INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY, Jan. 1986, p. 66-70 Vol. 36, No. 1 0020-7713/86/010066-05$02.00/0 Copyright 0 1986, International Union of Microbiological Societies

Halobacterium denitrificans sp. nov. an Extremely Halophilic Denitrifying Bacterium

G. A. TOMLINSON,~L. L. JAHNKE,~AND L. I. HOCHSTEIN~" Department of Biology, University of Santa Clara, Santa Clara, California 95053,l and National Aeronautics and Space Administration, Ames Research Center, Moflett Field, California 940352

Halobacterium denitrificans was one of several carbohydrate-utilizing, denitrifying, extremely halophilic bacteria isolated by anaerobic enrichment in the presence of nitrate. Anaerobic growth took place only when nitrate (or nitrite) was present and was accompanied by the production of dinitrogen. In the presence of high concentrations of nitrate @em,0.5%), nitrous oxide and nitrite were also detected. When grown aerobically in a mineral-saltsmedium containing 0.005 % yeast extract, H. denitrificans utilized a variety of carbohydrates as sources of carbon and energy. In every case, carbohydrate utilization was accompanied by acid production. A type culture has been deposited with the American Type Culture Collection, Rockville, Md. (ATCC 35960).

At present, the taxonomy of the halobacteria is controver- mud or brine. The bottles were filled with additional freshly sial. This reflects, in part, the recent characterization of new autoclaved medium, sealed with serum caps, and incubated metabolic types (9, 14, 19, 21, 28, 30) and the apparent in the dark at 37°C. After vigorous gas formation was biochemical monotony of the group (23). At one time five observed, fractions were transferred to bottles containing species were recognized. Two were distinguished from the freshly autoclaved YH medium, and the bottles were topped rest on the basis of gas production from nitrate, and one of offas described above, sealed with serum caps, and incubated these, Halobacterium marismortui, produced acid from sev- in the dark at 37°C until growth and gas production were eral carbohydrates (5). H. marismortui is no longer recog- observed. After three such transfers, material from the liquid nized as a valid species because of the absence of an cultures was streaked on YH agar plates (YH medium plus 2% extant-type culture (16). A numerical taxonomic analysis (4) Bacto-Agar [Difco Laboratories, Detroit, Mich.]). The plates recommended that only two halobacterial species be recog- were incubated in the dark at 37°C in Gas-Pak Jars (BBL nized, Halobacterium salinarium and Halobacterium Microbiology Systems, Cockeysville, Md.). After about 10 cutirubrum. Neither one produced gas from nitrate or acid days, several isolated colonies were streaked onto YH agar from carbohydrates. Of the currently recognized species, plates and again incubated anaerobically. Finally, represen- only Halobacterium mediterraneii (20) and Halobacterium tative colonies were transferred to YH agar slants and vallismortis (9) produce a gas when grown in the presence of incubated aerobically at 37°C. Each of these isolates was nitrate. Since some halobacteria acidify media containing subsequently tested for nitrate-dependent anaerobic growth, carbohydrates (9,20, 31) and a number of extreme halophiles and those which grew anaerobically only when nitrate was are capable of fermentative growth (15), the presence of gas present were saved for further study. We subsequently in a medium containing nitrate is not sufficient evidence to isolated a number of denitrifying, extremely halophilic conclude that nitrogen was produced or that growth took bacteria from the Guerro Negro salterns in Baja California place as a consequence of denitrification. using the same procedure. Recently, we isolated several bacteria, the characteristics Unless stated otherwise, the extreme halophiles were of which suggested that they were members of the genus grown in the dark at 37°C by the procedure described by Halobacterium. These organisms grew anaerobically, but Hochstein and Tomlinson (12). Two media were used: the only when nitrate was present. Such nitrate-dependent HYH medium, which was YH medium buffered at pH 6.7 anaerobic growth was accompanied by the production of with 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesul- nitrite, nitrous oxide, and dinitrogen. We believe that these fonic acid (HEPES) buffer, and a mineral salts-yeast extract organisms are denitrifying, extreme halophiles and that medium (MSY), which contained the following (in grams per strain S1 is sufficiently distinctive to warrant its character- liter): NaCl, 176.0; MgS04.7H20, 20.0; KCl, 2.0: K2HP04, ization and designation as Halobacterium denitriJicans. 0.1; (NHJ2S04, 1.0; CaC12.2H20, 0.1; FeS04.7H20, 0.005; 2-(N-morpholino)ethanesulfonic acid (MES) buffer, 19.5; MATERIALS AND METHODS yeast extract, 0.05. The pH was adjusted to 6.7 at room Enrichment cultures, using either mud or brine from temperature. Carbon sources were sterilized by filtration salterns located in the southern portion of San Francisco Bay, through a Nalgene filter (pore size, 0.2 pm) and added to were grown in 60-ml serum bottles containing a freshly autoclaved medium. Growth was measured with a Klett- autoclaved medium. The medium (designated YH medium) Summerson colorimeter with a red (no. 66) filter and cor- contained the following additions (in grams per liter): yeast rected when necessary, as described previously (24). Nitrite extract, 5.0; Hy-Case SF, 2.0, NaCl, 176.0; MgC12.6H20, was determined by the method of Showe and DeMoss (26). 20.0; KN03, 5.0; KCl, 2.0; CaC12-2H20, 0.1. The pH was Carbon dioxide, nitrous oxide, and dinitrogen were analyzed adjusted to 7.4 at room temperature (22°C) with a by gas chromatography with a Poropak Q column (11). low-sodium-error electrode. Bottles that were partially filled Ether-linked lipids were detected chromatographically (22) with freshly autoclaved medium were inoculated with either with cells grown aerobically in HYH medium and harvested in the early stationary phase. Cell dimensions were determined with a splitting eye piece * Corresponding author. (Vickers Instruments, Malden, Mass.). The biochemical

66 VOL. 36, 1986 H. DENITRIFICANS SP. NOV. 67

halodenitriJicans (lane 4). The mobility of the major constit- uent in the acid methanolysates from strain S1 and Halobacterium saccharovorum (lane 5) was similar. The following diphytanyl ethers were found in the polar lipid fraction from strain S1: phosphatidyl glycerol, phosphatidyl- glycerophosphate, sulfated diglycosyl glycerol diether, traces of diglycosyl glycerol diether. The principal quinones were MK-8 (76.9%) and MK-8(H2) (20.6%), which agrees with the observation of Collins et al. (3) that these are also the principal quinones found in other extremely halophilic bacteria. Traces of MK-7 and MK-7(H2) were also present. The major squalene derivative was tetrahydrosqualene; only traces of squalene were detected (B. J. Tindall, personal communication). At 37"C, strain S1 grew aerobically in HYM medium containing from 1.5 to 4.5 M NaCl, which was the highest concentration tested. When cells were placed in media containing less than 1.5 M NaCl, they assumed a spherical shape before they lysed. Lysis was essentially immediate when cells were placed in distilled water. Strain S1 was a relatively rapid-growing extreme halophile. A 3-h generation time was observed in HYH medium containing from 2 to 3 M NaCl. At higher salt concentrations, the generation time FIG. 1. Thin-layer chromatogram of acid methanolysates from increased to a maximum of 6 h in the presence of 4.5 M lyophilized cells. Lane 1, Strain S1; lane 2, fatty acid, methyl ester NaC1. standard; lane 3, E. coli; lane 4, P. halodenitrijicans; lane 5, H. A generation time of 2 h was observed when cells were saccharovorum. grown at 50°C in HYH medium containing 3 M NaCl. Growth was observed from 30°C (13-h generation time) to 55°C (4-h generation time). There was no growth at either 20 tests used for characterization were carried as described by or 60°C. Smibert and Krieg (27) with HY medium. Method 2 was used To demonstrate carbohydrate utilization, advantage was for all tests, except for gelatin liquefaction and hydrogen taken of the ability of strain S1 to grow in a mineral-salts sulfide production, for which method 1 was employed. The medium containing 0.005% yeast extract (MYS medium). antibiotic sensitivity of aerobically growing cells was tested The unsupplemented MYS medium supported a small on HYH agar plates with disks containing the following amount of growth, which in the presence of suitable carbon amounts of antibiotics: bacitracin (10 U), streptomycin (10 sources, such as D-glucose or pyruvate, was considerably pg), novobiocin (30 pg), penicillin G (10 U), polymyxin B (50 enhanced (Fig. 2). D-Fructose, maltose, sucrose, D-galac- U). Aphidicolin sensitivity was determined in liquid medium tose, lactate, D-gluconate, glycerol, acetate, fumarate, L- to which the antibiotic, dissolved in dimethyl sulfoxide, was malate, citrate, succinate, and a-ketoglutarate were also added just before inoculation. Dimethyl sulfoxide, at the used as the sole carbon and energy sources, whereas D- concentration added with aphidicolin (0.04% [vol/vol]), had ribose, D-mannose, and lactose were not (data not shown). no effect on growth. The effect of chloramphenicol and When MES buffer was omitted from carbohydrate- erythromycin was also determined in liquid medium. These containing MSY medium, there was less growth, and in antibiotics were added as methanolic solutions. Methanol those cases in which the sugars were utilized the final pH did not affect growth at the concentrations employed. was 4.9. Aphidicolin was obtained from Sigma Chemical Co., St. The growth of strain S1 was not affected by penicillin, Louis, Mo. Yeast extract was purchased from Difco Labo- streptomycin, or polymyxin B. Bacitracin and novobiocin ratories. Hy-Case SF, an acid-hydrolyzed casein, was ob- inhibited growth; the zones of inhibition were 13 and 15 mm, tained from the Humko-Sheffield Chemical Co., Norwich, respectively. In the case of novobiocin, growth beyond the N.Y. The fatty acid, methyl esters standard was purchased zone of inhibition was not pigmented, suggesting that this from Applied Science, Los Altos, Calif. antibiotic might be useful for probing the early steps of carotenoid biosynthesis. Chloramphenicol inhibited growth RESULTS at concentrations greater than 100 pg/ml, while erythromy- Colonies on aerobic plates were orange red. The pigment cin was inhibitory at concentrations greater than 400 pg/ml. was probably a-bacterioruberin since the visible absorption Aphidicolin, an inhibitor of eucaryotic DNA replication (29) spectrum of aqueous cell extracts had maxima located at and the growth of various halobacteria, but not eubacteria 473, 501, and 540 nm. These maxima agreed with those (6, 25, 29), inhibited the growth of strain S1 at a concentra- reported for the a-bacterioruberin-protein complex obtained tion of 20 pg/ml. by lysing extremely halophilic bacteria in distilled water (2). Strain S1 grew anaerobically in HYH medium in the When grown anaerobically, the colonies were white with a presence of nitrate or nitrite; no growth was observed when slight pinkish cast. either was omitted or when they were replaced with nitrous As shown in Fig. 1, the acid methanolysate prepared from oxide. The generation time of cells grown anaerobically at aerobically grown strain S1 (lane 1) contained several com- 37°C in the presence of 3 M NaCl was 7.5 h in the presence ponents, the mobilities of which were distinct from the fatty of nitrate and 10 h with nitrite. Anaerobic growth was acid-methyl esters standard (lane 2) and from the accompanied by vigorous gassing in the presence of nitrate. methanolysates of Escherichia coli (lane 3) and Paracoccus When the nitrate concentration was 0.1%, dinitrogen was 68 TOMLINSON ET AL. INT. J. SYST.BACTERIOL. produced concomitantly with exponential growth and was the only product of nitrate dissimilation detected (Fig. 3a). This contrasts with an earlier observation (13) that anaerobic growth in the presence of 0.5% nitrqte resulted in the production of dinitrogen, nitrite, and nitrous oxide. Acety- lene, which inhibits nitrous oxide reduction by denitrifying bacteria at concentrations ranging from 0.01 to 0.1 atm (1, 33), inhibited the reduction of nitrous oxide by strain S1. When strain S1 was grown anaerobically in the presence of 0.1% nitrate and 0.04 atm of acetylene, the reduction of nitrous oxide was completely inhibited, small quantities of nitrite were also detected (data not shown), the generation time increased from 7 to 23 h, and the growth yield de- creased by about 30% (Fig. 3b). The last two observations were unexpected since nitrous oxide did not support the growth of strain S1 and suggest that acetylene acts at a site(s) other than that (those) for nitrous oxidF reduction.

DISCUSSION The relatively high NaCl concentration required for growth, lysis at NaCl concentrations less than 1.5 M, the presence of glycerol diethers, and the sensitivity to aphidicolin indicated that strain S1 was a member of the genus Halobacterium. The absolute dependence of anaerobic growth on the presence of nitrate (or nitrite) and the concomitant production of dinitrogen with growth con- firmed that strain S1 grew by coupling growth to denitrifica- tion. (a 1 (b) I I 1 1 I I The ability to grow anaerobically is not a singular charac- .001 1

teristic among the halobacteria. Certain strains (designated Halobacterium halobium, H. cutirubrum, and H. salinarium) grow anaerobically by fermenting arginine to ornithine, and H. halobium can also grow anaerobially to a limited extent by a light-dependent process with preformed bacteriorhodopsin (10). Results of recent studies suggest that the ability of halobacteria to grow in the absence of added nitrate (i.e., fermentatively) may be a widely distributed property (14). Two closely related extreme halophiles (23) bear at least a superficial resemblance to strain S1. All produce acid from carbohydrates and gas from nitrate. One of these, the putative H. marismortui, differs from strain S1 in that H. marismortui produces acid from mannose and presumably grows best at 30°C (5). The extreme halophile isolated by Ginzburg et al. (8), and said to correspond to H. marismortui (7), differs from strain Sl in that nitrous oxide is the major product of denitrification, with only traces of dinitrogen and nitric oxide being detected (32). The other extreme halophile H. vallismortis produces gas when nitrate is present (9). Even though not all strains of H. vallismortis will grow anaerobically in the absence of nitrate (23), the likelihood that strain S1 and H. vallisrnortis are identical appears to be .001' I I I I 1 slight. They differ in their temperature optima for growth, 0 20 40 60 80 100 TIME (h) the minimum NaCl concentration at which growth takes FIG. 2. Aerobic growth of strain S1. Strain S1 was grown in place, and several biochemical properties (Table 1). mineral-salts medium containing 0.005% yeast extract and 0.25% The presence of tetrahydrosqualene as the principal (wthol) of the indicated carbon sources. Symbols: A, none; 0, squalene derivative in strain S1 suggests that it may belong glucose; @, pyruvate. A 1% inoculum was taken from a 48-h culture to a branch of the halobacteria that also includes H. mediter- grown in complex (HYH)medium. ranei and Halobacterium volcanii (B. J. Tindall, personal VOL.36, 1986 H. DENITRIFICANS SP. NOV. 69

TABLE 1. Distinguishing Characteristics of some gasogenic, Oxidase and catalase positive. Gelatin liquified, hydrogen . acid-producing, extremely halophilic bacteria sulfide produced from thiosulfate, and Tween 40 (palmitic Roperty H. val- H. medi- acid ester) hydrolyzed. Does not hydrolyze starch, urea, or Strain s1 lisrnortis" terraneih Tween 80 (oleic acid ester). Indole negative. Hydrolysis of Habitat. Solar evaporation ponds (salterns) in the San Gelatin Francisco Bay area of California and probably in other Starch salterns. Tween 40 Type strain. Strain S1 has been deposited as Tween 80 Halobacterium denitrijicans in American Type Culture Col- H2S from S203- lection, Rockville, Md. (ATCC 35960). lndole production Maximum growth rate" ACKNOWLEDGMENTS NaCl (%) 15 25 16 Temp ("C) 50 40 35 We are indebted to B. J. Tindall, Institut fur Mikrobiologie, Temp growth range ("C) 30-55 20-45 25-45 Rheinische Fredrich-Wilhelms Universitat, Bonn, Federal Republic of Germany, for the lipid analysis and helpful comments. 'I Gonzalez et al. (9). These studies were carried out with the technical assistance of " Rodriguez-Valera et al. (20). Steven Andersen and Mary Broderick. ' Shortest generation time. LITERATURE CITED 1. Balderston, W. L., B. Sherr, and W. J. Payne. 1976. Blockage by communication). Strain S1 and H. mediterranei share sev- acetylene of nitrous oxide reduction in Pseudomonas eral properties, such as the ability to use carbohydrates perfectomarinus. Appl. Environ. Microbiol. 31504-508. when grown in a simple mineral salts medium supplemented 2. Baxter, R. M. 1960. Carotenoid pigments of halophilic bacteria. with low concentrations of yeast extract, rapid growth, and Can. J. Microbiol. 6:417423. growth at relatively low NaCl concentrations (21). Whether 3. Collins, M. D., H. N. M. Ross, B. J. Tindall, and W. D. Grant. 1981. Distribution of isoprenoid quinones in halophilic bacteria. H. mediterranei grows anaerobically by denitrification is not J. Appl. Bacteriol. 50559-565. clear (20), and several other properties distinguish strain S1 4. Colwell, R. R., C. D. Litchfield, R. H. Vreeland, L. A. Kiefer, and H. mediterranei (Table 1). Strain S1 and H. volcanii are and N. E. Gibbons. 1979. Taxonomic studies of red halophilic clearly different in that the latter does not grow anaerobically bacteria. Int. J. Syst. Bacteriol. 29:379-399. in the absence of nitrate (17). 5. Elazari-Volcani, B. 1957. R. S. Breed, E. G. D. Murray, and Therefore, we conclude that strain S1 is different from N. R. Smith (ed.), Bergey's manual of determinative bacteriol- other validly described, extremely halophilic bacteria. In ogy, 7th ed, p. 207-212. The Williams & Wilkins Co., Baltimore. recognition of its ability to grow anaerobically by denitrifica- 6. Forterre, C., C. Elie, and M. Kohiyama. 1984. Aphidicolin strain S1 Halobacterium inhibits growth and DNA synthesis in halobacterial tion, we propose to designate archaebacteria. J. Bacteriol. 159:80&802. denitrif cans. 7. Ginzburg, M. 1978. Ion metabolism in whole cells of Description of Halobacterium denitrijicans sp. nov. (de- Halobacterium halobium and H. marismortui, p. 561-577. In ni-tri'fi-cans. M.L. part. adj. denitrijicans denitrifying). Ex- S. R. Caplan and M. Ginzburg (ed.), Energetics and structure of ponential-phase cells from liquid cultures are highly pleo- halophilic microorganisms. Elsevier, Amsterdam. morphic and disk shaped. They are from 0.8 to 1.5 pm wide 8. Ginzburg, M., L. Sachs, and B. Z. Ginzburg. 1970. Ion metab- and 2.0 to 3.0 pm long. Motility is not present. Colonies on olism in a Halobacterium. I. Influence of age of culture on complex medium are 2 to 3 mm in diameter, round, convex, intracellular concentrations. J. Gen. Physiol. 55187-207. entire, and orange red. Cells are gram-negative and ex- 9. Gonzalez, C., C. Gutierrez, and C. Ramirez. 1978. Halobacter- ium vallismortis sp. nov. An amylolytic and carbohydrate- tremely halophilic. Cells lyse at NaCl concentrations less metabolizing extremely halophilic bacterium. Can. J. Microbiol. than 1.5 M. Growth occurs between 1.5 M and at least 4.5 M 24:71&715. NaCl. Maximum growth rate between 2 and 3 M NaCl (at 10. Hartmann, R., H.-D. Sickinger, and D. Oesterhelt. 1980. 37°C). Temperature range for growth is 30 to 55"C, with Anaerobic growth of halobacteria. Proc. Natl. Acad. Sci. USA maximum growth rate at 50°C (in the presence of 3 M NaC1). 77:3821-3825. The pH range for growth is 6 to 8 (optimum, 6.7) at 37°C. 11. Hochstein, L. I., M. Betlach, and G. Kritikos. 1984. The effect of Cells are red because of the presence of a-bacterioruberin. oxygen on denitrification during steady-state growth of Polar lipids are diphytanyl-glycerol-ether derivatives. Paracoccus halodenitrificans. Arch. Microbiol. 137:74-78. Chemoorganotroph. H. denitrificans is a facultative 12. Hochstein, L. I., and G. A. Tomlinson. 1984. The growth of Paracoccus halodenitriJicans in a defined medium. Can. J. anaerobe capable of anaerobic growth only when nitrate (or Microbiol. 30:837-840. nitrite) is present. It reduces nitrate to nitrite, nitrous oxide, 13. Hochstein, L. I., and G. A. Tomlinson. 1985. Denitrification by and dinitrogen when grown in the presence of 0.5% nitrate. extremely halophilic bacteria. FEMS Microbiol. Lett. Only dinitrogen accumulates when it is grown in the pres- 27: 329-3 31. ence of 0.1% nitrate. Grows aerobically, but not anaerobi- 14. Javor, B., C. Requadt, and W. Stoeckenius. 1982. Box shaped cally, in a mineral-salts medium containing 0.005% yeast halophilic bacteria. J. Bacteriol. 151:1532-1542. extract, ammonium as the nitrogen source, and suitable 15. Javor, B. J. 1984. Growth potential of halophilic bacteria carbon and energy sources such as D-glucose, D-galactose, isolated from solar salt environments: carbon sources and salt D-fructose, maltose, sucrose, acetate, citrate, fumarate, requirements. Appl. Environ. Microbiol. 48:352-360. 16. Larsen, H. 1984. Family V. Halobacteriaceae Gibbons 1974, glycerol, lactate, a-ketoglutarate, malate, succinate or pyru- 269, p. 261-267. In N. R. Krieg (ed.), Bergy's manual of vate. In unbuffered mineral-salts-yeast extract medium, systematic bacteriology, vol. I, The Williams & Wilkins Co., sufficient acid is produced from utilizable carbohydrates and Baltimore. glycerol to change the pH from 6.7 to 4.9. 17. Mullakhanbhai, M. F., and H. Larsen. 1975. Halobacterium

Growth. Growth was inhibited by aphidicolin, bacitracin, volcanii spec. nov., a Dead Sea halobacterium with a moderate , chloramphenicol, erythromycin, and novobiocin. salt requirement. Arch. Microbiol. 104:207-214. 70 TOMLINSON ET AL. INT. J. SYST. BACTERIOL.

18. Nicholson, D. E., and G. E. Fox. 1983. Molecular evidence for a regulation of synthesis of nitrate reductase in Escherichia coli. close phylogenetic relationship among box-shaped halophilic J. Bacteriol. 951305-1313. bacteria, Halobacterium vallismortis, and Halobacterium 27. Smibert, R. M. and N. R. Krieg. 1981. General characterization. marismortui. Can. J. Microbiol. 2952-59. 1984, p. 407-443. Zn P. Gerhardt (ed.) Manual of methods for 19. Oren, A. 1983. Halobacterium sodomense, sp. nov., a Dead Sea general microbiology. American Society for Microbiology, halobacterium with an extremely high magnesium requirement. Washington, D .C. Int. J. Syst. Bacteriol. 33:381-386. 28. Soliman, G. S. H., and H. G. Truper. 1982. Halabacterium 20. Rodriguez-Valera, F., G. Juez, and D. J. Kushner. 1983. pharaonis sp. nov., a new extremely haloalkaliphilic Halobacterium mediterranei spec. nov., a new carbohydrate archaebacterium with a low magnesium requirement. Z. utilizing extreme halophile. Syst. Appl. Microbiol. 4:369-381. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe C 21. Rodriguez-Valera, F., F. Ruiz-Berraquero, and A. Ramos- 3:31&329. Cormenzana. 1980. Isolation of extremely halophilic bacteria 29. Spadari, S., S. Sala, and G. Pedrali-Noy. 1982. Aphidicolin: a able to grow in defined inorganic media with single carbon specific inhibitor of nuclear DNA replication in eukaryotes. sources. J. Gen. Microbiol. 119535-538. Trends Biochem. Sci. 7:29-32. 22. Ross, H. N. M., M. D. Collins, B. J. Tindall, and W. D. Grant. 30. Tindall, B. J., A. A. Mills, and W. D. Grant. 1980. An 1981. A rapid procedure for the detection of archaebacterial alkalophilic red halophilic bacterium with a low magnesium lipids in halophilic bacteria. J. Gen. Microbiol. 123:75-80. requirement from a Kenya soda lake. J. Gen. Microbiol. 23. Ross, H. N. M., and W. D. Grant. 1985. Nucleic acid studies on 116:257-260. halophilic archaebacteria. J. Gen. Microbiol. 131:165-173. 31. Tomlinson, G. A., and Hochstein, L. I. 1972. Studies on acid 24. Sadler, M., M. McAninch, R. Alico, and L. I. Hochstein. 1980. production during carbohydrate metabolism by extremely The intracellular Na+ and K+ composition of the moderately halophilic bacteria. Can. J. Microbiol. 18:1973-1976. halophilic bacterium, Paracoccus halodenitrijcans. Can. J. 32. Werber, M. M., and M. Mevarech. 1978. Induction of a dissim- Microbiol. 26:49&502. ilatory reduction pathway of nitrate in halobacterium of the 2.5. Schinzel, R., and K. J. Burger. 1984. Sensitivity of halobacteria Dead Sea. Arch. Biochem. Biophys. 186:60-65. to aphidicolin, an inhibitor of eukaryotic a-type DNA polymer- 33. Yoshinari, T., and R. Knowles. 1976. Acetylene inhibition of ase. FEMS Microbiol. Lett. 25187-190. nitrous oxide reduction by denitrifying bacteria. Biochem. Bio- 26. Showe, M. K., and J. A. DeMoss, 1968. Localization and phys. Res. Commun. 69:705-710.