Journal
of Biotechnology,
19 (1991)
159
159-172
0 1991 Elsevier Science Publishers B.V. 0168-1656/91/$03.50 ADONIS 0168165691001028 BIOTEC 00620
Influence of low-temperature storage and glucose starvation on growth recovery in Escherichia coli relA and reIA+ strains Michel van Bake1 ‘, Norbert Vischer ‘, RenC Tixador and Conrad Woldringh r ’ Universiiy
of Amsterdam, The Netherlands
Department of Molecular Cell Biology, and ’ UniversitP Paul Sabatier, Facultk Toulouse Cedex, France
2, Gilbert
section Molecular Cytology, de MJdecine Txdouse-Purpan,
Gasset ’ Amsterdam,
(Received 11 October 1990; revision accepted 16 December 1990)
summary To study the influence of microgravity on bacterial growth behavior during a space mission, the special experimental conditions and the hardware environment necessitate storage of cells at low temperature, and permit a relatively short experimental period. Before this experimental period, cells have to recover their condition of steady-state growth, because it is only in this condition that the growth behavior of the flight and ground populations can be adequately compared. To meet these requirements and to obtain cells which recover rapidly their steady-state growth, we analyzed the size and shape of Escherichia coli cells during storage at 4°C with and without previous glucose starvation of the cells. It appeared that cells stored at low temperature in the presence of glucose continued to increase in average mass and assumed ovoid shapes. In addition, upon restoration of maximal growth rate at 37O C, they continued to increase in size and showed a transient overshoot of their final steady-state value, which was reached after about 5 h. Cells previously starved for glucose, however, maintained their average size and rod-shape during low-temperature storage. Recovery of the starved cells was most rapid in the relA + strain which, contrary to the isogenic reL4 strain, showed no overshoot and reached its final steady-state size within 2 h. Correspondence ro: M.A.J.M. van Bakel, Department of Molecular Cellbiology, Section Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands.
160
Escherichia coli; Cold shock; Low-temperature shape change; R&status; Growth recovery
storage; Glucose starvation;
Cell
Introduction
Extensive biological experiments during space missions have suggested an adaptation of plant, bacterial, protozoan and animal cells to changes in the gravitational environment (Lapchine et al., 1986; Mennigmann and Lange, 1986; Moatti et al., 1986; Theimer et al., 1986; Cogoli et al., 1987). Regarding the effects on bacterial cells it was found that the maximal growth rate and the total yield of biomass of Bacillus subtilis were higher under microgravity conditions than at 1 g (Mennigmann and Lange, 1986), whereas in Escherichia cofi an increase in resistance to antibiotics was found under microgravity conditions (Tixador et al., 1985). For the International Microgravity Laboratory-l (IML-1) mission in 1990, two experiments with bacteria have been planned, in which bacterial cell proliferation will be investigated again. Without intending to furnish an explanation for the phenomenon, a physical factor such as microgravity could be envisaged as influencing bacterial growth in different ways. First, the change could only interact at the level of energy metabolism, simply enabling the cells to produce more biomass from the available nutrients. Second, and perhaps in addition to the first possibility, a change in rnicrogravity could influence the regulation of physiological processes such as protein synthesis, DNA replication or the division process. Whereas all of these processes influence both the size and the shape of the average cell in the population, only a change in protein synthesis regulation will affect the maximal growth rate. Examples of the effect of physical and chemical factors on either of these processes have been described in the literature, e.g. changes in temperature (Trueba et al., 1982b; Shaw, 1968; Gilbert et al., 1979; Broeze et al., 1978), osmolarity (Baldwin et al., 1987; Ingraham, 1987) and nutrient conditions (Schaechter et al., 1958; Nanninga and Woldringh, 1985; Woldringh et al., 1980). From the above studies, it has become clear that differences in cell or population properties can only be ascribed to the changes in the physical or nutritional environment under study, if the populations are fully adapted to their environment. In addition, the inherent variability of individual cells in any population, necessitates the screening of a great number of cells to estimate average cell properties. If a culture is fully adapted to its environment and has been growing exponentially for a sufficient period, the distribution of all intensive properties will be constant in time. Such a population has been said to be in the steady state of growth (Painter and Marr, 1968), which is usually attained after growing bacteria for 10 to 20 generations in a constant environment (Harvey et al., 1967), and is considered to be established in batch cultures when average cell mass (see Material and Methods) remains constant in time. Adequate comparison of two cultures, e.g. one growing in a space-flight and one on the ground, can only occur if the two populations are
161
growing in a steady state. Otherwise, differences are found which reflect the previous growth history of the cells or which represent a transient state in adaptation. Steady-state growth is achieved in batch cultures by inoculating fresh colonies from an agar plate in synthetic medium and keeping a low cell concentration (less than 5 x lo* cells per ml) by periodical dilution of the culture. To perform such an experiment during a space mission several limitations have to be met. First, cells have to be stored for an undefined period of time (about two weeks). Second, the experimental growth period is limited and, third, the cultivation is performed in specialized hardware which, in a simple system (Tixador et al., 1983), does not allow periodical dilution of the culture. Because of these limitations we decided to ‘freeze’ a steady-state population either by cooling or by glucose starvation intending to establish conditioned cells which quickly recover steady-state growth when supplied with fresh medium under space-mission conditions. In addition, a comparison was made between a reU and an isogenic relA + strain, because carbon-source starvation is known to induce stringent control (Molin et al., 1977). This so-called stringent response is thought to protect cells from producing incomplete proteins which adversely affect growth recovery under conditions of unbalanced synthesis. This protection is accomplished, amongst others, by inhibition of stable RNA synthesis and stimulation of intracellular proteolysis (Lamond and Travers, 1985). In this work therefore we analyzed the effect of storage at 4°C on cells of the two types, relA and reL4 +, with or without previous starvation for glucose. The measurements show that the use of a relA + strain and of glucose starvation before cooling to 4°C assures the most rapid recovery of steady-state growth. Materials and Methods Organisms and culture media E. coli MC4100 F- araD139 del(argF-lac)U169 rpsLI50 jlbB5301 ptsF25 deoC1 rbsR relA1 (Casadaban, 1976) and the isogenic relAI + strain were used. Test of the
relA phenotype was performed by determining the slow growth on agar plates supplemented with 100 pg ml-’ of serine, methionine and glycine. The growth medium used was M9 medium with 0.4% glucose (w/v) containing: 1 g 1-l NH,Cl, 7.5 g 1-l NaH,PO, .2H,O, 3 g 1-l KH,PO, and 0.5 g 1-l NaCl, 2 mg 1-l aneurine (vit. Bl), 0.5 mM MgSO,, and NaCl (final concentration 18 mM), which was added in order to keep the osmolarity 300 mOsm. The pH was brought to 7.4 with 1 N NaOH. In M9 medium without glucose the osmolarity was kept at 300 mOsm by adding NaCl up to a final concentration of 26 mM. Preparation of cells
Cells were cultured in M9 medium with 0.4% glucose in batch cultures. Samples were fixed with formaldehyde (final concentration 0.24%). Growth was monitored
162
by determination of the optical density at 450 nm (OD,,,) with a Gilford STASAR II spectrophotometer and cell number per ml (N) with an electronic particle counter equipped with a 30 pm aperture tube. As a measure of relative average cell mass, M was calculated as OD units per lo9 cells. Exponential growth was maintained by periodical dilution. Steady state growth in batch culture is defined as an exponentially growing population of cells, whose average cell mass remains constant. A steady-state growing culture (with an OD,,, of about 0.3) was cooled for 3 min on ice and stored at 4°C. Bacteria that were first starved for glucose by dilution of a steady-state growing culture with an OD,,, of about 0.3 in M9 medium without glucose, were also cooled on ice and stored at 4°C as soon as the optical density became constant (about 0.3). The absence of glucose was tested with glucose test strips (Glukotest; Boehringer, Mannheim). Restarting growth was performed by dilution of an either nonstarved or starved culture in M9 medium with glucose and aeration by shaking at 37°C. Preparation of cells for electron microscopy
Samples that were fixed with formaldehyde were additionally fixed with 0~0, (final concentration 0.1% w/v) and prepared for electron microscopy by agar filtration (Woldringh et al., 1977). Samples were photographed in a Philips EM 300 electron microscope. Length distributions were made from these photographs with a Macintosh programme called ‘Capture’ (developed by N. Vischer), which was especially developed for this purpose, using a tablet (Summagraphics MacTablet) for data input. Kolmogorov-Smirnov
test
The similarity of length distributions was tested by applying the KolmogorovSmimov test to cumulative distributions (Siegel, 1956). The cumulative frequency S is the sum of the relative frequencies and is given by: s(a) = C$+ X 100%; in which N, is the number of cells in class a and N is the total number of cells in the distribution. The maximum difference D,,,,, between the two distributions is given by: D max=~*(w-&(2);
in which S,(l) and S,(2) are the cumulative frequencies of the two distributions. The critical difference can be calculated as follows: Dcritica,= M x
163
in which n, and n2 are the total number of cells in both distributions. probability limit is given by (Y,then M’ppT;
If the
(4
If (Y= 0.05. then the M value will be 1.36.
Results Changes in average cell size
To obtain rapid recovery of steady-state growth conditions (within 5 h) after storage of E. coli cells at .4’C, we compared the growth behavior of a ret!,4 strain with that of an isogenic ret!,4+ strain. In order to ‘freeze’ the condition of steady-state growth, storage of the strains was either performed by rapid cooling of the cells in the exponential phase of growth or by starving the cells for glucose prior to cooling to 4°C. The two isogenic strains showed a difference in growth rate. In the glucose minimal M9 medium, the relA strain grew more rapidly in four independent experiments, having an average doubling time of 51 min, compared to 65 min for the reL4 + strain. The steady-state average cell mass (M) was similar for both strains, about 3.2. Although larger, this difference confirmed previous observations conceming another pair of isogenic relA/relA + strains, i.e. E. co/i NF161 and NF162 (unpublished results). In Fig. 1 the growth and division behavior of the reL4 strain is shown. Upon cooling to 4°C (time 0, Fig. lA), the average cell mass slightly decreased as a result of residual division. After a growth stop of about 2 d (50 h), average cell mass slowly increased again, resulting in a nearly 2-fold increase after 189 h of storage. During this period the percentage of constricting cells in the population rapidly increased from its steady-state value of 25% to about 40%. Restoration of maximal growth rate at 37°C after 189 h of storage (Fig. lB), showed a continuing increase in average mass, resulting in a 3.5-fold increase after 1 h at 37°C (t = 190 h, Fig. lB), compared to steady-state average cell mass. This overshoot was followed by a decrease in average cell mass, not reaching its steady-state value within 5 h (194 h in Fig. 1B) of incubation at 37°C. The percentage of constricting cells remained at the elevated level of about 40% during this period. The relA + strain showed a similar behavior under these conditions (data not shown), although average cell mass showed a smaller overshoot of about 2.5 times the steady-state value. In contrast with the ret% strain, a decrease in percentage of constricting cells occurred from steady-state 32% to about 20%. Upon restoration of growth, the percentage of constricting cells recovered the steady-state value. Fig. 2 shows the behavior of the relA cells during glucose starvation. Conforming to general experience (Ingraham et al., 1983) growth terminated abruptly at glucose depletion compared to cells normally entering the stationary phase at a much higher
164
1000
of Biotechnology,
19 (1991)
159
159-172
0 1991 Elsevier Science Publishers B.V. 0168-1656/91/$03.50 ADONIS 0168165691001028 BIOTEC 00620
Influence of low-temperature storage and glucose starvation on growth recovery in Escherichia coli relA and reIA+ strains Michel van Bake1 ‘, Norbert Vischer ‘, RenC Tixador and Conrad Woldringh r ’ Universiiy
of Amsterdam, The Netherlands
Department of Molecular Cell Biology, and ’ UniversitP Paul Sabatier, Facultk Toulouse Cedex, France
2, Gilbert
section Molecular Cytology, de MJdecine Txdouse-Purpan,
Gasset ’ Amsterdam,
(Received 11 October 1990; revision accepted 16 December 1990)
summary To study the influence of microgravity on bacterial growth behavior during a space mission, the special experimental conditions and the hardware environment necessitate storage of cells at low temperature, and permit a relatively short experimental period. Before this experimental period, cells have to recover their condition of steady-state growth, because it is only in this condition that the growth behavior of the flight and ground populations can be adequately compared. To meet these requirements and to obtain cells which recover rapidly their steady-state growth, we analyzed the size and shape of Escherichia coli cells during storage at 4°C with and without previous glucose starvation of the cells. It appeared that cells stored at low temperature in the presence of glucose continued to increase in average mass and assumed ovoid shapes. In addition, upon restoration of maximal growth rate at 37O C, they continued to increase in size and showed a transient overshoot of their final steady-state value, which was reached after about 5 h. Cells previously starved for glucose, however, maintained their average size and rod-shape during low-temperature storage. Recovery of the starved cells was most rapid in the relA + strain which, contrary to the isogenic reL4 strain, showed no overshoot and reached its final steady-state size within 2 h. Correspondence ro: M.A.J.M. van Bakel, Department of Molecular Cellbiology, Section Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The Netherlands.
160
Escherichia coli; Cold shock; Low-temperature shape change; R&status; Growth recovery
storage; Glucose starvation;
Cell
Introduction
Extensive biological experiments during space missions have suggested an adaptation of plant, bacterial, protozoan and animal cells to changes in the gravitational environment (Lapchine et al., 1986; Mennigmann and Lange, 1986; Moatti et al., 1986; Theimer et al., 1986; Cogoli et al., 1987). Regarding the effects on bacterial cells it was found that the maximal growth rate and the total yield of biomass of Bacillus subtilis were higher under microgravity conditions than at 1 g (Mennigmann and Lange, 1986), whereas in Escherichia cofi an increase in resistance to antibiotics was found under microgravity conditions (Tixador et al., 1985). For the International Microgravity Laboratory-l (IML-1) mission in 1990, two experiments with bacteria have been planned, in which bacterial cell proliferation will be investigated again. Without intending to furnish an explanation for the phenomenon, a physical factor such as microgravity could be envisaged as influencing bacterial growth in different ways. First, the change could only interact at the level of energy metabolism, simply enabling the cells to produce more biomass from the available nutrients. Second, and perhaps in addition to the first possibility, a change in rnicrogravity could influence the regulation of physiological processes such as protein synthesis, DNA replication or the division process. Whereas all of these processes influence both the size and the shape of the average cell in the population, only a change in protein synthesis regulation will affect the maximal growth rate. Examples of the effect of physical and chemical factors on either of these processes have been described in the literature, e.g. changes in temperature (Trueba et al., 1982b; Shaw, 1968; Gilbert et al., 1979; Broeze et al., 1978), osmolarity (Baldwin et al., 1987; Ingraham, 1987) and nutrient conditions (Schaechter et al., 1958; Nanninga and Woldringh, 1985; Woldringh et al., 1980). From the above studies, it has become clear that differences in cell or population properties can only be ascribed to the changes in the physical or nutritional environment under study, if the populations are fully adapted to their environment. In addition, the inherent variability of individual cells in any population, necessitates the screening of a great number of cells to estimate average cell properties. If a culture is fully adapted to its environment and has been growing exponentially for a sufficient period, the distribution of all intensive properties will be constant in time. Such a population has been said to be in the steady state of growth (Painter and Marr, 1968), which is usually attained after growing bacteria for 10 to 20 generations in a constant environment (Harvey et al., 1967), and is considered to be established in batch cultures when average cell mass (see Material and Methods) remains constant in time. Adequate comparison of two cultures, e.g. one growing in a space-flight and one on the ground, can only occur if the two populations are
161
growing in a steady state. Otherwise, differences are found which reflect the previous growth history of the cells or which represent a transient state in adaptation. Steady-state growth is achieved in batch cultures by inoculating fresh colonies from an agar plate in synthetic medium and keeping a low cell concentration (less than 5 x lo* cells per ml) by periodical dilution of the culture. To perform such an experiment during a space mission several limitations have to be met. First, cells have to be stored for an undefined period of time (about two weeks). Second, the experimental growth period is limited and, third, the cultivation is performed in specialized hardware which, in a simple system (Tixador et al., 1983), does not allow periodical dilution of the culture. Because of these limitations we decided to ‘freeze’ a steady-state population either by cooling or by glucose starvation intending to establish conditioned cells which quickly recover steady-state growth when supplied with fresh medium under space-mission conditions. In addition, a comparison was made between a reU and an isogenic relA + strain, because carbon-source starvation is known to induce stringent control (Molin et al., 1977). This so-called stringent response is thought to protect cells from producing incomplete proteins which adversely affect growth recovery under conditions of unbalanced synthesis. This protection is accomplished, amongst others, by inhibition of stable RNA synthesis and stimulation of intracellular proteolysis (Lamond and Travers, 1985). In this work therefore we analyzed the effect of storage at 4°C on cells of the two types, relA and reL4 +, with or without previous starvation for glucose. The measurements show that the use of a relA + strain and of glucose starvation before cooling to 4°C assures the most rapid recovery of steady-state growth. Materials and Methods Organisms and culture media E. coli MC4100 F- araD139 del(argF-lac)U169 rpsLI50 jlbB5301 ptsF25 deoC1 rbsR relA1 (Casadaban, 1976) and the isogenic relAI + strain were used. Test of the
relA phenotype was performed by determining the slow growth on agar plates supplemented with 100 pg ml-’ of serine, methionine and glycine. The growth medium used was M9 medium with 0.4% glucose (w/v) containing: 1 g 1-l NH,Cl, 7.5 g 1-l NaH,PO, .2H,O, 3 g 1-l KH,PO, and 0.5 g 1-l NaCl, 2 mg 1-l aneurine (vit. Bl), 0.5 mM MgSO,, and NaCl (final concentration 18 mM), which was added in order to keep the osmolarity 300 mOsm. The pH was brought to 7.4 with 1 N NaOH. In M9 medium without glucose the osmolarity was kept at 300 mOsm by adding NaCl up to a final concentration of 26 mM. Preparation of cells
Cells were cultured in M9 medium with 0.4% glucose in batch cultures. Samples were fixed with formaldehyde (final concentration 0.24%). Growth was monitored
162
by determination of the optical density at 450 nm (OD,,,) with a Gilford STASAR II spectrophotometer and cell number per ml (N) with an electronic particle counter equipped with a 30 pm aperture tube. As a measure of relative average cell mass, M was calculated as OD units per lo9 cells. Exponential growth was maintained by periodical dilution. Steady state growth in batch culture is defined as an exponentially growing population of cells, whose average cell mass remains constant. A steady-state growing culture (with an OD,,, of about 0.3) was cooled for 3 min on ice and stored at 4°C. Bacteria that were first starved for glucose by dilution of a steady-state growing culture with an OD,,, of about 0.3 in M9 medium without glucose, were also cooled on ice and stored at 4°C as soon as the optical density became constant (about 0.3). The absence of glucose was tested with glucose test strips (Glukotest; Boehringer, Mannheim). Restarting growth was performed by dilution of an either nonstarved or starved culture in M9 medium with glucose and aeration by shaking at 37°C. Preparation of cells for electron microscopy
Samples that were fixed with formaldehyde were additionally fixed with 0~0, (final concentration 0.1% w/v) and prepared for electron microscopy by agar filtration (Woldringh et al., 1977). Samples were photographed in a Philips EM 300 electron microscope. Length distributions were made from these photographs with a Macintosh programme called ‘Capture’ (developed by N. Vischer), which was especially developed for this purpose, using a tablet (Summagraphics MacTablet) for data input. Kolmogorov-Smirnov
test
The similarity of length distributions was tested by applying the KolmogorovSmimov test to cumulative distributions (Siegel, 1956). The cumulative frequency S is the sum of the relative frequencies and is given by: s(a) = C$+ X 100%; in which N, is the number of cells in class a and N is the total number of cells in the distribution. The maximum difference D,,,,, between the two distributions is given by: D max=~*(w-&(2);
in which S,(l) and S,(2) are the cumulative frequencies of the two distributions. The critical difference can be calculated as follows: Dcritica,= M x
163
in which n, and n2 are the total number of cells in both distributions. probability limit is given by (Y,then M’ppT;
If the
(4
If (Y= 0.05. then the M value will be 1.36.
Results Changes in average cell size
To obtain rapid recovery of steady-state growth conditions (within 5 h) after storage of E. coli cells at .4’C, we compared the growth behavior of a ret!,4 strain with that of an isogenic ret!,4+ strain. In order to ‘freeze’ the condition of steady-state growth, storage of the strains was either performed by rapid cooling of the cells in the exponential phase of growth or by starving the cells for glucose prior to cooling to 4°C. The two isogenic strains showed a difference in growth rate. In the glucose minimal M9 medium, the relA strain grew more rapidly in four independent experiments, having an average doubling time of 51 min, compared to 65 min for the reL4 + strain. The steady-state average cell mass (M) was similar for both strains, about 3.2. Although larger, this difference confirmed previous observations conceming another pair of isogenic relA/relA + strains, i.e. E. co/i NF161 and NF162 (unpublished results). In Fig. 1 the growth and division behavior of the reL4 strain is shown. Upon cooling to 4°C (time 0, Fig. lA), the average cell mass slightly decreased as a result of residual division. After a growth stop of about 2 d (50 h), average cell mass slowly increased again, resulting in a nearly 2-fold increase after 189 h of storage. During this period the percentage of constricting cells in the population rapidly increased from its steady-state value of 25% to about 40%. Restoration of maximal growth rate at 37°C after 189 h of storage (Fig. lB), showed a continuing increase in average mass, resulting in a 3.5-fold increase after 1 h at 37°C (t = 190 h, Fig. lB), compared to steady-state average cell mass. This overshoot was followed by a decrease in average cell mass, not reaching its steady-state value within 5 h (194 h in Fig. 1B) of incubation at 37°C. The percentage of constricting cells remained at the elevated level of about 40% during this period. The relA + strain showed a similar behavior under these conditions (data not shown), although average cell mass showed a smaller overshoot of about 2.5 times the steady-state value. In contrast with the ret% strain, a decrease in percentage of constricting cells occurred from steady-state 32% to about 20%. Upon restoration of growth, the percentage of constricting cells recovered the steady-state value. Fig. 2 shows the behavior of the relA cells during glucose starvation. Conforming to general experience (Ingraham et al., 1983) growth terminated abruptly at glucose depletion compared to cells normally entering the stationary phase at a much higher
164
1000
