CURRENT MICROBIOLOGY, Vol. 11 (1984), pp. 205-210 Current Microbiology �9 Springer-Verqag 1984

A Halotolerant Planococcus from Antarctic Dry Valley Soil Karen J. Miller I and Susan B. Leschine 2

tDepartment of Biological Chemistry, Harvard Medical School, Boston; and 2Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA

Abstract. A halotolerant Planococcus (strain A4a) was isolated from saline Antarctic Dry Valley soil. Planococcus strain A4a grew over wide ranges of temperature (0~176 and NaCl concentrations (0-2.0 M). When the NaC1 concentration of the growth medium was increased, the total intracellular free amino acid concentration increased; however, the intracellular potassium concentration did not increase. This result suggested that intracellular free amino acids functioned as compatible solutes for growth of Planococcus strain A4a at elevated NaCI concentrations. The halotolerant and psychrotolerant nature of Planococcus strain A4a would appear to provide it with the capacity for growth in the saline Antarctic Dry Valley soil environment from which it was isolated.

The ice-free Dry Valleys of Antarctica are charac- terized by low temperatures, low humidities, di- urnal freeze-thaw cycling, low annual precipitation, and high-velocity winds. The soils of these valleys are also generally saline [24]. A variety of microbial forms have been isolated from these soils including Gram-positive and Gram-negative rods and cocci, cyanobacteria, and fungi [2-7, 12, 22, 24]. The halotolerance of these isolates, however, has not been described in detail.

We report here the isolation and characteriza- tion of a halotolerant, psychrotolerant bacterium from soil from the Taylor Valley of Antarctica. The isolate was a Gram-positive, obligately aerobic, nonmotile species of Planococcus. A preliminary report of part of this work was presented at the Twenty-fourth Plenary Meeting of the Committee on Space Research [21].

Materials and Methods

Isolation of Planococcus strain A4a. Planococcus strain A4a was isolated from soil collected in Taylor Valley, Antarctica, by Robert E. Benoit of the Virginia Polytechnic Institute. The soil sample was stored at 70~ at the Virginia Polytechnic In- stitute, transported to the University of Massachusetts on dry ice, and then stored at -80~ until use.

Growth was enriched in 5TYE-HES broth. Medium 5TYE- HES contained HES salts (0.85 M NaC1, 0.04 M CaC12 �9 2H20, 0.2 M MgSO4 �9 7H20), 0.05% (wt/vol) yeast extract (Difco Labo- ratories, Detroit, M1), and 0.05% (wt/vol) trypticase peptone (Baltimore Biological Laboratories, Cockeysville, MD). The medium was prepared in 95% of the final volume without

CaC12 �9 2H20 added. The pH was adjusted to 7.2 using 0.1 M KOH prior to sterilization by autoclaving. A solution of CaClz' 2H20 was prepared (in 5% of the final volume) and autoclaved separately. The two solutions were combined after sterilization. Tubes (16 mm • 150 ram) containing 10 ml of medium were inoculated with soil (0.1 g) and incubated at 5~ without agitation. Initial growth was observed after two weeks incubation. Planococcus strain A4a was isolated on plates of agar medium (5TYE-HES, supplemented with 0.7% [wt/vol] noble agar [Difco Laboratories]) incubated at 5~

Growth and maintenance of cultures. Planococcus strain A4a was routinely maintained at room temperature in 5TYE-HES broth and 5TYE-Na broth (5TYE-Na media were the same as 5TYE- HES except that the NaCl concentration was varied). Cells were transferred to fresh medium every five days (5% vol/vol inocula). Cultures were also maintained at 5~ on plates of 5TYE-HES agar medium (medium 5TYE-HES, supplemented with 1.0% [wt/vol] noble agar [Difco Laboratories]). Cells were transferred to fresh agar medium every four weeks.

NNA broth was nutrient broth (Difco Laboratories) sup- plemented with 3% (wt/vol) NaC1. NNA agar was the same as NNA broth except that it contained 2% (wt/vol) Bacto agar (Difco Laboratories).

Growth rates were determined in 5TYE-Na broth media containing different concentrations of NaCI (0.0-1.5 M). These media (100 ml in 250-ml side-arm flasks) were inoculated with 5 ml of a culture grown in 5TYE-Na broth (containing 1.0 M NaC1) at room temperature for 36 h. Cultures were incubated at different temperatures on a gyratory shaker and growth was monitored turbidometrically at 660 nm using a Klett-Summerson colorimeter.

Planococcus citreus ATCC 14404 and Escherichia coli K12 ATCC e23716 were obtained from the American Type Culture Collection.

Address reprint requests to: Karen J. Miller, Department of Biological Chemistry, Harvard Medical School, Building C2, Room 447, 25 Shattuck Street, Boston, MA 02115, USA.

206 CURRENT MICROBIOLOGY, VOI. I1 (1984)

Determination of guanine + cytosine content of DNA. DNA from Planococcus strain A4a was purified by the method described by Marmur [19]. The guanine + cytosine composit ion of the puri- fied D N A was calculated from the thermal denaturat ion tempera- ture by the method described by Mandel and Marmur [18]. Escherichia coli K12 D N A was used as a standard.

Physiological and morphological characterization. Catalase, ox- idase, gelatin hydrolysis , coagulase, starch hydrolysis, and acid and alkaline phospha tase activities were determined as described by Smibert and Krieg [23]. Glucose utilization was determined by optical densi ty measu remen t s at 660 nm of cultures grown in 5TYE-HES medium supplemented with 1.0% (wt/vol) glucose as compared with cultures not supplemented with glucose. Anaerobic growth of Planococcus strain A4a was assessed by incubating cells in 5TYE-HES broth supplemented with 1.0% (wt/vol) glucose in a nitrogen a tmosphere and on plates of 5TYE-HES medium supplemented with 1.0% (wt/vol) glucose and 0.7% (wt/vol) noble agar in an a tmosphere of 5% CO2, 10% H2, and 85% N 2 within an anaerobic chamber (Coy Laboratory Products , Inc. , Ann Arbor, MI).

Staining for flagella was performed by the method of Kodaka et al. [15]. Planococcus citreus was cultured on the same medium for the same time period and used as a positive control.

Intracellular free amino acid and potassium determinations. Intra- cellular free amino acid concentra t ions were determined by the trichloroacetic acid extract ion procedure of Measures [20]. Planococcus strain A4a was grown at room temperature in 500 ml of 5TYE medium (0.05% [wt/vol] yeast extract, 0.05% [wt/vol] t rypticase peptone, 0.04 M CaC12 �9 2H20, pH 7.2) sup- p lemented with either 1.5 M NaCI or no added NaCI. Cell extracts were examined for free amino acid content with a Beckman model 119 amino acid analyzer. A known amount of norleucine was added to each sample as an internal standard.

Identification of free amino acids was based on retention time, relative ninhydrin reactivity as measured at 570 nm and 440 nm, and susceptibili ty to hydrolysis . Hydrolysis was performed by combining 0.5 ml of the trichloroacetic acid supernatant (containing 1300-2500 nmol of total free amino acids) with 0.5 ml of 12 N HCl. The solution was flushed with nitrogen and heated to 110~ for 14 h. The sample was dried under a s t ream of ni trogen at 50~ and resuspended in 0.6 ml of 0.1 N HC1. This solution was analyzed on the amino acid analyzer.

Intracellular potass ium determinat ions were performed in the same manne r as the intracellular amino acid analysis except that the trichloroacetic acid supernatants were examined for potass ium using a Perkin Elmer model 403 atomic absorption spect rophotometer .

Results

Morphology and growth characteristics. Cells of Planococcus strain A4a were examined by phase contrast microscopy. shaped, measured 1-1.5 curred most commonly

Cells were spherically /xm in diameter, and oc- in pairs, occasionally in

irregular groups of 3-10 cells, and as single cells. Nei ther spores nor motility were observed. Flagella also were not observed after specific staining of

o

0 6

{n c~

0 5

~3

o o4

"G 03

a= 0 2

~9 0 I

! [ I I q I i I

5 I 0 15 2 0 2 5 5 0 3 5 4 0 4 5

Tempera tu re (~

Fig. 1. Growth rate of Planococcus strain A4a as a function of incubation temperature. Cells were grown in 5TYE-Na medium containing different concentra t ions of NaC1. Growth rates were determined as described in Materials and Methods. �9 0.0 M NaCI; Q, 0.2 M NaCI; A, 0.6 M NaCI; ~', 1.0 M NaC1; [], 1.5 M NaCl.

cells. Cells stained Gram-positive, and the Gram- positive structure of the cell wall was confirmed by transmission electron microscopy [21]. Colonies grown on 5TYE-HES agar at 5~ and room tem- perature were 0.1-1.5 mm in diameter, circular, slightly convex, and orange in color. Colonies of similar size were observed on plates of NNA me- dium; however, colonies were pale orange to trans- lucent. Subsequent transfer of cells from these pale orange-translucent colonies onto plates of 5TYE- HES agar medium resulted in orange-pigmented colonies as described above.

Planococcus strain A4a was strictly aerobic. Growth occurred in the temperature range of 0~176 but did not occur at 45~ Growth occurred over a wide range of NaC1 concentrat ion (0-2.0 34). The optimal growth temperature at all NaC1 con- centrations was 25~ as indicated in Fig. 1. Growth rate was maximal in media that were not sup- plemented with NaC1. Planococcus strain A4a grew in 5TYE-HES media when any combination of two salt species was removed from the medium. If all three salts (0.2 M MgSO4, 0.04 M CaCI2, and 0.85 M NaCl) were removed from the medium, however, growth did not occur after three successive trans- fers. Although Planococcus strain A4a was rou- tinely cultured in medium 5TYE-HES that did not contain carbohydrates, growth was stimulated when the medium was supplemented with glucose. When a culture of Planococcus strain A4a grown in 5TYE-Na medium (containing 1.0 M NaC1) was inoculated into N N A broth (inoculum, 5% vol/vol),

K. J. Miller and S. B. Leschine: A Planococcus from Antarctic Dry Valley Soil 207

Table 1. Intracellular free amino acid content of Planococcus strain A4a"

Concentration (mM) b No added 1.5 M

Amino acid NaC1 NaC1

Glutamine 7.6 102.0 Glutamic acid 38.9 70.6 Aspartic acid 6.3 49.2 Lysine 6.0 31.7 Cysteine 14.8 24.5 Histidine 1.3 17.6 Citrulline 4.5 9.7 Arginine 0.4 7.9 Alanine 3.7 7.6 Glycine 3.5 6.8 Ornithine 1.0 3.8 Methionine 0.2 3.2 Leucine 1.2 2.8 Phenylalanine 1.6 1.9 Serine 0.1 1.8 y-Aminobutyric acid ND c 0.1 Threonine 1.1 1.3 Tyrosine 1.2 1.1 Isoleucine 0.5 0.5 Asparagine ND c ND ~ Proline 9.1 ND C Valine 3.5 ND"

Cells were grown at room temperature in 5TYE medium supplemented with either 1.5 M NaCI or no added NaC1. b The intracellular concentration (millimolar) of each amino acid was calculated by estimating the intracellular volume from the measured cell radius. c Not detected.

growth occurred; cells did not grow, however, when serially transferred in this medium.

Planococcus strain A4a was positive for gelatin hydrolysis and catalase activities, and negative for starch hydrolysis, coagulase, oxidase, and acid and alkaline phosphatase activities.

Guanine + cytosine content of the DNA. The gua- nine + cytosine content of the DNA of Planococ- cus strain A4a was 48.7 _+ 0.6 tool%.

Intracellular free amino acid and potassium concen- trations. The intracellular free amino acid concen- tration of Planococcus strain A4a cells grown in media containing two different NaCl concentrations is shown in Table 1. The intracellular free amino acid content of Planococcus strain A4a was found to increase when the NaC1 concentration of the growth medium was increased. In cultures not supplemented with NaC1, the total intracellular free amino acid concentration was calculated to be ap- proximately 110 raM. In these cells, glutamic acid

was the predominant free amino acid detected. When Planococcus strain A4a was grown in a medium containing 1.5 M NaC1, the intracellular concentration of the total free amino acids in- creased to approximately 340 raM. In these cells, glutamine was the predominant intracellular free amino acid detected.

Two unidentified ninhydrin-reactive peaks were also detected within the trichloroacetic acid ex- tracts of Planococcus strain A4a. The relative amount of both peaks increased significantly when Planococcus strain A4a was grown in media con- taining 1.5 M NaC1. Both peaks were absent from chromatograms after acid hydrolysis.

The intracellular potassium concentration of Planococcus strain A4a was calculated to be ap- proximately 640 mM in cells cultured in media not supplemented with NaC1 and 605 mM in cells cul- tured in media supplemented with 1.5 M NaC1. The potassium ion concentration of the growth medium was 1.I mM and t.3 mM for media containing no added NaC1 and 1.5 M NaCI, respectively.

D i s c u s s i o n

We have isolated a Gram-positive, obligately aero- bic, halotolerant, psychrotolerant, orange-pigment- ed coccus from Antarctic Dry Valley soil. The isolate was classified as a member of the genus Planococcus of the family Micrococcaceae. This classification was based on morphological and physiological characteristics and on the guanine + cytosine content of the DNA [13, 14]. The presence of significant levels (40 mol%) of phosphatidyl- ethanolamine within this isolate [21] (K. J. Miller, unpublished observations) is also consistent with this classification [11, 16, 17].

The halotolerant nature of Planococcus strain A4a may be related to the high level of intracellular potassium found in this bacterium. For example, Christian and Waltho [8] demonstrated a correlation between high intracellular potassium content and NaCI tolerance for a variety of nonhalophilic bac- teria. The intracellular potassium concentration cal- culated for Planococcus strain A4a in this study was similar to levels that have been reported for the halotolerant staphylococci [8-10]. The intracellular potassium concentration was unaffected by the con- centration of NaC1 in the growth medium in the range 0-1.5 M. The intracellular free amino acid content of Planococcus strain A4a, however, in- creased when the NaCI concentration of the growth medium was increased. Such an increase of intra-

208 CURRENT MICROBIOLOGY, Vol. 11 (1984)

cellular free amino acid content has been detected in a variety of microorganisms [20, 25]. Typically, Gram-negative bacteria accumulate glutamic acid while Gram-positive bacteria accumulate proline or y-aminobutyric acid in response to increased con- centrations of NaCl [20]. In Planococcus strain A4a, much of the detected increase in total free amino acid content was a result of increased glutamine. Glutamine may, therefore, serve as an intracellular compatible solute for Planococcus strain A4a. It is of interest that Staphylococcus aureus has also been found to accumulate glutamine when grown in media containing high NaC1 concen- trations [1]. Measures [20] has suggested that an advantage to the accumulation of proline or 7- aminobutyric acid by Gram-positive bacteria is that neither of these amino acids is highly charged at neutral pH and would, therefore, not require the presence of neutralizing cations. Similarly, gluta- mine is not highly charged at neutral pH.

Planococcus strain A4a was similar but not identical to Planococcus citreus. For example, mo- tility was never observed for Planococcus strain A4a in any of the growth media used in this study, whereas motile cells of P. citreus were detected in all growth media. Flagella were not detected after staining of Planococcus strain A4a, while they were observed after staining of P. citreus. Also, Planococcus strain A4a did not grow in NNA broth or on plates of NNA agar medium after two serial transfers, whereas P. citreus grew in these media after repeated transfers. It should be noted that nutrient agar supplemented with 1%--10% (wt/vol) NaC1 has been recommended for the isolation and routine maintenance of the planococci [14]. If this medium had been used in the enrichment and isola- tion procedures described in this study, Planococ- cus strain A4a would not have been isolated.

Planococci have not previously been described among the isolates from Dry Valley Antarctic soil; however, morphologically similar forms have been commonly described from these soils [3-7]. It would be interesting to determine whether the halotolerant planococci constitute a significant component of the microbial flora of the Dry Valley soils. Due to its halotolerance and psychrotoler- ance, Planococcus strain A4a would appear to be well adapted for growth in the saline Antarctic Dry Valley environment from which it was isolated. Planococcus strain A4a has been deposited with the American Type Culture Collection under the number 35671.

ACKNOWLEDGMENTS

We wish to thank E. Canale-Parola for helpful discussions. This work was partially supported by National Science Foundation grant DPP-8120605. K.J.M. was the recipient of a National Institutes of Health Research Service Award under grant GM07473 from the National Institute of General Medical Sci- ences. K.J.M. was a member of the Department of Biochemistry at the University of Massachusetts when this research was conducted.

Literature Cited 1. Anderson C. B., Witter, L. D. 1982. Glutamine and proline

accumulation by Staphylococcus aureus with reduction in water activity. Applied and Environmental Microbiology 43:1501-1503.

2. Bahareen, S., Bantle, J. A., Vishniae, H. S. 1982. The evolution of Antarctic Yeasts: DNA base composition and DNA-DNA homology. Canadian Journal of Microbiology 28:407-413.

3. Benoit, R. E., Hall, C. L. 1970. The microbiology of some Dry Valley soils of Victoria Land, Antarctica, pp. 697-701. In: Holdgate, M. W. (ed.), Antarctic ecology. New York: Academic Press.

4. Cameron, R. E. 1972. Microbial and ecologic investigations in Victoria Valley, Southern Victoria Land, Antarctica, pp. 195-260. In: Llano, G. A. (ed.), Antarctic terrestrial biology, Antarctic Research Series, vol. 20. Washington, DC: American Geophysical Union.

5. Cameron, R. E., King, J., David, C. 1968. Soil microbial and ecological studies in Southern Victoria Land. Antarctic Journal of the United States 3:121-123.

6. Cameron, R. E., King, J., David, C. N. 1970. Microbiology, ecology, and microclimatology of soil sites in Dry Valleys of Southern Victoria Land, Antarctica, pp. 702-716. In: Holdgate, M. W. (ed.), Antarctic ecology. New York: Academic Press.

7. Cameron, R. E., Morelli, F. A., Randall, L. P. 1972. Aerial, aquatic, and soil microbiology of Don Juan Pond, Antarc- tica. Antarctic Journal of the United States 7:254-258.

8. Christian, J. H. B., Waltho, J. A. 1961. The sodium and potassium content of non-halophilic bacteria in relation to salt tolerance. Journal of General Microbiology 25:97-102.

9. Christian, J. H. B., Waltho, J. A. 1962. Solute concentra- tions within cells of halophilic and non-halophilic bacte- ria. Biochimica et Biophysica Acta 65:506-508.

10. Christian, J. H. B., Waltho, J. A. 1964. The composition of Staphylococcus aureus in relation to the water activity of the growth medium. Journal of General Microbiology 35:205-218.

11. Goldfine, H. 1982. Lipids of prokaryotes: structure and distribution, pp. 1-44. In: Razin, S., Rottem, S. (eds.), Current topics in membranes and transport, vol. 17. New York: Academic Press.

12. Horowitz, N. H., Cameron, R. E., Hubbard, J. S. 1972. Microbiology of the Dry Valleys of Antarctica. Science 176:242-245.

13. Koeur, M. 1974. Planococcus, pp. 489-490. In: Buchanan, R. E., Gibbons, N. E. (eds.), Bergey's manual of deter- minative bacteriology, 8th edn. Baltimore: Williams and Wilkins.

14. Koeur, M., Schliefer, K. 1981. The genus Planococcus, pp.

K. J. Miller and S. B. Leschine: A Planococcus from Antarctic Dry Valley Soil 209

1570-1571. In: Starr, M. P., Stolp, H. S., Truper, H. G., Balows, A., Schlegel, H. G. (eds.), The prokaryotes, vol. 2. New York: Springer-Verlag.

15. Kodaka, H., Armfieid, A. Y., Lombard, G. L., Dowell, V. R. 1982. Practical procedure for demonstrating bacterial flagella. Journal of Clinical Microbiology 16:948-952.

16. Komura, I., Yamada, K., Komagata, K. 1975. Taxonomic significance of phospholipid composition in aerobic Gram- positive cocci. Journal of General and Applied Microbiol- ogy 21:97-107.

17. Lechevalier, M. P. 1977. Lipids in bacterial taxonomy: a taxonomist 's view. CRC Critical Reviews in Microbiology 5:109-210.

18. Mandel, M., Marmur, J. 1968. Use of ultraviolet absorbance-temperature profile for determining the gua- nine plus cytosine content of DNA, pp. 195-206. In: Grossman, L., Moldane, K. (eds.), Methods in enzymol- ogy, vol. 12. New York: Academic Press.

19. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from microorganisms. Journal of Molecular Biology 3:208-218.

20. Measures, J. C. 1975. Role of amino acids in osmoregulation

of non-halophilic bacteria. Nature 257:398400. 21. Miller, K. J., Leschine, S. B., Ituguenin, R. L. 1983. Char-

acterization of a halotolerant-psychrotolerant bacterium from Dry Valley Antarctic soil. Advances in Space Re- search 3:4347.

22. Siegel, B. Z., McMurty, G., Siegel, S. M., Chert, J., LaRock, P. 1979. Life in the calcium chloride environment of Don Juan Pond, Antarctica. Nature 280:828-829.

23. Smibert, R. M., Krieg, N. R. 1981. General characterization, pp. 409-443. In: Gerhardt, P., Murray, R. G. E., Costilow, R. N., Nester, E. W., Wood, W. A., Krieg, N. R., Phillips, G. B. (eds,), Manual of methods for general bacteriology. Washington, DC: American Society for Microbiology.

24. Tedrow, J. C. F., Ugolini, F. C. 1966. Antarctic soils, pp. 161-177. In: Tedrow, J. C. F. (ed.), Antarctic soils and soil forming processes, Antarctic Research Series, vol. 8. Washington, DC: American Geophysical Union.

25. Tempest, D. W., Meets, J. L., Brown, C. M. 1970. Influence of environment on the content and composition of micro- bial free amino acids. Journal of General Microbiology 64:171-185.