APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p. 3360-3366 Vol. 58, No. 100099-2240/92/103360-07$02.00/0Copyright © 1992, American Society for Microbiology

Lysogeny in Bradyrhizobium japonicum and Its Effect onSoybean Nodulationt

H. M. ABEBE,4 M. J. SADOWSKY, B. K. KINKLE,§ AND E. L. SCHMIDT*Departments ofMicrobiology and Soil Science, University ofMinnesota, St. Paul, Minnesota 55108

Received 6 February 1992/Accepted 5 August 1992

Rhizobiophage V, isolated from soil in the vicinity of soybean roots, was strongly lytic on Bradyrhizobiumjaponicum 123B (USDA 123) but only mildly lytic on strain 14-4, a chemically induced small-colony mutant of123. Numerous bacteriophage-resistant variants were isolated from 14-4 infected with phage V; two werestudied in detail and shown to be lysogenic. The two, 14-4 (V5) and 14-4 (V12), are the first reported examplesof temperate-phage infection in B. japonicum. Phage V and its derivative phages V5 and V12 were closelyrelated on the basis of common sensitivity to 0.01 M sodium citrate and phage V antiserum, phage immunitytests, and apparently identical morphology when examined by electron microscopy. However, the three phagesdiffered in host range and in virulence. Lysogens 14-4 (V5) and I4-4 (V12) were immune to infection by phagesV and V5 but not to infection by V12. Southern hybridization analysis confirmed the incorporation of phageV into the genomes of strains L4-4(V5) and 14-4(V12) and also demonstrated the incorporation of phage V intothe genome of a phage V-resistant derivative of USDA 123 designated 123 (V2). None of the three lysogens,14A4(V5), I4-4(V12), or 123B(V2), was able to nodulate soybean plants. However, Southern hybridizationprofile data indicated that phage V had not incorporated into any of the known B.japonicum nodulation genes.

Bacteriophages that infect rhizobia (rhizobiophages) werefirst reported by von Gerretsen et al. in 1923 (31) and havebeen isolated subsequently from all of the major groups ofrhizobia (27). The importance of rhizobiophages in theregulation and evolution of host populations in nature is notknown, but their potential to act as vehicles of geneticexchange has been demonstrated in vitro. Generalized trans-duction was observed for a number of phages amongstrains of Rhizobium meliloti (5, 6, 11, 12, 16, 25). Twovirulent rhizobiophages were shown to cotransduce auxo-trophic and antibiotic resistance markers in R leguminosa-rum bv. viceae (4); the same phages mediated transductionbetween strains of R. leguminosarum bv. trifolii and one-way transduction from R. leguminosarum bv. viceae to R.leguminosarum bv. trifolii. Generalized cotransduction ofantibiotic resistance markers has also been reported forBradyrhizobium japonicum (23). The only instance of spe-cialized transduction among rhizobia was that documentedforR meliloti by Svab et al. (28), who transferred a cysteinemarker at relatively low frequency.Systems now available to transfer genetic material be-

tween strains of B. japonicum are limited mainly to con-jugation, although Shah et al. (24) used generalized trans-duction to construct a linkage map for strain D211.Rhizobiophages capable of specialized transduction could beuseful for high-resolution fine-structure mapping as moredetailed linkage maps of B. japonicum are developed. Whilelysogeny has been observed in several groups of rhizobia(11, 17, 29), it has not been reported in the slow growers, thebradyrhizobia.

In a previous study (22), we isolated 12 phages from soil

* Corresponding author.t Paper 18713 in the Scientific Journal Series of the Minnesota

Agricultural Experiment Station, St. Paul.t Present address: Bioprocess Research and Development, The

Upjohn Company, Kalamazoo, MI 49001.§ Present address: Department of Biology, University of Cincin-

nati, Cincinnati, OH 45221.

and used them to characterize a group of 79 isolates of B.japonicum serocluster 123. We examined several phages inmore detail and noted that at least one of them, phage V, hadproperties suggestive of a temperate bacteriophage. Thepurpose of this report is to summarize evidence confirmingthe temperate nature of phage V and to call attention to thepromise of this first temperate phage of B. japonicum as atool with which to study the genetics of the bradyrhizobia.

MATERIALS AND METHODS

Bacteria. The two principal strains of B. japonicum used inthis study were members of serogroup 123. The wild-typestrain, 123B (USDA 123), was obtained from the RhizobiumCulture Collection of the U.S. Department of Agriculture,Beltsville, Md. It is lysed by phages A, I, J, V, Z, and W(22). Lysogeny was studied primarily in strain L4-4, asmall-colony mutant of 123B obtained by nitrous acid muta-genesis. Strains L4-4 (V5) and L4-4 (V12) were phageV-resistant variants of L4-4 derived in this study. All threestrains of L4-4 grew equally well as prototrophs on arabi-nose-ammonia mineral salts agar medium. Strains 123 (V1),a small, compact colony type; 123 (V2), an intermediate-sizecolony type; and 123 (V3), the larger colony size typical ofthe parent, were phage-resistant derivatives isolated fromthe phage V-infected culture suspension of USDA 123B.Cultures R3N20C, St. Paul 42, and Webster 48 were sero-cluster 123 strains isolated from Minnesota soils (22). B.japonicum USDA 110 and USDA 228 were obtained fromthe Beltsville Rhizobium Culture Collection. Cultures werepropagated in yeast extract-mannitol (YEM) (30) or yeastextract-maltose (17) broth or solid medium and maintainedon YEM slants. Serogroup 123-specific fluorescent antibody(FA) was used to ensure the purity of the cultures in allexperiments (22).

Bacteriophage. Rhizobiophage V was isolated from fieldsoil associated with soybean roots (22). Phage titrations wereperformed by the single-layer indicator method (2) usingphage suspensions diluted 10-fold in 0.1% yeast extract. A

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0.2-ml aliquot of the appropriate B. japonicum host wasmixed aseptically with a 0.1-ml phage suspension and al-lowed to equilibrate for 15 min at room temperature, afterwhich 3.0 ml of YEM soft (0.6%) agar (50°C) was added andsubsequently poured onto triplicate YEM agar plates. Phagetyping was carried out as previously reported (22).

Lysogenization. A log-phase, saline-washed cell suspen-sion, with or without preincubation in 0.01 M MgSO4, wasmixed with phage at a 1:1 ratio and equilibrated for 15 min.The infection mixture was then inoculated into 100 ml ofYEM broth and incubated on a rotary shaker for 6 days at25°C. Potential lysogens were detected from among thesurviving population by dilution plating on YEM agar.Isolated colonies obtained after 2 weeks were picked andstreaked for further isolation. Each of these strains wassuccessively streaked and reisolated four times duringgrowth in the presence of 0.01 M sodium citrate (10) orspecific antiserum (9) to eliminate free phage, phage pro-duced by cells committed to lysis, or abortive lysogens.

Serial passage of lysogen in the presence of antibody. A0.1-ml aliquot of a log-phase, saline-washed culture of L4-4(V12) was used to inoculate each of two 5-ml tubes of YEMbroth. To one tube was added 0.3 ml of undiluted phage Vantiserum. This tube and the untreated control tube wereallowed to stand for 20 min and then incubated for 12 to 18h on a shaker at 25°C. After incubation, each culture wascentrifuged aseptically for 15 min at 10,000 rpm to pellet thecells. The supernatants were collected separately in sterilevials and kept for phage testing. Each pellet was washedthree times in saline and suspended in 1.0 ml of YEM brothto provide a 0.1-ml inoculum for the next serial passage. Atotal of five serial passages were completed by following thisprocedure. The presence of phage in the supernatants ateach passage was assayed against sensitive host strain 123Bas described above. Plaques, if any, were visible after a 4- to5-day incubation period. Passage 5 of the L4-4 (V12) plusanti-phage V series was continued with a sixth and finalpassage made in the absence of antibody to determinewhether the phage production capability of L4-4 (V12) hadbeen retained during passage in the absence of free phage.

Adsorption of phage. Stationary-phase cultures were cen-trifuged, washed cells were adjusted to a density of about 3x 108 ml-' in 0.1% yeast extract, and 1.0 ml was added to a3.0-ml phage suspension with a titer of 1.3 x 109 PFU ml-1.After 15-, 30-, and 45-min incubation periods, a 1.0-mlsample was diluted in 4.5 ml of yeast extract-0.5 ml ofchloroform. The mixture was shaken vigorously for 15 min,and serial 10-fold dilutions were assayed for free phage bythe agar layer method (7) with L4-4 as the indicator host.

Spontaneously liberated phage. YEM broth was inoculatedwith 103 to 104 cells of L4-4 (V5) or L4-4 (V12) ml-1.Multiplication of both phages and bacteria was monitoreddaily until the stationary growth phase was reached. Plaquecounts were determined by the procedures cited above, andhost cell counts were done by dilution spread plating.Phage V antiserum preparation. Phage V was introduced

into 250 ml of a mid-log-phase yeast extract-maltose cultureof 123B at a multiplicity of infection of 1. After 15 min ofadsorption time, the culture was placed on a rotary shakerfor about 24 h at 25°C for complete lysis. Chloroform (2.5 ml)was added, and the mixture was returned to the shaker for 15min. The lysate was then centrifuged at 10,000 x g for 15 minat 4°C. The supernatant (about 109 PFU ml-1) was stored ina sterile flask in 0.5 ml of chloroform at 4°C. Phage wasconcentrated by centrifugation overnight (Beckman L8-8OM Ultracentrifuge) at 45,000 rpm and 4°C. The pellet was

suspended in 0.1% sterile yeast extract, passed through asterile 0.2-,um-pore-size membrane, and subsequently ti-trated. The yield was about 6 x 1011 PFU ml-'. Antibodyformation was initiated in young New Zealand White rabbitswith an antigen preparation consisting of a 4.0-ml phagesuspension mixed with 4.0 ml of Freund incomplete adju-vant. Twice weekly for 3 weeks, 1.0 ml of the vigorouslyshaken antigen suspension was distributed subcutaneouslyat the hind- and forequarters of each animal. Active antise-rum was obtained 1 month after the final injection.The antiviral activity of the serum was assayed by the

neutralization method of Adams (1) and expressed as a Kvalue calculated with the formula K = 2.3 x [(Dit) log(pOIP)], where K is the velocity constant, D is the finaldilution factor of the serum in the phage-serum mixture (500in this case), pO is the phage titer in PFU at time zero, andp is the phage count at time t (taken at 5-min intervals).

Electron microscopy. Rhizobiophage V was concentratedas described for antigen preparation. The pellet that resultedfrom ultracentrifugation was suspended in 0.1% yeast ex-tract to a titer of 1011 PFU ml-'. Some remaining cell debrisor fragments were removed by two cycles of high- andlow-speed centrifugation. Negative staining was performedwith uranyl acetate or potassium phosphotungstate. Prepa-rations were examined with a Philips transmission electronmicroscope at 80 kV.

Molecular analyses. To determine whether the phage-resistant 123B and L4-4 isolates had rhizobiophage V incor-porated into their genomes, we hybridized a phage V geneprobe to genomic DNAs from strains 123B, 123(VI),123(V2), 123(V3), L4-4, L4-4(V5), and L4-4(V12). Large-scale preparation of B. japonicum phage V was done byusing a modification of plate lysate method II of Sambrook etal. (20). After the 123B culture was grown to the mid-exponential growth phase in YEM medium, a 0.5-ml cellsuspension and enough phage V liquid lysate to give 3 x 105PFU per plate were added to 8.0 ml of overlay agar (0.5%agarose in YEM medium). The mixture was poured onto thesurface of a large (150-mm diameter) petri dish containingYEM bottom agar. Plates were incubated at 28°C for 2 or 3days, until plaques were almost confluent. Typically, thelarge-plate procedures produced lysates containing about 6x 109 PFU/ml.Phage V was purified from lysates by using a modification

of the procedures of Sambrook et al. (20). A 125-ml aliquotof phage lysate was warmed to room temperature, and 1.0 p.gof DNase I per ml and 1.0 pg of RNase A per ml were added.The solution was incubated for 30 mir -t 25°C, and NaCl wasadded to a final concentration of 1.0 M. The solution wasincubated on ice for 1 h and centrifuged at i0,000 x g for 10min at 4°C. Solid polyethylene glycol (PEG 8000) was addedto the supernatant to give a final concentration of 10%(wtlvol), and the solution was incubated on ice for 1 h. Theprecipitate was recovered by centrifugation at 10,000 x g for10 min at 4°C, and the pelleted material was allowed todissolve overnight at 4°C in 2 ml of SM buffer (20). Thephage solution was extracted once with an equal volume ofchloroform. The aqueous phase was transferred to a cleantube, and EDTA, pronase E, and sodium dodecyl sulfatewere added to final concentrations of 20 mM, 50 ,ug/ml, and0.5% (wt/vol), respectively. The solution was incubated for 1h at 37°C and sequentially extracted with phenol, phenol-chloroform, and chloroform. The aqueous solution was

dialyzed overnight against TE buffer (10 mM Tris [pH 8.0],1.0 mM EDTA), and sodium acetate was added to a finalconcentration of 0.3 M. Phage nucleic acid was precipitated

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with 2 volumes of ethanol at room temperature for 30 min.Nucleic acids were recovered by centrifugation at 12,000 xg for 10 min at 4°C, washed with 70% ethanol, dried invacuo, and suspended in 100 ,ul of TE buffer. To determinewhether the recovered nucleic acids were composed of RNAor DNA and whether the preparation was free of genomicDNA contaminants, the nucleic acid sample was digestedwith DNase-free RNase (Bethesda Research Laboratories,Gaithersburg, Md.), DNase I, and BamHI and digestionproducts were separated by horizontal gel electrophoresisusing Tris-borate-EDTA buffer (20).

Total genomic DNAs from phage-infected and uninfectedstrains were prepared as previously described (19). DNA(0.5 ,ug) was transferred to nitrocellulose membranes byusing a Dot Blot Manifold (Schleicher & Schuell, Inc.,Keene, N.H.) (9) and hybridized to 32P-labelled probes asdescribed previously (19).To examine phage V integration in the B. japonicum

genome further, we hybridized the phage V DNA probe toEcoRI-digested genomic DNAs from lysogenized [L4-4(V5),L4-4(V12), and 123(V2)] and phage-resistant [123(V1) and123(V3)] serogroup 123 strains. Genomic DNAs from phage-resistant, nodulation-defective isolates [L4-4(VS), L4-4(V12), and 123(V2))] and from phage-resistant, nodulation-competent isolates [123(Vi) and 123(V3)] were digested withEcoRI, transferred to Nytran membrane, and hybridized toHindIII-digested, 32P-labelled cosmid pR32 DNA. CosmidpR32 contains the nodDlYABC, nodSU, nodIJ, nodD2,nodZ, and nolA genes from B. japonicum I110 (18).

Plant infection assays. Soybean seeds (cv. Hodgson 78)were surface sterilized, germinated for 2 days in sterile petridishes, and then placed in surface depressions in autoclavedsand contained in 6-in. (15-cm)-diameter pots. Three seedswere planted per pot, and four replicate pots per treatmentwere used. Inoculation treatments were carried out bydispensing 1.0 ml of a stationary-phase broth culture (about10' cells) of either strain L4-4, L4-4 (V5), L4-4 (V12) 123B,123 (V), 123 (V2), or 123 (V3) directly on the germinatedseed and then covering the seed immediately with sterilesand. A final treatment consisted of uninoculated seeds.Plants were provided with a 0.1 x nitrogen-free nutrientsolution during growth in a Conviron EY-15 plant growthchamber. Plants were harvested after 35 days, and rootsystems were washed free of sand and examined for rootnodules. All nodules were collected and serotyped individ-ually by using serogroup 123-specific FA to confirm thatnodules were derived from the inoculant. For the L4-4control and L4-4 lysogens, rhizosphere samples were as-sayed for inoculant rhizobia by quantitative immunofluores-cence using serogroup 123-specific FA (21). The entireexperiment was repeated by following the same protocol.

RESULTS

Examination of a group of rhizobiophages isolated fromsoil (22) focused on one designated phage V when coloniesappeared in plaques of certain serogroup 123 B. japonicumhosts. In particular, the interactions of rhizobiophage V withB. japonicum L4-4 suggested the possibility of lysogeniza-tion. The differential responses of strains L4-4 and 123B toinfection with phage V are shown in Fig. 1. A mid-log-phaseculture of wild-type strain 123B was extensively lysed within24 h of phage inoculation, but analogous treatment of strainL4-4 led to only slightly reduced optical density of theculture. Dilution plating of L4-4 phage V-infected culturesdisclosed numerous variants in colony morphology, 40 of

C60-* . * L4-4+V*60

20 - 123B+V

O 2 4 6 8 10Days

FIG. 1. Effect of rhizobiophage V on the growth of B. japonicum123B (USDA 123) and L4-4 in yeast extract-maltose broth. Thearrow indicates time of phage infection.

which were isolated, restreaked for purity, and verifiedindividually to be 123 strains by immunofluorescence and tobe completely resistant to infection when used as a lawnspotted with phage V.Of the total of 40 phage V-resistant strains of L4-4, 8 were

cultured and the supernatant of each was tested for infectiv-ity on lawns of strain L4-4, the supernatant-producing (ho-mologous) strain, and the 7 other (heterologous) variantstrains. Included also was phage V produced in the super-natant of strain 123B. Phage V formed plaques only on thelawn of L4-4. Supernatants of each of the eight phageV-resistant isolates tested were infective on strain L4-4 butvaried in infectivity to heterologous resistant isolates. Atleast five different host range patterns were observed. Hostrange similarities, however, did not extend to plaque mor-phologies, as supematant plaques varied somewhat on dif-ferent host lawns. Phage V12 was of special interest becauseits plaque morphology was consistent. Strains L4-4 (V5),producing phage V5, and L4-4 (V12), forming phage V12,were selected for further tests of lysogeny. Superinfectiontests to evaluate immunity more precisely indicated that hostL4-4 was only weakly immune to phages V and V5, whilehosts L4-4 (V5) and L4-4 (V12) were strongly immune (notlysed) by these same phages. None of the hosts, however,including L4-4 (V12), proved to be immune when superin-fected with phage V12.A more extended host range study, summarized in Table

1, showed further differences between phages V5 and V12and that neither of the derivative phages had as wide a hostrange as phage V. Phage V12, like V but unlike V5, wasinfective to B. japonicum 110 and 62, both of which repre-sent serogroups outside of serocluster 123.Lysogenic bacteria retained the capacity to produce phage

when grown in the presence of agents which inactivate freephage, whereas phage production by pseudolysogens orcarrier strains is diluted by bacterial growth in the absence ofreinfection by free phage. Inactivation of phage V wasaccomplished with either 0.01 M sodium citrate or phage Vantiserum. The kinetics of inactivation are shown in Fig. 2.Neutralization by anti-phage V was essentially the same for

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TABLE 1. Susceptibility of B. japonicum isolates not derivedfrom strain L4-4 to rhizobiophage V and derivative

phages V5 and V12

Plaque formationaB. japonicum isolate Serogroup

V V5 V12

L4-4 123 + + +123B 123 + + +StP42 123 + + +Ble4N19 123 + + +ISDN8 123 + + +PRC49 123 + - -PA3 127 + - -W162 129 + - +R3N20c 129 + - -USDA 110 110 + - +USDA 62 62 + - +

a +, distinct plaques;-, no plaques; +, faint plaques.

phages V5 and V12 as for phage V. K values (1) at 20 minwere 90.3 for V, 101.1 for V5, and 91.7 for V12, reflecting theserological relatedness of the derivative phages to rhizo-biophage V. Serial subculture of strain L4-4 (V12) in thepresence of antiserum to phage V eliminated free phageduring each of five passages. Whereas no plaques wereformed in the presence of antiserum, a counterpart controlculture carried through five passages under the same proto-col in the absence of antibody formed plaques too numerousto count. When the antiserum-treated line was transferred in

I0

O-

D 8 ~

0

7

6-

Time (min)FIG. 2. Effects of sodium citrate and phage V antiserum on

infectivity of phage V from an infected culture of strain 123B.Symbols: El, 0.01 M sodium citrate present; *, 10-1 anti-V serumpresent; -- -, distilled water controls; , experimental.

101

_ O

0)n=

a-)0-J

-J

0Cy 4J

2

2 4 6 0 2Time (Days)

4 6 8

FIG. 3. Spontaneous induction of rhizobiophage during growthof B. japonicum lysogens LA-4 (V5) and IA-4 (V12). Symbols on theleft: 0, L4-4 (V5); 0, phage V5. Symbols on the right: *, L4-4(V12); El, phage V12.

a sixth passage in the absence of antiserum, growth resultedin the formation of several hundred plaques on each assayplate. Thus, L4-4 (V12) had the characteristics of a lysogenrather than a phage carrier, since it retained the capacity toproduce phage under conditions that prevent reinfection in acarrier system.During log-phase growth of either L4-4 (V5) or L4-4 (V12),

the rate of bacterial growth and the rate of release of theirrespective phages were the same (Fig. 3). The phage-to-hostratio in each case was 10-2. The pattern in Fig. 3 meets oneof the criteria of lysogeny, namely, that host multiplicationand phage release maintain the same constant rate duringbalanced growth (26).The results of adsorption experiments as a further test of

lysogenicity are summarized in Table 2. These data showthat both susceptible strain L4-4 and resistant strains L4-4(V5) and L4-4 (V12) adsorbed between 50 and 75% of thephage V particles to which they were exposed over a 30-minperiod. The inability of phage V to lyse phage V-inducedvariants of L4-4 was not due to inability of the phage toadsorb to the bacteria. No discernible differences were seenamong phages V, V5, and V12 when they were examined byelectron microscopy. Basic morphological features (Fig. 4)include a hexagonal head and a short, noncontractile taildevoid of fibers or a base plate. The phages fall withinmorphological group C (3), along with coliphages T3 and T7,and resemble numerous other rhizobiophages (2, 6, 13, 14).

Plant infection tests comparing lysogens L4-4 (V5) andL4-4 (V12) with uninfected L4-4 demonstrated a markedconsequence of the lysogeny. Neither lysogen was capableof nodulating soybeans. Plants inoculated with L4-4 (V5) andL4-4 (V12) were stunted with yellow leaves, whereas thoseinoculated with L4-4 were larger and dark green and obvi-ously fixed nitrogen. Examination of the root systems dis-closed abundant normal nodules typical of serogroup 123-

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TABLE 2. Adsorption of rhizobiophage V to strains L4-4, LA-4(V5), and L4-4 (V12) as determined by assay of unadsorbed

phage after different periods of virus-cell contact'

% of phage adsorbed by:Time (min)

LA-4 L4-4 (V5) L4-4 (V12)

15 67.9 52.8 62.130 73.1 50.9 65.245 74.6 61.9 57.9

a Titers of unadsorbed phage were 2.5 x 108 to 4.5 x 108 PFU ml-[.

soybean interactions on L4-4-inoculated plants. All nodulesfrom these plants were determined by immunofluorescenceto be singly infected with strain L4-4. Uninoculated controlplants had zero to three nodules per plant, none of whichwere 123 FA positive. Roots of the lysogen-inoculatedstrains were devoid of true nodules but had occasional smallswellings, perhaps indicative of incipient but aborted nod-ules or nodule primordia. Failure of the lysogens to nodulatewas not due to absence of the inoculant strain in the rootzone, as rhizospheres of all treatments contained >108serogroup 123 FA-reacting cells per g as determined byquantitative immunofluorescence.

Results of the plant nodulation assays for the phageV-resistant derivatives of strain 123B indicated that onlyisolate 123(V2) was Nod- on soybean plants. Plants inocu-lated with 123(V2) were chlorotic and had numerous small(<1.0-mm) root hypertrophies on the root systems. Whilethe ability of isolate 123(V3) to nodulate was not affected(compared with the wild-type control), the nodulation abilityof 123(V1) was partially altered and it produced a fewscattered lateral root nodules on the soybean plants.The phage V-derived nucleic acid sample proved to be

digested totally by DNase I and was resistant to RNasedigestion, indicating that the recovered nucleic acids werecomposed of DNA. The double-stranded nature of the phageDNA was demonstrated when the sample was cut into sixfragments with BamHI. We estimate that the phage genomeis about 45 kb long. Since a large number of restrictionfragments was not seen following BamHI digestion, weconcluded that the phage V DNA preparation was essen-tially free of contaminating genomic DNA from host phage-propagating strain 123B.Dot blot hybridization results (Fig. 5) indicated that hy-

bridization homology was found only with genomic DNAfrom phage-resistant isolates L4-4(V5), L4-4(V12), and123(V2). The phage V DNA probe did not hybridize tophage-resistant, nodulation-proficient strain 123(Vi) or123(V3). Since isolates L4-4(B5), L4-4(V12), and 123(V2) allfailed to form nodules on soybean plants, our results suggestthat there was a relationship between integration of phage Vand nodulation ability. No hybridization homology was seenwith parent strain L4-4 or USDA 123 or with negativecontrol strain St. Paul 42, R3N20c, USDA 110, or Webster48. To determine whether bradyrhizobia from other serolog-ical groups had phage V integrated in their genomes, wehybridized phage V DNA to genomic DNAs fromBradyrhizobium strains USDA 4, USDA 6, USDA 31,USDA 38, USDA 46, USDA 61, USDA 62, USDA 76,USDA 94, USDA 110, USDA 122, USDA 123, USDA 124,USDA 127, USDA 129, USDA 130, USDA 135, and USDA228. The phage V probe failed to hybridize to genomic DNAfrom any of these bradyrhizobia, indicating that the strainsdo not contain prophage. In addition, these results further

FIG. 4. Electron micrographs of rhizobiophage V. Panels: a,Empty phage particles attached to host fragment; b and c, intactphage. Magnifications: a and b, x135,000; c, x190,000.

confirm that our DNA probe preparation was not contam-inated with Bradyrhizobium genomic DNA. Figure 6 showsthat the phage V probe hybridized to three EcoRI fragments(23.3, 15.74, and 6.2 kb) in all three lysogenized serogroup123 isolates. No hybridization homology with the phage-resistant, nodulation-competent isolates or any of the con-trol strains tested was seen.The relationship between the presence of phage V in

lysogens L4-4(V5), L4-4(V12), and 123(V2) and the strains'inability to nodulate soybeans could not be accounted for byintegration of phage V into one or more of the nodulationgenes in these strains (Fig. 7). Comparison of the hybridiza-tion profiles shows that cosmid pR32 hybridized to thesame-size EcoRI fragments in DNAs from nodulation-com-

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1 2 3 4

FIG. 5. Dot blot hybridization of rhizobiophage V DNA probe togenomic DNAs from wild-type, phage-resistant, and lysogenized B.japonicum strains. Spots: la, 123B; 2a, LA-4; 3a, L4-4(V5); 4a,LA-4(V12); lb, 123(V1); 2b, 123(V2); 3b, 123(V3); 4b, R3N20c; Sb,St. Paul 42; 6b, USDA 110; 7b, Webster 48.

petent and -deficient isolates (as well as control parentstrains L4-4 and USDA 123). A similar result was foundwhen BamHI-digested genomic DNA was used (data notshown).

DISCUSSION

The two variants derived from rhizobiophage V-infectedB. japonicum serogroup 123 strain L4-4 are the first reportedlysogens in the slowly growing rhizobia. Strains L4-4 (VS)and L4-4 (V12) met such standard criteria of lysogeny asspontaneous induction of phage, immunity to reinfectionwith the inducing phage, and retention of lysogenic proper-ties during growth in the presence of phage-inactivatingagents. Broth cultures of L4-4 (V5) anid L4-4 (V12) alwayscontained plaque-forming particles which were released atrates proportional to the growth rate of the lysogens (Fig. 3).The ratio of phage released to lysogen numbers during loggrowth was 10-, well within the range of values cited for

_2 Irl "I r *- _.'C 7 s C -- Y A A / E-other lysogenL4-4 (V12) wrather than reby the lysogeLysogenes

B. japonicumV and hosttogether witkfiles (Table 2'

23.3 >-15.17 -

6.2 >

FIG. 6. Souto EcoRI-digenized, and wilc123(V3); 2, 12.228; 7, LA-4; 8molecular size

21.2 >

5A

4.3 >-

3.5 >

1.3 >-

*0fF--i n W$0Q~~~~~~~~~~~~~~~~-7 ,

FIG. 7. Southern hybridization of a nodulation gene probe(pR32) to EcoRI-digested genomic DNAs from wild-type, phage-resistant, and lysogenized B. japonicum serogroup 123 strains.Lanes: 1, L4-4; 2, 123B; 3, L4-4(V5); 4, L4-4(V12); 5, 123(Vl); 6,123(V2); 7, 123(V3). The numbers on the left are molecular sizes, inkilobases.

is (1). Failure of phage V to infect L4-4 (V5) and were clearly related. All were inactivated by sodium citrate,as a function of immunity (prophage formation) and by antiserum prepared against phage V. The K valuesDsistance, since the phage was adsorbed as well determined for the three phages indicated that they were2ns as by susceptible strain L4-4 (Table 2). indistinguishable serologically. Similarly, electron micro-is-induced modifications of both phage V and scopic examination revealed no discernible differences be-t L4-4 were observed. The interaction of phage tween phage V and its derivatives.L4-4 generated a number of host variants, Differences between variant phages V5 and V12 werevariant phages with different host range pro- evident not only in host range but also in virulence. Super-

). The phages studied in detail, V, V5, and V12, infection with V5 showed ready lysis of nonlysogenic indi-cator strain L4-4 but did not affect lysogenic strain L4-4 (V5)or L4-4 (V12). Phage V12, in contrast, appeared more

1 2 3 4 5 6 7 8 9 virulent, lysing not only L4-4 and ILA-4 (V5) but also L4-4(V12), the lysogen from which V12 was liberated spontane-ously. Why the prophage-coded repressor failed to provideimmunity to V12 is not clear, but a reasonable analogy maybe found in the relationship between Escherichia coli K-12(+) and the virulent mutants which this lysogenic strainspontaneously released (8). One mutant, lambda clear (c),did not lyse E. coli K-12 (+), but the other mutant, lambdavirulent (vir), induced lysogenic E. coli K-12 (+). All threelambda phages formed plaques on nonlysogenic E. coli K-12.The absence of immunity against V12 might be attributed tothe fact that virulent mutants may induce the development ofiVprobe related prophages. In contrast, phage V5 may have beensted genomic DNAs from phage-resistant, lysoge- ..,-e .gaponic seroclustere13stans. Lanes: 1, weakly virulent (like lambda clear) and failed to induceJ-type B. japonicum serocluster 123Z strains. Lanes: , eihe I,- V)o IA4(13(V2); 3, 123(V1); 4, 123B; 5, Webster 48; 6, USDA either IA-4 (Vs) or L4-4 (V12).

, L4-4(V12); 9, L4-4(V5). The numbers on the left are Hybridization studies with the phage V probe not onlys, in kilobases. confirmed the lysogenic nature of strains L4-4(V5) and

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L4-4(V12) but also showed that phage V-induced lysogeny isnot limited to nitrous acid mutant strain IA-4. The phageV-resistant derivative of strain 123B, 123(V2), proved also tobe a lysogen. Other serogroup 123 strains, however, includ-ing two other phage V-resistant derivatives of USDA 123,were not lysogenized. Moreover, the lack of hybridizationhomology between the phage V probe and genomic DNAfrom Bradyrhizobium strains in other serological groupssuggests that phage V has the capacity to lysogenize only alimited number of strains. The fact that phage V doesintegrate into the genomes of some strains offers hope for thedevelopment of a B. japonicum-specific transducing systemwhich would greatly facilitate genetic studies. Integration ofphage V into the genome of a phage-resistant isolate in eachcase was accompanied by the inability of that isolate tonodulate soybean plants. The reason for the Nod- pheno-type is not known; however, it is apparently not due tointegration of phage V into several of the known Bradyrhizo-bium nodulation genes.

ACKNOWLEDGMENTS

We gratefully acknowledge the help of H. C. Tsien and D. L.Anderson with electron microscopy preparations and examinations.

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