [L.M.W.], alcohol soluble protein, lipid, hot TCA soluble material, and residue protein) were monitored to determine the variability in cellular composition as a function of the environment. Special emphasis was placed on the quantitative relationship between incorporation of sulfur into protein and bulk protein synthesis and conditions which might alter the sulfur content of protein. It was found that sulfur metabolism is restricted predominantly to the production and utilization of protein precursors. The protein synthesis inhibitor chloramphenicol caused an immediate halt to both bulk protein synthesis and sulfur incorporation into protein, accompanied by a rapid swelling of L.M.W. organic sulfur pools, in both bacteria. Incorporation of exogenous sulfur into protein was rapid due to the very small size of the L.M.W. pool. No significant deviation from the ratio of protein-S:bulk protein determined for unperturbed exponential growth was observed as a function of carbon limitation, nitrogen limitation, treatment with chloramphenicol, or during lag and stationary phases. However, the concentration of sulfate in the growth medium exerted a strong influence on the sulfur content of both whole cells and isolated protein. At concentrations less than 500 μM (P. halodurans) or 100 μM (A. luteo-violaceus) the weight % S in protein was proportional to the silate concentration in the medium. Since the sulfate concentration is invariably high in seawater (25mM), data from sulfur-limited growth were not included in the analysis of compositional variability. Under all the conditions examined, the incorporation of sulfur into protein provided the best measurement of protein synthesis and cell growth, with a very low coefficient of variation for the protein-S:bulk protein ratio (less than 16%). The mean true weight % S in protein, 1.07 (P. halodurans) and 0.92 (A. luteoviolaceus) agrees well with the 1.1% predicted from analyses of sequenced proteins. (3) The method used for the analysis of sulfur incorporation into protein was tested with mixed natural populations of marine bacteria in enrichment culture and 13 isolates from the Sargasso Sea to establish the variability of the protein-S:bulk protein ratio among marine bacteria. The mean true weight % S in protein, 1.09, and the operational weight % S in protein, 0.93, have coefficients of variation of 13.1 and 15.1%, respectively. The values are similar to those obtained with the two marine bacteria studied in detail and to that predicted from protein composition studies. Therefore sulfur incorporation into protein measures protein synthesis in marine bacteria within a small degree of error. (4) The method was applied to unenriched natural populations of marine bacteria in waters of the continental shelf, slope, and Sargasso Sea. Time-course incorporation measurements revealed along lag period at the shelf and slope stations, whereas incorporation of sulfur into protein began immediately in the Sargasso Sea. However, long term incubations confirmed that the potential for bacterial protein synthesis decreases in an off-shore transect. These observations were confirmed by simultaneous incorporation studies using labeled ammonia, phosphate, and organic carbon compounds. The potential protein synthesis measured in the unenriched samples provides evidence suggesting that bacterial biomass may be an important contributor to marine food webs.