Vol. 55, No. 3APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1989, p. 645-6480099-2240/89/030645-04$02.00/0Copyright © 1989, American Society for Microbiology

Isolation and Characterization of a Bacteriophage Lytic forDesulfovibrio salexigens, a Salt-Requiring,

Sulfate-Reducing BacteriumKAZUO KAMIMURA* AND MICHIO ARAKI

Government Industrial Research Institute, Chugoku, Ministry of International Trade and Industry, 2-2-2, Hiro-Suehiro,Kure, Hiroshima, 737-01, Japan

Received 28 June 1988/Accepted 23 November 1988

A bacteriophage that lysed Desulfovibrio salexigens cells was isolated from marine sediments and prelimi-narily characterized by electron microscopy and electrophoretic analysis of structural proteins and genomicnucleic acid. The bacteriophage had an icosahedral head and a long flexible tail, and the buoyant density of thebacteriophage particles was 1.468 g/ml in cesium chloride. The particles consisted of a double-stranded DNAmolecule about 33 kilobase pairs long and at least 11 structural proteins.

Sulfate-reducing bacteria (SRB) are anaerobes that carryout dissimilatory sulfate reduction. In this process, thesulfate ion acts as does oxygen in conventional respiration,i.e., as an electron acceptor for the dissimilation of organicmatter (10). SRB can be found in almost all natural environ-ments, such as soils, fresh and marine waters, hot springs,geothermal areas, oil and natural gas wells, estuarial muds,and sewage. They are well known to cause heavy corrosionin many structural materials under anaerobic conditions (15)and are also known to be constitutive bacteria in marinemicrobial fouling. Seawater is generally supplied with oxy-gen through air, and this environment appears to be unfa-vorable for anaerobic bacteria. Nevertheless, active partic-ipation of SRB in corrosion of metals exposed to seawater iswell recognized (10, 12-14).We have studied marine bacterial fouling communities

formed on metal plates and have isolated bacteriophages thatlyse aerobic heterotrophic fouling bacteria and found thedifference in fouling bacterial communities between metalplates by using the host specificity of bacteriophage (4, 5).Although there have been numerous reports on the physiol-ogy and ecology of SRB, there are no published reports onbacteriophage in this important group of bacteria. The iso-lation of bacteriophage will be important for further under-standing of the ecology and molecular biology of SRB. Thispaper is the first description of the isolation and preliminarycharacterization of a bacteriophage that lyses the salt-re-quiring, sulfate-reducing bacterium Desulfovibrio salexi-gens.

MATERIALS AND METHODSBacterial strain and growth. D. salexigens (NCIMB 8308)

was kindly provided by T. Nakahara of the National Chem-ical Laboratory for Industry, MITI, Japan. For routineculture and maintenance of this strain, media B and Cdescribed by Postgate (10) were prepared with seawater. Fordetection and isolation of bacteriophage, medium C wasmodified by the omission of ferrous sulfate so that detectionof plaques was not obscured by blackening of the plates withFeS. Agar was added to 1.5% for plates and to 0.7% for thesoft-agar overlay. Cells were grown at 30°C in anaerobic

* Corresponding author.

GasPak jars (BBL Microbiology Systems, Cockeysville,Md.) containing hydrogen plus a carbon dioxide generatorenvelope and palladium catalyst.

Isolation of bacteriophage. Samples of blackened muddysediments indicating production of H2S were collected nearthis institute at the seashore of Nagahama harbor, SetoInland Sea, at low tide. Samples were suspended in auto-claved seawater and centrifuged at 4,000 x g for 10 min. Thesupernatants were mixed with an equal volume of double-strength medium B. Enrichment mixtures were prepared byinoculation of the supernatants into a log-phase culture of D.salexigens and incubation of the culture at 30°C for 48 h in ananaerobic jar. Single plaques were isolated from the enrich-ment sample as described by Adams (1) except that aconcentrated cell suspension was used in soft agar. Whencells of log-phase cultures were embedded in the soft agar inthe usual manner, only very small colonies appeared. There-fore, cells precipitated by centrifugation and then suspendedin medium C were used to prepare a uniform lawn fordetection of plaques.The bacteriophage titer was determined as PFU by the

soft-agar overlay technique described above. For prepara-tion of bacteriophage stock, a log-phase culture was infectedwith one plaque and incubated overnight. After centrifuga-tion at 12,500 x g for 10 min, the supernatant was filteredthrough a membrane filter (pore size, 0.2 ,um; MilliporeCorp., Bedford, Mass.). Bacteriophage stock was stored at40C.

Purification of bacteriophage particles. Bacteriophage par-ticles in the phage stock were precipitated by addition ofpolyethylene glycol 6000 to 10% (wt/vol). The mixture waskept at 4°C overnight and then centrifuged at 4,000 x g for 10min for recovery of bacteriophage particles. The precipitatewas suspended in AAM solution (6) containing 1% ammo-nium acetate and 10 mM magnesium chloride. The materialobtained by membrane filtration was centrifuged at 100,000x g for 1 h for recovery of bacteriophage particles. Theprecipitate was suspended in 20 ml of AAM solution andthen mixed with 16 g of cesium chloride. The visible bandthat formed after centrifugation at 100,000 x g for 20 h at20°C in an angle rotor (Hitachi SCP85H 7OTi) was collectedand dialyzed in AAM solution at 4°C. The purified bacterio-phage preparations were stored at -200C.

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FIG. 1. Plaques formed on soft-agar overlay. An enrichmentsample containing plaque-forming activity was diluted, and 1 ml ofeach dilution was plated with D. salexigens cells by the soft-agaroverlay technique. The plates were incubated at 30°C in anaerobicjars.

Electron microscopy. Bacteriophage particles were nega-tively stained with 2% phosphotungstate solution and ob-served with a transmission electron microscope (JEOL2000-FX) with an accelerating voltage of 120 kV. Magnifica-tion was calibrated with carbon grating (Ladd Co.).

Analysis of nucleic acid and structural proteins. The nucleicacid in the bacteriophage was analyzed by staining withacridine orange according to the method of Hidaka (3). The

nucleic acid was purified according to the method ofLawrence et al. (7) and then treated with DNase, RNase,and a variety of restriction endonucleases at 37°C in buffersprovided by the supplier (Wako Pure Chemical Industries,Ltd.). The samples were loaded onto 1% agarose gelsprepared in running buffer containing 40 mM Tris, 5 mMsodium acetate, and 1 mM EDTA (pH 8.0). After electro-phoresis at 5 V/cm for 4 h, the gel was stained with 0.5 ,ug ofethidium bromide per ml. DNA bands were visualized with aUV transilluminator.

Structural proteins were separated on 10% sodium dode-cyl sulfate-polyacrylamide gels. Purified bacteriophage par-ticles were suspended in a solution containing 2% sodiumdodecyl sulfate, 50% glycerol, 5% mercaptoethanol, and 20mM phosphate buffer (pH 7.2) and disrupted by boiling at100°C for 10 min. Electrophoresis was carried out accordingto the method of Weber and Osborn (16), and the gel wasfixed and stained with silver according to the method ofOakley et al. (9).Buoyant density determination. A 1-ml sample of purified

bacteriophage preparation was suspended in 25 ml of 45%(wt/wt) cesium chloride in AAM solution and centrifuged at100,000 x g for 20 h at 20°C in the angle rotor. The gradientwas fractionated in a density gradient fractionator (modelDGF-U; Hitachi) and scanned for UV A260 peaks with a UVspectrophotometer (model UVIDEC-100-VI; Jasco). Thedensity of each fraction was measured by a densitometer(model DMA 60 in combination with a DMA 601 remote cell;Anton Paar KG).

FIG. 2. Electron micrograph of negatively stained bacteriophage particles lysing D. salexigens. Bar, 100 nm.

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D. SALEXIGENS BACTERIOPHAGE 647

FIG. 3. (A) Agarose gel electrophoresis of purified genomicDNA from bacteriophage lysing D. salexigens Lanes: 1 Hindllldigests of lambda DNA used as fragment length standards (molec-

ular masses in kilobase pairs); 2, genomic DNA of D. salexigens

phage; 3, D. salexigens phage DNA digested with Hindlll. (B)

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of struc-tural proteins of the bacteriophage particle. Lanes: 1, structural

proteins; 2, molecular mass (in kilodaltons) standards.

RESULTS

Isolation of bacteriophage lysing D. salexigens cells. Noplaques were observed when filtrates obtained from suspen-sions of blackened sediments or seawater samples weredirectly examined by the soft-agar overlay technique. Someplaques with halos around clear centers were observed afterincubation of filtrates from the enrichment culture for 2 dayson the soft-agar overlay (Fig. 1).The culture of D. salexigens infected with well-isolated

plaques gave titers of 108 to 109 PFU/ml. The plaque-formingmaterial was filtered out through a membrane filter andconcentrated by centrifugation at 100,000 x g for 60 min.Morphology and buoyant density of bacteriophage. Elec-

tron microscopic observation of the concentrated samplerevealed regular particles with heads of hexagonal shape andlong tails, which were typical features (Fig. 2). The bacte-riophage particles were morphologically similar to coliphagelambda, a member of the family Styloviridae (8). The headwas icosahedral, with a diameter of approximately 51 to 61nm. No collar was visible. The long and noncontractile tailwas flexible. Striation of the tail was not clear. Someparticles appeared to have spikes at the ends of the tails.Tails were 165 to 194 nm in length and 10 to 15 nm in width.Buoyant density was about 1.468 g/ml in cesium chloride.

Characterization of genomic nucleic acid. The genomicnucleic acid of purified bacteriophage particles fluorescedgreen under UV light, which suggested that the nucleic acidwas double-stranded DNA. This possibility was confirmedby enzymatic digestion of extracted nucleic acid. The ex-tracted nucleic acid was treated with DNase, RNase, and11 restriction endonucleases and analyzed by agarose gelelectrophoresis. The nucleic acid was digested by DNase,HindIII, Hinfl, and Sall. Gel patterns of HindIII digests are

shown in Fig. 3A. The bacteriophage genome was digestedinto two fragments with Sall and at least 13 with HindIll.Numerous small fragments were observed after digestionwith Hinfl. EcoRI, XhoIII, SmaI, BamHI, KpnI, HaeIII,ApaI, and PstI had no recognition sites on the genomic DNAof the bacteriophage. The genome was estimated to be about33 kilobase pairs in length.

Structural proteins of bacteriophage particles. The struc-tural proteins of purified bacteriophage particles were ana-lyzed by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. Two major and at least nine minor bands weredetected (Fig. 3B). The molecular weights of two majorstructural proteins were determined to be about 56,000 and38,000, respectively. The other proteins had molecularweights of 77,000, 74,000, 60,000, 44,000, 34,000, 32,000,31,000, 24,000, and 21,000.

DISCUSSION

The soft-agar overlay technique, which has been widelyused to detect and isolate bacteriophages, was used in thisstudy to detect a bacteriophage that lyses D. salexigens.Detection of the bacteriophage requires preparation of auniform lawn, which necessitates the concentration of log-phase cells. It has been reported that small amounts ofoxygen inhibit the growth of SRB (10). It seems probablethat the cells generate a reducing environment, facilitatingbacterial growth and bacteriophage infection.Some plaques were observed on soft agar plated with

filtrates obtained from enrichment cultures. On the otherhand, seawater samples, suspensions of blackened sedi-ments, and filtrates obtained from cultures without addedseawater or sediments did not produce plaques. Theseresults suggest that the concentration of plaque-formingmicrobes is very low in seawater and sediments and that theisolated bacteriophage is virulent.

Clear lysates prepared from the plaque-forming agentusually contained 109 to 1011 PFU of bacteriophage particlesper ml (5). However, lysates of D. salexigens cells infectedwith a single plaque gave titers of only about 108 to 109PFU/ml. Complete lysis of the cultures was not observed. Itseems that the bacteriophage has a long latent period and asmall burst size.The bacteriophage particle has a head with a distinct

hexagonal shape and a noncontractile tail, characteristics ofmembers of the family Styloviridae (8). A typical member ofStyloviridae is the lysogenic bacteriophage lambda, whichconsists of about 11 proteins (2). The largest of theseproteins (37.5 kilodaltons) is the major constituent of thehead, accounting for 57% of total protein and about 95% ofthe head protein. The second protein (32.5 kilodaltons) is theprincipal constituent of the tail and accounts for 19% of totalprotein and 90% of the tail protein. The isolated bacterio-phage particle consisted of two major and nine minor pro-teins. The main component was similar in molecular weightto the major head protein of lambda. Therefore, the proteinof 38 kilodaltons is believed to be the major constituent ofthe head, and the protein of 56 kilodaltons appears to be theprincipal constituent of the tail.

This is the first report on a bacteriophage that lyses asalt-requiring SRB, D. salexigens. The bacterial strain usedin this study was originally isolated by Postgate and Camp-bell (11) from dried mud samples from Ain-el-Braghi, a sulfurlake near El Agheila, Libya. However, the bacteriophageinfecting this strain was isolated from the muddy marinesediment collected at the seashore of Seto Inland Sea. This

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648 KAMIMURA AND ARAKI

fact indicates that SRB closely related to the host strain usedin this study are likely present in natural seawater. There-fore, it may be useful to examine the phage sensitivity toother SRB strains. Since bacteriophage that lyses SRB isknown to be present in natural seawater, the ecologicaleffect of the bacteriophage on the population dynamics ofmarine SRB should be investigated.

ACKNOWLEDGMENTS

This work was supported by a grant from the Ministry ofInternational Trade and Industry (MITI) of Japan.

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Inc., New York.2. Buchwald, M., H. Murialdo, and L. Siminovitch. 1970. Morpho-

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3. Hidaka, T. 1975. Identification of the type of nucleic acid inmarine bacteriophages with acridine orange staining. Mem. Fac.Fish. Kagoshima Univ. 24:133-138.

4. Kamimura, K., and M. Araki. 1984. Scanning electron micro-scopic observation of bacteria attached to titanium and alumi-num alloy plates. Fuchaku Seibutsu Kenkyu (Mar. Fouling)5:19-28.

5. Kamimura, K., and M. Araki. 1985. Bacteriophages lysing themarine fouling bacteria. Fuchaku Seibutsu Kenkyu (Mar. Foul-ing) 5:17-25.

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8. Matthews, R. E. F. 1982. Classification and nomenclature ofviruses. Intervirology 17:4-199.

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10. Postgate, J. R. 1984. The sulphate-reducing bacteria, 2nd ed.Cambridge University Press, Cambridge.

11. Postgate, J. R., and L. L. Campbell. 1966. Classification ofDesulfovibrio species, the nonsporulating sulfate-reducing bac-teria. Bacteriol. Rev. 30:732-738.

12. Salvarezza, R. C., and H. A. Videla. 1980. Passivity breakdownof mild steel in sea water in the presence of sulfate reducingbacteria. Corrosion 36:550-554.

13. Sanders, P. F., and W. A. Hamilton. 1985. Biological andcorrosion activities of sulphate-reducing bacteria in industrialprocess plant, p. 47-68. In S. C. Dexter (ed.), Proceedings of theinternational conference on biologically induced corrosion,Gaithersburg, Md. NACE, Houston, Tex.

14. Stoecker, J. G. 1984. Guide for the investigation of microbio-logically induced corrosion. Mater. Perform. 23:48-55.

15. Tatnall, R. E. 1981. Fundamentals of bacteria induced corro-sion. Mater. Perform. 20:32-38.

16. Weber, K. and M. J. Osborn. 1969. The reliability of molecularweight determinations by dodecyl sulfate-polyacrylamide gelelectrophoresis. J. Biol. Chem. 244:4406-4412.

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