Vol. 57, No. 1
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1991, p. 272-276 0099-2240/91/010272-05$02.00/0 Copyright C 1991, American Society for Microbiology
Intracellular Accumulation of Potassium and Glutamate Specifically Enhances Survival of Escherichia coli in Seawater MICHEL J. GAUTHIER,'* GILLES N. FLATAU,1 DANIEL LE RUDULIER,2 RENE L. CLEMENT,' AND MARIA-PILAR COMBARRO COMBARRO3 Institut National de la Sante et de la Recherche Medicale, U. 303, 1 avenue Jean Lorrain, 06300 Nice,1 and Laboratoire de Biologie Vegetale et Microbiologie, Faculte des Sciences et des Techniques, Universite de Nice-Sophia Antipolis, Parc Valrose, 06034 Nice,2 France, and Department of Microbiology and Parasitology, University of Santiago de Compostela,
Santiago de Compostela, Spain3 Received 6 September 1990/Accepted 5 November 1990
The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a signfficant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments. In oligotrophic seawater, enteric bacteria rapidly lose their ability to grow on bacteriological media with a low osmolarity (7, 11, 31). This loss in culture ability is generally, though perhaps incorrectly, considered to reflect the overall decrease in survival of the cells in seawater. Loss of culturability can be temporarily blocked by preventing the osmotic down-shock when counting viable cells either through the use of saline culture media (11) or through the addition of glycine betaine (GB) to seawater samples (28) before counting, even though the protection provided by GB is probably limited because of the inhibition of ProP (low affinity) and ProU (high affinity) GB transport systems in nutrient-free seawater (13). Recent studies have shown that the evolution of enteric bacteria towards a nonculturable state in seawater partly depends on the conditions under which they were grown or maintained prior to their transfer to seawater (12). Thus, cells preadapted to high osmolarity are highly resistant to seawater, which emphasizes the fundamental role of osmoregulating mechanisms in their survival under marine conditions (23). It is therefore important to better understand
During the treatment, particular emphasis was placed on variations in cellular potassium and glutamate, whose roles in osmoregulation are well known (see reviews in references 3, 8, and 16). MATERIALS AND METHODS Bacterial strain and growth conditions. All tests were performed with Escherichia coli MC4100, a strain derived from E. coli K-12 (5). Stock cultures were maintained in liquid nitrogen. For survival tests, cells were grown in mineral medium M9 (19) supplemented with NaCl (0.5 M) (Na cells) or NaCl (0.5 M) and GB (2 mM) (NaGB cells) or not supplemented (A cells). Growth was checked spectrophotometrically at 600 nm with a Uvikon 720LC spectrophotometer (Kontron, Paris, France) and if necessary converted to bacterial dry weight according to a calibration curve. Mineral chemicals were from Carlo Erba (Farmitalia, Paris, France), and organic chemicals were from Sigma Chemical Co. (St. Louis, Mo.). Survival tests. Survival tests were performed in microcosms made up of 250-ml Erlenmeyer flasks containing 100 ml of natural seawater collected along a rocky shore (Cape of Nice, France) and filtered through membranes (pore size, 0.22 ,m; Millipore Corp., Bedford, Mass.). Microcosms were inoculated with A, Na, or NaGB cells. Cells were harvested at the end of the logarithmic phase of growth (Awo = 0.6) and washed three times by centrifugation (10,000 x g, 20 min, 20°C) with 30 ml of distilled water (pH 7.4 with 0.01 N NaOH), distilled water supplemented with KCI (5 or 80 mM), or seawater. In some experiments, A, Na, and NaGB cells were submitted to a rapid osmotic down-shock in distilled water (for 20 min) and then washed twice (20 min
the cellular processes controlling this resistance. Furthermore, the conditions experienced by cells prior to their arrival in the sea need to be considered. For example, during their transport in wastewater, they could undergo structural or metabolic modifications due to osmotic down-shock which might significantly influence their later behavior in seawater. The goal of the present study was to examine the consequences of an osmotic down-shock on cells grown at low or high osmolarity and their subsequent behavior in seawater. *
Corresponding author. 272
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K+ AND GLUTAMATE ENHANCE E. COLI SURVIVAL IN SEAWATER
each) with KCl solutions (5
or
80 mM)
or seawater.
Other
TABLE 1. Effect of osmotic down-shock on potassium, glutamate, and Dragendorff-positive compound contents of E. coli MC4100 cellsa
tests were performed with Na cells submitted to osmotic
down-shock and washed for 40 to 60 min in KCI (80 mM) solutions containing glutamate (1, 10, or 50 mM) or an analogous substrate: glutamine, glutarate, aspartate, or succinate (50 mM) (Sigma). After being washed, cell pellets were finally suspended in 2 ml of sterile seawater, and microcosms were inoculated with these cell suspensions to a density of 1 x 106 to 5 x 106 CFU/ml. They were then incubated in the dark at room temperature (23 to 25°C). Culturable-cell counts. All microcosms were sampled immediately after inoculation (To) and then daily during the following 6 or 7 days. Bacterial counts were made in triplicate by the membrane filtration technique (Millipore filters; pore size, 0.45 ,im) on nutrient agar (Difco Laboratories, Detroit, Mich.). The number of CFU was counted after a 24-h incubation at 37°C. For each experiment, the viability loss of cells was expressed as the following ratios: CFU at To to CFU after 2 days and CFU at To to CFU after 6 or 7 days of incubation. Since they derived from numbers of culturable cells, these ratios could however restrictively represent loss of culturability, depending on whether unculturable cells were dead or had evolved towards the viable but nonculturable state (7, 31). Culturable-cell counts performed during this study do not provide any information on this point. Potassium measurements. The K+ content of cells was measured by the method of Meury et al. (21). Aliquots of cell suspensions were filtered on Millipore filters (pore size, 0.45 ,um) and washed with NaCl solutions containing 5 g more of NaCl per liter than that in the filtered suspension. Filters were placed in glass tubes filled with 5 ml of 0.1 M HNO3. K+ was determined with an atomic absorption spectrophotometer (Philips PU 9200). Results were expressed as micromoles of K+ per gram of dried cells. Potassium concentration was also measured in supernatants from cells washed with distilled water and was expressed as micromoles of K+ per milliliter of supernatant. Glutamate measurements. The glutamate content of cells was measured by using the enzymatic assay described by Beutler and Michal (2). Results were expressed as micromoles of glutamate per gram of dried cells. Protein measurements. The protein content of cells was measured by the method of Lowry et al. (17) after their complete solubilization by heating to 90°C for 10 min in 1 M NaOH. The standard curve was established with bovine serum albumin.
273
Amt of the following in E. coli cells:
Growth medium
WahDaedff Potassium Wash Glutamate Dragendorif(p.mol/gb) positive (Rmol/gb)
M9
Seawater Distilled water
14.5 7.2
267 86
M9Na
Seawater Distilled water
27 6.4
4,278 400
M9NaGB
Seawater Distilled water
22.6 9
315 102
compounds 0.055 ND
(Rf)'
0.032 ND 0.055, 0.24 (GB)d ND
a Cells were previously grown in M9 medium supplemented with NaCl and GB or in unsupplemented M9 medium. b Dried cells. ' From thin-layer chromatograms on silica gel plates. ND, Not detected. d Two spots were detected, one of them being glycine betaine (GB).
Thin-layer chromatography of cell extracts. The presence of Dragendorff-positive compounds in A, Na, and NaGB cells before and after osmotic down-shock was monitored by thin-layer chromatography. Cell pellets (30 to 50 mg, dry weight) were extracted three times with 10 ml of ethanol (70% in distilled water). Ethanolic extracts were dried and fractionated on silica gel plates (60F 254 Kieselgel; Merck, Darmstadt, Federal Republic of Germany; thickness, 0.25 mm), with ascending chromatography in methanol-acetoneformic acid-hydrochloride (90:10:20:0.25). Plates were then sprayed with Dragendorff reagent (Sigma); Dragendorffpositive compounds appear orange on a yellow background. Reproducibility and statistical analysis of results. All tests were performed at least in duplicate, and some were performed in triplicate. A Student's t test (29) was performed in some cases to define the significance level of observed differences. RESULTS
Effects of osmotic down-shock on intracellular solutes. When submitted to an osmotic down-shock, E. coli MC4100
TABLE 2. Potassium content and viability loss in seawater of E. coli MC4100' Na cells
A cells Wash
K+ content
(pLmol/g)b
Seawaterd
8
Viability loss after': 7 days 6,530 110,000
2 days
K+
Viability loss afterc:
content
(p.mol/g)b
13,400 4,730
21
2 days 1.1
7 days 12
2.8 156 135,000 143,000 9 145 122,000 173,000 29 122 138 87,000 128,000 11.5 54 51 59,000 2,545 a E. coli MC4100 was grown in M9 medium at low (A cells) or high (Na cells) osmolarity (0.5 M NaCI), submitted to an osmotic down-shock (distilled water,
Distilled water KCl (5 mM) KCl (80 mM) Seawater
6.7 10.2 31.3 13.3
20 min), and then washed with distilled water, KCI solutions, or seawater. b In washed cells (dry weight) before transfer to seawater microcosm. c Viability loss is defined as the ratio of CFU at To to CFU after 2 or 7 days of incubation in seawater. d Without previous osmotic down-shock (reference).
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274
TABLE 4. Effect of osmotic down-shock and reload with potassium and glutamate on subsequent survival in seawater of E. coli MC4100 previously grown at high osmolarity (Na cells) Viability loss after":
TABLE 3. Loss of viability in seawater of E. coli MC4100' Mean viability loss (±SD) afterb:
Temperature ('C) of wash
2
days
6
days
400 (± 113) 50 (± 21) 14 (± 9.6)
2.7 (± 1) 1.9 (± 2) 1.3 (± 1.6)
4 20 37
a E. coli MC4100 was grown in M9 medium and washed with seawater (45 min) at different temperatures before transfer to seawater. b See Table 2, footnote c.
cells grown in M9A, M9Na, and M9NaGB media showed significant losses of intracellular K+ and glutamate (Table 1). K+ loss was higher for Na cells (76%) than for A or NaGB cells (50 and 60%, respectively). Na cells initially contained more K+ than the other two cell types because of the increase of K+ transport at high osmolarity (9) and the K+ release after accumulation of GB (3). In the same way, glutamate loss was higher for Na cells (90%) than for other cells (68%). Osmotic down-shock also led to a loss of Dragendorff-positive compounds, including GB accumulated in NaGB cells, as previously observed by Poccard and Le Rudulier (24a). Influence of osmotic down-shock and potassium accumulation on survival in seawater. The behavior of A and Na cells in seawater was highly influenced by the osmotic downshock and by the washing technique used prior to their transfer into seawater (Table 2). As previously reported (11, 23), A cells were much more sensitive to seawater than Na cells even when they had not undergone osmotic shock (reference cells). After the osmotic down-shock, they were slightly more sensitive, and washing in seawater or in KCI solutions significantly increased their resistance. With regard to the viability loss of A and Na cells not previously submitted to osmotic down-shock, the effect of hypoosmotic stress was much higher on Na cells, which lost most of their resistance to seawater after a 20-min wash in distilled water. Subsequent washing in 80 mM KCl or in seawater resulted in the partial recovery of this resistance. For both cell types, increased resistance was linked to an increase in intracellular K+ concentration. A loss of intracellular K+ could explain the increase in the sensitivity of cells washed at 4°C (Table 3), since E. coli cells lose a high amount of K+ at low temperature (10). Influence of glutamate accumulation on survival. Washing
- 60- A
Wash 2 days
Seawaterb Distilled water KCI (80 mM) + 1 mM glutamate + 10 mM glutamate + 50 mM glutamate
7 days
1
19
386
170,000
38 41 7
3,970 2,586 30
Table 2, footnote c. "See b
Reference cells not submitted to previous osmotic down-shock.
Na cells sensitized by osmotic down-shock in KCI solutions (80 mM) containing increasing concentrations of glutamate before transfer to seawater led to the almost total recovery of their resistance to seawater at the highest glutamate concentration (50 mM) (Table 4). This recovery could depend on the speed at which these two compounds accumulate, compared with wash time. This speed was then measured in sensitized Na cells suspended in a KCI (80 mM)glutamate (50 mM) solution over increasing periods prior to their transfer to seawater. K+ transport was rapid initially, possibly according to a logarithmic function of incubation time (Fig. 1). Glutamate was accumulated more slowly and linearly with time. The protection provided by a de novo accumulation of K+ in Na cells after osmotic down-shock was exponentially linked to the concentration of this ion in cells before their transfer to seawater (Fig. 2). This relationship was significant after both 2 (P c 0.05) and 7 (P c 0.01) days in seawater. Such a relationship was not significant for glutamate even though this compound was obviously required to achieve maximum resistance (Table 4). In fact, it has been shown that glutamate is synthesized by E. coli as a counterion in response to intracellular accumulation of K+, whose transport is activated at high osmolarity (3). This condition was obviously not achieved in our experiments, as reload of cells with K+ and glutamate was performed at low osmolarity for an arbitrary ratio of K+ to glutamate, which could explain the lack of significant correlation observed between the protection provided by K+ and that provided by
glutamate.
-@6000-
B
-o
=.
40-
202
30-
E
E
E U
~.4000-
co
2000
20 a
10
CD
r = 0.996
Can. [ ,01
,1
I
Hours
10
-
~~~~~~~~r=0.996
Cu 0
Hours
FIG. 1. K+ (A) and glutamate (B) accumulation with time by E. coli MC4100 grown in M9Na medium, submitted to osmotic down-shock in distilled water, and suspended in a KCI (80 mM)-glutamate (50 mM) solution.
K+ AND GLUTAMATE ENHANCE E. COLI SURVIVAL IN SEAWATER
VOL. 57, 1991
275
TABLE 5. Efficiency of different monovalent cations, glutamate, and glutamate analogs in restoring resistance to seawater
in E. coli MC4100' 4o Co10 0
103
r
0.980
100
10
1 0
20
3'0 4'0 (gmol/g dry cells)
.50
Intracellular potassium FIG. 2. Loss of viability in seawater, after 2- and 7-day incubation periods, of E. coli MC4100 grown in M9Na medium, submitted to osmotic down-shock, and provided with different concentrations of K+ ions in the suspension medium. Arrows indicate the viability loss for Na cells not submitted to osmotic down-shock after 2 (A) and 7 (B) days in seawater.
Specificity of K+ and glutamate in cell protection. The specificity of K+ and glutamate in restoring the protection lost after osmotic down-shock in Na cells was tested by replacing one or the other of these elements by analogous compounds: sodium (Na+) and rubidium (Rb+) for K+ and glutamine, glutarate, aspartate, and succinate for glutamate (Table 5). Neither Na+ nor Rb+ was able to provide similar resistance. In the same way, none of the glutamate analogs could replace glutamate to restore resistance in cells after osmotic down-shock. DISCUSSION Apart from the antagonistic activity of purification factors less specific to marine environments (see reviews in references 1 and 6), survival of E. coli in seawater primarily depends on its capacity to overcome osmotic shock (11, 23) and nutrient starvation (15). Survival is strongly influenced by previous growth conditions and the physiological state of cells during transfer to seawater (12). Thus, the history of their growth in the enteric environment and in wastewaters could be important. It has been previously demonstrated that preadaptation to a high osmolarity provides E. coli with a high level of resistance to seawater (23). The present results show that this resistance is mainly due to the intracellular accumulation of compounds transported or synthesized at high osmolarity which are rapidly eliminated by osmotic down-shock. Many cell components, such as periplasmic proteins (24) or mineral and organic cytoplasmic elements (16, 21, 22), are released by E. coli when a sharp drop in external osmotic pressure occurs. Some of these compounds are directly linked to regulation of osmotic balance, e.g., K+ (10, 21, 22) and GB (16). Our results show that intracellular glutamate concentration is also sensitive to osmotic shock and decreased in E. coli at low osmolarity. The K+ content of cells at the time of their transfer to seawater is a determinant in their later survival in this fluid. We have previously reported the protective role played by Trk and Kdp K+ transport systems on this bacterium (12). The highly resistant Na cells have a high K+ content, and the loss of K+ during distilled water shock is followed by a drastic loss of resistance. Such sensitized cells partly remore or
Viability loss in seawater afterc:
Preincubation (40 min) solutionsb
2 days
7 days
Seawater Distilled water
1.2 9
12 128,000
KCI-glutamate NaCI-glutamate RbCl-glutamate
3 8 7
54 3,100 2,540
KCI-aspartate KCI-glutamine KCI-glutarate KCI-succinate
4.7 3.8 5.5 3.7
5,640 3,540 2,650 2,890
a E. coli MC4100 was previously grown at high osmolarity (M9Na medium) and submitted to osmotic down-shock (distilled water, 20 min). b Cells were first washed for 20 min in distilled water and then for 40 min in distilled water containing monovalent cations (80 mM) and glutamate or analogs (50 mM), except for cells washed in seawater, which were not previously submitted to osmotic down-shock. c See Table 2, footnote c.
cover their resistance by increasing their K+ intracellular concentration. It is noteworthy that the protective effect is specific to K+; Na+ and Rb+ are not able to protect cells from decay in seawater. This could be due to their intrinsic inefficacy in maintaining cell homeostasis in seawater or result from the lack of transport of these ions by E. coli at high osmolarity (10). It is well known that E. coli cells regulate their turgor pressure with rapid K+ influx and efflux following changes of external osmolarity (21, 22). However, at high concentrations, K+ is not compatible with normal
metabolism since it modifies cytoplasmic pH and ionic strength (3, 8). This could explain the transient aspect of protection afforded both A and Na cells by washing in KCI solutions (Table 2) and the complementary effect of glutamate. In most procaryotes examined, the intracellular concentration of glutamate also increases in response to hyperosmotic shock (20, 26, 30). According to Tempest et al. (30),
activation of glutamate synthesis by glutamate dehydrogenase at high osmolarity causes a release of protons and an alkalinization of the cytoplasm. Its synthesis through glutamate synthase is also stimulated by high concentrations of K+, and it has been suggested that this ion is a signal for the regulation of glutamate synthesis during osmotic stress (20). In any event, accumulation of glutamate helps to reestablish cellular homeostasis in E. coli at high osmolarity by maintaining the membrane potential through neutralization of a part of accumulated K+ ions. Furthermore, potassium glutamate has been considered as a signal for the activation of several systems involved in the osmotic response of enteric bacteria (4, 25). Influence of glutamate on cell survival in seawater as observed in this study concurs with data from the literature. It appears very efficient and completes the protection provided by K+ by almost totally restoring the resistance lost by Na cells during hypoosmotic shock and partially restored by K+. It is currently being studied whether this influence could result from a better regulation of cellular pH. The protective effect seems specific to glutamate, but it is possible that the ineffectiveness of structural analogs used in our experiments was due to the lack of their transport under the selected experimental
GAUTHIER ET AL.
276
conditions, since their intracellular accumulation was not verified. Practically, this work demonstrates that in vitro studies on survival of enteric bacteria in seawater by using microcosms require a rigorous control of cell washout conditions, particularly for K+ concentrations in the liquids or buffers used. In addition, it does not appear advisable to wash cells at a low temperature (4°C), as is frequently done in such experiments.
From an environmental point of view, these results suggest that the K+ and glutamate contents of media in which E. coli cells are grown and transported to the sea can influence their subsequent survival in marine environments. It is also possible that the presence of glutamate in some marine compartments such as sediments or eutrophic waters (20, 30), together with the relatively high potassium content of seawater (10 mM) (18), favors survival of enteric bacteria, as has already been suggested for GB and other organic osmolytes (13, 14). It remains to be proven, however, whether this amino acid is actually transported by enteric bacteria in this environment as has already been demonstrated for GB (13). This is worth investigating since it has been shown that osmotic stress inhibits transport of carbohydrates in E. coli (27). Another source of glutamate could be its in situ synthesis from natural nutritive substrates, but this also remains to be proven under marine conditions. Additional studies are thus necessary to understand and objectively evaluate the influence of K+ and glutamate on survival of E.
coli in natural marine environments. ACKNOWLEDGMENTS We thank H. Olagnero for her technical assistance. This work was partly supported by grants from the Institut Franqais de Recherche pour l'Exploitation de la Mer (IFREMER, Paris, France) and from the World Health Organization within the framework of the Long-Term Program of Pollution Monitoring and Research in the Mediterranean Sea (MED POL Phase II). REFERENCES 1. Aubert, M., M. J. Gauthier, J. Aubert, and P. Bernard. 1980. Les systemes d'information des micro-organismes marins. Rev. Int, Oceanogr. Med. 60-61:37-106. 2. Beutler, H. O., and G. Michal. 1974. Colorimetric method for the determination of L-glutamic acid in foodstuffs, p. 1708-1713. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press, Inc., New York. 3. Booth, I. R., J. Cairney, L. Sutherland, and C. F. Higgins. 1988. Enteric bacteria and osmotic stress: an integrated homeostatic system. J. Appl. Microbiol. Symp. Suppl. 17:35S-49S. 4. Booth, I. R., and C. F. Higgins. 1990. Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress? FEMS Microbiol. Rev. 75:239-246. 5. Casadaban, M. J. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104:541-555. 6. Chamberlin, C. E., and R. Mitchell. 1978. A decay model for enteric bacteria in natural waters, p. 325-348. In R. Mitchell (ed.), Water pollution microbiology. John Wiley & Sons, Inc., New York. 7. Colwell, R. R. 1987. From counts to clones. J. Appl. Bacteriol.
Symp. Suppl. 63:1S-6S. 8. Csonka, L. N. 1989. Physiological and genetic responses of bacteria to osmotic stress. Microbiol. Rev. 53:121-147. 9. Epstein, W. 1986. Osmoregulation by potassium transport in Escherichia coli. FEMS Microbiol. Rev. 39:73-78. 10. Epstein, W., and S. G. Schultz. 1965. Cation transport in Escherichia coli. V. Regulation of cation content. J. Gen.
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APPL. ENVIRON. MICROBIOL. 11. Gauthier, M. J., P. M. Munro, and S. Mohajer. 1987. Influence of salts and sodium chloride on the recovery of Escherichia coli from seawater. Curr. Microbiol. 15:5-10. 12. Gauthier, M. J., P. M. Munro, and V. A. Breittmayer. 1989. Influence of prior growth conditions on low nutrient response of Escherichia coli in seawater. Can. J. Microbiol. 35:379-383. 13. Gauthier, M. J., and D. Le Rudulier. 1990. Survival in seawater of Escherichia coli cells grown in marine sediments containing glycine betaine. Appl. Environ. Microbiol. 56:2915-2918. 14. Ghoul, M., T. Bernard, and M. Cormier. 1990. Evidence that Escherichia coli accumulates glycine betaine from marine sediments. Appl. Environ. Microbiol. 56:551-554. 15. Kjelleberg, S., M. Hermanson, and P. Marden. 1987. The transient phase between growth and nongrowth of heterotrophic bacteria with emphasis of the marine environment. Annu. Rev. Microbiol. 41:25-49. 16. Le Rudulier, D., A. R. Strom, A. M. Dandekar, L. T. Smith, and R. C. Valentine. 1984. Molecular biology of osmoregulation. Science 224:1064-1068. 17. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 18. Lyman, J., and R. H. Fleming. 1940. Composition of seawater. J. Mar. Res. 3:134-146. 19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual, p. 86-95. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. Measures, J. C. 1975. Role of amino acids in osmoregulation of nonhalophylic bacteria. Nature (London) 257:398-400. 21. Meury, J., A. Robin, and P. Monnier-Champeix. 1985. Turgorcontrolled K+ fluxes and their pathways in Escherichia coli. Eur. J. Biochem. 151:613-619. 22. Meury, J., and A. Kepes. 1981. The regulation of potassium fluxes for the adjustment and maintenance of potassium levels in Escherichia coli. Eur. J. Biochem. 119:165-170. 23. Munro, P. M., M. J. Gauthier, V. A. Breittmayer, and J. Bongiovanni. 1989. Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater. Appl. Environ. Microbiol. 55:2017-2024. 24. Oliver, D. B. 1987. Periplasm and protein secretion, p. 56-69. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. 24a.Poccard, J. A., and D. Le Rudulier. Personal communication. 25. Ramirez, R. M., W. S. Prince, E. Bremer, and M. Villarejo. 1989. In vitro reconstitution of osmoregulated expression of proU of Escherichia coli. Proc. Natl. Acad. Sci. USA 86:11531157. 26. Richey, B., D. S. Cayley, M. C. Mossing, C. Kolka, C. F. Anderson, T. C. Ferrar, and M. T. Record. 1987. Variability in the intracellular ionic environment of Escherichia coli: differences between in vitro and in vivo effect of ion concentrations on protein-DNA interactions and gene expression. J. Biol. Chem. 262:7157-7164. 27. Roth, W. G., M. P. Leckie, and D. N. Dietzler. 1985. Osmotic stress drastically inhibits active transport of carbohydrates by Escherichia coli. Biochem. Biophys. Res. Commun. 126:434441. 28. Roth, W. G., M. P. Leckie, and D. N. Dietzler. 1989. Restoration of colony-forming activity in osmotically stressed Escherichia coli by betaine. Appl. Environ. Microbiol. 54:3142-3146. 29. Schwartz, D. 1980. Methodes statistiques A l'usage des medecins et des biologistes, p. 173-187. Flammarion, Paris. 30. Tempest, D. W., J. L. Meers, and C. M. Brown. 1970. Influence of environment on the content and composition of microbial free amino acid pools. J. Gen. Microbiol. 64:171-185. 31. Xu, H.-S., N. Roberts, F. L. Singleton, R. W. Atwell, D. J. Grimes, and R. R. Colwell. 1982. Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb. Ecol. 8:313-323.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1991, p. 272-276 0099-2240/91/010272-05$02.00/0 Copyright C 1991, American Society for Microbiology
Intracellular Accumulation of Potassium and Glutamate Specifically Enhances Survival of Escherichia coli in Seawater MICHEL J. GAUTHIER,'* GILLES N. FLATAU,1 DANIEL LE RUDULIER,2 RENE L. CLEMENT,' AND MARIA-PILAR COMBARRO COMBARRO3 Institut National de la Sante et de la Recherche Medicale, U. 303, 1 avenue Jean Lorrain, 06300 Nice,1 and Laboratoire de Biologie Vegetale et Microbiologie, Faculte des Sciences et des Techniques, Universite de Nice-Sophia Antipolis, Parc Valrose, 06034 Nice,2 France, and Department of Microbiology and Parasitology, University of Santiago de Compostela,
Santiago de Compostela, Spain3 Received 6 September 1990/Accepted 5 November 1990
The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a signfficant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments. In oligotrophic seawater, enteric bacteria rapidly lose their ability to grow on bacteriological media with a low osmolarity (7, 11, 31). This loss in culture ability is generally, though perhaps incorrectly, considered to reflect the overall decrease in survival of the cells in seawater. Loss of culturability can be temporarily blocked by preventing the osmotic down-shock when counting viable cells either through the use of saline culture media (11) or through the addition of glycine betaine (GB) to seawater samples (28) before counting, even though the protection provided by GB is probably limited because of the inhibition of ProP (low affinity) and ProU (high affinity) GB transport systems in nutrient-free seawater (13). Recent studies have shown that the evolution of enteric bacteria towards a nonculturable state in seawater partly depends on the conditions under which they were grown or maintained prior to their transfer to seawater (12). Thus, cells preadapted to high osmolarity are highly resistant to seawater, which emphasizes the fundamental role of osmoregulating mechanisms in their survival under marine conditions (23). It is therefore important to better understand
During the treatment, particular emphasis was placed on variations in cellular potassium and glutamate, whose roles in osmoregulation are well known (see reviews in references 3, 8, and 16). MATERIALS AND METHODS Bacterial strain and growth conditions. All tests were performed with Escherichia coli MC4100, a strain derived from E. coli K-12 (5). Stock cultures were maintained in liquid nitrogen. For survival tests, cells were grown in mineral medium M9 (19) supplemented with NaCl (0.5 M) (Na cells) or NaCl (0.5 M) and GB (2 mM) (NaGB cells) or not supplemented (A cells). Growth was checked spectrophotometrically at 600 nm with a Uvikon 720LC spectrophotometer (Kontron, Paris, France) and if necessary converted to bacterial dry weight according to a calibration curve. Mineral chemicals were from Carlo Erba (Farmitalia, Paris, France), and organic chemicals were from Sigma Chemical Co. (St. Louis, Mo.). Survival tests. Survival tests were performed in microcosms made up of 250-ml Erlenmeyer flasks containing 100 ml of natural seawater collected along a rocky shore (Cape of Nice, France) and filtered through membranes (pore size, 0.22 ,m; Millipore Corp., Bedford, Mass.). Microcosms were inoculated with A, Na, or NaGB cells. Cells were harvested at the end of the logarithmic phase of growth (Awo = 0.6) and washed three times by centrifugation (10,000 x g, 20 min, 20°C) with 30 ml of distilled water (pH 7.4 with 0.01 N NaOH), distilled water supplemented with KCI (5 or 80 mM), or seawater. In some experiments, A, Na, and NaGB cells were submitted to a rapid osmotic down-shock in distilled water (for 20 min) and then washed twice (20 min
the cellular processes controlling this resistance. Furthermore, the conditions experienced by cells prior to their arrival in the sea need to be considered. For example, during their transport in wastewater, they could undergo structural or metabolic modifications due to osmotic down-shock which might significantly influence their later behavior in seawater. The goal of the present study was to examine the consequences of an osmotic down-shock on cells grown at low or high osmolarity and their subsequent behavior in seawater. *
Corresponding author. 272
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each) with KCl solutions (5
or
80 mM)
or seawater.
Other
TABLE 1. Effect of osmotic down-shock on potassium, glutamate, and Dragendorff-positive compound contents of E. coli MC4100 cellsa
tests were performed with Na cells submitted to osmotic
down-shock and washed for 40 to 60 min in KCI (80 mM) solutions containing glutamate (1, 10, or 50 mM) or an analogous substrate: glutamine, glutarate, aspartate, or succinate (50 mM) (Sigma). After being washed, cell pellets were finally suspended in 2 ml of sterile seawater, and microcosms were inoculated with these cell suspensions to a density of 1 x 106 to 5 x 106 CFU/ml. They were then incubated in the dark at room temperature (23 to 25°C). Culturable-cell counts. All microcosms were sampled immediately after inoculation (To) and then daily during the following 6 or 7 days. Bacterial counts were made in triplicate by the membrane filtration technique (Millipore filters; pore size, 0.45 ,im) on nutrient agar (Difco Laboratories, Detroit, Mich.). The number of CFU was counted after a 24-h incubation at 37°C. For each experiment, the viability loss of cells was expressed as the following ratios: CFU at To to CFU after 2 days and CFU at To to CFU after 6 or 7 days of incubation. Since they derived from numbers of culturable cells, these ratios could however restrictively represent loss of culturability, depending on whether unculturable cells were dead or had evolved towards the viable but nonculturable state (7, 31). Culturable-cell counts performed during this study do not provide any information on this point. Potassium measurements. The K+ content of cells was measured by the method of Meury et al. (21). Aliquots of cell suspensions were filtered on Millipore filters (pore size, 0.45 ,um) and washed with NaCl solutions containing 5 g more of NaCl per liter than that in the filtered suspension. Filters were placed in glass tubes filled with 5 ml of 0.1 M HNO3. K+ was determined with an atomic absorption spectrophotometer (Philips PU 9200). Results were expressed as micromoles of K+ per gram of dried cells. Potassium concentration was also measured in supernatants from cells washed with distilled water and was expressed as micromoles of K+ per milliliter of supernatant. Glutamate measurements. The glutamate content of cells was measured by using the enzymatic assay described by Beutler and Michal (2). Results were expressed as micromoles of glutamate per gram of dried cells. Protein measurements. The protein content of cells was measured by the method of Lowry et al. (17) after their complete solubilization by heating to 90°C for 10 min in 1 M NaOH. The standard curve was established with bovine serum albumin.
273
Amt of the following in E. coli cells:
Growth medium
WahDaedff Potassium Wash Glutamate Dragendorif(p.mol/gb) positive (Rmol/gb)
M9
Seawater Distilled water
14.5 7.2
267 86
M9Na
Seawater Distilled water
27 6.4
4,278 400
M9NaGB
Seawater Distilled water
22.6 9
315 102
compounds 0.055 ND
(Rf)'
0.032 ND 0.055, 0.24 (GB)d ND
a Cells were previously grown in M9 medium supplemented with NaCl and GB or in unsupplemented M9 medium. b Dried cells. ' From thin-layer chromatograms on silica gel plates. ND, Not detected. d Two spots were detected, one of them being glycine betaine (GB).
Thin-layer chromatography of cell extracts. The presence of Dragendorff-positive compounds in A, Na, and NaGB cells before and after osmotic down-shock was monitored by thin-layer chromatography. Cell pellets (30 to 50 mg, dry weight) were extracted three times with 10 ml of ethanol (70% in distilled water). Ethanolic extracts were dried and fractionated on silica gel plates (60F 254 Kieselgel; Merck, Darmstadt, Federal Republic of Germany; thickness, 0.25 mm), with ascending chromatography in methanol-acetoneformic acid-hydrochloride (90:10:20:0.25). Plates were then sprayed with Dragendorff reagent (Sigma); Dragendorffpositive compounds appear orange on a yellow background. Reproducibility and statistical analysis of results. All tests were performed at least in duplicate, and some were performed in triplicate. A Student's t test (29) was performed in some cases to define the significance level of observed differences. RESULTS
Effects of osmotic down-shock on intracellular solutes. When submitted to an osmotic down-shock, E. coli MC4100
TABLE 2. Potassium content and viability loss in seawater of E. coli MC4100' Na cells
A cells Wash
K+ content
(pLmol/g)b
Seawaterd
8
Viability loss after': 7 days 6,530 110,000
2 days
K+
Viability loss afterc:
content
(p.mol/g)b
13,400 4,730
21
2 days 1.1
7 days 12
2.8 156 135,000 143,000 9 145 122,000 173,000 29 122 138 87,000 128,000 11.5 54 51 59,000 2,545 a E. coli MC4100 was grown in M9 medium at low (A cells) or high (Na cells) osmolarity (0.5 M NaCI), submitted to an osmotic down-shock (distilled water,
Distilled water KCl (5 mM) KCl (80 mM) Seawater
6.7 10.2 31.3 13.3
20 min), and then washed with distilled water, KCI solutions, or seawater. b In washed cells (dry weight) before transfer to seawater microcosm. c Viability loss is defined as the ratio of CFU at To to CFU after 2 or 7 days of incubation in seawater. d Without previous osmotic down-shock (reference).
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TABLE 4. Effect of osmotic down-shock and reload with potassium and glutamate on subsequent survival in seawater of E. coli MC4100 previously grown at high osmolarity (Na cells) Viability loss after":
TABLE 3. Loss of viability in seawater of E. coli MC4100' Mean viability loss (±SD) afterb:
Temperature ('C) of wash
2
days
6
days
400 (± 113) 50 (± 21) 14 (± 9.6)
2.7 (± 1) 1.9 (± 2) 1.3 (± 1.6)
4 20 37
a E. coli MC4100 was grown in M9 medium and washed with seawater (45 min) at different temperatures before transfer to seawater. b See Table 2, footnote c.
cells grown in M9A, M9Na, and M9NaGB media showed significant losses of intracellular K+ and glutamate (Table 1). K+ loss was higher for Na cells (76%) than for A or NaGB cells (50 and 60%, respectively). Na cells initially contained more K+ than the other two cell types because of the increase of K+ transport at high osmolarity (9) and the K+ release after accumulation of GB (3). In the same way, glutamate loss was higher for Na cells (90%) than for other cells (68%). Osmotic down-shock also led to a loss of Dragendorff-positive compounds, including GB accumulated in NaGB cells, as previously observed by Poccard and Le Rudulier (24a). Influence of osmotic down-shock and potassium accumulation on survival in seawater. The behavior of A and Na cells in seawater was highly influenced by the osmotic downshock and by the washing technique used prior to their transfer into seawater (Table 2). As previously reported (11, 23), A cells were much more sensitive to seawater than Na cells even when they had not undergone osmotic shock (reference cells). After the osmotic down-shock, they were slightly more sensitive, and washing in seawater or in KCI solutions significantly increased their resistance. With regard to the viability loss of A and Na cells not previously submitted to osmotic down-shock, the effect of hypoosmotic stress was much higher on Na cells, which lost most of their resistance to seawater after a 20-min wash in distilled water. Subsequent washing in 80 mM KCl or in seawater resulted in the partial recovery of this resistance. For both cell types, increased resistance was linked to an increase in intracellular K+ concentration. A loss of intracellular K+ could explain the increase in the sensitivity of cells washed at 4°C (Table 3), since E. coli cells lose a high amount of K+ at low temperature (10). Influence of glutamate accumulation on survival. Washing
- 60- A
Wash 2 days
Seawaterb Distilled water KCI (80 mM) + 1 mM glutamate + 10 mM glutamate + 50 mM glutamate
7 days
1
19
386
170,000
38 41 7
3,970 2,586 30
Table 2, footnote c. "See b
Reference cells not submitted to previous osmotic down-shock.
Na cells sensitized by osmotic down-shock in KCI solutions (80 mM) containing increasing concentrations of glutamate before transfer to seawater led to the almost total recovery of their resistance to seawater at the highest glutamate concentration (50 mM) (Table 4). This recovery could depend on the speed at which these two compounds accumulate, compared with wash time. This speed was then measured in sensitized Na cells suspended in a KCI (80 mM)glutamate (50 mM) solution over increasing periods prior to their transfer to seawater. K+ transport was rapid initially, possibly according to a logarithmic function of incubation time (Fig. 1). Glutamate was accumulated more slowly and linearly with time. The protection provided by a de novo accumulation of K+ in Na cells after osmotic down-shock was exponentially linked to the concentration of this ion in cells before their transfer to seawater (Fig. 2). This relationship was significant after both 2 (P c 0.05) and 7 (P c 0.01) days in seawater. Such a relationship was not significant for glutamate even though this compound was obviously required to achieve maximum resistance (Table 4). In fact, it has been shown that glutamate is synthesized by E. coli as a counterion in response to intracellular accumulation of K+, whose transport is activated at high osmolarity (3). This condition was obviously not achieved in our experiments, as reload of cells with K+ and glutamate was performed at low osmolarity for an arbitrary ratio of K+ to glutamate, which could explain the lack of significant correlation observed between the protection provided by K+ and that provided by
glutamate.
-@6000-
B
-o
=.
40-
202
30-
E
E
E U
~.4000-
co
2000
20 a
10
CD
r = 0.996
Can. [ ,01
,1
I
Hours
10
-
~~~~~~~~r=0.996
Cu 0
Hours
FIG. 1. K+ (A) and glutamate (B) accumulation with time by E. coli MC4100 grown in M9Na medium, submitted to osmotic down-shock in distilled water, and suspended in a KCI (80 mM)-glutamate (50 mM) solution.
K+ AND GLUTAMATE ENHANCE E. COLI SURVIVAL IN SEAWATER
VOL. 57, 1991
275
TABLE 5. Efficiency of different monovalent cations, glutamate, and glutamate analogs in restoring resistance to seawater
in E. coli MC4100' 4o Co10 0
103
r
0.980
100
10
1 0
20
3'0 4'0 (gmol/g dry cells)
.50
Intracellular potassium FIG. 2. Loss of viability in seawater, after 2- and 7-day incubation periods, of E. coli MC4100 grown in M9Na medium, submitted to osmotic down-shock, and provided with different concentrations of K+ ions in the suspension medium. Arrows indicate the viability loss for Na cells not submitted to osmotic down-shock after 2 (A) and 7 (B) days in seawater.
Specificity of K+ and glutamate in cell protection. The specificity of K+ and glutamate in restoring the protection lost after osmotic down-shock in Na cells was tested by replacing one or the other of these elements by analogous compounds: sodium (Na+) and rubidium (Rb+) for K+ and glutamine, glutarate, aspartate, and succinate for glutamate (Table 5). Neither Na+ nor Rb+ was able to provide similar resistance. In the same way, none of the glutamate analogs could replace glutamate to restore resistance in cells after osmotic down-shock. DISCUSSION Apart from the antagonistic activity of purification factors less specific to marine environments (see reviews in references 1 and 6), survival of E. coli in seawater primarily depends on its capacity to overcome osmotic shock (11, 23) and nutrient starvation (15). Survival is strongly influenced by previous growth conditions and the physiological state of cells during transfer to seawater (12). Thus, the history of their growth in the enteric environment and in wastewaters could be important. It has been previously demonstrated that preadaptation to a high osmolarity provides E. coli with a high level of resistance to seawater (23). The present results show that this resistance is mainly due to the intracellular accumulation of compounds transported or synthesized at high osmolarity which are rapidly eliminated by osmotic down-shock. Many cell components, such as periplasmic proteins (24) or mineral and organic cytoplasmic elements (16, 21, 22), are released by E. coli when a sharp drop in external osmotic pressure occurs. Some of these compounds are directly linked to regulation of osmotic balance, e.g., K+ (10, 21, 22) and GB (16). Our results show that intracellular glutamate concentration is also sensitive to osmotic shock and decreased in E. coli at low osmolarity. The K+ content of cells at the time of their transfer to seawater is a determinant in their later survival in this fluid. We have previously reported the protective role played by Trk and Kdp K+ transport systems on this bacterium (12). The highly resistant Na cells have a high K+ content, and the loss of K+ during distilled water shock is followed by a drastic loss of resistance. Such sensitized cells partly remore or
Viability loss in seawater afterc:
Preincubation (40 min) solutionsb
2 days
7 days
Seawater Distilled water
1.2 9
12 128,000
KCI-glutamate NaCI-glutamate RbCl-glutamate
3 8 7
54 3,100 2,540
KCI-aspartate KCI-glutamine KCI-glutarate KCI-succinate
4.7 3.8 5.5 3.7
5,640 3,540 2,650 2,890
a E. coli MC4100 was previously grown at high osmolarity (M9Na medium) and submitted to osmotic down-shock (distilled water, 20 min). b Cells were first washed for 20 min in distilled water and then for 40 min in distilled water containing monovalent cations (80 mM) and glutamate or analogs (50 mM), except for cells washed in seawater, which were not previously submitted to osmotic down-shock. c See Table 2, footnote c.
cover their resistance by increasing their K+ intracellular concentration. It is noteworthy that the protective effect is specific to K+; Na+ and Rb+ are not able to protect cells from decay in seawater. This could be due to their intrinsic inefficacy in maintaining cell homeostasis in seawater or result from the lack of transport of these ions by E. coli at high osmolarity (10). It is well known that E. coli cells regulate their turgor pressure with rapid K+ influx and efflux following changes of external osmolarity (21, 22). However, at high concentrations, K+ is not compatible with normal
metabolism since it modifies cytoplasmic pH and ionic strength (3, 8). This could explain the transient aspect of protection afforded both A and Na cells by washing in KCI solutions (Table 2) and the complementary effect of glutamate. In most procaryotes examined, the intracellular concentration of glutamate also increases in response to hyperosmotic shock (20, 26, 30). According to Tempest et al. (30),
activation of glutamate synthesis by glutamate dehydrogenase at high osmolarity causes a release of protons and an alkalinization of the cytoplasm. Its synthesis through glutamate synthase is also stimulated by high concentrations of K+, and it has been suggested that this ion is a signal for the regulation of glutamate synthesis during osmotic stress (20). In any event, accumulation of glutamate helps to reestablish cellular homeostasis in E. coli at high osmolarity by maintaining the membrane potential through neutralization of a part of accumulated K+ ions. Furthermore, potassium glutamate has been considered as a signal for the activation of several systems involved in the osmotic response of enteric bacteria (4, 25). Influence of glutamate on cell survival in seawater as observed in this study concurs with data from the literature. It appears very efficient and completes the protection provided by K+ by almost totally restoring the resistance lost by Na cells during hypoosmotic shock and partially restored by K+. It is currently being studied whether this influence could result from a better regulation of cellular pH. The protective effect seems specific to glutamate, but it is possible that the ineffectiveness of structural analogs used in our experiments was due to the lack of their transport under the selected experimental
GAUTHIER ET AL.
276
conditions, since their intracellular accumulation was not verified. Practically, this work demonstrates that in vitro studies on survival of enteric bacteria in seawater by using microcosms require a rigorous control of cell washout conditions, particularly for K+ concentrations in the liquids or buffers used. In addition, it does not appear advisable to wash cells at a low temperature (4°C), as is frequently done in such experiments.
From an environmental point of view, these results suggest that the K+ and glutamate contents of media in which E. coli cells are grown and transported to the sea can influence their subsequent survival in marine environments. It is also possible that the presence of glutamate in some marine compartments such as sediments or eutrophic waters (20, 30), together with the relatively high potassium content of seawater (10 mM) (18), favors survival of enteric bacteria, as has already been suggested for GB and other organic osmolytes (13, 14). It remains to be proven, however, whether this amino acid is actually transported by enteric bacteria in this environment as has already been demonstrated for GB (13). This is worth investigating since it has been shown that osmotic stress inhibits transport of carbohydrates in E. coli (27). Another source of glutamate could be its in situ synthesis from natural nutritive substrates, but this also remains to be proven under marine conditions. Additional studies are thus necessary to understand and objectively evaluate the influence of K+ and glutamate on survival of E.
coli in natural marine environments. ACKNOWLEDGMENTS We thank H. Olagnero for her technical assistance. This work was partly supported by grants from the Institut Franqais de Recherche pour l'Exploitation de la Mer (IFREMER, Paris, France) and from the World Health Organization within the framework of the Long-Term Program of Pollution Monitoring and Research in the Mediterranean Sea (MED POL Phase II). REFERENCES 1. Aubert, M., M. J. Gauthier, J. Aubert, and P. Bernard. 1980. Les systemes d'information des micro-organismes marins. Rev. Int, Oceanogr. Med. 60-61:37-106. 2. Beutler, H. O., and G. Michal. 1974. Colorimetric method for the determination of L-glutamic acid in foodstuffs, p. 1708-1713. In H. U. Bergmeyer (ed.), Methods of enzymatic analysis. Academic Press, Inc., New York. 3. Booth, I. R., J. Cairney, L. Sutherland, and C. F. Higgins. 1988. Enteric bacteria and osmotic stress: an integrated homeostatic system. J. Appl. Microbiol. Symp. Suppl. 17:35S-49S. 4. Booth, I. R., and C. F. Higgins. 1990. Enteric bacteria and osmotic stress: intracellular potassium glutamate as a secondary signal of osmotic stress? FEMS Microbiol. Rev. 75:239-246. 5. Casadaban, M. J. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J. Mol. Biol. 104:541-555. 6. Chamberlin, C. E., and R. Mitchell. 1978. A decay model for enteric bacteria in natural waters, p. 325-348. In R. Mitchell (ed.), Water pollution microbiology. John Wiley & Sons, Inc., New York. 7. Colwell, R. R. 1987. From counts to clones. J. Appl. Bacteriol.
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