JOURNAL OF BIOSCIENCE AND BIOENGWEERING Vol. 88, No. 3, 342-344. 1999

Effect of Hypergravitational Stress on Microbial Cell Viability NAOTO YOSHIDA,‘* TAKUMI MINAMIMURA,’ TERUTOYO YOSHIDA,* AND KIHACHIRO OGAWA’

Department of Biological Resource Sciences,’ and Department of Fisheries,2 Faculty of Agriculture, Miyazaki University, 1-I Gakuen Kibanadai-Nishi, Miyazaki-shi 889-2192, Japan

Received 14 January 1999/Accepted 10 June 1999

Cell of Escherichia coli B, Thiobacillus intermedius 13-1, Bacillus amyloliquefaciens IF014141, Staph- ylococcus aureus IID975, and Saccharomyces cerevisiae Kyokai no. 7 cultivated in nutrient media were subjected to hypergravitational stress for a period of between 1 and 24 h at 450,000 X g. The E. coli, B. amyloliquefaciens and S. cerevisiae cells showed survival rates of 38.5x, O.OOS%, and 14.7x, respectively, after gravity treatment for 24 h as determined by their ability for colony formation, whereas a survival rate of S. cerevisiae cells of almost 100% was observed as determined using the methylene blue reduction test. E. coli cells, either in the logarithmic growth phase or cultivated in minimum media, were more sensitive to gravita- tional stress than those either in the stationary phase or cultivated in nutrient media.

[Key words: gravitational stress, mechanical stress]

Little is known about the resistance of bacteria and yeast cells to mechanical stress other than that to ultra- sonic vibration (l), shearing forces in grinding with abra- sives (2), high magnetic field (3), microgravity (4-6), and the effect of high pressure (7). Several studies have sug- gested that bacteria respond in various way to environ- mental stress as a mechanism for survival. Recently, rice (Oryza sativa L. Var. Nipponbare) seeds, seedlings and suspensions of callus were subjected to hypergravitational stress ranging from 150,000 to 450,000 xg. The sus- pensions of callus and dehulled seeds showed 32% and 15% survival rates, respectively, after gravity treatment at 450,000 x g for 6 h, whereas all the seedlings died (8). However, the effects of mechanical hypergravitational stress have not yet been fully investigated in microorgan- isms. These studies on the response of microorganisms to gravity may be useful for understanding the protective mechanisms in microorganisms against various environ- mental stresses, and lead to the finding of noble genes which could confer stress resistance. In this paper, we report on the effects of hypergravity at 450,000 xg on the viability of Escherichia coli B and Thiobacillus inter- medius 13-1 (gram-negative bacteria), Bacillus amyloli- quefaciens IF014141 and Staphylococcus aureus IID (gram-positive bacteria), and Saccharomyces cerevisiae Kyokai no. 7 (eucaryotes).

E. coli in LB or M9 minimum medium, B. amyloli- quefaciens, T. intermedius, and S. aureus in LB medium (9), and S. cerevisiae in YPD medium were cultivated for 24 to 48 h at 30°C on a rotatory shaker (150rpm). E. coli cells were harvested in either the logarithmic phase or the stationary phase of growth. Cells were har- vested by centrifugation at 3000 xg for 10 min and washed twice with physiological saline. The cells were resuspended in physiological saline and adjusted to a density 2 x log cells/ml. A polyallomer bell-top type cen- trifuge tube (Beckman, 16x 45 mm), and fixed angle rotor (Beckman, 70.1 Ti), whose tube angle is 24”, was used. The neck of the tube was dried and completely sealed by a hand-held tube topper after the bacterial cell suspension was filled to the base of the tube stem. The maximum and minimum radial distance of the fixed an-

* Corresponding author.

gle rotor is 82.0 or 40.5 mm, respectively. The relative centrifugal field at the radial distance of 82.0 mm is 450,000 x g, where the rotor rotates at 70,000 rpm. Each of the cell suspensions (2 x log/ml physiological saline) was placed in sterilized centrifuge tubes and centrifuged at 450,000 x g for various durations ranging from 1 to 24 h at 4°C. The viability of the cells was determined by counting the number of colony-forming units (CFUs) on LB plates for E. coli, B. amyloliquefaciens, T. inter- medius, and S. aureus or on a YPD plate for S. cere- visiae. The survival rates (S) of cells was calculated by cornparity the number of CFUs of gravity-treated cells (T) and non gravity treated cells (C) using the follow- ing equation: S=(T/C) x 100. There was a significant decrease in the viability of all samples as the duration of treatment increased. The viabilities of E. coli, B. amyloli- quefaciens and S. cerevisiae cells in the stationary phase were 38.5%, 0.005% and 14.7%, respectively, after grav- ity treatment at 450,OOOxg for 24 h (Figs. lA, 2, and 3A). E. coli cells cultured to the stationary-phase in M9 minimum medium (Fig. lB), and to the mid log-phase to a culture density of 0.5 (ODsso) in LB medium (Fig. IC) were much less resistant, with survival rates of only 5.1% and 4.5%, respectively, after gravity treatment for 24 h. Figure IC also shows that the cells of E. coli cultured to the mid log-phase in LB medium were most sensitive, with survival rates decreased to 22-37% after gravity treatment for only 1 h. The survival rates of B. amyloliquefaciens cells were 45.6-76.3X and 0.1% after gravity treatment for 1 h and 6 h, respectively, indicating that B. amyloliquefaciens cells are extremely sensitive to hypergravitational stress (Fig. 2). Table 1 compares the survival rates of the other gram-positive and gram-nega- tive bacteria examined in this study, after gravity treat- ment at 450,000~ g for 6 h. The survival rates of the gram-positive and gram-negative bacteria were 0.12- 9.30% and 27.0-77.0x, respectively, indicating that gram-negative bacteria (including E. coli and T. inter- medius) are relatively more resistant to hypergravitational stress than gram-positive bacteria (including B. amylo- liquefaciens and S. aureus).

When examining the effect of gravity on cells, the damage to or destruction of the cells during the ex- perimental procedure by cell to cell contact or contact of

342

VOL. 88, 1999 NOTES 343

Survival rate (%)

IOOI A A

60 60-

60 60-

40 40-

20 ZO-

00 0 5 10 15 20 25

Time(h)

FIG. 1. Effect of gravity (450,OOOxg) on the survival rate of E. coli B (A), stationary phase cells in LB medium; (B), stationary phase cells in M9 minimum medium; (C), mid-log phase cells in LB medium. The survival rate of cells in each phase was determined by counting the number of colony-forming units on LB plates.

cells with the inner tube surface can not be ignored. Thus, E. cofi cells were suspended in physiological saline containing 15% gelatin (Wako Pure Chemical Industries Ltd.) at the cell density of 2 x log cells/ml. The E. coli cell suspension was then packed into a centrifuge tube and rapidly cooled on ice to allow the cells to become embedded in gelatin. Since the cells are immobilized in the gelatin, it is assumed that they do not receive physi- cal injury lesions such as contiguities. After gravity treat- ment at 450,000 x g for 24 h at 4°C the gelatin surround-

Survival rate (%1

O\ 0 5 10 15 20 25

Time(h)

FIG. 2. Effect of gravity (450,000 X g) on the survival rate of B. atnyloliquefaciens IF014141.

Survival

Ob 25

Od 25

Time(h)

FIG. 3. Effect of gravity (450,000 X g) on the survival rate of S. cerevisiae Kyokai no. 7. The survival rate detected by (A), their ability for colony formation; (B), methylene blue reduction test.

ing the cells was liquefied by incubation at 37°C for 10min. The viability of the cells was determined by counting the number of CFU on an LB plate. E. coli cells were dispersed homogeneously in the inner tube, and their precipitate was not confirmed after gravity treatment. As shown in Table 2, the viable cell count of cells embedded and not embedded in gelatin were 45.1% and 3&O-45.0%, respectively, thus a marked difference was not observed. On the other hand, when the cell den- sity of E. coli was changed from 2 x lo8 to 2 x 106/ml, and the cells were subjected to treatment at 450,OOOxg for 24 h, the viable cell count was before and after 45%, indicating that there was no relationship in the cell con- centration. Therefore, it was concluded that the destruc- tion of the cells due to the contiguities of the cell or the contiguities with the cell and the inner tube surface could be ignored. This result shows that the cells die only under the influence of hypergravitational stress.

The viability of yeast cells has for many years been assessed by the ability of viable, as opposed to non viable cells, to take up the dye methylene blue and reduce it to the colorless leucomethylene blue (10). Non viable cells, in contrast, being unable to reduce the dye, stain blue. Thus the viability of S. cerevisiae was determined in this study by the methylene blue reduction test. Gravity-treat- ed and untreated yeast cell suspensions were diluted 5

TABLE 1. The survival rate of gram-positive or gram-negative bacteria after gravity treatment at 450,000 X g for 6 h

Bacterial strains Survival rate (%)

Gram-positive bacteria B. amyloliquefaciens IF014141 0.12- 0.13 S. aureus IID 4.80- 9.30

Gram-negative bacteria E. coli B 27.0 -77.0 T. intermedius 13-1 47.5 -54.8

344 YOSHIDA ET AL.

TABLE 2. The survival rate of E. co/i B after gravity treatment at 450,000 X g for 24 h when embedded in gelatin and for

different cellular concentrations

Treatment Cell cont. Survival rate (cells /ml) r?o

Physiological saline 2x109 38.0-45.0 Physiological saline containing 15% gelatin 2 x lo9 45.1 Physiological saline 2X 10s 48.0 Physiological saline 2x 107 45.1 Physiological saline 2x106 43.0

times with distilled water to adjust the cell density to lOOcells/ml. To an aliquot of each of the diluted cell suspensions, an equal volume of 0.02% methylene blue was added and the mixture incubated for 10min at 25°C a viable cell count was performed on a hemocyto- meter. Stained and unstained yeast cells were counted under a light microscope. Although, S. cerevisiae cells were resistant to gravitational stress, and showed a 97.3% survival rate as determined by the methylene blue reduc- tion test, their ability for colony formation was markedly decreased to 14.7% after a 24-h gravity treatment (Fig. 3B), suggesting that even though the cells were not viable, their enzyme systems were still active after the gravity treatment.

B. amyloliquefaciens was cultured in LB medium to the stationary phase at 30°C. The medium was replaced with 0.4M or 0.8 M of saccharide solutions of either sucrose, mannitol, or glycerol, and subjected to a 3-h grav- ity treatment at 450,000 x g. Each of the cell suspensions was diluted and plated onto LB plates. The viability of the cells treated with saccharides was determined by counting the number of CFUs on the LB plates. Cells treated with only physiological saline were concurrently examined as controls. The effect of pretreatment with saccharides on the viability of gravity-treated B. amyloli- quefaciens cells was investigated. Table 3 shows the results of centrifugation experiments with B. amyloli- quefaciens cells treated with saccharides, and control cells not treated with saccharides. The cell viability of B. amyloliquefaciens pretreated with either sucrose or man- nitol was 91.0-96.3% lower than that of the control cells. We concluded that pretreatments with sucrose and mannitol were not effective for cell protection. In the case of Oryza sativa, as a result of pretreatment with 0.6 M of sucrose, an increase from 0 to 13.6% in the sur- vival rate of suspensions of callus subjected to strong gravitational stress at 450,000 x g has been reported (8). However, there was no increase in the survival rate of B. amyloliquefaciens cells after pretreatment with 0.4 or 0.8M of either sucrose or mannitol. On the other hand, the survival rate increased to 40% when the cells were treated with 0.4 M glycerol, suggesting that glycerol effects cell stabilization, a known phenomenon applied for the storage of bacteria in lO-50% glycerol. Sucrose and mannitol may not be taken up intracellularly because their molecular weights are larger than that of glycerol, whereas glycerol is often taken up intracellular- ly, when the cell is exposed to hypergravity.

In this study, we used E. coli and T. intermedius, which have fairly thin cell walls as gram-negative bacte- ria, B. amyloliquefaciens and S. aureus, which have thick peptidoglycan layer as gram-positive bacteria, and S. cerevisiae whose cell wall is composed of chitin and man- nan as a eucaryote. E. coli and T. intermedius cultured

TABLE 3. Effect of saccharide pretreatment on the survival rate of B. atnyloliquefuciens IF014141 subjected to gravitational

stress at 450,000 x g for 3 h - - --_I_-_-. ~~.--.-~

Sugar cont. Survival rate (%I

Non pretreatment (physiological saline) Treatment with

Sucrose 0.4 M 0.8 M

Mannitol 0.4 M 0.8 M

Glycerol 0.4 M 0.8M

43.x

3.75 2.33 0.27 0.95

61.2 45.2

in LB medium to the stationary phase were more resistant to gravitational stress than B. amyloliquefa- ciens, S. aureus and S. cerevisiae, suggesting that the thickness of the cell wall does not play any role in resistance to gravity. The morphology of the cells sub- jected to gravity treatment at 450,000 Xg for 24 h was compared to that of non-treated cells by electron microscopic examination. No change in the shape of E. coli and B. amyloliquefaciens was observed following gravity treatment by either scanning or transmission elec- tron microscopy (data not shown). It has been reported that organelle stratification occurs in the cells of stato- cytes in the root cap of Lepidium sativum (11). In bac- terial cells subjected to gravitational force, chromosomal DNA may be concentrated in either intracellular pole. It is likely that the distribution of chromosomal DNA is an important factor for some metabolic activities and cell division for the subsequent growth of microorganisms after exposure to hypergravitational stress. Plasmolysis or partial destruction of the cell wall may also occur due to hypergravitational stress.

REFERENCES

1. Sachs, F.: Sensory transduction, p. 241-260. Rockefeller Univ. Press, New York (1992).

2. Martbtac, B.: Thermodynamics of membrane receptors and channels, p. 327-352. CRC, Boca Raton (1993).

3.

4.

5.

6.

7.

8.

9.

10.

11.

Mahdi, A:, Gowland, P. A., Mansfield, P., Coupiand, R. E., and Lloyd, R. G.: The effects of static 3.0 T and 0.5 T magnet- ic fields and the echo-planar imaging experiment at 0.5 T on E. coli. Br. J. Radiol., 67, 983-987 (1994). Hatton, J. P., Lewis, M. L., Roquefeuil, S. B., Chaput, D., Cazenave, J. P., and Schmitt, D.A.: Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cul- ture in microgravity. J. Cell. Biochem., 70, 252-267 (1998). Bouloc. P. and D’Ari. R.: Escherichiu coli metabolism in space. J. Gen. Microbioi., 137, 2839-2843 (1991). Kacena. M. A.. Leonard. P. E.. Todd. P.. and Luttaes. M. W.: Low gravity and inertial kffects’on the’ growth of E. >oh and B. subtilis in semi-solid media. Aviat. Space Environ. Med., 68, 1104-1108 (1997). Raso, J., Barbosa-Canovas, G., and Swanson, B. G.: Sporula- tion temperature affects initiation of germination and inactiva- tion bv high hydrostatic pressure of Bacillus cereus. .I. -4~1. Microbial., 85,-17-24 (1997). Kwon, S. T. and Oono, K.: Gravity responsible protein and mRNA related to the survival of rice (Oryzu mtiva I..) from gravity stress. Jpn. J. Genet., 67, 321-334 (1992). Maniatis, T., Fritsch, E. F., and Sambrook, J.: Molecular clon- ing a laboratory manual, p. 68-73. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). Rose, A. H.: Growth and handling of yeasts. Methods Cell Biol., 12, l-16 (1975). Sievers, A. and Caspers, L.: The effect of centrifugal accelera- tion on the polarity of statocytes and on the graviperception of cress root. Planta, 157, 64-70 (1983).