Biol6gica Dr. "Bernabe Bloj," Chacabuco 461, 4000 San Miguel de Tiucumdn, Argentina. Received 10 December 1987/Accepted 19 May 1988. The unsaturated ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1988, p. 2107-2111
Vol. 54, No. 8
0099-2240/88/082107-05$02.00/0 Copyright C) 1988, American Society for Microbiology
Influence of Unsaturated Fatty Acid Membrane Component on Sensitivity of an Escherichia coli Fatty Acid Auxotroph to Conditions of Nutrient Depletion EDDY M. MASSA,* ALBERTO LOPEZ VINALS, AND RICARDO N. FARiAS Departamento de Bioquimica de la Nutrici6n, Instituto Superior de Investigaciones Biol6gicas, and Instituto de Quimica Biol6gica Dr. "Bernabe Bloj," Chacabuco 461, 4000 San Miguel de Tiucumdn, Argentina Received 10 December 1987/Accepted 19 May 1988
The unsaturated fatty acid auxotroph Escherichia coli AK7 was provided with either oleic acid (cis 18:1) or linolenic acid (cis 18:3) to vary the degree of unsaturation of cell membrane lipids. The susceptibility of oleic acid- and linolenic acid-grown cells to starvation at 37°C in 154 mM NaCl was compared following the decline in the number of CFU by plating the cells on agar medium. The decline in CFU was faster for linolenic acidthan for oleic acid-grown cells, but it was not indicative of cell death, since culturable CFU was recovered after respirable substrate was added to the starved cell suspension. Cell envelope microviscosity (determined by fluorescence polarization) of oleic acid- and linolenic acid-grown cells was equal in the presence of a respirable substrate, but in its absence the microviscosity of linolenic acid-grown cells was lower than that of oleic acid-grown cells. The results suggest that cell envelope microviscosity is an important factor in determining the sensitivity of E. coli to conditions of nutrient depletion.
The capacity of bacteria to survive under unfavorable conditions is of singular importance in microbial ecology. Coliform bacteria in natural aquatic environments exhibit a rapid decline when they are enumerated by the agar plate technique. However, although a die-off is indicated by plate count, neither the total number of cells nor the active cell count declines (9, 20, 28). In fact, the evidence indicates that enteric bacteria, including Escherichia coli, remain alive in environmental waters with various salinities long after they cease to be cultivable on laboratory media (9, 20, 28). The nonculturable state results from morphological and physiological changes that are considered to be an adaptation mechanism whereby bacteria survive conditions of nutrient starvation (19). In this report, we show that the sensitivity of E. coli to conditions of nutrient depletion is altered by changing the unsaturated fatty acid (UFA) membrane component. The UFA auxotroph E. coli AK7 (28) was provided with either oleic acid (cis 18:1) or linolenic acid (cis 18.3) to vary the degree of unsaturation of the cell membrane lipids. When starved at 37°C in 154 mM NaCl, linolenate-grown cells lost their ability to form colonies on agar medium faster than oleate-grown cells did. The difference appeared to be related to a change in the microviscosity of the cell envelope.
at 37°C in either minimal salt medium M (1 g of NH4Cl, 0.5 g of NaCl, 0.2 g of MgSO4 7H20, 3 g of KH2PO4, and 6 g of Na2HPO4 per liter), with 0.5% glycerol as the carbon and energy source, or in the complex medium LB (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl per liter). These media were supplemented with 0.04% polyoxyethylene-20-cetyl ether (Brij 58) and 0.01% oleate or linolenate (potassium salt), which were added from a 20% stock solution in 80% ethanol. Bacterial cultures were grown to the exponential phase (optical density at 560 nm, 0.200 to 0.400), harvested by centrifugation (6,000 x g, 10 min) at room temperature, washed 3 times, and suspended in 154 mM NaCl (optical density at 560 nm, about 0.200). Incubations. The cell suspensions were incubated at 37°C in a shaking water bath under the conditions described for each experiment (see Fig. 1 to 3 and Table 1). Anaerobic incubations were performed in Thunberg tubes, with the substrate succinate separated in the side arm until anaerobiosis was achieved by four successive cycles of evacuating the gas phase with an aspirator vacuum pump and replacing it with nitrogen bubbled through a solution of 0.1 M sodium -
sulfite. CFU. The number of CFU was determined by diluting the cellular suspensions in 154 mM NaCl and plating the samples onto LB agar (for cells which were grown on LB agar) or medium M-0.4% glycerol agar (for cells which were grown in medium M-glycerol) supplemented with 0.04% Brij 58 and 0.01% oleate or linolenate (potassium salt). The plates were incubated at 37°C for 24 to 48 h before colonies were counted. It should be noted that the fraction of CFU at a given incubation time varied from one experiment to another, and for that reason the study of oleic and linolenic acid-grown cells was carried out in parallel so that the results were comparable. The data from two independent experiments (Fig. 2) represent the general results obtained in several other experiments. Lipid hydroperoxide assay. The lipid hydroperoxide con-
MATERIALS AND METHODS Bacterial strain, growth conditions, and cell suspensions. The mutant E. coli AK7 (zfa::TnlO mutant of strain K1060 [26]) was obtained from D. de Mendoza. This strain is resistant to tetracycline and is derived from the auxotroph E. coli K1060 (fabB fadE lacl), which can neither synthesize nor degrade UFAs (6, 17, 22), and therefore, the UFA in the membrane lipids is the one that is supplied in the growth medium (10, 12, 23, 29). In cells grown at 37°C in medium supplied with oleic or linolenic acid, the content of oleate or linolenate is 52 or 37% of the total fatty acids, respectively (29). The UFA auxotroph E. coli AK7 was grown aerobically *
Corresponding author. 2107
2108
tent of the bacterial cells was determined by the iodometric assay described by Buege and Aust (1). Fluorescence polarization. Cell envelope microviscosity was estimated by fluorescence polarization measurements carried out at 37°C with a spectrofluorometer (SLM 4800). Excitation was made at 350 nm, and the emission was read through a 0-51 filter (Corning Glass Works, Corning, N.Y.). Corrections for background fluorescence and scattered light were made by using cell suspension blanks which contained no probe; these were examined under the same conditions as the samples. Fluorescence polarization (P) was calculated as P = (I - IL)/(Ill + I,), where II, and 11 represent the intensity, corrected for scatter, of the emission polarized parallel and perpendicular to the vertically polarized excitation beam, respectively. Fluorescence polarization values are expressed as the mean + standard deviation of at least six measurements. For the experiment for which the results are shown in Table 1, cells grown in the minimal medium described above were washed 3 times and suspended in medium M (without NH4Cl) at room temperature, to give an optical density at 560 nm of 0.200. The cell suspensions were fractionated into aliquots of 2 ml, which were used for the fluorescence polarization measurement. Each sample was labeled by adding 3 p.1 of 1 mM trimethylammonium diphenylhexatriene in dimethyl formamide and then preincubated for 5 min at 37°C in the thermostated cuvette holder of the spectrofluorometer, to ensure-thermal equilibrium before the fluorescence polarization measurement was performed. The additions of glyceroj, KCN, or both were done immediately before labeling.
RESULTS AND DISCUSSION Behavior of oleic acid- and linolenic acid-supplemented cells under growing conditions. Oleic cells and linolenic cells indicate cells'grown on medium supplemented with oleic acid or linolenic acid, respectively. At 37°C the growth curves of E. coli AK7 in either rich (LB medium) or minimal (medium M-0.5% glycerol) medium supplemented with linolenic acid were identical to those in medium supplemented with oleic acid. In minimal medium M with a limiting amount of the carbon and energy source (0.03% glycerol), the doubling time and growth yield (estimated from the final turbidity of the cultures) for linolenic cells were equal to those for oleic cells. Furthermore; exponential-phase cultures of E. coli AK7 supplemented with oleic or linolenic acid had the same susceptibility toward the detergent sodium dodecyl sulfate (Fig. 1), implying that the function of the outer membrane as a permeability barrier (2, 3, 7, 16) was similar for both types of cells. CFU decline under starvation conditions. Although the behaviors of oleic and linolenic cells were comparable under growing conditions, linolenic cells exhibited a faster decline in the number of CFU compared with oleic cells when starved at 37°C in 154 mM NaCI (Fig. 2). The number of CFU at each incubation time was independent of the kind of UFA provided in the agar plates, as shown in experiment 1, Fig. 2; and therefore, in all the other experiments only oleate-supplemented agar plates were used, because oleic acid is less expensive than linolenic acid. The same decline in'CFU of linolenic cells was observed when the NaCl concentration was lowered to 107 mM in order to make the osmolarity of the incubation medium equal to that of the LB growth medium (200 mosM) or when the incubation medium was 154 mM KCI instead of NaCl (data not
APPL. ENVIRON. MICROBIOL.
MASSA ET AL.
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1 2 34 5 1 2 3 4 5 Time (hours) FIG. 1. Sensitivity of oleic (A) and linolenic (B) cells to the detergent sodium dodecyl sulfate. At the points indicated by the arrows, 50% sodium dodecyl sulfate was added to exponentialphase cultures in LB medium to give final concentrations of 0% (0), 1% (x), 2.5% (@), 5% (LI), 10% (A), and 20% (A). The optical density of each culture was read periodically by pouring the culture into a steril, reading tube connected to the top of the incubation Erlenmeyer flask through a ground glass joint.
It has been reported (8) that the survival of E. 0oli in river water (determined by the plate technique on nutrient agar) is dependent on temperature, with survival at 4°C > 25°C >
37°C. The experiment for which the results are shown in Fig. 2 was also performed with cells grown at 37°C and then incubated in an ice bath for 4 h. Under these conditions, the CFU of oleic and linolenic cells remained 100%, indicating
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Incubation time (hr) FIG. 2. CFU of oleic (closed symbols) and linolenic (open symbols) cells as a function of incubation time. Cells which were grown in LB medium were incubated at 37°C in 154 mM NaCI; samples were removed at various intervals, diluted, and plated. The CFU at time zero was taken as 100%. The data from two independent experiments are shown. In experiment 1 (solid lines), oleic and linolenic cells were plated onto medium supplemented with either oleate (circles) or linolenate (triangles). In experiment 2 (broken lines), oleic and linolenic cells were plated only onto medium supplemented with oleate.
Vol. 54. 1988
E. COLI MEMBRANE FATTY ACIDS AND STARVATION-SURVIVAL
that the phenomenon shown in Fig. 2 is temperature dependent. Lipid peroxidation. Since the rate of CFU decline increased with increasing unsaturation of the fatty acid supplement, as does the propensity to lipid peroxidation (14. 27), assays of potential lipid oxidation products were performed with linolenic cells (see above). No increase in the lipid hydroperoxide content was observed after 4 h of cell incubation under the conditions of the experiment (Fig. 2). and therefore, the CFU loss was not related to the peroxidative damage of membrane UFAs. CFU in the presence of a substrate. No decline in the number of CFU was observed when oleic and linolenic cells were incubated at 37°C for 3 h in 154 mM NaCl in the presence of sodium succinate. To test whether succinate must be metabolized in order to prevent the CFU decline, linolenic cells that were grown on LB medium were incubated at 37°C in 154 mM NaCl for 3 h in the presence and absence of 20 mM sodium succinate under aerobic and anaerobic conditions (see above). The CFU was 2 and 5% for cells incubated in the absence of the substrate under aerobic and anaerobic conditions, respectively. The CFU remained at 100% in the presence of succinate under aerobic conditions, but anaerobic conditions greatly blocked the effect of succinate, since in this case the CFU was 20% (the block was incomplete, possibly because of traces of air remaining in the system since anaerobiosis was not strictly controlled). These results indicate that the loss of colonyforming ability is prevented or delayed when the cells are able to respire (to oxidize the substrate). Recovery of CFU. Coliforms starved in environmental waters with various salinities rapidly evolve toward a viable but nonculturable state, as demonstrated previously for Vibr-io cholerae (28), Salmnoniella en,teritidis (20), and E. coli (9, 28). Recovery of culturability on solid medium was possible by the addition of nutrients within a short period after apparent die-off (0 CFU/ml), as estimated by plate count techniques (20). However, longer periods of exposure to the aquatic environments appear to require conditions other than simple nutrient addition for the resumption of cell growth and division (9, 20). It was interesting to test whether linolenic cells which lost their colony-forming ability during incubation in 154 mM NaCl could recover their culturability in the presence of a substrate. The experiment was performed with cells grown in medium M-glycerol, to induce the enzymatic systems involved in the metabolism of the substrate glycerol (15), and the number of CFU was determined by plating the cells on medium M-glycerol agar supplemented with oleate. The results are summarized in Fig. 3. Loss of the colony-forming ability was prevented when glycerol was added to the medium at the beginning of the incubation. Furthermore, when glycerol was added to the medium after 3 h of incubation, at which time the CFU was only 0.4%, CFU were recovered in 30 min (but not immediately after glycerol addition). Respiration of a substrate seemed to be required in order to maintain or recover the colony-forming ability, since 5 mM KCN blocked the effect of added glycerol and also increased the rate of CFU decline in the absence of an external substrate, possibly by inhibiting the oxidation of endogenous substrates. The data presented in Fig. 3 indicate that although most of the cells lost their colony-forming ability during the 3 h of incubation in 154 mM NaCl, they did not die and were able to recover their culturability in the presence of an oxidizable substrate. These results raise an important question. Glycerol added to the starved cell sus-
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Incubation Time (hours) FIG. 3. Recovery of CFU in the presence of glycerol. Linolenic cells which were grown in medium M-glycerol were incubated at 37°C in 154 mM NaCI in the presence (0) or absence (a) of 0.4%c glycerol. The arrows indicate the time of glycerol addition. Samples were removed to determine the number of CFU at 0. 3, and 3.5 h of
incubation.
pension restored culturability on medium M-glycerol agar plates. Why, then, do starved cells fail to grow on plates of medium M plus glycerol? Since the agar plates were routinely supplemented with potassium oleate-Brij 58, we investigated whether the loss of culturability of starved cells is related to the presence of the detergent Brij 58 in the agar plates or whether starved cells are unable to recover their colony-forming ability in the presence of the detergent. Both of these possibilities were ruled out by an experiment, the protocol of which was similar to that of the experiment shown in Fig. 3. in which (i) starved linolenic cells plated onto M-glycerol medium supplemented with oleate but without Brij 58 presented the same decline in CFU observed with the agar plates containing Brij 58, and (ii) the addition of 0.04% Brij 58 or 0.04%Y Brij 58-0.01% potassium oleate just before the addition of glycerol to the starved cell suspension did not prevent the recovery of CFU. Therefore, the phenomenon is independent of the presence of the detergent Brij 58. At this time we do not have any explanation for the data shown in Fig. 3. The answer to this question will provide useful information regarding the loss of culturability and the starvation response. Cell envelope microviscosity. Manipulation of the unsaturated fatty acyl chain component of the membrane may lead to changes in membrane fluidity (4; D. de Mendoza and R. N. Farias, in R. C. Aloia, C. C. Curtain, and L. M. Gordon, ed., Advances in Membrlane Fluidity, vol. 3, in press). It has been reported (5) that the microviscosity of the UFA auxotroph E. (oli K1060 was lower when the UFA membrane component was linolenate than when it was oleate, which was estimated from the extent of fluorescence polarization of the probe molecule 1,6-diphenyl-1,3,5,-hexatriene dissolved in the membrane. We performed fluorescence polarization measurements with our oleic and linolenic cells, but 1,6diphenyl-1,3,5-hexatriene did not incorporate into the cells (since no increase in fluorescence intensity was observed compared with blanks for scattered light) that were incubated with the probe for 1 to 2 h at 37°C. Therefore, we used the hydrophobic fluorescent probe trimethylammonium diphenylhexatriene, which incorporates very rapidly and has specific localization in the plasma membranes of whole living cells (13). The fluorescence polarization value for linolenic cells was lower than that for oleic cells in the absence of added substrate, but became equal to that for oleic cells in
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MASSA ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Fluorescence polarization of trimethylammonium diphenylhexatriene incorporated into oleic and linolenic cells" Fluorescence polarization (P) of:
Addition
Oleic cells
None Glycerol (0.4%) Glycerol (0.4%)-KCN (5 mM) KCN (5 mM)
0.353 0.362 0.322 0.315
± ± ± ±
0.013 0.010 0.014 0.011
6.
Linolenic cells
0.316 0.356 0.315 0.325
± ± ± ±
0.016 0.020 0.015 0.013
a Conditions were as described in the text.
7.
8. 9.
the presence of glycerol (Table 1). KCN prevented the effect of glycerol on the fluorescence polarization value of linolenic cells and decreased the fluorescence polarization value of oleic cells. The difference of 10% between the fluorescence polarization of oleic and linolenic cells may reflect significant changes in microviscosity, since in paraffin oil a 7% change in fluorescence polarization corresponds to a viscosity change of about 20% (21), and variations of this magnitude in membrane microviscosity have been shown to result in large functional changes (11, 18). The data in Table 1 are interesting, since they demonstrate that cell envelope microviscosity depends on metabolic conditions and show that the microviscosities of oleic and
linolenic cells are equal in the presence of a respirable substrate but are different in its absence. This fact may be connected to our observation that the behavior of oleic and linolenic cells is comparable under growing conditions but not under starvation conditions. From the data in Table 1, at the beginning of the incubation at 37°C and in the absence of substrates, when the CFU was 100%, the microviscosity of linolenic cells was lower than that of oleic cells; this might determine the difference in the kinetics of the loss of colony-forming ability. It is reasonable to presume that the altered physical properties of the lipid matrix lead to changes in protein-lipid interactions, which would, in turn, modify the membrane processes associated with cell homeostasis. Membrane lipid composition or microviscosity has been implicated in many bacterial processes (31) and seems to play a major role in determining the sensitivity of E. coli to hyperthermia (5), hyperoxia (12), and irradiation (24, 25, 29, 30). Our data suggest that membrane microviscosity is involved in the response of bacterial cells to conditions of nutrient limitation. ACKNOWLEDGMENTS This study was supported by a grant from Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) and Consejo de Ciencia y Tdcnica de la Universidad Nacional de Tucuman. A. L6pez Vinials is a fellow and R. N. Farfas and E. M. Massa are career investigators of CONICET.
10. 11.
12.
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16. 17. 18.
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nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8:313-323. 29. Yatvin, M. B., J. J. Gipp, and W. H. Dennis. 1979. Influence of unsaturated fatty acids, membrane fluidity and oxygenation on the survival of an E. coli fatty acid auxotroph following yirradiation. Int. J. Radiat. Biol. 35:539-548.
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