JOURNAL OF BACTERIOLOGY, Jan. 1982, p. 114-1220021-9193/82/010114-09$02.00/0

Vol. 149, No. 1

Physical and Genetic Characterization of Symbiotic andAuxotrophic Mutants of Rhizobium meliloti Induced by

Transposon TnS MutagenesisHARRY M. MEADE, SHARON R. LONG, GARY B. RUVKUN, SUSAN E. BROWN, AND FREDERICK

M. AUSUBEL*Cellular and Developmental Group, Department ofBiology, Biological Laboratories, Harvard University,

Cambridge, Massachusetts 02138

Received 26 May 1981/Accepted 26 August 1981

We have physically and genetically characterized 20 symbiotic and 20 auxotro-phic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa(Medicago sativa), isolated by transposon TnS mutagenesis. A "suicide plasmid"mutagenesis procedure was used to generate TnS-induced mutants, and bothauxotrophic and symbiotic mutants were found at a frequency of 0.3% amongstrains containing random TnS insertions. Two classes of symbiotic mutants wereisolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules whichfailed to fix nitrogen (Fix-). We used a combination of physical and geneticcriteria to determine that in most cases the auxotrophic and symbiotic phenotypescould be correlated with the insertion of a single TnS element. Once the TnSelement was inserted into the R. meliloti fenome, the frequency of its transposi-tion to a new site was approximately 10- and the frequency of precise excisionwas less than 109. In approximately 25% of the mutant strains, phage Mu DNAsequences, which originated from the suicide plasmid used to generate the TnStranspositions, were also found in the R. meliloti genome contiguous with TnS.These latter strains exhibited anomalous conjugation properties, and therefore wecould not correlate the symbiotic phenotype with a TnS insertion. In general, wefound that both physical and genetic tests were required to fully characterizetransposon-induced mutations.

Van Vliet et al. (21) and Beringer et al. (2)have recently described a general method forintroducing transposons into the genomes of awide variety of gram-negative bacteria. A broadhost-range plasmid carrying both phage Mu anda transposon is conjugated from Escherichia coliinto the recipient gram-negative bacterium.Many nonenteric recipients are not killed byzygotic induction of prophage Mu. Instead, theMu-containing plasmid fails to replicate stably,thus permitting direct selection for the transposi-tion of the transposon from the Mu-containing"'suicide plasmid" to a recipient genome. Usingthis procedure, Beringer et al. (2) obtained andgenetically characterized a variety of auxotro-phic and symbiotic mutants of Rhizobium legu-minosarum, R. trifolii, and R. phaseoli causedby the insertion of the kanamycin/neomycinresistance-conferring transposon TnS. This tech-nique has also recently been successfully usedby Duncan (6) to isolate carbohydrate metabo-lism mutants ofR. meliloti L5-30 and by Rolfe etal. (16, 17) to obtain symbiotic mutants of R.trifolii strains SU329 and SU843.We have adopted the TnS suicide plasmid

technique of Beringer et al. (2) to isolate neomy-cin-resistant mutants of R. meliloti which aredefective in nodulation or nitrogen fixation orboth, or which are auxotrophic. In this paper wedescribe the physical analysis of these mutantsby using the Southern (20) gel transfer andhybridization technique. In combination withclassical genetic analysis, the physical analysisdemonstrates that some, but not all, of thesymbiotic and auxotrophic mutant phenotypescan be correlated with a newly acquired transpo-son insertion. In approximately 25% of thecases, phage Mu sequences were found to trans-pose in concert with TnS, and in other cases, themutant phenotypes could not be correlated witha transposon insertion event.

MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains

and plasmids used in (but not constructed during thecourse of) this study are listed in Table 1. Some of theauxotrophic and symbiotically defective mutants of R.meliloti isolated during the course of this study arelisted in Table 2. R. meliloti 1021, the symbioticeffective parent strain in which TnS transpositions

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TABLE 1. Bacterial strains and plasmids

Strain Genotype Relevant phenotype reference

E. coli1830 nal pro met (pJB4JI) Strr Ger, pJB4JI donor (2)W3115 thi (pGM102) pGM102 donor This paperEC102 thipro leu arg hspR hspM r,f Rifr This paper

R. meliloti:2011 Wild type Prototrophic, symbiotically effective J. Denarie1021 str Streptomycin-resistant derivative of 2011 This paper3359 trp his pyr rif str spc nov Same as genotype (12)3390 trp his pan rif str spc nov Same as genotype (12)1408 trp cys str spc rif Same as genotype This paper

PlasmidspJB4JI pPHI::Mu::Tn5 TnO donor (2)pHM5 pJC307::TnS Source of TnS DNA H. MeadepGM102 tet bla RP4 derivative which does not confer Nmr J. DenariepHM4021 pGM102::TnS Carries TnS This paperpHM4022 pGM102tet::Tn5 Does not confer Tcr This paper

were isolated, is a spontaneous streptomycin-resistant notype by inoculation onto alfalfa seedlings. Plantsmutant of R. meliloti 2011. were prepared by sterilizing seeds of alfalfa (varietyMedia. LB medium is 1% tryptone (Difco Labora- "Iroquois") in ethanol and sodium hypochlorite, soak-

tories, Detroit, Mich.), 0.5% yeast extract, and 0.5% ing in water overnight, and planting on nitrogen-freeNaCl (pH 7.2). The minimal basal salts medium (M9) agar (22) slants in 18-by-iS0-mm tubes. Individualused for the screening of R. meliloti auxotrophs has TnS-containing strains were suspended in sterile waterbeen described (13). LB and M9 media were supple- and added to duplicate tubes containing 1-week-oldmented with neomycin (Nm) (50 Lg/ml), streptomycin germinated seedlings; approximately 1 ml of suspen-(Sm) (500 pg/ml), novobiocin (Nov) (50 ,g/ml), spec- sion, containing at least 106 bacteria, was added totinomycin (Spc) (50 ,g/ml), rifamycin (Rif) (100 pg/ each tube. Inoculated plants were grown for 4 to 6ml), gentamicin (Gm) (50 ,ug/ml), or tetracycline (Tc) weeks in a growth chamber (25°C, 16-h light at 200 ft-c(10 ,ug/ml), or any combination of these, when appro- [ca. 2,150 lx]). They were examined for the presencepriate. LB and M9 media were solidified with 1.5% or absence of nodules and for symptoms of nitrogenagar (Difco). starvation, which indicate ineffective symbiosis. Ni-

G*netic techniques. Bacterial conjugations were per- trogenase activity was measured in the nodules byformed as described (12), except that pGM102 was capping each tube with a serum stopper, injecting 1 mlused to mobilize the donor R. meliloti chromosome of acetylene, and withdrawing a 0.5-ml sample frominstead of RP4. Derivatives of R. meliloti 1021 which the tube after 12 h to measure reduction of acetylene tocontained TnS transpositions and which failed to grow ethylene by gas chromatographic analysis (8).on unsupplemented M9 medium (see text) were Isolation of DNA. Total DNA was isolated from R.screened by the method of Holliday (9) on M9 medium meliloti strains according to the following procedure.to identify specific auxotrophic requirements. The A 5-ml saturated culture grown in LB at 30°C wasreversion frequencies of presumptive TnS-induced suspended in 25 ml of 1.0 M NaCl, shaken on a wrist-auxotrophs were determined by growing cells in LB to action shaker for 1 h at 4°C, suspended in 25 ml of coldsaturation (approximately 5 x 109 cells per ml), wash- TES buffer (Tris, 0.01 M, pH 8.0; 0.025 M EDTA; 0.15ing, concentrating 10-fold, and then plating on minimal M NaCI), centrifuged, and suspended in 5 ml of coldmedium (M9). TE buffer (TES without NaCl). A 0.5-mi sample of

Nodulatlon and nitrogenase assays. R. meliloti strains lysozyme solution (2 mg of Isozyme per ml in TE) waswith TS insertions were screened for symbiotic phe- added to the resuspended cells. After 15 min of

TABLE 2. Mutants obtained by TnO mutagenesisStraina Phenotype Genotype Reversion frequency1023 Met- Nmr met-1023::TnS str 2.8 x 1081033 Gly- Nmr gly-1033 (TlS)b str lo-101102 Arg- Nmr arg-1102::TnS str 10-101126 Nod- Nmr nod-1126::Mu TOS str NTc1029 Nod' Fix- Nmr fix-1029::TnS str NTc

a All strains are derived from R. meliloti 1021.b TnS and Gly- not linked; see Table 3.C NT, Not tested. Revertants cannot be selected directly.

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incubation at 37°C, 0.6 ml of a Sarkosyl-pronase (10oSarkosyl and 5-mg/ml pronase in TE) solution wasadded, and the mixture was incubated for an additional1 h at 37°C. The lysed cells were extracted at leasttwice with 5 ml of phenol (saturated with 0.01 M Tris,pH 8.0) and then with 5 ml of chloroform. The aqueousphase was brought to 0.3 M ammonium acetate, andthe DNA was precipitated with 0.54 volumes of iso-propanol at room temperature. The precipitated DNAwas removed with a glass rod and then dissolved in 2ml of Tris (0.01 M, pH 8.0)-EDTA (0.001 M).Phage Mu DNA was a gift from G. Riedel. Super-

coiled plasmid DNA was prepared by the clearedlysate technique followed by ethidium bromide-cesiumchloride equilibrium centrifugation (5).

Restriction endonudeases. Restriction endonucleaseEcoRI was purified according to the procedure of P.Myers, Cold Spring Harbor Laboratories (personalcommunication). XhoI was purchased from BethesdaResearch Laboratories, Rockville, Md., and used ac-cording to the manufacturer's specifications.

Southern hybridizations. Restriction endonuclease-digested DNAs were subjected to agarose gel electro-phoresis as described (14) and transferred to nitrocel-lulose as described by Southern (20) withmodifications by Botchan et al. (3). Purified DNAswere labeled with 32P by the nick translation method(11, 15) as described (14). To remove hybridized DNA,nitrocellulose filters were washed at room temperaturefor 15 min in 0.02 M NaOH and then neutralized with0.05 M Tris-hydrochloride, pH 7.0.

RESULTSTransposon TnS confers neomycin resistance to

R. melloti. Transposon TnS (1) confers neomy-cin and kanamycin resistance and was chosenfor these studies because the R. meliloti strainswe study are naturally sensitive to relatively lowlevels of neomycin (20 ,ug/ml) and because TnSexhibits little site specificity and in generalcauses polar mutations (19). To utilize TnS togenerate insertion mutations in R. meliloti, itwas first necessary to establish that TnS confersneomycin/kanamycin resistance to R. meliloti.

E. coli W3115 carrying plasmid pHM4021(Tcr; carrying TnS) was conjugated with R.meliloti 1021 (Sm'). Tcr Smr R. meliloti exconju-gants were selected and then tested for resist-ance to neomycin. All Tcr exconjugants testedwere resistant to 100 jig of neomycin per ml. Incomparison, growth of strain 1021 is inhibited by20 ,ug of neomycin per ml, at which level sponta-neous resistant mutants arise at a frequency of10-7. pHM4021 replicates stably in R. meliloti1021 and can be conjugated from this strain toappropriate E. coli recipients at a frequency of10-4 per 1021 donor.Suicide plasmid mutagenesis of R. meliloti. To

obtain TnS transpositions to R. meliloti repli-cons (chromosome and indigenous plasmid[s])we adopted the suicide plasmid techniques de-scribed by Van Vliet et al. (21) and Beringer etal. (2) to mutagenize Agrobacterium tumefa-

ciens and R. leguminosarum, respectively. Inparticular, we used the P-type plasmid pJB4JI,constructed by Beringer et al. (2), which confersgentamicin resistance and which carries TnSinserted into prophage Mu. Because plasmidpJB4JI does not replicate when transferred to R.meliloti, neomycin-resistant exconjugantsshould contain TnS transpositions to the Rhizo-bium genome.

E. coli 1830 (containing pJB4JI) was conjugat-ed with R. meliloti 1021 (see Materials andMethods for details), and Nmr Smr exconjugantswere obtained at frequencies ranging from 10-6to 2 x 10-5 in different experiments. On theaverage, 99% of the Nmr exconjugants weresensitive to gentamicin, and these were candi-dates for strains containing Tn5 transpositions toR. meliloti replicons.

Physical analysis of presumptive TnS transposi-tion strains for phage Mu DNA sequences. Utiliz-ing the Southern gel transfer and hybridizationprocedure (20), we constructed a partial restric-tion map of pJB4JI surrounding the site of TnSinsertion and corroborated genetic evidence pre-sented by Beringer et al. (2) that TnS is insertedinto prophage Mu (data not shown). If, aftertransfer of pJB4JI to R. meliloti, Nmr Gmsexconjugants were due to the transposition ofTnS to R. meliloti replicons and the subsequentloss of pJB4JI, then Nmr exconjugants shouldnot contain Mu DNA sequences. To test thisprediction, we isolated total DNA from 24 NmrGms exconjugants selected at random, digestedthe DNAs with the restriction endonucleaseXhoI, and hybridized the restricted DNAs with32P-labeled Mu DNA by the Southern blottingand hybridization procedure. Eleven of the 24DNAs contained significant amounts of MuDNA sequences as illustrated in Fig. 1A, whichshows strong hybridization to six of nine testedDNAs. (The weak hybridization seen in lane 6may not be due to Mu sequences because thisstrain did not contain TnS; see discussion of Fig.1B below.) Examination of this figure revealsthat among the strains containing Mu sequences,different-sized DNA fragments containing Musequences were present. When the 32P-labeledMu DNA was melted off the filter (see Materialsand Methods for details) and the filter wasrehybridized with 32P-labeled TnS DNA, it wasfound in the case of each strain containing MuDNA that Mu DNA and TnS DNA hybridized toat least one DNA fragment in common (Fig. 1B).In the five other cases (not shown) in which MuDNA was found in the Nmr Gm' exconjugants,both Mu DNA and TnS also hybridized to atleast one DNA fragment in common. Theseresults indicate that in those strains containingMu sequences, TnS and Mu sequences are stillcontiguous.

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A B1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

FIG. 1. Cotransposition of phage Mu and Tn5 se-quences from the suicide plasmid to the R. melilotigenome. Total DNA was isolated from R. melilotistrains chosen at random from neomycin-resistant,gentamicin-sensitive exconjugants of a suicide plasmidmating between E. coli 1830(pJB4JI) and R. meliloti1021. Purified DNA was digested with XhoI, and theresulting fragments were subjected to agarose gelelectrophoresis and transfer to nitrocellulose sheets asdescribed in the text. The filter was successivelyhybridized with 32P-labeled Mu and then 32P-labeledColE1::TnS DNA. Each lane contains DNA from adifferent presumptive TnS-containing strain. (A) Auto-radiogram of the filter hybridized with 32P-labeled MuDNA. (B) Autoradiogram of the same nitrocellulosesheet, washed with NaOH (see Methods) and rehy-bridized with 32P-labeled ColE1::TnS DNA. Beforerehybridization, an autoradiogram of the sheet wasmade to verify that the Mu32P-DNA had been removed(data not shown). One strain (lane 6) does not appearto contain Tn5 (see Fig. 2 and 3) and is probably aspontaneous Nmr mutant. The remaining strains (ex-cept lane 1) contain two internal XhoI fragments ofTnS (labeled A and B in Fig. 2). Lane 1 appears tocontain a deleted form of Tn5. Lanes 1 through 5 and 7contain at least one band which also hybridized to MuDNA; these bands are indicated with small arrows.Lane 7 contains an "extra" boundary fragment (seeFig. 2 and text) between TnS and adjacent DNAsequences which cannot be readily explained.

The results presented in this section indicatethat in the 11 Mu-containing strains one of twoevents had occurred: (i) concerted transposi-tions of TnS with Mu sequences, or (ii) repliconfusion of a partially deleted pJB4JI plasmid withthe R. meliloti genome. If Mu and TnS aretransposing together in these strains, differentMu sequences appear to have transposed indifferent strains because we could not detect anyobvious pattern in the particular Mu sequenceswhich did transpose.The results presented in this section also

indicate that approximately half of the presump-tive TnS transposition strains contain no detect-able Mu sequences and probably contain genu-ine TnS transpositions. The strains carryingbona fide Tn5 insertions are examined in moredetail in the next section.

Physical analysis of presumptive bona fide TnStranspositions. In the experiment described inthe previous section, 12 of 24 Nmr Gms R.meliloti exconjugants from a cross between E.coli 1830(pJB4JI) and R. meliloti 1021 did notcontain Mu DNA sequences and were candi-dates for bona fide TnS transpositions. If TnStransposed from pJB4JI to new locations on theR. meliloti genome, it should have acquired newrestriction sites on both sides which are differentin independent transposition strains. Therefore,the DNA sequences surrounding TnS in 12strains with symbiotic defects were character-ized using the Southern gel transfer and hybrid-ization method. The strategy employed in thisexperiment is illustrated in Fig. 2.Because TnS (5.7 kilobases) contains no rec-

ognition site for EcoRI (10), an EcoRI digest oftotal cellular DNA from a strain carrying a singleTn5 element should contain a single EcoRI frag-ment that will hybridize with a 3 P-labeled Tn5DNA probe. Figure 3A shows an autoradiogram

The 11 Mu-containing strains were analyzedfor the presence of plasmids by using a rapidscreening method (7), and in all cases no plas-mids could be detected, in contrast to strain1021(RP4), in which RP4 was readily observed.This result indicates that these strains did notarise as the result of deletions of Mu and genta-micin resistance genes to produce derivatives ofpJB4JI capable of replication in R. meliloti.One of the 24 Nmr exconjugant DNAs tested

in this experiment did not appear to contain Tn5(Fig. iB, lane 6). This strain is probably aspontaneous Nmr mutant. Examination of lane 6in Fig. 1A reveals weak hybridization to the MuDNA probe. This latter result is difficult toexplain although it may be due to the transposi-tion of a very limited amount of Mu DNA fromthe suicide plasmid, independent ofTnS transpo-sition.

x

j TN5

R X X XI .j i

X RI Chromosome

X EcoRi fragmen

e A oi^ , B D Xho ragments

FIG. 2. Representational map of Tn5 and region ofinsertion. Sites for the restriction enzymes EcoRI (R)and XhoI (X) are shown for TnS and for a hypotheticalgenomic site where Tn5 has inserted. Cleavage of totalDNA with EcoRI will yield one fragment containingTnS for each independent insertion. Digestion withXhoI will produce for each insertion two fragmentsinternal to Tn5 (A and B) and two border fragments (Cand D) containing an end of Tn5 and part of theadjacent genome.

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A1 2 3 4 5 6 7 8

-MO- _

B 1 2 3 4

40

FIG. 3. Restriction and hybridizatioinine non-Mu-containing strains with TnSonstrting a single TnS insertion. (A) Hyl32P-labeled ColE1::TnS to EcoRI digestsmutant strains. See the text and legenddetails. (B) Hybridization of labeled Idigests of DNA from the same mutant sstrain contains a doublet of internal fragi

nated A and B in Fig. 2). A different sfragments (C and D in Fig. 2) is appastrain. In lane 7, an extra band is visible v

likely due to a partial digest.

of a Southern filter blot of eight EcoDNAs from presumptive bona fide Tsition strains hybridized withColEl: :Tn5 DNA. Because each lato contain a single hybridization ban(that each strain carries a single Tn5that the Tn5 element is inserted inEcoRI fiagments in different strainsTo corroborate and extend the

tained with EcoRI, total cellular DNsame TnS transposition strains showiwas cleaved with XhoI. XhoI cleavetimes, once near the center and onceof the terminally repeated DNA sequends of the element (10). This resifragments containing TnS sequences.fragments (labeled A and B in Fig. 2)to TnS and will be the same wher4inserted. The other two fragmentsidentical 0.5-kilobase TnS segmentsthe "left" and "right" portions of thiXhoI fragment in which the TnS iThese two boundary fragments (C ax2) will vary in size, depending on theparticular XhoI fragment into whictserted and on the location of TnSfragment, and are likely to be differeindependent TnS insertion. Comparhybridization patterns in the differeFig. 3B clearly shows that TnS isdifferent locations in the eight differUsing the hybridization procedure dthe legend of Fig. 3, we have examin480 independent presumptive Tn.strains, and in all cases we conclud

5 6 7 8 strains carried a single TnS insertion, except inthe case illustrated in Fig. 1B, lane 6, in whichno TnS element was found.

Isolation of TnS-induced auxotrophic and sym-biotic mutants. The above physical analysis indi-

m" cated that the suicide plasmid pJB4JI, despitesome attendant problems with transposition ofMu DNA sequences, also generated some genu-

__m4IU~SII ine TnS transpositions in R. meliloti. Therefore,we sought to use TnS to generate mutants withsymbiotic defects. In addition, we sought TnS-induced auxotrophic mutants of R. meliloti be-cause their more precisely defined phenotype

analysis of would allow us to answer some technical ques-bridization of tions about the Tn5 mutagenesis procedure as

ofDNA from described below.to Fig. 1 for The procedure outlined above was used tors to XhoI obtain TnS transpositions in R. meliloti 1021; astrains. Each total of 6,000 Nmr exconjugants were purified byments (desig- streaking for single colonies on LB-SmNm agarset of border and were then test-streaked for residual genta-rent in each micin resistance (on LB-Gm agar) and auxotro-which is most phy (on M9-Nm agar). To identify TnS-induced

symbiotic mutants, the Nmr Gm' prototrophicexconjugants (approximately 5,900) were

RI-digested screened on individual alfalfa plants (see Materi-FnS transpo- als and Methods).32P-labeled Of the 6,000 presumptive TnS-containingane appears strains tested, 20 were auxotrophs, a frequencyd, it is likely of0.3%. Four of the auxotrophs were Met-, fiveelement and were deficient in more general sulfur metabolismito different functions (supplemented by thiosulfate or cyste-

ine, or both, as well as methionine), two wereresults ob- Arg-, and the other nine were deficient inIA from the different functions. The distributions of auxo-n in Fig. 3A trophs may indicate nonrandomness of TnS in-s TnS three sertion or may reflect a high number of geneswithin each involving sulfur metabolism in R. meliloti; this isences at the currently being investigated further. The proper-ults in four ties of a few of the auxotrophs are listed in TableTwo of the 2.are internal We also found at least 50 symbiotic mutantsever TnS is among the 6,000 presumptive TnS insertions.contain two The symbiotic mutants were grouped into twoattached to categories, Nod- and Fix-: in the first casee particular nodules were not formed (although in someis inserted. cases some abnormal swellings or other rootid D in Fig. reactions seemed to occur); the Fix- phenotypea size of the is defined by the formation of nodules which failh TnS is in- to reduce acetylene. In both cases, nitrogenwithin this starvation symptoms in the host plant were-nt for each apparent. The number of Nod- mutants foundison of the was 4 (0.07%); the number of Fix- mutants.nt lanes in found was 46 (0.08%).inserted at The 50 symbiotic mutants were subjected to a,ent strains. series of genetic and physical tests which areiescribed in described in detail below. On the basis of theseed a total of tests, the mutations causing the lesions in the 4S insertion Nod- and 16 of the 46 Fix- mutants appear toled that the correlate with the insertion of Tn5. Although

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the remaining 30 Fix- mutants contained a sin-gle TnS insertion, we were able to demonstratethat the Fix- phenotype of these strains was notdue to the insertion ofTnS but rather to the site-specific integration of an endogenous R. melilotiinsertion sequence into a cluster of essential"'Fix" genes closely linked to the structuralgenes for nitrogenase. We were able to demon-strate the existence of this endogenous R. meli-loti insertion sequence because of the previouscloning of the R. meliloti structural genes fornitrogenase (18). Characterization of this endog-enous insertion sequence and the Fix- muta-tions caused by its site-specific transpositionwill be the subject of a separate publication. Theproperties of a representative Fix- mutant and arepresentative Nod- mutant are listed in Table3.The 20 auxotrophic, 4 Nod-, and 16 Fix-

mutants which, in our initial screen, did notcorrelate with the transposition of an endoge-nous insertion sequence were tested for thepresence of Mu DNA sequences by using theprocedures described for Fig. 1. Three of theauxotrophs, two of the Nod- mutants, and threeof the Fix- mutants contained residual Mu DNAsequences, a significant proportion, althoughnot as high as in the Nmr exconjugants as awhole. Hybridization expenrments similar tothose illustrated in Fig. 1 indicated that theresidual Mu sequences in these strains are stillcontiguous with Tn5 (data not shown).

All 40 of these auxotrophic and symbioticmutants were also tested for the number of TnSelements present in the genome of each strain byusing the hybridization procedures outlined inFig. 3. All 40 mutants contained a single TnSelement (data not shown).

Genetic Ikage of TnS to auxotrophic andsymbiotic mutations. To verify that the insertionof TnS was indeed the cause of the symbioticand auxotrophic mutations, we determined thelinkage of the TnS-conferred neomycin resist-

ance phenotype to the auxotrophic or symbioticphenotype. This was done by conjugating themutant TnS-containing strains with appropriaterecipient strains, selecting or screening for Nmrexconjugants, and then testing these for auxo-trophy or symbiotic deficiency, according to thedonor's mutant phenotype. In these experi-ments, genome mobilization was mediated bythe promiscuous P-type plasmid, pGM102. Theresults of some of these experiments are listed inTable 3. It was found that in all but one casetransfer of Nmr to the recipient strain wasaccompanied by transfer of the auxotrophic orsymbiotic phenotype. This was found both whenNmr exconjugants were selected directly andwhen they were obtained by first selecting an-other marker and then screening for Nmr. Thisestablishes that the Tn5 insert and the mutantphenotype are at least in close genetic linkageand suggests that the TnS insertion is the causeof mutation. In one case (R. meliloti 1033 Gly-),however, the Nmr exconjugants were not Gly-.Moreover, in those cases in which the donorstrains contained Mu DNA sequences, no Nmrexconjugants were recovered. At present, wehave no explanation for this finding. This pointsout the desirability of conducting thorough ge-netic tests on putative Tn5-caused mutationsbefore concluding that the mutant phenotype iscaused by the transposon insertion.

Genetic characterization of TnS insertions. Todetermine whether TnS introduced into the R.meliloti genome via the suicide plasmid pJB4JIcould function subsequently as a transposon inR. meliloti, we tested the ability of several bonafide TnS elements in R. meliloti to excise pre-cisely and to transpose to new locations. In E.coli, precise excision occurs at frequencies rang-ing from 10-5 to 10-6 (1), and transpositionoccurs at frequencies of to- (1).

We tested for precise excision by selecting forprototrophic revertants of the 20 TnS-induced R.meliloti auxotrophic mutants. The reversion fre-

TABLE 3. Genetic linkage of Tn5 with auxotrophic or symbiotic phenotypesRelevant Selected % of selected % of Nmr exconjugants

Donor' donor Recipient- Shenectyed colonies which wit dofNmexoruuphnotyphenotype ~~~phenotyped wr m b with donor phenotype'phenotype were Nmr

1023(pGM102) Met- 3390 Pan+ 74 1001023(pGM102) Met- 3359 Nml 100 991033(pGM102) Gly- 3390 Pan+ 12 01033(pGM102) Gly- 3359 Nm' 100 01102(pGM102) Arg- 3359 Trp+ 1 1001102(pGM102) Arg- 3359 Nml 100 1001029(pGMl02) Nod+ Fix- 3390 Pan+ 60 NT1029(pGM102) Nod+ Fix- 1048 Nmr 100 100

aAll strains were R. meliloti.b At least % were tested in each case.I Al Nmr exconjugants were tested. NT, Not tested.d Pan+ and Trp+ are growth without pantothenate and tryptophan, respectively.

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quencies ranged from 10-8 to less than 10-10,and in the case ofall strains except one, all oftherevertants obtained (if any) were still Nmr. Anal-ysis of several revertants by the Southern trans-fer and hybridization technique showed complexpatterns of DNA rearrangements and deletionsin different revertants (data not shown). Weconcluded from these results that when TnStransposes from pJB4JI to the R. melilotigenome, subsequent precise excision of TnSdoes not occur or is extremely rare. (A detailedstudy of prototrophic revertants of Tn5-inducedauxotrophs will be published separately.)

In contrast to the failure to obtain preciseexcision of TnS when the original TnS transposi-tion event occurred in R. meliloti, when Tn5 wasinserted into the tet gene of pGM102 whenresident in E. coli and the pGM102tet: :TnS(pHM4022) plasmid was subsequently trans-ferred to R. meliloti, precise excision, as mea-sured by the frequency of Tcr colonies, occurredat a frequency of 10 . This result may indicatethat TnS transposition events in R. meliloti arefundamentally different from those in E. coli.We also tested the transposition frequency of

TnS from a locus in the R. meliloti genome toplasmid pGM102. R. meliloti 1023(pGM102)(Met: TnS) was conjugated with E. coli EC102.The ratio of Nmr Tcr E. coli exconjugants to Tcrexconjugants was 2 x 10-8, indicating a transpo-sition frequency of TnS to pGM102 of approxi-mately 10-8 in R. meliloti. This rate of transposi-tion is four to five orders of magnitude less thanthat observed in E. coli (1). The apparent lowrate of transposition of TnS from a locus in theR. meliloti genome is consistent with our findingthat among approximately 100 R. meliloti strainscontaining TnS that we tested, each one con-tained a single TnS element.The results reported in this section indicate

that TnS mutagenesis in R. meliloti mediated bythe suicide plasmid pJB4JI results in very stablemutants which should prove useful in manygenetic manipulations.

DISCUSSIONWe have successfully used a suicide plasmid

technique to isolate a series of TnS-inducedsymbiotic and auxotrophic mutants of R. meli-loti RmlO21. This was accomplished by screen-ing individual clones of R. meliloti containingpresumptive TnS transpositions on individualalfalfa plants or on appropriate media. However,physical analysis of the genomes of these pre-sumptive mutants revealed that a significantfraction of the mutants were not the result ofsimple TnS transposition events. In one class ofmutants, representing 20% of those tested, TnSand phage Mu sequences appear to have trans-

posed in a concerted fashion from the suicideplasmid to the R. meliloti genome. Another classof mutants, representing 65% of the Fix- mu-tants tested (not discussed in this paper), are theresult of the transposition of an endogenous R.meliloti insertion sequence into symbiotic genes.We were able to distinguish these latter twoclasses of mutants from genuine TnS transposi-tion mutants by using both genetic and physicaltests, neither of which alone would have beensufficient for fully characterizing the mutants.The suicide plasmid we used (pJB4JI) to gen-

erate TnS transpositions carries TnS insertedinto prophage Mu. It is possible that the locationof TnS inside of Mu is responsible for theconcerted transposition of Mu and TnS se-quences, and that a Mu-containing suicide plas-mid in which TnS is inserted outside of Mu mayeliminate the cotransposition events. Also, itmay be that the behavior of Mu depends on therecipient strain into which the suicide plasmid isconjugated.The results presented in this paper indicate

that once TnS has transposed in R. meliloti itdoes not readily transpose to a new location, nordoes is excise precisely. These defects could bedue to the fact that a TnS-encoded transpositionrepressor is more effective in R. meliloti than inE. coli and that initial transposition events occurin a repressor-free cytoplasm analogous to zy-gotic induction. Alternatively, these defectscould be due to the lack of host-specific transpo-sition factors(s) in R. meliloti which are suppliedin trans by the suicide plasmid vector or byprophage Mu. This latter possibility is unlikelybecause a plasmid containing a ColEl replicon,the tra genes of RP4, and TnS can be conjugatedinto R. meliloti and used to generate TnS trans-positions at a comparable frequency to pJB4JI(H. Meade, unpublished data). A missing trans-position factor in R. meliloti could result in astructural defect in TnS elements which hadtransposed within R. meliloti such that subse-quent transposition and excision events wereimpaired. A different type of explanation for thetransposition defects of TnS in R. meliloti is thata host factor essential for an early step intransposition is missing in R. meliloti, resultingin a situation in which early transposition eventsoccur in E. coli, a transposition intermediatesurvives conjugation into R. meliloti, and thetransposition event is completed in R. meliloti.This latter model would allow transpositionevents to occur just once in the primary R.meliloti exconjugant cells.One major consequence ofthe cotransposition

ofMu + TnS sequences was that Mu-containingstrains could not be used as TnS donors inconjugation experiments because no Nmr ex-conjugants could be obtained. In fact, before the

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physical analysis was performed indicating thepresence of Mu sequences, these strains wereconsidered to be an anomalous class, resistant togenetic analysis. Explanations for the failure toobtain Nmr exconjugants from these strains in-clude the possibilities that Mu sequences are notreadily mobilized or that they kill the recipientcells.Another disadvantage resulting from cotrans-

position of Mu + TnS is that the mutant genecontaining the Mu-TnS transposition cannot bereadily cloned. In the case of bona fide TnStranspositions, the EcoRI fragment into whichTnS has inserted can be easily cloned in E. colibecause TnS does not contain an EcoRI site andbecause TnS-containing fragments can be select-ed for directly. Mu, however, contains twoEcoRI sites, and when TnS and Mu have trans-posed together, the EcoRI fragment containingTn5 may not contain host DNA flanking TnS.This prevents use of a method we have recentlydeveloped for the replacement of a wild-typegene in R. meliloti with a homologous clonedDNA fragment containing TnS (18), whose suc-cess depends on cloned host sequences flankingboth sides of TnS.

It appears that pJB4JI behaves differently invarious species of Rhizobium. For example,when three strains of R. trifolii were used asrecipients for pJB4JI, one yielded no TnS-con-taining exconjugants, whereas TnS-containingsymbiotic mutants were found in the other two(17). Such differences have also been observedin R. meliloti: strain 102F51 (Nitragin Co., Mil-waukee, Wis.) was found not to yield TnS mu-tants from a pJB4JI conjugation (H. Meade,unpublished data). Another example of variationin TnS behavior after a suicide plasmid mutagen-esis is in R. leguminosarum, where a chromo-somal TnS insert (ade-92::TnS) was successfullyused as a source ofTnS for new transpositions toan indigenous plasmid (4). The apparent fre-quency of transposition was between 10-4 and 3x 10-6, within one or two orders of magnitudeof the frequency found in E. coli. Thus in R.leguminosarum, TnS insertions generated bypJB4JI do not appear to have a pronounceddefect for subsequent transpositions.

Although it is apparent from our results thatthere are problems in using suicide plasmidpJB4JI to generate TnS transpositions in R.meliloti, approximately half of the presumptiveauxotrophic and symbiotic mutants isolated ap-pear to be the result of legitimate TnS transposi-tion events. Thus, pJB4JI can be used success-fully in R. meliloti given that the mutations aresubjected to the physical and genetic tests de-scribed here. Moreover, the legitimate mutantsobtained are unusually stable for transposon-induced mutations and should prove to be very

useful in the study of the R. meliloti-alfalfasymbiosis.

ACKNOWLEDGMENTSWe thank J. Beringer for providing plasmid pJB4JI, and R.

Hyde for preparing the manuscript.H.M.M. and S.R.L. were supported by postdoctoral fellow-

ships from the National Science Foundation and from theNational Institutes of Health. This work was supported byNational Science Foundation grant PCM78-06834 awarded toF.M.A.

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