- 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).