APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1987, p. 410-415 Vol. 53, No. 20099-2240/87/020410-06$02.00/0Copyright ©3 1987, American Society for Microbiology

Transposon Mutagenesis of Azospirillum brasilense and Azospirillumlipoferum: Physical Analysis of Tn5 and Tn5-Mob Insertion Mutants

MARK VANSTOCKEM, KRIS MICHIELS, JOS VANDERLEYDEN,* AND AUGUST P. VAN GOOLF.A. Janssens Memorial Laboratory of Genetics, University of Leuven, B-3030 Heverlee, Belgium

Received 14 July 1986/Accepted 10 November 1986

Tn5-induced insertion mutants were generated in Azospirillum brasilense Sp7 and A. lipoferum SpBrl7 bymating with Escherichia coli strains carrying suicide plasmid vectors. The sources of TnS were the suicideplasmids pGS9 and pSUP2021. Kanamycin-resistant Azospirillum colonies appeared from crosses with E. coliat maximum frequencies of 10-7 per recipient cell. Transposon Tn5 also conferred streptomycin resistance onAzospirillum colonies as was observed earlier for Rhizobium sp. Eight TnS-induced Kmr Smr A. brasilense Sp7mutants with reduced nitrogen-fixing capacity were isolated. The potential use of TnS-Mob for labeling andmobilization of Azospirillum-indigenous plasmids was demonstrated by isolating Tn5-Mob insertions in themegaplasmids of A. brasilense Sp7.

Bacteria of the genus Azospirillum are free-living di-azotrophs that can readily be isolated from the rhizosphereand roots of forage and grain grasses (5). It has beenpostulated that biological nitrogen fixation by Azospirillumsp. in association with roots may contribute significantamounts of nitrogen to the plant, explaining beneficial effectson crop yield (3, 19). However, results from field experi-ments and physiological studies carried out since 1975 haveled to the conclusion that Azospirillum spp. promote cropyield only under certain environmental and soil conditionsand that the net beneficial effect, when observed on the plantupon Azospirillum inoculation, is the result of many physi-ological properties, either positive or negative in associationwith plants, of the bacterial partner (for a review, seereferences 20 and 21). Similar conclusions were formulatedat the 3rd Workshop on Azospirillum, Bayreuth, FederalRepublic of Germany, June 1985 (13).To study the various components of this associative

bacterium-plant system, it is necessary to develop the mo-lecular genetics of Azospirillum spp. So far, little is knownabout the genetics of these bacteria or the molecular biologyof their association with plants (for a review, see references6 and 7). The most recent development in Azospirillumgenetics is the ability to produce mutants impaired in nitro-genase activity by site-directed transposon mutagenesis,allowing study of the nifgenes in Azospirillum spp. (22, 32).This methodology was applied, taking advantage of the factthat homology with Klebsiella pneumoniae nifHDK geneswas detected in Azospirillum sp. (24). Recently, DNA ho-mology with Rhizobium meliloti nod and hsn genes andAgrobacterium chromosomal virulence (chv) genes was de-tected in various Azospirillum strains (9, 18), allowing us toinitiate a study on the molecular biology of Azospirillum-plant associations.A major drawback in the genetic analysis of Azospirillum

sp. is the lack of a suitable system for at-random transposonmutagenesis like that successfully applied in the geneticanalysis of other gram-negative soil bacteria like Rhizobiumspp. and Pseudomonas spp. (1, 30). Initial attempts with thesuicide plasmid pJB4JI (1) were reported (8). This plasmid

* Corresponding author.

appears to be stable in Azospirillum spp. (8, 31). Introduc-tion of another plasmid of the same incompatibility group aspJB4JI in Azospirillum transconjugants containing pJB4JIallowed the selection of some Kmr auxotrophs (8).

In the present study, pGS9, a suicide plasmid composed ofa pl5A-type replicon and an N-type bacterial mating system(26), and pSUP2021 and pSUPS011, suicide plasmids com-posed of a ColEl replicon and a Mob region of IncP-typeplasmids (29, 30), were used to transfer Tn5 or TnS-Mob intoAzospirillum strains. All of the vectors used transfer Tn5 orTnS-Mob at frequencies sufficient to allow isolation of inser-tion mutants.(A preliminary account of part of this work was presented

at the 3rd Workshop on Azospirillum, Bayreuth, FederalRepublic of Germany, 20-21 June 1985, by Vanstockem etal. [35].)

MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and

plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli strains were

maintained on LB agar and grown in LB broth supplementedwith the appropriate antibiotics. Concentrations of antibiot-ics used for E. coli were 100 ,ug of ampicillin or carbenicillinper ml, 25 Fg of kanamycin per ml, and 25 ,ug of chloram-phenicol per ml. Azospirillum strains were maintained onyeast extract-peptone (YEP) agar and grown in YEP broth orminimal medium (MMAB) at 30°C. The composition ofMMAB was as follows: K2HPO4 (3 g/liter), NaH2PO4 (1g/liter), NH4Cl (1 g/liter), MgSO4 7H20 (0.3 g/liter), KCl(0.15 g/liter), CaCl2 2H20 (0.01 g/liter), FeSO4 7H20(0.0025 g/liter), sodium malate (5 g/liter), biotin (0.005g/liter), and microelements. Tn5-induced mutants were se-lected on MMAB agar supplemented with 25 ig of kanamy-cin per ml.

Bacterial matings. Azospirillum strains were grown over-night at 30°C in YEP broth to a density of 108 CFU/ml. E.coli donor cells were grown at 37°C overnight, diluted10-fold, and grown for another 4 h. Samples of donor andacceptor cells (1:5 ratio) were centrifuged and suspended in10 mM MgSO4 solution. In some experiments, acceptorcells, before being mixed with donor cells, were heat

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TRANSPOSON MUTAGENESIS IN AZOSPIRILLUM SPP. 411

TABLE 1. Bacterial strains and plasmids

Strain or Relevant properties Source orplasmid reference

E. coli strainsWA803 met thi 26S17-1 pro thi hsdR hsdM+ recA Tpr Smr (RP4 2-Tc::Mu-Km::Tn7) integrated 30SM10 thi thr leu suIII Kmr (RP4 2-Tc::Mu) integrated 30HB101 F- hsdS20 (rB- mB) recA13 ara-14 proA2 lacYl galK2 rpsL20 (Smr) 4

xyl-5 mtl-l supE44 X-LE392 F- hsdR514 (rk- Mk-) supE44 supF58 lacYl galK2 gal722 metBI M.-D. Chilton

trpR55 X

Azospirillum strainsA. brasilense Sp7 Apr ATCC 29145A. lipoferum SpBrl7 Apr ATCC 29709A. brasilense 7317 Apr nifD::TnS Kmr C. Elmerich

PlasmidspSUP2021 Apr Cmr Tcr Kmr (Tn5) ColEl replicon tra mob' Tn5 donor 30pSUP5011 pBR325::Tn5-Mob Apr Cmr Kmr (Tn5) tra mob' TnS-Mob donor 29pGS9 Cmr Kmr (TnS) p1SA replicon N-tra Tn5 donor 26pUC19 Apr ColEl replicon 36

shocked for 5 min at 51°C to eliminate possible interferenceof the restriction system. Nevertheless, no significant differ-ences in transfer frequencies were observed between heatshocked and nontreated cells. Mating mixtures were spreadeither on nitrocellulose filters or directly on YEP (plus 10mM MgSO4) plates and incubated for various times at 30°Cin a humid atmosphere. Filters containing only donor or

recipient cells were included for proper controls. Conjuga-tion and control patches were resuspended by vortexing,washed several times when appropriate, diluted, and spreadon selective plates.

Isolation of Azospirillum Nif mutants. Kmr exconjugantswere selected on nitrogen-free MMAB solidified withagarose and supplemented with 25 ,ug of kanamycin per ml.Plates were incubated at 30°C for 4 days in an atmospherecontaining about 1% 02 in the gas phase. Small and translu-cent colonies were replica plated on nutrient agar supple-mented with 25 jig of kanamycin per ml, MMAB agar

supplemented with 25 jig of kanamycin per ml, and MMAB(1 mM NH4+) supplemented with 25 jig of kanamycin per ml.Colonies showing the same growth pattern as wild-typeAzospirillum spp. on the three media were selected fornitrogenase activity estimations in semisolid medium.

Assay for nitrogen fixation. Nitrogenase activity in wholecells was determined under conditions of nitrogenasederepression as described by Gauthier and Elmerich (11).DNA isolation. Single colonies of E. coli and Azospirillum

spp. were screened for the presence of plasmid DNA by therapid detection method of Kado and Liu (12). Azospirillumtotal DNA was prepared by a modification of the method ofMarmur (15) by using 2-ml cultures. Plasmid DNA from E.coli was prepared by using the cleared-lysate procedure (2).When used as a hybridization probe, plasmid DNA was

further purified by centrifugation through cesium chloride-ethidium bromide density gradients.DNA hybridizations. DNA hybridizations were conducted

overnight on nitrocellulose (0.45-jim pore size; Schleicher &Schuell B6) filters or Gene Screen (Dupont Co.) nylonmembranes. Colony filter hybridizations and DNA dot blothybridizations were done as described by Maniatis et al.(14). Southern-blotted DNA hybridizations were done as

described by Silhavy et al. (28). [a-32P]dCTP-labeled probes(specific activity, >5 x 107 cpm/,jg of DNA) were obtained

by using the nick translation kit of Amersham. Filters wereautoradiographed at -80°C by using Fuji RX films andintensifying screens (Kyokko special).Recombinant DNA cloning. Genomic DNA samples (2 ,ug)

of TnS-induced Kmr A. brasilense Sp7 transconjugants weredigested to completion with EcoRI and ligated with 0.5 to 1,ug of EcoRI-cleaved and alkaline phosphatase (1 U/,ug)-treated pUC19 DNA. Ligation reactions were carried out for16 h at 15°C. Transformation was as described by Maniatis etal. (14), with E. coli HB101 competent cells. Transformationmixtures were spread on LB agar containing 25 jig ofkanamycin per ml.

RESULTSIsolation of TnS and TnS-Mob mutants of A. brasilense Sp7

and A. lipoferum SpBrl7. The most-used Azospirillum strainsare A. brasilense Sp7 (ATCC 29145) and A. lipoferumSpBrl7 (ATCC 29709). They can be grown on minimalmedium (34) and are naturally resistant to carbenicillin (10).In matings between E. coli WA803(pGS9), E. coli S17-1(pSUP2021), or E. coli S17-1(pSUP5011) and A. brasilenseSp7 or A. lipoferum SpBr17 (Kmr), Azospirillum exconju-gants were isolated on MMAB containing 25 jg of kanamy-cin per ml at maximum frequencies ranging from 10-8 to 1O-7per recipient cell (Table 2). It was our experience thattransfer frequencies were influenced to a large extent by themating conditions and physiological state of donor andacceptor cells. Typical mating conditions are outlined inMaterials and Methods. The Azospirillum strains used as

TABLE 2. Tn5 and Tn5-Mob mutagenesis of Azospirillum strains

Mating no. and Acceptor strain No. of Kmrdonor strain transconjugantsa

1 S17-1(pSUP2021) A. brasilense Sp7 7 x 10-82 S17-1(pSUP2021) A. brasilense Sp7 1.3 x 10-73 S17-1(pSUP2021) A. lipoferum SpBrl7 2.2 x 10-94 WA803(pGS9) A. brasilense Sp7 2.8 x 10-85 WA803(pGS9) A. lipoferum SpBrl7 1.5 x 10-96 S17-1(pSUP5011) A. brasilense Sp7 8 x 10-77 S17-1(pSUP5011) A. brasilense Sp7 3 x 10-7

" Transconjugants were selected for Kmr on MMAB. The transfer frequen-cies are given per final number of recipient cells.

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412 VANSTOCKEM ET AL.

acceptor cells are very sensitive to kanamycin (2 to 5 ,ug/ml),and spontaneous mutants to 25 p.g of kanamycin per mlcould never be detected in control experiments.

It was observed earlier that TnS carries a streptomycinresistance gene that is not expressed in E. coli K-12 butconfers streptomycin resistance to Methylobacterium or-ganophilum (16) and Rhizobium spp. (23, 27). Therefore, wewanted to test whether Tn5 Azospirillum transconjugants areresistant to streptomycin. The kanamycin and streptomycinresistance levels of strains used in the mating experimentsand some selected bona fide TnS Azospirillum transconju-gants were estimated by determination of the MICs of thetwo antibiotics (Table 3). In Azospirillum spp., the MIC forkanamycin increased at least 250-fold. The MIC for strepto-mycin in Azospirillum spp. increased at least 5- (A.brasilense) to 50 (A. lipoferum)-fold when the parent strainand a Tn5-containing derivative were compared. Previouslyit was determined that in E. coli the MIC for streptomycinremained unchanged when the parent and a TnS-containingderivative were compared (23). Subsequently it was foundthat all TnS Azospirillum transconjugants tested did growwell on solid complete medium containing 200 ,ug of kana-mycin per ml and 150 ,ug of streptomycin per ml. Finally, itcould be demonstrated that, in crosses between E. coliSM1O(pSUP2021) and A. brasilense Sp7, kanamycin andstreptomycin Azospirillum exconjugants can be isolated atfrequencies of 10' per recipient cell on complete mediumcontaining 200 pg of kanamycin per ml and 150 ±g ofstreptomycin per ml. About 0.7% of the Kmr Smr colonieswere auxotrophs.

Physical analysis of Tn5 mutants from A. brasilense Sp7 andA. lipoferum SpBrl7. The presence of TnS in Kmr Smrtransconjugants was shown by colony hybridization. A totalof 200 Kmr Smr colonies chosen randomly from matingexperiments 1 to 5 (Table 2) were transferred on nitrocellu-lose filters and hybridized with 32P-labeled ColEl: :TnSDNA. The DNAs of all of the colonies tested showed astrong positive hybridization signal. To check for the ab-sence of vector sequences, 20 Kmr Smr transconjugantsobtained from mating experiment number 1 (Table 2) weretested in a colony hybridization experiment with 32P-labeledpBR322 DNA (part of pSUP2021). The results indicate thatthere was no pSUP2021 vector survival in Azospirillum spp.Furthermore, screening of Kmr Smr transconjugants onmedium containing 50 ,ug of chloramphenicol per ml allowedus to determine that, of 200 colonies tested, none hadacquired pGS9- or pSUP2021-encoded chloramphenicol re-sistance and confirmed the absence of vector DNA.

TABLE 3. Kanamycin and streptomycin MICs for E. coli andAzospirillum spp.

Strain Location of MIC (pug/ml) of":Tn5 if present Streptomycin Kanamycin

E. coli WA803(pGS9) Plasmid 10 NDE. coli LE392(pSUP2011) Plasmid 10 ND

A. brasilense Sp7 100 2A. brasilense Sp7-1 Chromosome >500 >500A. lipoferum SpBr17 10 2A. lipoferum SpBrl7-1 Chromosome >500 >500

a MICs were determined in YEP broth with increasing concentrations ofeach antibiotic. The starting cell number was kept at 103 cells per ml. Growthof the bacteria in the test tubes was evaluated visually after 48 h. The MIC fora given strain was determined in at least three independent experiments. ND,Not done.

X0co r*_

CM cm QLo O ~~~0) ft.

FIG. 1. Physical analysts of TnS Azospirillum transconjugants.Sail-restricted total DNAs of independent clones were transferredto nitrocellulose and hybridized with 32P-labeled pBR322::TnS. C16,C17, C18, C26, C27, and C28 are randomly chosen transconjugants.Sp7 is the wild-type strain, and 7317 is a TnS insertion mutantobtained by site-directed mutagenesis.

To determine the status of TnS in KMr SMr mutants, aSouthern blot of Sail-digested total DNA from six transcon-jugants was probed with 32Plabeled pBR322: :Tn5 DNA. Tn5contains a single Sall recognition site. Sall digests ofgenomic DNA containing one Tn5 insert should thereforegive rise to two hybridizing bands corresponding to fusionfragments of TnS DNA and flanking genomic DNA. All ofthe mutants tested contained only two Sall fragments hy-bridizing with a 32P-labeled TnS probe, and all hybridizingbands were unique for each particular clone tested (Fig. 1).This suggested that TnS transposes at random in the genomeof Azospirillum spp. This was also suggested by the fact thatvarious TnS-induced auxotrophic mutants of A. brasilensecould be identified (C. Elmerich, unpublished data).

Isolation and characterization of TnS-induced Nifr mutantsof A. brasilense. A total of 200 KMr SMr A. brasilense Sp7potentialNifXmutants, isolated as outlined in Materials andMethods, were assayed for acetylene reductase activity(ARA). Three clones with less than 5% of the wild-type (Sp7)ARA and five clones with less than 20% of the wild-typeARA were isolated. The first three clones (numbers 59, 143,and 170) were selected for further analysis. Growth testswere performed on MMAB-agarose containing the followingdifferent nitrogen sources: 20 mM NH4', 1 mM NH4', 10mM N037, 5 mM glutamate, 0.1% histidine, 0.1% proline,and 0.1% arginine. The three mutants showed good growthon all media, indicating that these mutants were not impairedin their basic nitrogen metabolism.EcoRt-digested total DNAs of these mutants were hybrid-

ized with 32P-labeledColEl:p:TnSDNA. EcoRI-digested totalDNA of A. brasilense Sp'7 strain 7317 was included as acontrol. Strain 7317 is an A. brasilense Sp7 Nif mutantobtained by site-directed transposon mutagenesis withTn.inserted in a 6.7-kilobase-pair (kbp) EcoRI fragment carryingthe nifrDK genes (23). The three mutants showed a singlehybridizing band containingTn3 DNA (Fig. 2). The hybrid-izing EcoRI fragments appeared to be of similar sizes foreach mutant, whereas the hybridizing EcoRi fragment inclone 7317 was clearly different. EcoRI-digested total DNAof each mutant was ligated with EcoRs-digested pUCt9

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TRANSPOSON MUTAGENESIS IN AZOSPIRILLUM SPP. 413

._ ._ ._ CO Lf~~~~~ ,f

129.

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y- N XC) v 0 0e .0X0 0

0.CN N N N N N N N N CO00 0a.0 0 0

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FIG. 2. Southern blot analysis of EcoRI-digested total cellularDNA from three Tn5 Nif mutants of Sp7 (lanes 1 to 3). Blots werehybridized with (32P-labeled) ColE1::TnS. Lanes 4 and 5 contain7317 and Sp7 DNAs, respectively. The numbers on the left indicatekilobase pairs.

DNA and transferred to competent E. coli HB101 cells.Kanamycin-resistant clones were selected, and their plasmidDNAs were digested with EcoRI or XhoI. All recombinantplasmids contained a 9.9-kbp EcoRI insert carrying intactTnS and 4.2-kbp of flanking Azospirillum DNA. Moreover,the XhoI digest, which generates fusion fragments, demon-strated that the three clones most likely represent the samemutation (data not shown).

Physical analysis of TnS-Mob insertion mutants of A.brasilense Sp7. All Azospirillum strains so far tested containplasmid DNA. A. brasilense Sp7 contains five plasmids withmolecular masses of 46, 90, 115, and >300 (n = 2)megadaltons (MDa) (10). The plasmid DNA bands of 90 and115 MDa are the most intense on ethidium bromide-stainedagarose gels. The two plasmids with molecular masses of>300 MDa show very weak fluorescent bands on ethidiumbromide-stained agarose gels and are referred as mega-plasmids a and b in this paper. All of these plasmids arecryptic so far. Simon (29) described a very elegant systemallowing simultaneous labeling and mobilization of crypticplasmids. The potential of TnS-Mob was demonstrated bymobilizing R. meliloti plasmids. We demonstrated earlier inthis study that TnS-Mob insertion mutants of Azospirillumsp. could be obtained in matings between E. coli S17-l(pSUP5O11) and Azospirillum spp. (Table 2). TnS-Mobinsertion mutants were found to be chloramphenicol sensi-tive, indicating the absence of pSUPSO11 vector DNA, anddid not show increased streptomycin resistance, in contrastto Tn5 insertion mutants, suggesting intactness of the Tn5-Mob construct. The mob region of the plasmid RP4 inte-grated in TnS inactivates the streptomycin resistance-encoding gene of TnS (29). Eighty randomly chosen TnS-Mob insertion mutants of A. brasilense Sp7 were analyzed.Cell lysates were examined by agarose electrophoresis (12),and the DNA was transferred to nitrocellulose filters. South-ern blots were hybridized with 32P-labeled ColEl: :TnSDNA. An example of 10 clones is shown in Fig. 3. In all ofthe mutants, hybridizing bands were seen either at the levelof linear DNA (chromosomal) or at the level of megaplasmida or b (molecular mass, >300 MDa). Mutants showing a

FIG. 3. Detection of TnS-Mob insertions in TnS-Mob A.brasilense Sp7 transconjugants. (A) Agarose gel electrophoresis ofcell lysates of putative Tn5-Mob insertion mutants. (B) Southernblot hybridizations with 32P-labeled ColE1::TnS. Sp7 is included asa negative control. pl a/b indicates the position of the twomegaplasmids with molecular masses of >300 MDa, and chr indi-cates the position of the linear (chromosomal) DNA. Mutants aredesignated with a number; C indicates mutants with Tn5-Mobinserted in the chromosomal DNA, and P indicates mutants withTnS-Mob inserted in megaplasmid DNA.

hybridizing band on megaplasmid DNA also contained ahybridizing band on linear DNA, most likely due to break-down of plasmid DNA during cell lysis. A surprising phe-nomenon was observed in some of the TnS-Mob insertionmutants analyzed. Clones number C27 and C29 lost the115-MDa indigenous plasmid of A. brasilense Sp7 (Fig. 3A).This phenomenon was only observed in some transconju-gants containing TnS-Mob in the chromosome of Azospiril-lum spp. Nine clones with TnS-Mob inserted in megaplasmidDNA were chosen randomly and subjected to further phys-ical analysis. Sall-digested total DNAs of these clones werehybridized with a 32P-labeled TnS probe. TnS-Mob containsa single Sall recognition site. The results are shown in Fig. 4.

rIf. I= 4cXt IftCN NI

TM (0 G(0

ILr.

0 a

a 4*

FIG. 4. Physical analysis of TnS-Mob Azospirillum transconju-gants. SalI-restricted total DNAs of independent clones were trans-ferred to nitrocellulose and hybridized with 32P-labeledpBR322::Tn5 DNA. Sp7 is the wild-type strain. C47 is a transcon-jugant with TnS-Mob inserted in the chromosome. All other lanescontain DNAs of individual transconjugants with Tn5-Mob insertedin the megaplasmid DNA.

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414 VANSTOCKEM ET AL.

Interestingly, of nine clones, only four different hybridizingpatterns were obtained, suggesting that Tn5-Mob (and prob-ably also TnS) insertions in Azospirillum megaplasmid DNAdo not occur at random.Some of the mutants with TnS-Mob inserted either in the

chromosome or in megaplasmid DNA (a or b) were used todetermine the copy number of the megaplasmids. We com-pared the intensities of the signals obtained by hybridizationof DNAs of Tn5-Mob chromosomal and megaplasmid inser-tion mutants with 32P-labeled TnS probe in a dot blothybridization experiment (Fig. 5). The two classes of Tn5-Mob insertion mutants displayed the same hybridizationintensity, indicating that the megaplasmids are present atone copy per Azospirillum chromosome.

DISCUSSION

The objective of this research was to determine whetherat-random transposon mutagenesis could be applied in ge-netic studies of Azospirillum spp. In heterospecific matingswith E. coli cells carrying suicide plasmid vector pGS9 orpSUP2021, Tn5 insertion mutations of A. brasilense and A.lipoferum could be routinely obtained at frequencies of i0'per recipient cell. The yield of Kmr transconjugants waslower than that in matings between the same donors and R.meliloti or Agrobacterium tumefaciens (25, 27). We believethat the rather low frequency of Tn5 transfer in matings withAzospirillum sp. is due to the specific genetic makeup of thisgenus. The high G+C content (70 to 75% G+C) of the genusAzospirillum could possibly interfere with the transpositionmechanism (17). Nevertheless, Tn5 appears to be a goodchoice for transposon mutagenesis in Azospirillum sp. sincethe frequency of spontaneous Kmr mutations is extremelylow and Tn5-encoded streptomycin resistance is expressedin Azospirillum spp. These observations allow good selec-tion for Tn5-induced Kmr Smr Azospirillum colonies ob-tained in heterospecific matings with E. coli donor cells.

Physical analysis of Tn5-induced Kmr Azospirillum colo-nies revealed that Tn5 was inserted randomly and that in allof the mutants studied only one copy of Tn5 per Azospirillumgenome was present, suggesting that secondary transposi-tion does not occur frequently. In 20 randomly chosen Tn5mutants, survival of suicide plasmid vector DNA could notbe detected by hybridization, and microbiological tests al-lowed us to determine that, if vector survival occurs at all, itoccurs in less than 0.5% of the mutants studied.Transposon mutagenesis in A. brasilense worked equally

well with either intact Tn5 or a TnS-Mob construct. Isolationof A. brasilense Sp7 strains with indigenous plasmids taggedwith Tn5 or TnS-Mob will allow us to start genetic analysis ofAzospirillum plasmid DNA either by plasmid curing or byplasmid mobilization. The observation that Azospirillumspp. contain large plasmids allows speculation that functionsimportant for Azospirillum-plant interactions are plasmidborne, in analogy with Rhizobium spp. Interestingly, wenoticed that about 10% of the Tn5-Mob insertion mutants ofA. brasilense had lost a specific indigenous plasmid (115MDa). In these mutants, Tn5-Mob was found to be locatedon the chromosome. Several hypotheses can be postulatedto explain this phenomenon. It could be postulated that thephenomenon does not reflect plasmid loss but rather integra-tion of the plasmid into the chromosome after TnS-Mobintegration in the chromosome, the particular plasmid, orboth. Secondly, it could be postulated that Tn5-Mob in thesemutants is integrated in a chromosomal region involved inreplication of this particular plasmid DNA. However, this

-r~.. (0 r~ 00 0

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400 *

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FIG. 5. Quantitative dot blot hybridization analysis of Tn5-Mobinsertion mutants. Dots with 200 and 800 ng of total DNA of sixchromosomal (C) and four megaplasmid (P) Tn5-Mob insertionmutants were hybridized with 32P-labeled ColE1::Tn5 DNA. Dotswith 200, 400, 800, 1,200, and 1,600 ng of strain Sp7 and 7317 DNAswere included as controls.

hypothesis implies that the Azospirillum chromosome con-tains hot spots for Tn5-Mob integration, which is in conflictwith earlier observations on TnS integration (see Results). Inthis context it is worthwhile to mention that loss of the same115-MDa plasmid was occasionally observed in Smr clonesof A. brasilense Sp7 obtained by chemical mutagenesis (C.Elmerich, personal communication) and in a spontaneousRifr clone of A. brasilense Sp7 (this laboratory). Clearly, thisphenomenon should be investigated in greater detail.

Finally, Tn5-induced insertion mutants of A. brasilensewere tested for an altered phenotype by screening for Nif-mutants. These mutants are very valuable in the geneticanalysis of the nif genes in Azospirillum spp. since DNAhomology with cloned nif genes from other nitrogen-fixingbacteria is limited to the nifHDK genes (22). Among eightselected mutants with reduced ARA, three clones werestudied in greater detail. They most likely represent the samemutation, based on the results of biochemical and moleculargenetic analyses. This class of mutants (numbers 59, 143,and 170) is clearly different from nifHDK-type mutantsbecause of their residual ARA. Moreover, in these mutantsTn5 is inserted in a 4.2-kbp EcoRI fragment, whereas thenifHDK genes of A. brasilense Sp7 are carried by a 6.7-kbpEcoRI fragment (22). Growth on minimal medium with 1 or20 mM NH4' predicts that these mutants are not impaired inglutamate or glutamine synthesis. Even so, growth on syn-thetic media with other defined inorganic or organic nitrogensources indicates that this class of mutants is affected in agene (or set of genes) involved in nitrogen fixation ratherthan in a regulatory gene involved in basic nitrogen metab-olism.

ACKNOWLEDGMENTS

This research was supported by grants from the Fonds voorKollektief Fundamenteel Onderzoek (F.K.F.O. 2.0013.85) and theCEC (contract no. TSD-A-255-B [R.S.]). Part of this work wascarried out at the Institut Pasteur, Paris, France, in collaborationwith C. Elmerich, with financial support of the NFWO (K.M.) andthe Onderzoeksfonds K.U. Leuven (J.V.). K.M. is a recipient of afellowship from the NFWO.We thank A. Puhler for providing strains, K. Vlassak for use of

his gas chromatography facilities, and G. Volckaert and S.Horemans for helpful discussions. We are also grateful to C.Elmerich for helpful suggestions and discussions during the courseof this work.

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TRANSPOSON MUTAGENESIS IN AZOSPIRILLUM SPP. 415

ADDENDUM

During the preparation of this manuscript, a publication bySingh and Klingmuller (33) appeared, describing the use ofpGS9 as a suicide plasmid vector for transposon mutagensisin Azospirillum sp. brasilense.

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