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Bacteria starved for prolonged periods develop increased protection against lethal temperatures
FEMS Microbiology Ecology 101 ( !992 ) 229-236 © 1992 Federation of European Microbiological Soc~eties 0168-6496/92/$05.00 Published by Elsevier
2~
FEMSEC 00407
Bacteria starved for prolonged periods develop increased protection against lethal temperatures •~ s a J o u p e r - J a a n a
a, A m a n d a
E. G o o d m a n b a n d Staffan Kjelleberg ~
Department o f General and Marine Microbiologb', Gfiteborg. Sweden, and b School o f Microbiolog). Unit'ersio"o f New South Wales. Kensington, New South Wales, Australia Received 13 February 1992 Revision received i i June 1992 Accepted 19 June 1992
Key words: Vibrio; De novo protein synthesis; Cross-protection; Non-growth; Heat stress
1. SUMMARY A marine Vibr/o sp. DW1 and two Escherichia coli strains, K165 (htpR-) and Sc122 (htpR ÷) were submitted to heat stress after different times of starvation. All three bacterial strains developed starvation-mediated cross protection against heat. While two hours (Vtbr/o sp. DWl) and 24 hours (E. coli) of starvation gave near maximal protection, prolonged periods of non-growth offered "ncreased protection. Chloramphenicoi was added, at different times of starvation, to investigate the dependence on de novo protein synthesis for survival after heat stress during prolonged starvation. An obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found. Starvation-induced cross protection against heat may be dependent on protein synthesis in the initial phase
Correspondence to: A, Jouper-Jaan, Department of General and Marine Microbiology, Carl Skottsbergs Gata ~2, S-413 19 G6teborg, Sweden.
of starvation while after prolonged starvation the continous protection offered is suggested not to be mediated by de novo protein synthesis at these times.
2. INTRODUCTION Bacteria in most natural environments live under feast and famine conditions [1]. T o be able to survive under these diverse conditions, bacteria have developed a flexible physiology. While some bacteria differentiate into resistant structures such as endospores and myxospores [2] during starvation and stress conditions, most species appear to survive extended periods of starvation without major morphological alterations [3]. The apparent morphogenesis is generally that o f formation of ultramicrocells. Starved marine Vibrio can be as small as 0:03/zm 3, while growing cells are several/~m 3 in volume [4], Bacteria that encounter a nutrient-poor environment may respond by undergoing a series of physiological rearrangements [5]. Based on the
230
induction of stress reguions and the pattern of macromolecular synthesis, different phases of the starvation-induced response by non-differentiating bacteria can be identified. Analyses of two-dimensional gel electrophoresis maps of pulse labelled polypeptides have made it possible to recognize sequential and temporal shifts of the synthesis of starvation-induced proteins [5-7]. This response is clearly evident during relatively longterm starvation, 48 h, by marine vibrios [5]. Interestingly, there also appears to be a significant overlap between proteins induced by different starvation and stress conditions. For example, several of the starvation-induced proteins have been shown to be induced by heat stress [5-7]. DnaK is one of about 20 proteins in the heatshock regulon that demonstrates an increased synthesis when cells are heat shocked. Interestingly, in Vibrio sp. S14, Sti (starvation-induced protein) 51 has recently been identified as an Hsp 70 protein by probing with antibodies against the E. coli DnaK protein (unpublished observation). In E. coli the DnaK protein affects ti~e synthesis of both Cst (carbon starvation) and Pex (post-exponential) proteins [8], the induction of which has been inferred to be essential for starvation survival and cross protection against a range of stress conditions during carbon starvation [91. In this investigation, we addressed the question whether bacteria pre-starved in sea-water for longer periods remain resistant to or change their pattern of resistance to a secondary stress condition. Heat was selected as a model stress condition. No long term, i.e. one week, starvation studies of cross protection of either marine or non-marine bacteria have been performed. Short-term starvation studies have so far demonstrated that the marine strain Vibrio sp. S14 has a maximal heat resistance after about 6 h of starvation [10], while in E. coli the maximal heat resistance was found to develop by 4 h [6]. We studied the pre-starvation-induced cross protection in a true marine bacterium Vibrio sp. D W l and in E. coli. The E. coli strains we selected for this study are Sc122 (htpR +) and K165 (htpR-) [11]. The gene product of htpR (rpoH), sigma-32, serves as an alternative sigma factor for the transcription of the member genes
of the heat shock regulon [12]. The mutant K165 (htpR-) lacks the ability to produce heat shock proteins above 42°C. The E. coli cells were heat-shocked at 50°C, a temperature at which they can not grow and where only heat-shock proteins are synthesized [13]. The Vibrio sp. DW1 cells were heat-shocked at 37°C, a temperature at which they can not grow. To investigate possible cross-protection effects on E. coli cells of the high sodium chloride content in the artificial sea-water used to suspend the bacteria in this experiment, we also prestarved and heat shocked the cells in a 0.9% saline solution. This paper demonstrates that prolonged periods of non-growth offer increased protection, in addition to the rapid cross protection that develops during the initial phase (first few hours) of starvation, against heat stress. We also suggest that the protection acheived after prclonged starvation, in this study a period of one week, may be independent of de novo protein synthesis at these times.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacterial strains The marine Vibrio sp. DW1 has been described by Dawson et al. [14]. The E. coli strains, Sc122 (htpR +) and K165 ( h t p R - ) were described by Cooper and Ruettiger [11]. K165 carries a temperature suppressor mutation and can not synthesize heat shock proteins above 42°(2. 3.2. Culture and starvation conditions The bacteria were grown in shaken flasks in 20 ml Lewins medium. This medium contains 5 g yeast extract, 5 g tryptone, 1 g Tris-HCI, 0.1 g sodium glycerophosphate and 1000 ml of a nine salt solution (nss) or 1000 ml of a modified nine salt solution (mnss). p H was adjusted to 7.6. nss and mnss were identical except for the sodium chloride concentration. The nine salt solutions contain 17.6 g NaCl (nss) or 4.86 g NaCl (mass) and 1.47 g Na2SO4, 0.08 g NaHCO3, 0.25 g KCI, 0.04 g KBr, 1.87 g MgCI 2 • 2 H 2 0 , 0.008 g S r C ! 2 • 6
ii¸
H 2 0 , 0.008 g H3BO 3. The salts were dissolved in 1000 mi Mi!li-Q water. Vibrio sp. D W l may be defined as a true marine bacterium based on its requirement for NaCI for growth. Vibrio sp. D W l cells were therefore grown and starved in nss based solutions only. E. coli cells were grown and starved in both nss and mnss. All strains were starved under static conditions. The bacteria were grown and starved at 26°C. Vibr/o sp. D W l was recovered after starvation and heat-shock on agar plates with Lewins medium (nss) at 26°C overnight. The E. coli strains were recovered on agar plates with Lewins medium (mnss), at 37°C overnight. The drop-plate method was used for enumeration of surviving cells [15].
shock. The Cm pre-exposure was done to prevent the cells from synthesizing starvation-induced heat-shock :proteins prior to the individual sampiing at various times of starvation. The ratio o f die-off rates were calculated by using the slope co,~stant of the die-off curve as follows: (Cm treated cells/cells starved for 0 h ) / ( C m untreated ceils/cells starved for 0 h). 3.5. Rate o f protein synthesis
The determination of the rate of protein synthesis was performed as described by Nystr6m et al. [16]. Cm was also added as a control that the antibiotic was still inhibiting protein synthesis during prolonged starvation.
3.3. Heat stress E. coil cells and Vibrio sp. DW1 cells were
4. RESULTS
harvested in mid-exponential phase. The cells were washed once and transferred to 100 mi of starvation medium to give a cell density-of approximately 2 x l0 T ceils per ml. Immediately after the cells were suspended in the starvation medium, a 2-ml sample was taken and exposed to temperatures which ceased the growth of the bacteria. In the initial round of experiments these temperatures were found to be 37~C for Vibr/o sp. D W l and 50°C for E. coll. Samples were also taken and exposed to heat after 2 h, as well as 22 h, two and nine days of starvation. P'tbr/o sp. DW1 was heat exposed for 120 min and the viability was determined every 15 rain during this treatment. E. coli was heat exposed for 180 min and the viability was determined every 30 min.
The marine Vibr/o sp. D W I demonstrated a high degree of survival during nutrient-deprived conditions. ARer nine days of multiple (energy and nutrien0 starvation, 77% of the initial population was found to be culturable (Fig. 1). In our mnss system which contains 0.9% salts, about 50% of the initial population of E. coil Sc122 and E. coli K165 survived nine days o f starvation. In nss, in which the salt concentration equals Z17%, 52% of the initial population of E. coil K165 survived nine days of starvation while only 17%
140 120! 1
3. 4. Protein synthesis inhibition
In another series of experiments, Vibr/o sp. DW1 cells were exposed to the protein-synthesis inhibitor chloramphenicol (Cm), 1 0 0 / z g / m t , for a period of 2 h only prior to heat stress. Viability of DW1 cells was severly impaired by Cm addition. E. coil cells were exposed to 100/zg C m / m l , for 2 h immediately after the onset of the starvation period and for a period of 18 h, before heat stress at 22 h, two and seven days of starvation, respectively. After the exposure for Cm, the cells were washed several times with nss prior to heat
i
t£ 0~
i
~ # b T'm~eo f ~ (days) Fig. 1. Percent survivingcells during nine days of starvation. ( A ) Fibtio sp. DWL (11) E. coil Se122 starved in nss, (O) E. coil Sc122 starved in mnss, (0)E. coil K165starvedin nss and ~ (o) E. coli K165 starved in mnss. Mean values of at least three parallel experimentsfor each data point are shown. :
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of the initial population of E. coli Sc122 survived the nine days of starvation (Fig. 1). Vibrio sp. DWI achieved maximum resistance against lethal heat stress, 37°C, after two days of starvation (Fig. 2A). However, after 2 h of starvation the heat resistance was already increased. The die-off rate at 2 h was decreased by 57% compared to the die-off rate at 0 h (data not shown), while the die-off rate at 48 h was decreased by 72% compared to that of the onset of starvation (Fig. 2A). When the starving E. coli cells were exposed to 50°C, both strains were found to tolerate heat better the longer they were starved, in nss (Fig. 2B,D), the protection offered
for E. coli Sc122 and K165 was not as pronounced as in mnss (Fig. 2C,2E). E. coli K165 cells cannot grow at temperatures above 42°C. One hour exposure at such temperatures leads to a complete loss in viability [12]. The mutant K165 cells achieved the same tolerance as did the wild-type Sc122 cells ior h;~la temperature exposure. By adding chloramphenicol (Cm) to the starvation regimes prior to heat stress we attempted to resolve whether de novo protein synthesis during prolonged starvation was important for the development of heat resistance. The E. coli cells were exposed to Cm for a period of 18 h prior to the sampling at 22 h~ two and seven days
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I
t
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l
0.1
0.001
I
0
30
I
I
I
t
90 120 150 180 Time of heat exposure(rain) 60
D 0
E
30
60 90 120 150 180 Time of heat exposure (rain)
210
Fig. 2. Percent surviving Vibrio sp. DWI cells (A), E. coli Sc122 (htpR + ) cells in nss (B) and in mnss (C), and E. co!i K165 ( h t p R - ) cells in nss (D)and in mnss (E), after heat-shock at the following times of starvation; onset of the starvation (e), 22 h (B), two ( • ) and nine ( • ) days of starvation. Each experiment was repeated at least twice to confirm reproducibility. Representative results are presented.
~3 3
(Fig. 3). In the first round of experiments, Cm was not washed away prior to heat exposure. In those experiments, a ~ r y low number of viable Cm-treated l/ibm sp. DWl cells could be detected and E. coil Cm-treated cells died faster than untreated ceils during the first two days of starvation while after nine days of starvation there were no significant differences between the Cmtreated and untreated cells (data not shown). In order to further assess the significance o f protein synthesis during long-term starvation, we measured the rate of [3H]-lcucine incorporation. The rate of leucine incorporation in I/'duio sp. DW1 was reduced by 93% after the cells h a d been starved for nine days. In E. coli Sc122 and K165 this reduction was 99% and 87% at that time (Table 1). Cm addition after one week of starvation reduced the rate of protein synthesis by 83% for V'tbr/o sp. D W I . The corresponding figures E. coil Sc122 and K165 were 75% and 28%, respectively. For all strains, the rate of protein synthesis on the ninth day of starvation was as low as the protein synthesis observed at the onset of starvation after Cm addition ( T a b l e 1). Similar data were given when the C m concentration was increased to 200/.tg/ml.
2.5 2 ? i.5
11.5 2
22 48 168 Time of sire-ration 0mars)
Fig. 3. Ratio o f the die-off rates of heat-shocked Cm-tteated and untreated cells after different times of starvation. A value > 1.0 denotes a greater die-off rate for Cm-treated cells. The ratio of die-off rates after different times of starvation are shown by black bars for Vlbrio sp. DW1, double hatched bars for E. coil Sc122 and single hatched bars for E. coil K165. Mean values and standard deviations of at least three parallel experiments for each data point: are sho,~vn.
of starvation. Starved E. coil K165 ,:ells displayed a reduced viability as a result of heat stress after Cm addition at the onset of starvation. E. coli Sc122 cells were most effected by this treatment after one day of starvation. Essentially, when the E. coli cells had been starved for a week there were very little differences in the ratio of die-off rates between treated and non-treated cells (Fig. 3). Vibr/o DW1 was exposed to Cm for 2 h. The cells showed a complete loss in viability after long periods of Cm incubations. The V/br/o D W l ce!!s that were exposed for the 2-h Cm treatment did not die off faster than the untreated cells as a result of heat exposure. The ratio of the die-off rake of heat shocked Cm-treated and untreated cells was less than 1.0 at all times of starvation
5. DISCUSSION This paper addresses the development of heat resistance during prolonged starvation of Vibrio and E. coil cells and investigates whether de novo protein synthesis mediates such a cross protection.
Table 1 Relative rate (%) of [-~H]-Ieucine incorporation Time of starvation
Bacterial strains l:tbtio sp. DWI - Cm
0h 9 days
10~ 7.0
a
E. Coil Sc122 + Cm
3.9 1.2
b
- Cm
100 0.4
a
E. co/i K165 + Cm
7.7 0.3
b
-- Cm a
+ Cm
100 13
15 3 ;6
b
The rate of incorporation at the onset of starvation, 0 h, is given the value 100. The data are mean values from two separate experiments. a - C m , no Cm added to the starvation regimes. b + C m , Cm added to the starvation regimes.
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Marine vibrios maintain viability when the cells encounter prolonged starvation conditions [17,18]. Several studies have also addressed the ability of E. coil cells to survive in sea-water [19,20] and to resist other forms of stress [6]. The survival of E. coli in sea-water seems to be strain dependent and the ability to survive is related to the ability to osmoregulate [21]. The difference in the long term survival potential between the two E. coli strains, Sc122 and K165 (Fig. 1), in artificial seawater reported in this study is surprising as the strains are considered to be identical under room temperature conditions [11]. While it is clear that 17,. coli cells may survive during prolonged exposure in artificial sea-water with 2.17% as well as 0.9% salt, we have at present no explanation for the difference between the strains in their activity to form colony-forming units when suspended in 2.17% salt containing artificial sea-water. The E. coil cells were also heat stressed in both low (0.9%) and high (2.17%) salt solution to ensure that t h e heat resistance developed during starvation was not due to a secondary effect of salt stress. We found that when E. coil cells were in their normal habitat (0.9% salt solution) they developed a better protection against heat stress (Fig. 2C,E). It has been demonstrated for both ,2-. coli and I4brio cells that an on-going protein synthesis and the expression of starvation specific proteins are necessary for prolonged survival during multiple as well as carbon starvation [5,22]. The molecular and physiological adaptation of I,qbrio cells during starvation has recently been reviewed. It is suggested that a subclass of the starvation specific genes that are induced during the differentiation program of starvation is essential for the prolonged survival of starving Vibrio cells [10]. It has also been suggested that starvation-induced proteins in both Vibr/o and E. coli cells may mediate the cross protection against ~. range of stress conditions during the survival phase of starvation [6,23,24]. This study demonstrates that Vibrio sp. DW1 and E. coil cells, starved f o r two and nine days respectively, are considerably more heat'resistent than cells starved for less than two days (Figs. 2A-E). This protection may conceivably be a
result of the induction of heat-shock proteins during starvation. The gene product of htpR (rpoH), Sigma-32, that promotes the synthesis of the heat shock proteins has a half-life of I min at 30°C and of 4 min at 42°(2. Continuous synthesis of Sigma-32 is required for induction of the heatshock proteins [25,26]. We believe that the similarity in response to high temperature exposure of the wild-type and the mutant E. coli cells rules out the possibility that cross protection against heat stress at various times of starvation reflects the induction of the heat-shock regulon. VanBogelen et al. [27] induced the synthesis of heatshock proteins by exposure to nalidixic acid, puromycin, and H 2 0 2 for 15 min, but did not obtain thermotolerance. Thermotolerance was acquired, however, when the cells were exposed to an elevated temperature, ethanol, CdCI 2 and prolonged treatment by H 2 0 2. It was concluded that thermotolerance is developed by processes other than the htpR-dependent induction of heat-shock proteins. In support of the notion that the starvation-induced increased tolerance against heat stress found in this study is not due to the ability to synthesize heat-shock proteins, we have recently established that the starvation induced synthesis of the heat-shock protein DnaK in starving Vtbr/o sp. DW1 does not correlate with the development of cross protection against heat (unpublished results). In E. coli K12, the carbon starvation-induced synthesis of heat-shock proteins is reported to occur during the initial 6 h of starvation [28]. Based on the decrease in the rate of protein synthesis (Table 1) and rather similar protection against heat stress for both Cm-treated and untreated cells after prolonged starvation (Fig. 3), it may be suggested that while the de novo protein synthesis during the initial phase of starvation plays a role for the development of increased heat resistance, relatively long-term starved cells of the strains included in this study may develop heat resistance via an additional, de novo protein synthesis independcnt response. In fact, an obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found in this study. Vibrio sp. DWl was not effected by the interruptions in protein syn-
235
thesis prior to heat stress at any time of starvation, while E. coli Sc122 showed a great dependence of protein synthesis between 4 and 22 h of starvation for developing heat resistance (Fig. 3). However, protein synthesis dependent development of thermal protection was not achieved during the first 2 h of starvation. After 22 h of starvation, E. coil Sc122 cells did not depend on de novo protein synthesis for developing increased thermotolerance. In support of the latter finding, it has been shoval that non-differentiating bacteria shut down the metabolism and become smaller and coccoid during prolonged starvation. It has also been argued that these cells enter a state of dormancy, and that starving vegetative non-differentiating bacteria transform themselves into a more spore-like structure [5]. In addition to very low levels of endogenous metabolism and protein synthesis, a condensed nucleoid also signifies such cells [4]. Furthermore, two dimensional gel electrophoresis analysis of pulse labelled polypeptides at various times of starvation of WdTr/o sp. $14, revealed that very few new starvation-specific proteins are induced after 72 h (3 days) of starvation, while a total o f 66 of the starvation-induced proteins were identified within 48 h of energy and nutrient starvation. It may therefore be suggested that the processes that allow for the development of the resistant phenotype may partly be a result of proteins synthesized prior to the onset of starvation. In summary, enhanced resistance against heat stress was detected not only for the initial 24 h of starvation, which may be similar to the resistance previously reported for Vtbr/o sip. $14 [5] and E. coil K12 cells [6], but also during the entire period of nine days of starvation. WhOe shortterm starvation gives a very significant degree of protection, prolonged periods of non-growth in fact offer increased protection although after different time periods for the different strains, and for E. coli strains, depending on the salinity of the starvation regime. Both de novo protein synthesis dependent and, with time of starvation, de novo protein synthesis independent starvationinduced protection against heat are suggested to take place in these strains.
:~•i ••
•~ i •
ACKNOWLEDGEMENTS We thank Ruth VanBogelen for providing the E. coil strains and for sharing her experience from working with these strains. We thank Agn e t a Danielsson for technical assistance. This study was supported by a grant from the Swedish Natural Science Research Council Amanda E Goodman was funded in part by an Australian Research Council Fellowship.
REFERENCES Ill Kjeheberg,S., N~n~m. T., Albertson~N. ar,d P~rdh, K. (1990) S p ~ a l Issue. Nutrient Limitation: Global Resp~].~'.s and Procaryotic :Development. [2j H ~ , D.A. and Chafer, K.F. (1989) Genctics of Bacterial Diversity. Academic P r e ~ London. [3] Amy, P.S. and Morita, R.Y. 0983) Starvatlo,vsurvival patterns of sixteen freshly isolated open-oc~n bacteria~ Appl. Environ. M ~ . 45, 1109-1115.
[4] Moyer, C.L and Morita, R.Y. (1989) Effect of growth rate and statwafion-survival on cellular DNA, RNA, and protein of a psychrophilic marine bacterium. AppL Environ. Microbiol. 55~ 2710-2716.
[5] Nystrfm, T., Fl~rdh, K- and K~elteberg,S. (1~0) Responses to multiple-nutrient starvation in marine trdn~,o ~p. Strain CCJ.JG 1.5956. J. Bacterio!. 172, 7f~-7097. 16] Jenkins, D.E., Schuitz, J.E. and Matin, A. (1988) Starvation-induced cross protection against heat o r H 2 0 2 challenge in Escher/dda co//. J. Bacteliol. 170, 3910-3914. [7] Spector, M.P, Aliabadi, Z., Gonzalez, T. and Foster, J.W. (1986) Global control in Sa/mond/a typ/dmut/um: Two-dimenfiona! eha~rophorctic anab~sis of starvation-, anaerobiosis-, and heat shock-inducible proteins. J. BacterioL 168, 420-424. ~8] Spence, J., Cegieisga, A. and Geofgop~ak~, C. (1990) Role of Escherichia coli heat-shock proteins DnaK and HtpG (C62,5) in response to nutritional deprivation..L Bacteriol. 172, 7157-7166. [9] Matin, A., Auger, E.A., Blum, P.H. and Schultz, J.E. (1989) Genetic basis of stat~ation survival in nondifferentiatittg bacteria. Annu. Rev. MicrobioL 43, 293-316. [10] NysttSm, T , Albertson, N.H. Fl~rdh, K. and Kjelleberg, S. (1990) Physiological and molecular adaptation to starvation iV the marine Ftbr/o sp. S14. FEMS MicrobioL Ecol. 74, |29-140. [1t] Cooper, S. and Reuettinger, T. (1975)A temperature sensitive nonsense mutation affecting the synthesis of a major protein of Escherichia coil K12. Mol. gen. Genet2 139, 167-176. [12] NeidhardL F.C. and VanBogelen, R.A. (1981) Positive regulatory gene for temperature-controlled proteins in
236 Escherichia coll. Biochem. Biophys. Res. Comm. 100, 894-900. [13] Neidhardt, F.C., VanBogelen, R.A. and Vaughn, V. (1984) The genetics and regulation of heat-shock proteins. Ann. Rev. Genet. 18, 295-329. [14] Dawson, M,P., Humphrey, B. and Marshall, K.C. (1981) Adhesion: a tactic in the survival strategy of a marine Vibrio during starvation, Curr. Microbiol. 6, 930-940. [15] Hoben, H.J. and Somesegaran, P. (1982) Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl. Environ. Microbiol. 44, 1246-1247. [16] Nystr6m, T., M~rd6n, P. and Kjelleberg, S. (1986) Relative changes in incorporation rates of lencine and methaionine during starvation survival of two bacteria isolated from marine waters. FEMS Microbiol. Ecol. 38, 285-292. [17] Amy, P.S., Pauling, C. and Morita, R.Y. (1983) Starvation-survival processes of a marine Vibrio. Appl. Environ. Microb. 45, 1041-1048. [18] Hoff, K.A. 0989) Survival of Vd~r/o Anguillamm and Fibrio sabnonicida at different salinities, Appl. Environ. Microbiol. 55, 1775-1786. [19] Sorensen, S.S. (1991) Survival of Escherichia coil KI2 in seawater. FEMS MicrobioL Ecol. 85, 161-168. [20] Munro, P.M., Gauthier. MJ. and Laumopd, F.M. (1987) Changes in Escherichia coli starved ~:nseawater or grown in seawater-wastewater mixtures. Appl. Environ. Microbiol. 53, 1476-1481. [21] Munro, P.M., Gauthier, M.J.. Breittmayer, V.A. and
Bongiovanni, J. (1989) Influence of osmoregulation proces,ses on starvation survival of Escherichia coli in seawater. App. Environ. MicrobioL 55, 2017-2024. [22] Reeve, C.A., Amy, P.S. and Matin, A. (1984) Role of protein synthesis in the survival of carbon-starved Escherichia coil K-12. J. Bacteriol. 160, 1041-1046. [23] Nystr6m, T., OIsson, R.M. and Kjeileberg, S. (1992) Sur~val, stress resistance and alterations in protein expression in the marine Vibrio sp. S14 during starvation fol different individual nutrients. AppL Environ. MicrobioL 58, 55-65. [24] Jenkins, D.E., Chaisson, S.A. and Matin, A. (1990) Starvation-induced cross protection against osmotic challenge in Eschetichia coll. J. Bacteriol. 172, 2779-2781. [25] Straus, D.B., Walter, W.A. and Cross, C.A. (1987) The heat-shock response of E. coil is regulated by changes in the concentration of sigma-32. Nature 329, 348-351. [26] Yura, T., Kawasaki, Y., Kusukawa, N., Nagai. H., Wada, C. and Yano, R. (1990) Roles and regulation of the heat-shock sigma factor sigma-32 in Escherichia coll. Antonie van Leeuwenhoek. 58, 187-190. [27] VanBogelen, R,A, Acton, M.A. and Neidhardt, F.C. (1987) Induction of the heat-shock regulon does not produce thermotolerance in Escherichia coll. Gen. Develop. 1,525-531. [28] Jenkins, D.E., Auger, E.A. and Matin, A. (1992) Role of RpoH, a heat shock regulator protein, in Eschetichia coil carbon starvation protein synthesis and survh,al. J. Bacteriol. 173, 1992-1996.
2~
FEMSEC 00407
Bacteria starved for prolonged periods develop increased protection against lethal temperatures •~ s a J o u p e r - J a a n a
a, A m a n d a
E. G o o d m a n b a n d Staffan Kjelleberg ~
Department o f General and Marine Microbiologb', Gfiteborg. Sweden, and b School o f Microbiolog). Unit'ersio"o f New South Wales. Kensington, New South Wales, Australia Received 13 February 1992 Revision received i i June 1992 Accepted 19 June 1992
Key words: Vibrio; De novo protein synthesis; Cross-protection; Non-growth; Heat stress
1. SUMMARY A marine Vibr/o sp. DW1 and two Escherichia coli strains, K165 (htpR-) and Sc122 (htpR ÷) were submitted to heat stress after different times of starvation. All three bacterial strains developed starvation-mediated cross protection against heat. While two hours (Vtbr/o sp. DWl) and 24 hours (E. coli) of starvation gave near maximal protection, prolonged periods of non-growth offered "ncreased protection. Chloramphenicoi was added, at different times of starvation, to investigate the dependence on de novo protein synthesis for survival after heat stress during prolonged starvation. An obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found. Starvation-induced cross protection against heat may be dependent on protein synthesis in the initial phase
Correspondence to: A, Jouper-Jaan, Department of General and Marine Microbiology, Carl Skottsbergs Gata ~2, S-413 19 G6teborg, Sweden.
of starvation while after prolonged starvation the continous protection offered is suggested not to be mediated by de novo protein synthesis at these times.
2. INTRODUCTION Bacteria in most natural environments live under feast and famine conditions [1]. T o be able to survive under these diverse conditions, bacteria have developed a flexible physiology. While some bacteria differentiate into resistant structures such as endospores and myxospores [2] during starvation and stress conditions, most species appear to survive extended periods of starvation without major morphological alterations [3]. The apparent morphogenesis is generally that o f formation of ultramicrocells. Starved marine Vibrio can be as small as 0:03/zm 3, while growing cells are several/~m 3 in volume [4], Bacteria that encounter a nutrient-poor environment may respond by undergoing a series of physiological rearrangements [5]. Based on the
230
induction of stress reguions and the pattern of macromolecular synthesis, different phases of the starvation-induced response by non-differentiating bacteria can be identified. Analyses of two-dimensional gel electrophoresis maps of pulse labelled polypeptides have made it possible to recognize sequential and temporal shifts of the synthesis of starvation-induced proteins [5-7]. This response is clearly evident during relatively longterm starvation, 48 h, by marine vibrios [5]. Interestingly, there also appears to be a significant overlap between proteins induced by different starvation and stress conditions. For example, several of the starvation-induced proteins have been shown to be induced by heat stress [5-7]. DnaK is one of about 20 proteins in the heatshock regulon that demonstrates an increased synthesis when cells are heat shocked. Interestingly, in Vibrio sp. S14, Sti (starvation-induced protein) 51 has recently been identified as an Hsp 70 protein by probing with antibodies against the E. coli DnaK protein (unpublished observation). In E. coli the DnaK protein affects ti~e synthesis of both Cst (carbon starvation) and Pex (post-exponential) proteins [8], the induction of which has been inferred to be essential for starvation survival and cross protection against a range of stress conditions during carbon starvation [91. In this investigation, we addressed the question whether bacteria pre-starved in sea-water for longer periods remain resistant to or change their pattern of resistance to a secondary stress condition. Heat was selected as a model stress condition. No long term, i.e. one week, starvation studies of cross protection of either marine or non-marine bacteria have been performed. Short-term starvation studies have so far demonstrated that the marine strain Vibrio sp. S14 has a maximal heat resistance after about 6 h of starvation [10], while in E. coli the maximal heat resistance was found to develop by 4 h [6]. We studied the pre-starvation-induced cross protection in a true marine bacterium Vibrio sp. D W l and in E. coli. The E. coli strains we selected for this study are Sc122 (htpR +) and K165 (htpR-) [11]. The gene product of htpR (rpoH), sigma-32, serves as an alternative sigma factor for the transcription of the member genes
of the heat shock regulon [12]. The mutant K165 (htpR-) lacks the ability to produce heat shock proteins above 42°C. The E. coli cells were heat-shocked at 50°C, a temperature at which they can not grow and where only heat-shock proteins are synthesized [13]. The Vibrio sp. DW1 cells were heat-shocked at 37°C, a temperature at which they can not grow. To investigate possible cross-protection effects on E. coli cells of the high sodium chloride content in the artificial sea-water used to suspend the bacteria in this experiment, we also prestarved and heat shocked the cells in a 0.9% saline solution. This paper demonstrates that prolonged periods of non-growth offer increased protection, in addition to the rapid cross protection that develops during the initial phase (first few hours) of starvation, against heat stress. We also suggest that the protection acheived after prclonged starvation, in this study a period of one week, may be independent of de novo protein synthesis at these times.
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacterial strains The marine Vibrio sp. DW1 has been described by Dawson et al. [14]. The E. coli strains, Sc122 (htpR +) and K165 ( h t p R - ) were described by Cooper and Ruettiger [11]. K165 carries a temperature suppressor mutation and can not synthesize heat shock proteins above 42°(2. 3.2. Culture and starvation conditions The bacteria were grown in shaken flasks in 20 ml Lewins medium. This medium contains 5 g yeast extract, 5 g tryptone, 1 g Tris-HCI, 0.1 g sodium glycerophosphate and 1000 ml of a nine salt solution (nss) or 1000 ml of a modified nine salt solution (mnss). p H was adjusted to 7.6. nss and mnss were identical except for the sodium chloride concentration. The nine salt solutions contain 17.6 g NaCl (nss) or 4.86 g NaCl (mass) and 1.47 g Na2SO4, 0.08 g NaHCO3, 0.25 g KCI, 0.04 g KBr, 1.87 g MgCI 2 • 2 H 2 0 , 0.008 g S r C ! 2 • 6
ii¸
H 2 0 , 0.008 g H3BO 3. The salts were dissolved in 1000 mi Mi!li-Q water. Vibrio sp. D W l may be defined as a true marine bacterium based on its requirement for NaCI for growth. Vibrio sp. D W l cells were therefore grown and starved in nss based solutions only. E. coli cells were grown and starved in both nss and mnss. All strains were starved under static conditions. The bacteria were grown and starved at 26°C. Vibr/o sp. D W l was recovered after starvation and heat-shock on agar plates with Lewins medium (nss) at 26°C overnight. The E. coli strains were recovered on agar plates with Lewins medium (mnss), at 37°C overnight. The drop-plate method was used for enumeration of surviving cells [15].
shock. The Cm pre-exposure was done to prevent the cells from synthesizing starvation-induced heat-shock :proteins prior to the individual sampiing at various times of starvation. The ratio o f die-off rates were calculated by using the slope co,~stant of the die-off curve as follows: (Cm treated cells/cells starved for 0 h ) / ( C m untreated ceils/cells starved for 0 h). 3.5. Rate o f protein synthesis
The determination of the rate of protein synthesis was performed as described by Nystr6m et al. [16]. Cm was also added as a control that the antibiotic was still inhibiting protein synthesis during prolonged starvation.
3.3. Heat stress E. coil cells and Vibrio sp. DW1 cells were
4. RESULTS
harvested in mid-exponential phase. The cells were washed once and transferred to 100 mi of starvation medium to give a cell density-of approximately 2 x l0 T ceils per ml. Immediately after the cells were suspended in the starvation medium, a 2-ml sample was taken and exposed to temperatures which ceased the growth of the bacteria. In the initial round of experiments these temperatures were found to be 37~C for Vibr/o sp. D W l and 50°C for E. coll. Samples were also taken and exposed to heat after 2 h, as well as 22 h, two and nine days of starvation. P'tbr/o sp. DW1 was heat exposed for 120 min and the viability was determined every 15 rain during this treatment. E. coli was heat exposed for 180 min and the viability was determined every 30 min.
The marine Vibr/o sp. D W I demonstrated a high degree of survival during nutrient-deprived conditions. ARer nine days of multiple (energy and nutrien0 starvation, 77% of the initial population was found to be culturable (Fig. 1). In our mnss system which contains 0.9% salts, about 50% of the initial population of E. coil Sc122 and E. coli K165 survived nine days o f starvation. In nss, in which the salt concentration equals Z17%, 52% of the initial population of E. coil K165 survived nine days of starvation while only 17%
140 120! 1
3. 4. Protein synthesis inhibition
In another series of experiments, Vibr/o sp. DW1 cells were exposed to the protein-synthesis inhibitor chloramphenicol (Cm), 1 0 0 / z g / m t , for a period of 2 h only prior to heat stress. Viability of DW1 cells was severly impaired by Cm addition. E. coil cells were exposed to 100/zg C m / m l , for 2 h immediately after the onset of the starvation period and for a period of 18 h, before heat stress at 22 h, two and seven days of starvation, respectively. After the exposure for Cm, the cells were washed several times with nss prior to heat
i
t£ 0~
i
~ # b T'm~eo f ~ (days) Fig. 1. Percent survivingcells during nine days of starvation. ( A ) Fibtio sp. DWL (11) E. coil Se122 starved in nss, (O) E. coil Sc122 starved in mnss, (0)E. coil K165starvedin nss and ~ (o) E. coli K165 starved in mnss. Mean values of at least three parallel experimentsfor each data point are shown. :
232
of the initial population of E. coli Sc122 survived the nine days of starvation (Fig. 1). Vibrio sp. DWI achieved maximum resistance against lethal heat stress, 37°C, after two days of starvation (Fig. 2A). However, after 2 h of starvation the heat resistance was already increased. The die-off rate at 2 h was decreased by 57% compared to the die-off rate at 0 h (data not shown), while the die-off rate at 48 h was decreased by 72% compared to that of the onset of starvation (Fig. 2A). When the starving E. coli cells were exposed to 50°C, both strains were found to tolerate heat better the longer they were starved, in nss (Fig. 2B,D), the protection offered
for E. coli Sc122 and K165 was not as pronounced as in mnss (Fig. 2C,2E). E. coli K165 cells cannot grow at temperatures above 42°C. One hour exposure at such temperatures leads to a complete loss in viability [12]. The mutant K165 cells achieved the same tolerance as did the wild-type Sc122 cells ior h;~la temperature exposure. By adding chloramphenicol (Cm) to the starvation regimes prior to heat stress we attempted to resolve whether de novo protein synthesis during prolonged starvation was important for the development of heat resistance. The E. coli cells were exposed to Cm for a period of 18 h prior to the sampling at 22 h~ two and seven days
100 8 ._~ ~ N ~
to 1 0.I
8, o.o! I
I
|
A
tO0 8
tO l
.u
0.1
8. 0.01 t
I
l
I
I
t
100
.> ~.
l
0.1
0.001
I
0
30
I
I
I
t
90 120 150 180 Time of heat exposure(rain) 60
D 0
E
30
60 90 120 150 180 Time of heat exposure (rain)
210
Fig. 2. Percent surviving Vibrio sp. DWI cells (A), E. coli Sc122 (htpR + ) cells in nss (B) and in mnss (C), and E. co!i K165 ( h t p R - ) cells in nss (D)and in mnss (E), after heat-shock at the following times of starvation; onset of the starvation (e), 22 h (B), two ( • ) and nine ( • ) days of starvation. Each experiment was repeated at least twice to confirm reproducibility. Representative results are presented.
~3 3
(Fig. 3). In the first round of experiments, Cm was not washed away prior to heat exposure. In those experiments, a ~ r y low number of viable Cm-treated l/ibm sp. DWl cells could be detected and E. coil Cm-treated cells died faster than untreated ceils during the first two days of starvation while after nine days of starvation there were no significant differences between the Cmtreated and untreated cells (data not shown). In order to further assess the significance o f protein synthesis during long-term starvation, we measured the rate of [3H]-lcucine incorporation. The rate of leucine incorporation in I/'duio sp. DW1 was reduced by 93% after the cells h a d been starved for nine days. In E. coli Sc122 and K165 this reduction was 99% and 87% at that time (Table 1). Cm addition after one week of starvation reduced the rate of protein synthesis by 83% for V'tbr/o sp. D W I . The corresponding figures E. coil Sc122 and K165 were 75% and 28%, respectively. For all strains, the rate of protein synthesis on the ninth day of starvation was as low as the protein synthesis observed at the onset of starvation after Cm addition ( T a b l e 1). Similar data were given when the C m concentration was increased to 200/.tg/ml.
2.5 2 ? i.5
11.5 2
22 48 168 Time of sire-ration 0mars)
Fig. 3. Ratio o f the die-off rates of heat-shocked Cm-tteated and untreated cells after different times of starvation. A value > 1.0 denotes a greater die-off rate for Cm-treated cells. The ratio of die-off rates after different times of starvation are shown by black bars for Vlbrio sp. DW1, double hatched bars for E. coil Sc122 and single hatched bars for E. coil K165. Mean values and standard deviations of at least three parallel experiments for each data point: are sho,~vn.
of starvation. Starved E. coil K165 ,:ells displayed a reduced viability as a result of heat stress after Cm addition at the onset of starvation. E. coli Sc122 cells were most effected by this treatment after one day of starvation. Essentially, when the E. coli cells had been starved for a week there were very little differences in the ratio of die-off rates between treated and non-treated cells (Fig. 3). Vibr/o DW1 was exposed to Cm for 2 h. The cells showed a complete loss in viability after long periods of Cm incubations. The V/br/o D W l ce!!s that were exposed for the 2-h Cm treatment did not die off faster than the untreated cells as a result of heat exposure. The ratio of the die-off rake of heat shocked Cm-treated and untreated cells was less than 1.0 at all times of starvation
5. DISCUSSION This paper addresses the development of heat resistance during prolonged starvation of Vibrio and E. coil cells and investigates whether de novo protein synthesis mediates such a cross protection.
Table 1 Relative rate (%) of [-~H]-Ieucine incorporation Time of starvation
Bacterial strains l:tbtio sp. DWI - Cm
0h 9 days
10~ 7.0
a
E. Coil Sc122 + Cm
3.9 1.2
b
- Cm
100 0.4
a
E. co/i K165 + Cm
7.7 0.3
b
-- Cm a
+ Cm
100 13
15 3 ;6
b
The rate of incorporation at the onset of starvation, 0 h, is given the value 100. The data are mean values from two separate experiments. a - C m , no Cm added to the starvation regimes. b + C m , Cm added to the starvation regimes.
234
Marine vibrios maintain viability when the cells encounter prolonged starvation conditions [17,18]. Several studies have also addressed the ability of E. coil cells to survive in sea-water [19,20] and to resist other forms of stress [6]. The survival of E. coli in sea-water seems to be strain dependent and the ability to survive is related to the ability to osmoregulate [21]. The difference in the long term survival potential between the two E. coli strains, Sc122 and K165 (Fig. 1), in artificial seawater reported in this study is surprising as the strains are considered to be identical under room temperature conditions [11]. While it is clear that 17,. coli cells may survive during prolonged exposure in artificial sea-water with 2.17% as well as 0.9% salt, we have at present no explanation for the difference between the strains in their activity to form colony-forming units when suspended in 2.17% salt containing artificial sea-water. The E. coil cells were also heat stressed in both low (0.9%) and high (2.17%) salt solution to ensure that t h e heat resistance developed during starvation was not due to a secondary effect of salt stress. We found that when E. coil cells were in their normal habitat (0.9% salt solution) they developed a better protection against heat stress (Fig. 2C,E). It has been demonstrated for both ,2-. coli and I4brio cells that an on-going protein synthesis and the expression of starvation specific proteins are necessary for prolonged survival during multiple as well as carbon starvation [5,22]. The molecular and physiological adaptation of I,qbrio cells during starvation has recently been reviewed. It is suggested that a subclass of the starvation specific genes that are induced during the differentiation program of starvation is essential for the prolonged survival of starving Vibrio cells [10]. It has also been suggested that starvation-induced proteins in both Vibr/o and E. coli cells may mediate the cross protection against ~. range of stress conditions during the survival phase of starvation [6,23,24]. This study demonstrates that Vibrio sp. DW1 and E. coil cells, starved f o r two and nine days respectively, are considerably more heat'resistent than cells starved for less than two days (Figs. 2A-E). This protection may conceivably be a
result of the induction of heat-shock proteins during starvation. The gene product of htpR (rpoH), Sigma-32, that promotes the synthesis of the heat shock proteins has a half-life of I min at 30°C and of 4 min at 42°(2. Continuous synthesis of Sigma-32 is required for induction of the heatshock proteins [25,26]. We believe that the similarity in response to high temperature exposure of the wild-type and the mutant E. coli cells rules out the possibility that cross protection against heat stress at various times of starvation reflects the induction of the heat-shock regulon. VanBogelen et al. [27] induced the synthesis of heatshock proteins by exposure to nalidixic acid, puromycin, and H 2 0 2 for 15 min, but did not obtain thermotolerance. Thermotolerance was acquired, however, when the cells were exposed to an elevated temperature, ethanol, CdCI 2 and prolonged treatment by H 2 0 2. It was concluded that thermotolerance is developed by processes other than the htpR-dependent induction of heat-shock proteins. In support of the notion that the starvation-induced increased tolerance against heat stress found in this study is not due to the ability to synthesize heat-shock proteins, we have recently established that the starvation induced synthesis of the heat-shock protein DnaK in starving Vtbr/o sp. DW1 does not correlate with the development of cross protection against heat (unpublished results). In E. coli K12, the carbon starvation-induced synthesis of heat-shock proteins is reported to occur during the initial 6 h of starvation [28]. Based on the decrease in the rate of protein synthesis (Table 1) and rather similar protection against heat stress for both Cm-treated and untreated cells after prolonged starvation (Fig. 3), it may be suggested that while the de novo protein synthesis during the initial phase of starvation plays a role for the development of increased heat resistance, relatively long-term starved cells of the strains included in this study may develop heat resistance via an additional, de novo protein synthesis independcnt response. In fact, an obvious de novo protein synthesis mediated induction of protection against heat stress during starvation was not found in this study. Vibrio sp. DWl was not effected by the interruptions in protein syn-
235
thesis prior to heat stress at any time of starvation, while E. coli Sc122 showed a great dependence of protein synthesis between 4 and 22 h of starvation for developing heat resistance (Fig. 3). However, protein synthesis dependent development of thermal protection was not achieved during the first 2 h of starvation. After 22 h of starvation, E. coil Sc122 cells did not depend on de novo protein synthesis for developing increased thermotolerance. In support of the latter finding, it has been shoval that non-differentiating bacteria shut down the metabolism and become smaller and coccoid during prolonged starvation. It has also been argued that these cells enter a state of dormancy, and that starving vegetative non-differentiating bacteria transform themselves into a more spore-like structure [5]. In addition to very low levels of endogenous metabolism and protein synthesis, a condensed nucleoid also signifies such cells [4]. Furthermore, two dimensional gel electrophoresis analysis of pulse labelled polypeptides at various times of starvation of WdTr/o sp. $14, revealed that very few new starvation-specific proteins are induced after 72 h (3 days) of starvation, while a total o f 66 of the starvation-induced proteins were identified within 48 h of energy and nutrient starvation. It may therefore be suggested that the processes that allow for the development of the resistant phenotype may partly be a result of proteins synthesized prior to the onset of starvation. In summary, enhanced resistance against heat stress was detected not only for the initial 24 h of starvation, which may be similar to the resistance previously reported for Vtbr/o sip. $14 [5] and E. coil K12 cells [6], but also during the entire period of nine days of starvation. WhOe shortterm starvation gives a very significant degree of protection, prolonged periods of non-growth in fact offer increased protection although after different time periods for the different strains, and for E. coli strains, depending on the salinity of the starvation regime. Both de novo protein synthesis dependent and, with time of starvation, de novo protein synthesis independent starvationinduced protection against heat are suggested to take place in these strains.
:~•i ••
•~ i •
ACKNOWLEDGEMENTS We thank Ruth VanBogelen for providing the E. coil strains and for sharing her experience from working with these strains. We thank Agn e t a Danielsson for technical assistance. This study was supported by a grant from the Swedish Natural Science Research Council Amanda E Goodman was funded in part by an Australian Research Council Fellowship.
REFERENCES Ill Kjeheberg,S., N~n~m. T., Albertson~N. ar,d P~rdh, K. (1990) S p ~ a l Issue. Nutrient Limitation: Global Resp~].~'.s and Procaryotic :Development. [2j H ~ , D.A. and Chafer, K.F. (1989) Genctics of Bacterial Diversity. Academic P r e ~ London. [3] Amy, P.S. and Morita, R.Y. 0983) Starvatlo,vsurvival patterns of sixteen freshly isolated open-oc~n bacteria~ Appl. Environ. M ~ . 45, 1109-1115.
[4] Moyer, C.L and Morita, R.Y. (1989) Effect of growth rate and statwafion-survival on cellular DNA, RNA, and protein of a psychrophilic marine bacterium. AppL Environ. Microbiol. 55~ 2710-2716.
[5] Nystrfm, T., Fl~rdh, K- and K~elteberg,S. (1~0) Responses to multiple-nutrient starvation in marine trdn~,o ~p. Strain CCJ.JG 1.5956. J. Bacterio!. 172, 7f~-7097. 16] Jenkins, D.E., Schuitz, J.E. and Matin, A. (1988) Starvation-induced cross protection against heat o r H 2 0 2 challenge in Escher/dda co//. J. Bacteliol. 170, 3910-3914. [7] Spector, M.P, Aliabadi, Z., Gonzalez, T. and Foster, J.W. (1986) Global control in Sa/mond/a typ/dmut/um: Two-dimenfiona! eha~rophorctic anab~sis of starvation-, anaerobiosis-, and heat shock-inducible proteins. J. BacterioL 168, 420-424. ~8] Spence, J., Cegieisga, A. and Geofgop~ak~, C. (1990) Role of Escherichia coli heat-shock proteins DnaK and HtpG (C62,5) in response to nutritional deprivation..L Bacteriol. 172, 7157-7166. [9] Matin, A., Auger, E.A., Blum, P.H. and Schultz, J.E. (1989) Genetic basis of stat~ation survival in nondifferentiatittg bacteria. Annu. Rev. MicrobioL 43, 293-316. [10] NysttSm, T , Albertson, N.H. Fl~rdh, K. and Kjelleberg, S. (1990) Physiological and molecular adaptation to starvation iV the marine Ftbr/o sp. S14. FEMS MicrobioL Ecol. 74, |29-140. [1t] Cooper, S. and Reuettinger, T. (1975)A temperature sensitive nonsense mutation affecting the synthesis of a major protein of Escherichia coil K12. Mol. gen. Genet2 139, 167-176. [12] NeidhardL F.C. and VanBogelen, R.A. (1981) Positive regulatory gene for temperature-controlled proteins in
236 Escherichia coll. Biochem. Biophys. Res. Comm. 100, 894-900. [13] Neidhardt, F.C., VanBogelen, R.A. and Vaughn, V. (1984) The genetics and regulation of heat-shock proteins. Ann. Rev. Genet. 18, 295-329. [14] Dawson, M,P., Humphrey, B. and Marshall, K.C. (1981) Adhesion: a tactic in the survival strategy of a marine Vibrio during starvation, Curr. Microbiol. 6, 930-940. [15] Hoben, H.J. and Somesegaran, P. (1982) Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl. Environ. Microbiol. 44, 1246-1247. [16] Nystr6m, T., M~rd6n, P. and Kjelleberg, S. (1986) Relative changes in incorporation rates of lencine and methaionine during starvation survival of two bacteria isolated from marine waters. FEMS Microbiol. Ecol. 38, 285-292. [17] Amy, P.S., Pauling, C. and Morita, R.Y. (1983) Starvation-survival processes of a marine Vibrio. Appl. Environ. Microb. 45, 1041-1048. [18] Hoff, K.A. 0989) Survival of Vd~r/o Anguillamm and Fibrio sabnonicida at different salinities, Appl. Environ. Microbiol. 55, 1775-1786. [19] Sorensen, S.S. (1991) Survival of Escherichia coil KI2 in seawater. FEMS MicrobioL Ecol. 85, 161-168. [20] Munro, P.M., Gauthier. MJ. and Laumopd, F.M. (1987) Changes in Escherichia coli starved ~:nseawater or grown in seawater-wastewater mixtures. Appl. Environ. Microbiol. 53, 1476-1481. [21] Munro, P.M., Gauthier, M.J.. Breittmayer, V.A. and
Bongiovanni, J. (1989) Influence of osmoregulation proces,ses on starvation survival of Escherichia coli in seawater. App. Environ. MicrobioL 55, 2017-2024. [22] Reeve, C.A., Amy, P.S. and Matin, A. (1984) Role of protein synthesis in the survival of carbon-starved Escherichia coil K-12. J. Bacteriol. 160, 1041-1046. [23] Nystr6m, T., OIsson, R.M. and Kjeileberg, S. (1992) Sur~val, stress resistance and alterations in protein expression in the marine Vibrio sp. S14 during starvation fol different individual nutrients. AppL Environ. MicrobioL 58, 55-65. [24] Jenkins, D.E., Chaisson, S.A. and Matin, A. (1990) Starvation-induced cross protection against osmotic challenge in Eschetichia coll. J. Bacteriol. 172, 2779-2781. [25] Straus, D.B., Walter, W.A. and Cross, C.A. (1987) The heat-shock response of E. coil is regulated by changes in the concentration of sigma-32. Nature 329, 348-351. [26] Yura, T., Kawasaki, Y., Kusukawa, N., Nagai. H., Wada, C. and Yano, R. (1990) Roles and regulation of the heat-shock sigma factor sigma-32 in Escherichia coll. Antonie van Leeuwenhoek. 58, 187-190. [27] VanBogelen, R,A, Acton, M.A. and Neidhardt, F.C. (1987) Induction of the heat-shock regulon does not produce thermotolerance in Escherichia coll. Gen. Develop. 1,525-531. [28] Jenkins, D.E., Auger, E.A. and Matin, A. (1992) Role of RpoH, a heat shock regulator protein, in Eschetichia coil carbon starvation protein synthesis and survh,al. J. Bacteriol. 173, 1992-1996.