Graduate Thesis Or Dissertation

Effects of salinity and temperature changes on the induction of glutamic dehydrogenase in the marine psychrophilic bacterium, Vibrio marinus MP-1

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  • In this study the relationship between salinity and temperature changes on microbial growth, enzyme induction and substrate uptake were investigated. The obligately psychrophilic bacterium, V. marinus MP-1 was grown for 48 hours at 15 C in a glucose-ammonium medium (GAM) containing 0.4 M NaCl. Log-phase cells were harvested and shifted to a glutamate medium (GM) at either 4, 15, 20 or 25 C and containing 0.26, 0.4, 0.6 or 0.8 M NaCI. It was found that at any one temperature investigated (with the exception of 25 C), an increase in the amount of NaC1 in GM from 0.26 to 0.4 M resulted in an increase in shifted cell growth. A further increase in NaCI to 0.6 or 0.8 M reduced the net cellular growth. Of the four salinities tested, 0.4 M NaCl was determined as being optimal for growth. Growth temperature profile studies in a polythermostat revealed an optimal growth temperature of 12-13 C and a maximum of 20-22 C in GAM (0.4 M NaCI), GM (0.4 M NaCl), and marine broth (MB). Cells shifted to GM at 4 C showed only low levels of induction of glutamic dehydrogenase (GDH) after ten hours in any NaCl concentration. Increasing the shift temperature to 15 C rapidly increased the induction rate of GDH with a maximum at 0.4 M NaCI (56-fold increase in ten hours). A temperature increase to 20 C in the shift medium produced lower rates of induction than at 15 but a maximum in 0.4 M NaCl was maintained (35-fold increase in ten hours). Cells shifted to GM at 25 C initially began to produce GDH but ceased to do so within 2-4 hours since all cells died within two hours after shifting. Uptake of ¹⁴C-glutamate by shifted cells, at the various temperatures and salinities tested, revealed a maximal rate of uptake and net incorporation of label into cells at 0.26 M NaCI at 15 C. Further increases in salinity (0.4, 0.6 and 0.8 M) or alterations in temperature (4, 20, 25 C) reduced the uptake rate and net incorporation of the isotope. One exception, however, occurred at 25 C where the maximal rate of uptake and net incorporation occurred in 0.6 M. Measurement of uptake and incorporation of ¹⁴C-proline into protein by shifted cells at various salinities and temperatures revealed essentially the same pattern as in the ¹⁴C-glutamate studies, except that maxima for both determinations occurred at 20 C. Radiorespirometry revealed that increasing salt concentrations from 0.26 M to 0.8 M reduces cell respiration at 15 C but does not prevent it. It also showed that 2% of the ¹⁴C-glutamate available to the cells was taken up after one hour incubation in GM (0.4 M NaCl) at 15 C, Of the total amount available, only 0.2% (12% of that taken up) was respired as ¹⁴CO These data are interpreted to mean that the induction of GDH is directly related to the growth rate of the organism in GM. The growth rate at any one temperature, however, is salinity dependent. Suboptimal NaCl concentrations may well be affecting some integral part of the cells structural integrity necessary for GDH synthesis, since a NaCl concentration of 0.26 M allowed a maximal rate of entry and accumulation of ¹⁴C-glutamate into cells at 15 C, as well as a maximal rate of protein synthesis and respiration, but produced suboptimal growth rates. It would appear that growth rate limitation by lowered NaCl concentrations is not a direct consequence of any of these processes. In addition, since 0.4 M NaCl allowed maximal GDH induction and produced a maximal growth rate, it seems likely that this concentration of NaCl enhances GDH induction, thereby producing a maximal rate of growth. NaC1 concentrations greater than 0.4 M generally seem to retard GDH induction by inhibiting substrate uptake, protein synthesis, and respiration, thus inhibiting growth. Metabolic inhibitor studies revealed that energy generation is necessary for ¹⁴C-glutamate uptake at 15 C in GM (0.4 M NaCl). The chloride ion (Cr) seems to be required for the uptake of ¹⁴C-glutamate at 15 C in GM (0.4 M NaCI).
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