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Applied and Environmental Microbiology, June 2000, p. 2422-2429, Vol. 66, No. 6
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Effect of Temperature and Salinity Stress on Growth
and Lipid Composition of Shewanella gelidimarina
David S.
Nichols,1,*
June
Olley,1
Horacio
Garda,2
Rodolfo R.
Brenner,2 and
Tom A.
McMeekin1,3
School of Agricultural
Science1 and Antarctic
CRC,3 University of Tasmania, Hobart,
Tasmania 7001, Australia; and Instituto de Investigaciones
Bioquimicas de la Plata, UNLP-CONICET, Facultad de Ciencias
Médicas, La Plata, Argentina2
Received 14 October 1999/Accepted 22 March 2000
 |
ABSTRACT |
The maximum growth temperature, the optimal growth temperature, and
the estimated normal physiological range for growth of Shewanella
gelidimarina are functions of water activity (aw), which can be manipulated by changing the concentration of sodium chloride. The growth temperatures at the boundaries of the normal physiological range for growth were characterized by increased variability in fatty acid composition. Under hyper- and hypoosmotic stress conditions at an aw of 0.993 (1.0% [wt/vol] NaCl)
and at an aw of 0.977 (4.0% [wt/vol] NaCl) the
proportion of certain fatty acids (monounsaturated and branched-chain
fatty acids) was highly regulated and was inversely related to the
growth rate over the entire temperature range. The physical states of
lipids extracted from samples grown at stressful aw values
at the boundaries of the normal physiological range exhibited no abrupt
gel-liquid phase transitions when the lipids were analyzed as
liposomes. Lipid packing and adaptational fatty acid composition
responses are clearly influenced by differences in the
temperature-salinity regime, which are reflected in overall cell
function characteristics, such as the growth rate and the normal
physiological range for growth.
| |
INTRODUCTION |
Antarctic sea ice is characterized
by the presence of a unique bacterial community that is dominated by
psychrophilic bacteria (2, 3, 12). Nichols et al.
(25) have discussed the potential role of the
temperature-salinity regime in selecting psychrophilic bacteria in sea
ice. It has been suggested that understanding the physiological
response of psychrophilic bacteria to combined temperature-salinity
stress is very important for understanding the bacterial sea ice
community. The importance of fluctuations in salinity that affect the
composition of microbial populations in estuarine and brackish water
ecosystems has been recognized (7, 30), and such
fluctuations are very important when the survival and viability of
psychrophilic marine bacteria are considered (12, 20, 21,
34). Changes in salinity similar to those observed in estuarine
and brackish water environments also occur in Antarctic coastal waters
and sea ice and are associated with the annual ice formation and
melting cycle. However, researchers have attempted to relate changes in
physicochemical parameters to a physiological mechanism for growth in
only a few studies of psychrophilic bacteria (8, 11, 21, 26,
35).
Workers have proposed a number of models to describe the effect of
temperature and/or salinity on bacterial growth. Most of these models
are empirical and seek solely to summarize observations of bacterial
growth under various conditions. Many are based on the Arrhenius
equation and utilize Arrhenius kinetics to describe bacterial growth in
response to temperature. The concept of a normal physiological range
(NPR) for bacterial growth is derived from modelling of the bacterial
growth rate by using Arrhenius models. Over a defined range of
temperature, the growth rates of all bacteria obey Arrhenius kinetics,
and this temperature range is designated the NPR. The boundaries of the
NPR are defined at both high and low temperatures by deviations in the
bacterial growth rate response from Arrhenius kinetics (22).
Deviations in the bacterial growth rate outside this range have been
explained by the postulated inactivation of one or more enzymatic
processes essential for growth, although no single mechanism has been
discovered yet (22).
In this study a temperature gradient incubator was used to investigate
the effects of temperature-salinity regimes on the growth, lipid phase,
and fatty acid composition of the psychrophilic sea ice bacterium
Shewanella gelidimarina. Square root type models were used
to establish limits for growth, while an Arrhenius model was used to
estimate the NPR.
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MATERIALS AND METHODS |
Bacterial strains.
S. gelidimarina ACAM
456T was first described by Nichols et al. (24)
and was fully described by Bowman et al. (4). S. gelidimarina was maintained on slopes of Zobell's agar
(42) and was cultured in Zobell's broth prior to experiments.
Temperature gradient incubator experiments.
The effect of
salinity on the rate of growth of S. gelidimarina at 20°C
was determined by using a temperature gradient incubator (Toyo Kagaku
Sangyo, Tokyo, Japan). The growth medium used was Zobell's broth;
however, it contained distilled water rather than seawater. Sixteen
incubation tubes were prepared with water activity (aw)
values of 1.00 to 0.892 (0 to 15% [wt/vol] NaCl) by adding NaCl to
individual tubes. One incubation tube was also prepared by using
nominal seawater salinity (3.5%, wt/vol), and another tube was
prepared with natural seawater (aw, 0.982). The tubes were
inoculated with an actively growing culture, and growth was monitored
by measuring percent transmittance at 540 nm until growth was complete.
Cultures were shaken via oscillatory motion of the incubator through a
60° arc at a rate of 60 oscillations min
1. Growth rates
at each temperature were calculated by fitting a modified Gompertz
function to the data (18). The growth temperature for each
incubation tube was determined in triplicate by using a Fluke model 51 K/J thermometer and a Fluke model 80PK-1 thermocouple after growth
ceased. The calculated growth rate data were then fitted to the
modified four-parameter square root model of Miles et al.
(19) for use with aw data by using UltraFit software.
The effect of temperature on the rate of growth of S. gelidimarina was determined by using a temperature gradient
incubator. Preparations (20 ml) were incubated at temperatures ranging
from 
2 to 28°C at approximately 1°C intervals. Modified Zobell's
broth was prepared with aw values of 0.993 (1.0%
[wt/vol] NaCl), 0.986 (2.5% [wt/vol] NaCl), and 0.977 (4.0%
[wt/vol] NaCl). Tubes were inoculated with an actively growing
culture, and growth was monitored as described above. The calculated
square roots of growth rate data were then fitted to the model of
Ratkowsky et al. (29) by using UltraFit software. Growth
rate data were also fitted to the model of Rosso et al. (32)
in order to estimate the optimal growth temperature
(Topt). The latter model contains
Topt as a parameter, and thus standard errors
can be determined. The two models were found to give similar results
for other parameters. Significance values were generated by performing
a Student's t test.
Sampling and harvesting of cultures.
The study to determine
the effect of temperature on the rate of growth of S. gelidimarina at two stress-inducing salinities was repeated, and
growth was monitored to ensure that the previous experimental
conditions were replicated. When individual incubation tubes reached
percent transmittance values of 27 to 30, corresponding to the
mid-exponential phase, the cultures were immediately harvested by
filtration onto glass fiber filters (Whatman) which previously had been
heated at 400°C for 24 h. Sample filters were then frozen at
20°C until lipids were analyzed. The initial fatty acid composition data set was used as a guide for choosing lipid phase transition samples. Temperatures of 15.0 and 4.0°C (aw, 0.993) and
13.5 and 5.0°C (aw, 0.977) were selected based on abrupt
changes in the ratio of branched-chain fatty acids to monounsaturated
fatty acids. Due to the amount of lipid required, samples used for
lipid phase transition measurements were grown in duplicate 100-ml
portions of appropriate-salinity media at these temperatures. The
cultures were harvested by centrifugation at 8,750 × g
for 15 min, and each cell mass was washed in an isotonic saline
solution. The cell mass that was collected was extracted directly as
described below.
Lipid extraction and analysis.
Filters were extracted by
using a direct transesterification procedure (6) that
yielded fatty acid profiles comparable to those obtained with the
solvent extraction method of Bligh and Dyer (1, 41).
Briefly, filters were placed in screw cap test tubes containing 3 ml of
methanol-chloroform-hydrochloric acid (10:1:1, vol/vol/vol). The tubes
were heated at 80°C for 1 h and then cooled to room temperature.
After Milli-Q water (1 ml) was added, the resultant fatty acid methyl
esters were extracted three times with 1.5 ml of hexane-chloroform
(4:1, vol/vol). Duplicate samples of biomass which was used for lipid
phase analysis were extracted directly by using the method of Bligh and
Dyer to obtain a total solvent extract. One sample was stored in
chloroform prior to analysis as described below, and the other sample
was transesterified as described above for filter samples in order to
correlate the results with the gradient incubator results.
Fatty acid methyl esters were analyzed by using a Hewlett-Packard model
5890 II gas chromatograph and a model 5970A mass selective
detector
equipped with a cross-linked methyl silicone (film thickness,
0.33 µm) fused-silica capillary column (internal diameter, 0.22
mm;
length, 50 m). The operating conditions were similar to those
described by Nichols et al. (
23). Fatty acid methyl esters
in
all of the samples were identified by comparing the component
spectra to spectra of known standards. Double bond positions and
the
geometry of monounsaturated isomers of selected samples were
determined
by producing and analyzing dimethyl disulfide adducts
(
27).
The data presented below are the sums of the values for
individual
fatty acid methyl esters identified in each
category.
Lipid phase transition measurements.
Solvent was evaporated
from the total solvent extract material (0.2 mg), and 0.5 ml of buffer
A (0.1 M Tris-HCl, 0.16 M NaCl; pH 8.0) was added. After 15 min of
incubation at room temperature, samples were vortexed for 2 min, and
suspensions were extruded through a 100-nm-pore-size filter by using a
Liposofast extruder (Anestin Inc., Ottawa, Ontario, Canada). Extruded
liposomes (0.5 ml) were mixed with 0.5 ml of a 2 µM fluorescent probe
suspension (1,6-diphenyl-1,3,5-hexatriene [DPH] or
6-lauroyl-2,2-dimethylaminoaphthalene [laurdan]) in buffer A, and the
preparation was vortexed before it was incubated at room temperature
for 30 min. Samples were diluted 2.5-fold with buffer A prior to
analysis. All data were obtained by using a model SLM 4800 spectrofluorometer and cuvettes (1 by 1 cm). The steady-state
fluorescence anisotrophy (rS), lifetime (
),
and differential polarized phase lifetime (
) of DPH were measured
by using an excitation wavelength of 361 nm and observing the total
emission at wavelengths of >389 nm through a sharp cutoff filter (type
KV389). For and measurements the exciting light was
modulated sinusoidally in amplitude at 18 or 30 MHz with a Debye-Sears
modulator and was polarized with a Glan-Thompson polarizer. For measurements, emission was observed through a Glan-Thompson polarizer
oriented 55° to the vertical. The phase shift and demodulation of the
emitted light were measured relative to
1,4-bis(5-phenyloxazol-2-yl)benzene in ethanol and were used to compute
the phase and modulation lifetimes. was determined from the
phase shift between the parallel and perpendicular components of the
emission observed between vertically and horizontally oriented emission
polarizers. Data were interpreted by using the hindered wobbling
rotation model (16, 40). The rotational correlation time
(R) and the limiting anisotropy
(r
) were calculated as described previously
(9).
Laurdan fluorescence intensity and generalized polarization (GP)
spectra were determined as described by Garda et al. (
10).
All spectra were corrected for any background signal by subtracting
the
signal obtained with unlabelled samples. An excitation wavelength
of
360 nm (emission intensity spectra) and an emission wavelength
of 430 nm (excitation intensity spectra) were used. GP spectra
were determined
by determining the excitation intensity spectra
at 440 nm
(I
440) and 490 nm (I
490) for emission and the
emission
intensity spectra at excitation wavelengths of 340 nm
(I
340) and
410 nm (I
410). GP data for the
excitation and emission bands were
obtained by using the
(I
440 - I
490)/(I
440 + I
490) and (I
410 I
340)/(I
410 + I
340) ratios,
respectively.
| |
RESULTS |
Growth rate.
S. gelidimarina grew at a narrow range of
salinities, which is characteristic of marine bacteria (Fig.
1). The minimum aw for growth
was 0.974 ± 0.004 (4.6% [wt/vol] NaCl). This bacterium required NaCl for growth, and the maximum aw was 0.996 ± 0.002 (0.7% [wt/vol] NaCl). No growth was detected in 10 of the
16 incubation tubes in which the aw values were outside
this range during the experiment. The optimum aw for growth
under experimental conditions was 0.986 (2.5% [wt/vol] NaCl).
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FIG. 1.
Plot of growth rate (1/Generation time [min]) versus
aw for S. gelidimarina grown in Zobell's broth
containing NaCl at different concentrations. The growth temperature was
20°C.
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The response of the growth rate of
S. gelidimarina to
temperature depended on the salinity (Fig.
2 and Table
1). The maximum
growth temperatures
(
Tmax) of
S. gelidimarina at the
three a
w values tested were significantly different
(
P < 0.001) from each
other; higher
Tmax values were observed at lower
a
w values. The
Topt values were also
significantly different (
P < 0.001) from
each other,
and the highest
Topt was observed at an
a
w of 0.986.
The theoretical minimum growth temperatures
(
Tmin) under different
conditions were not
significantly different (
P > 0.05) (Table
1). However,
a significant increase (
P < 0.05) in the theoretical
growth range was observed when the data for an a
w of 0.986 and
an a
w of 0.977 were compared due to an increase in the
Tmax under
low-a
w conditions.
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FIG. 2.
Plots of growth rate (1/ Generation time
[min]) versus temperature for S. gelidimarina grown
in Zobell's broth at three aw values. Symbols:  ,
aw of 0.993 (1.0% [wt/vol] NaCl); +, aw of
0.986 (2.5% [wt/vol] NaCl);  , aw of 0.977 (4.0%
[wt/vol] NaCl).
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Fatty acid composition.
The fatty acid compositions of
S. gelidimarina cultures grown at aw
values of 0.993 and 0.977 at all of the temperatures at which growth
occurred are shown in Fig. 3. The total
values are the sum of values for the following major components:
saturated fatty acids (12:0, 13:0, 14:0, 15:0, 16:0, 17:0, and
18:0), monounsaturated fatty acids (14:15c, 15:16c,
15:18c, 16:17c, 17:18c, 18:17c, and 18:19c),
branched-chain fatty acids (i13:0, i14:0, and i15:0), and
polyunsaturated fatty acids (PUFA) (18:43, 20:43, and 20:53). There were generally higher proportions of saturated and
monounsaturated fatty acids in samples grown at an aw of
0.977, while higher proportions of branched-chain fatty acids and PUFA
were present in samples grown at an aw of 0.993. The
percentage of saturated fatty acids increased linearly as the
temperature increased at both aw values, while the
proportions of monounsaturated components revealed the opposite
trend (Fig. 3). For both aw values the proportion of branched-chain fatty acids responded to temperature in a parabolic manner centered around a temperature range (ca. 9 to 11°C for an
aw of 0.993; 10 to 12°C for an aw of 0.977)
slightly below the Topt (Fig. 3 and Table 1). A
constant percentage of PUFA was present at both aw values
until the temperature was around Topt.
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FIG. 3.
Plots of S. gelidimarina fatty acid
compositions for the entire biokinetic temperature range at an
aw of 0.993 (1.0% [wt/vol] NaCl) for two data sets ( and  ) and at an aw of 0.977 (4.0% [wt/vol] NaCl). The
data points for aw 0.977 represent means based on values in
three data sets, and standard deviations are indicated by error bars.
(a and b) Total saturated fatty acids at an aw of 0.993 (a)
or 0.977 (b). (c and d) Total monounsaturated fatty acids at an
aw of 0.993 (c) or 0.977 (d). (e and f) Total
branched-chain fatty acids at an aw of 0.993 (e) or 0.977 (f) (g and h). Total polyunsaturated fatty acids at an aw
of 0.993 (g) or 0.977 (h).
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Of note were the distinct areas of compositional variability in the
fatty acid temperature profiles at both a
w values. At
an
a
w of 0.977 large increases in the standard deviations were
apparent for the values for monounsaturated fatty acids, branched-chain
fatty acids, and PUFA at temperatures from 4 to 6°C and at 13°C
(Fig.
3); this was clearly shown by the variance in the proportions
of
branched-chain and monounsaturated fatty acids (Fig.
4). At
an a
w of 0.993 the two
data sets for the pre-
Topt range (9 to
10°C)
(Fig.
3) implied that there was a similar level of increased
variability.
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FIG. 4.
Variances in the percentages of branched-chain and
monounsaturated fatty acids for the entire biokinetic temperature range
for S. gelidimarina grown at an aw of 0.977, based on data from three data sets. Open bars, branched-chain
components; cross-hatched bars, monounsaturated components; shaded
bars, branched-chain and monounsaturated components.
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NPR.
Estimates of the NPR were obtained from the linear
portions of the Arrhenius plots, as shown in Fig.
5 (for an aw of 0.993, ca. 1 to 12°C; for an aw of 0.977 ca. 2 to 15°C). The
percentage of monounsaturated fatty acids plus branched-chain fatty
acids was inversely related to the Arrhenius curve at both salinities. The minimum proportion of monounsaturated fatty acids plus
branched-chain fatty acids observed occurred at the temperature at
which the growth rate was maximal, Topt. The
temperatures at which fatty acid compositional variability occurred at
both an aw of 0.993 and an aw of 0.977 (Fig. 3
and 4) corresponded to sample temperatures that were immediately inside
the boundaries of the NPR (e.g., for an aw of 0.977, variable region at 4 to 6°C and boundary at 2°C and variable region
at 13°C and boundary at 15°C) (Fig. 4 and 5). When the
proportions of branched-chain and monounsaturated fatty
acids were added (Fig. 4), the variability in the data for the
temperature ranges shown in Fig. 3 decreased markedly for the data
obtained at an aw of 0.977.
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FIG. 5.
Arrhenius plots of growth rate ( ) versus temperature
for S. gelidimarina at two salinities. Also shown are data
for the percentage of total monounsaturated and branched-chain fatty
acids versus temperature. (a) Growth at an aw of 0.993 (1.0% [wt/vol] NaCl). The data from two fatty acid data sets are
indicated by different symbols ( and ). (b) Growth at an
aw of 0.977 (4.0% [wt/vol] NaCl). , means based on
three fatty acid data sets. The error bars indicate standard
deviations.
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Lipid phase transition.
Large (100-ml) cultures of S. gelidimarina were grown under the conditions described above that
produced perturbations in the total fatty acid composition for the
lipid phase analyses (at an aw of 0.993, 4.0 and 15.0°C;
at an aw of 0.977, 5.0 and 13.5°C). While the increased
culture volume resulted in minor changes in the fatty acid composition,
the overall trends apparent from the gradient incubator sample data
were supported (data not shown). The temperature dependence of the DPH
rS for the lipids in samples was determined at 1 to 24°C (Fig. 6). No
abrupt change in slope was observed, indicating that there was no
abrupt phase transition in this temperature range. Liposomes from
cultures grown at low temperatures also exhibited lower
rS values, and the slopes were not altered by
the aw. However, the growth temperature did have a
significant effect on liposome lipid properties. The r values indicated that the lipids from
S. gelidimarina cells grown at higher temperatures were more
ordered than the lipids from cells grown at a low temperature (Table
2). An increase R was observed for the lipids from samples
grown at 15°C compared with the lipids from samples grown at 4°C at
an aw of 0.993. However, the R of
lipids from samples grown at 13.5°C was less than the
R of lipids from samples grown at 5°C at an
aw of 0.977 (data not shown).
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FIG. 6.
(a) Temperature dependence of the
rS of DPH in liposomes made with total lipid
extracts from S. gelidimarina grown under the following
conditions: aw of 0.993 (1.0% [wt/vol] NaCl) and 4.0°C
(), aw of 0.993 (1.0% [wt/vol] NaCl) and 15.0°C
(), aw of 0.977 (4.0% [wt/vol] NaCl) and 5.0°C
( ), and aw of 0.977 (4.0% [wt/vol] NaCl) and 13.5°C
(). (b) Fluorescence excitation and emission spectra of laurdan in
liposomes grown under the following conditions: aw of 0.993 (1.0% [wt/vol] NaCl) and 4.0°C
( ), aw of 0.993 (1.0% [wt/vol] NaCl) and 15.0°C
(---), aw of 0.977 (4.0% [wt/vol] NaCl)
and 5.0°C (······), and aw of 0.977 (4.0%
[wt/vol] NaCl) and 13.5°C (-··-).
(c) GP spectra for laurdan in liposomes grown under the following
conditions: (1.0% [wt/vol] NaCl) and 4.0°C (), aw
of 0.993 and 15.0°C (), aw of 0.977 (4.0% [wt/vol]
NaCl) and 5.0°C (), and aw of 0.977 (4.0% [wt/vol]
NaCl) and 13.5°C ().
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TABLE 2.
Effect of NaCl concentration on the
r of DPH in lipids from S. gelidimarina grown under different conditions
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The emission spectra of laurdan in the liposomes obtained from
S. gelidimarina total lipid extracts predominantly exhibited
a blue
band at ~435 nm (Fig.
6), which is characteristic of lipid
bilayers
in the gel state or in the liquid ordered state normally
found in
membranes containing cholesterol (
28). However, a relaxed
emission shoulder at ~490 nm, which is characteristic of the liquid
state, was also present. The shoulder was less pronounced when
we
examined the lipids from samples grown at higher temperatures
(13.5 and
15°C), indicating that there was a higher degree of
lipid packing in
liposomes in these samples than in samples grown
at the lower
temperatures (4.5 and 5°C). The degree of relaxed
emission from the
lipids from samples grown at low temperatures
also increased as the
analysis temperature increased. These spectral
changes could be
quantified by using the GP parameter, which related
the relative
fluorescence intensities at the two wavelengths.
Lower GP values
indicated that the degree of probe mobility was
higher or the degree of
lipid packing was lower (Table
3). The
wavelength dependence of the GP parameter on the excitation and
emission bands at 4.5 and 14.3°C is shown in Fig.
6. The GP values
for all of the samples decreased as the wavelength along the excitation
band increased and increased as the wavelength along the emission
band
increased. This pattern is characteristic of lipids in the
liquid
crystalline or liquid ordered state and not the gel state
(
28). It is therefore probable that a large proportion of
the
lipid domains in the liposomes from all of the samples had
characteristics
of the liquid ordered state and that a small proportion
of liquid
crystalline lipid domains was also present.
| |
DISCUSSION |
S. gelidimarina exhibited a narrow salinity range for
growth, which is typical of marine bacteria.
Tmax was also a function of salinity. This
phenomenon has been observed for other marine bacteria, including
Moritella marinus (21) and Vibrio
anguillarum (11). However, the major effect of high
salinity on the growth temperature range was evident only when the
temperature was greater than Topt, and salinity
had little effect on the growth rate at low temperatures (<10°C) or
on Tmin. It should be stressed that the square
root models used to fit cardinal temperatures in this study yield
estimates of these parameters that are more accurate than the estimates
obtained from Arrhenius plots, which tend to be curvilinear at
suboptimal growth temperatures in the region up to 18°C greater than
Tmin (18). However, as shown in Fig. 5, when growth of the bacterium does not extend significantly into this
suboptimal temperature region, the natural log of growth rate (ln
k) remains directly proportional to the reciprocal of absolute temperature (1/temperature (K) within the NPR. Using an
Arrhenius plot to estimate the NPR for growth of psychrophilic bacteria
is therefore appropriate.
An increase in salinity was accompanied by an increase in the
proportion of saturated fatty acids in S. gelidimarina,
which correlated with a higher degree of lipid order at temperatures greater than 10°C in liposomes, compared to liposomes from cells grown at a low salinity. The liposomes from cells grown at a high salinity and a high temperature exhibited greater retention of lipid
packing with increased temperature than the corresponding low-salinity
liposomes (Fig. 6). Concurrently, the high-salinity, low-temperature
liposomes exhibited greater retention of lipid packing when they were
analyzed at a high temperature, while the high-salinity,
high-temperature liposomes also exhibited a greater increase in packing
when they were analyzed at a low temperature (Table 3). These findings
imply that growth at a high salinity resulted in a lipid composition
that could increase membrane packing in liposomes and a tendency to
retain a higher degree of packing at high temperatures. This effect was
correlated with the ability of cells to withstand higher growth
temperatures without a detrimental effect on low-temperature growth.
The implication is that a loss of lipid packing in the native membrane
may have been the limiting factor for the growth of S. gelidimarina at high temperatures, while a more tightly packed
membrane did not inhibit growth at low temperatures.
The bulk salinity of Antarctic sea ice has been reported to range from
0.4 to 0.9% in November and December (33), the
spring-summer period when maximal heterotrophic activity occurs
(14). At this time brine salinities are likely to be low due
to the flushing effect of ice melt and saline depletion resulting from
the preceding period of high-salinity brine drainage over winter
(12). Thus, S. gelidimarina probably experiences
low-salinity conditions during the period when maximal metabolic
activity occurs. While the maximal growth rate apparently is not
adapted to this environmental characteristic, the estimated NPR is
shifted to lower temperatures at an aw of 0.993. The
generally higher proportion of branched-chain fatty acids and PUFA in
samples at an aw of 0.993 is also correlated with greater
stability of physical lipid characteristics at low salinity values.
These results suggest that there may be preferential membrane
adaptation to low-salinity conditions. However, the similarity of the
fatty acid profiles suggests that similar adaptive strategies with
respect to temperature occur at high and low salinity values.
Suutari and Laakso (36) and Suutari et al. (37)
studied changes in the fatty acid compositions of two bacilli and a
yeast, respectively, over their characteristic temperature ranges for growth. A close temperature sampling regime, such as the one used in
this study, revealed similar trends in the percentages of certain fatty
acid components, which were species specific, within narrow temperature
ranges near the limits for growth. The results of these studies
foreshadowed the results of the present study. However, in neither
study did the researchers replicate the data sets to assess
compositional variability, nor did they consider the correlation of
fatty acid composition with the NPR of the organisms studied.
When S. gelidimarina was studied, there were two regions
where there was increased variation in fatty acid composition over the
growth temperature range at an aw of 0.977, as shown by the increased standard deviation of data points at 4 to 6°C and at 13°C
(Fig. 3 and 4). Similar variation was implied by the scatter of data
points in the two data sets obtained at an aw of 0.993. The
temperature at which the variations occurred was 2°C inside the
estimated boundaries of the NPR at both high and low temperatures. Furthermore, the percentage of total monounsaturated and branched-chain fatty acids was closely correlated with the response of the growth rate
to temperature, and there was little variation in the three data sets,
in contrast to the data obtained for the individual branched-chain and
monounsaturated components (Fig. 3 and 4). This implies that the
proportion of total monounsaturated and branched-chain fatty acids is
strongly regulated in response to temperature and/or cell function.
These observations also imply that the physiological roles of
branched-chain and monounsaturated fatty acids in S. gelidimarina are interchangeable; that is, at transitional
temperatures around the boundaries of the NPR, the proportions of
individual branched-chain and monounsaturated fatty acids are not
crucial to cell function as long as the total proportion is adequate.
These observations indicate that there is a physiological basis for the
NPR for bacterial growth. The fact that the deviation for the
relationship between relative cell yield and temperature is similar to
the deviation for the growth rate of Escherichia coli has
led to the suggestion that the transition of a catabolic enzyme(s) from
an active state to an inactive state may define the NPR
(31). The number of isozymes involved in the initiation of
fatty acid biosynthesis (predominantly 
-ketoacyl-acyl carrier protein synthase I [KAS-I] through KAS-III), which play an important role in branched-chain fatty acid synthesis versus monounsaturated fatty acid biosynthesis (13, 17), provides a tempting
hypothesis for the observed changes and variability in fatty acid
composition near the estimated boundaries of the NPR. It has been
suggested that the fatty acid desaturation activity and the
availability or utilization of potential fatty acid chain primers
explain the variations in the fatty acid compositions of Bacillus
subtilis and Bacillus megaterium near their temperature
extremes for growth (37). While the exact in vivo roles of
KAS isozymes in the selection of fatty acid chain primer molecules in
the genus Shewanella remain unclear, it is probable that a
number of KAS isozymes exhibit overlapping in vitro activities for the
initiation of branched-chain fatty acid synthesis versus
monounsaturated fatty acid biosynthesis (17, 38, 39). It is
also known that the KAS enzymes of E. coli (particularly
KAS-II) are thermally regulated (17), while KAS-III has
recently been identified as the central determinant of branched-chain
fatty acid production (5). In addition, other fatty acid
biosynthetic enzymes are sensitive to NaCl (15). One
possible explanation for the observed regulation of branched-chain and
monounsaturated fatty acid composition by temperature and salinity is
the function of different KAS isozymes.
Studies on the in situ effects of temperature and salinity variations
on psychrophilic and psychrotrophic populations have not been
performed. However, previous evidence and the results of this study
indicate that these physicochemical parameters may be selective. Lipid
packing and adaptational fatty acid composition responses are clearly
influenced by the temperature-salinity regimen, which may be reflected
in overall cell function characteristics, such as the growth rate and
the NPR. In future studies researchers must also address the potential
role of the frequency, duration, and rate of salinity variations in
bacterial cell function.
| |
ACKNOWLEDGMENTS |
This work was supported by the Australian Research Council (in
Australia) and by CONICET and CIC grants (in Argentina).
Tom Ross is thanked for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Agricultural Science, University of Tasmania, GPO Box 252-54, Hobart,
Tasmania 7001, Australia. Phone: 61 3 62 261831. Fax: 61 3 62 262642. E-mail: D.Nichols{at}utas.edu.au.
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Top
Abstract
Introduction
