Vol. 54, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1988, p. 55-610099-2240/88/010055-07$02.00/0Copyright C) 1988, American Society for Microbiology

Cloning and Mapping of a Novel Nodulation Region fromBradyrhizobium japonicum by Genetic Complementation of a

Deletion MutantMATTHIAS HAHN AND HAUKE HENNECKE*

Mikrobiologisches Institut, Eidgenossische Technische Hochschule, ETH-Zentrum, CH-8092 Zurich, Switzerland

Received 6 April 1987/Accepted 1 October 1987

The phenotypes of a set of Bradyrhizobium japonicum 110 mutants with large deletions in the region ofsymbiotic gene cluster I were tested. The majority of the mutants showed a delayed nodulation on soybean and,by mixed-infection experiments, were found to be strongly reduced in their competitiveness. Phenotypiccomparison of mutants with different deletion endpoints allowed a preliminary localization of two genomicregions, called nod-i and nod-2, which were required for normal nodulation on soybean. Loss of nod-i was

found to result in a Nod- phenotype on cowpea, mung bean, and siratro. A recombinant cosmid was identifiedwhich fully restored nodulation ability of a mutant lacking nod-i. Using Tn5-containing derivatives andsubclones of this cosmid for complementation, we delimited the nod-i region to a DNA segment of 3.1 to 3.5kilobase pairs.

Formation of the nitrogen-fixing root-nodule symbiosisbetween bacteria of the genera Rhizobium and Bradyrhizo-bium and legumes is a process which requires sequential andcoordinated gene expression in both partners. Within thegenus Bradyrhizobium, detailed information about the orga-nization of symbiotic genes is available only for Bradyrhizo-bium japonicum 110. In this organism, two gene clustershave been found: cluster I contains at least nine genesinvolved in nitrogen fixation (nijD, nifK, nifE, nifN, nifS,nifB, nifH, fixB, and fixC [6, 8]); cluster II contains thecommon nodulation (nod) genes (nodA, nodB, nodC, nodD,nodI, and nodJ) and the genes nifA and fixA (17, 18, 21).Very little is known about genes which determine the hostspecificity of B. japonicum. Recently, a locus was identifiedclose to the common nod genes which appeared to bespecifically required for nodulation of siratro (21).

Previously, we isolated B. japonicum mutants with largedeletions in and around cluster I (11, 15). Within the delet-able region, many repeated sequences (RS) were mapped(10). One of them, called RSa, was identified as an insertionsequence-like element (15). Several of the deletions havebeen shown to be generated by recombinational fusion ofdistant RSa copies (10).

In this paper a phenotypic investigation of selected dele-tion mutants is presented. The study revealed the presenceof at least two DNA regions which are required for normalsoybean nodulation. One region was cloned and character-ized in more detail. The results indicate that the presence ofthis novel nod region partially contributes to the host rangeof B. japonicum.

MATERIALS AND METHODSBacterial strains. Bradyrhizobium and Rhizobium strains

used in this study are listed in Table 1. The B. japonicumdeletion mutants listed in Table 2 have been describedpreviously (10, 15). Construction of B. japonicum A4199 isdetailed below and in Fig. 1. Escherichia coli strains used forTnS mutagenesis and triparental matings were as describedpreviously (4, 5), except that strain MC1061 was used

* Corresponding author.

instead of strain HB101. Plasmid constructs were also trans-formed into MC1061 (Table 1). Bradyrhizobium and Rhizo-bium strains were grown in PSY medium (24). E. coli strainswere grown as described previously (20).Recombinant DNA techniques. Recombinant DNA exper-

iments were performed by using established procedures (20).TnS mutagenesis of pL30-11B was done as described byDitta (4). TnS-containing cosmids were mapped by digestionof miniscale plasmid preparations with EcoRI, ClaI, BglII,Hindlll, and BamHI.

Heterologous hybridization. Total DNA isolation andSouthern blot hybridization were performed as describedpreviously (15), except that the hybridization temperaturewas 58°C and after hybridization the filter was washed in 5 xSSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate. As radioactive nod-i probe forthe experiment shown in Fig. 4, a 3.8-kilobase (kb) BglII-BamHI fragment was purified from a subclone carrying the6.2-kb BamHI fragment of pL30-11B.

Construction of B. japonicum A4199. The starting materialfor construction of strain A4199 (Fig. 1) was the cosmidpL38-12F, which carries 22.3 kb ofB.japonicum DNA in thevector pLAFR1 (7, 10). By restriction with XhoI and liga-tion, 17.5 kb of the insert DNA was removed and replacedby a 2.35-kb XhoI fragment of TnS coding for kanamycinresistance (Kin) (14). The resulting plasmid was transferredinto B. japonicum 110spc4 by triparental conjugation (5).Marker exchange was achieved after growing the transcon-jugants under kanamycin selection and screening for tetra-cycline-sensitive derivatives (loss of vector). The genomicstructure of the resulting deletion mutant, A4199, was con-firmed by Southern blot hybridization.

Construction of plasmids. Plasmids (see Fig. 3) were con-structed in the following ways. pRJ4327 was obtained bydigestion of cosmid pL30-11B with HindIII and ligation,thereby eliminating 13.8 kb of the insert DNA. pRJ4348 wasisolated by ligation of the 11-kb EcoRI fragment of pL30-11Bwith pLAFRl cut with EcoRI. pRJ4350 was obtained bycloning the 6.2-kb BamHI fragment of pL30-11B intopRK290 (5) linearized with BglII. For the construction ofpRJ4350-a, pRJ4350 was linearized with BglII and ligated

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56 HAHN AND HENNECKE

TABLE 1. Bacterial strains used

Strain Description Source or reference

B. japonicum110 spc4 Spcr wild-type derivative 24A3 nifD:::Tn5 mutant of 110 spc4 961-A-76 Wild-type isolate Rhizobium Germplasm

Resource Center(NifTAL), Paia, Hawaii

"Bradyrhizobium" sp.ANU289 Parasponia strain, wild type P. Gresshoff32H1 Crotolaria strain, wild type Nitragin Co., Milwaukee, Wis.

R. frediiUSDA 191 str Strr wild-type derivative E. R. AppelbaumUSDA 191C3 Sym plasmidfree derivative of USDA 191 str E. R. Appelbaum

E. coliMC1061 A(lacIPOZYA)X7 hsdR 2

with a 1.9-kb BamHI fragment coding for Kmr. This frag-ment was derived from a 1.9-kb BamHI-HindIII fragmentpresent in pKC7 (23), whose HindlIl site had been convertedinto a BamHI site by filling in and ligation with a BamHIlinker. pRJ4350-b was constructed by linearizing pRJ4350and HindIII and ligating it with a 3.35-kb HindIII fragmentfrom TnS encoding Kmr. pRJ4354 was obtained by ligation ofthe 7-kb BamHI fragment of pL30-11B::TnS-7 coding forKmr into the BglII site of pRK290.

Plant infection tests. Soybean seeds (Glycine max (L.)Merr. cv. Williams) were provided by Jaques Seed Com-pany, Prescott, Wis. Seeds from cowpea (Vigna unguiculatacv. Red Caloona), mung bean (Vigna radiata), and siratro(Macroptilium atropurpureum) were generously supplied byW. D. Broughton.

Surface sterilization of the seeds was done as describedpreviously (12). Seedlings of soybean, cowpea, and mungbean were inoculated and grown as described previously

(12). One-day-old siratro seedlings were transferred intoglass tubes (200 by 25 mm) containing 20-ml agar slants withJensen mineral solution (26). The seedlings were inoculatedwith 0.3 ml of B. japonicum suspension (ca. 108 cells inH20). The tubes were held closed by cotton stoppers to keepthe plants sterile during growth. All plants were grown ingrowth chambers as described previously (9).

Competition experiments were performed by mixing thestrain to be analyzed (Fix-) with a Fix' reference strain. Asreference strain, deletion mutant AE1-7dl (Table 2) waschosen, which itself was strongly reduced in its competitive-ness compared with the wild-type strain (see Results). Sus-pensions of two strains were washed in sterile H20, adjustedto the same density, and mixed in the desired ratio beforeinoculation of soybean. Cell counts were checked by platingserial dilutions of the suspensions. Acetylene reductionactivity in nodules of mixed-infected plants was measuredafter 20 days. Specific activities were calculated as micro-

TABLE 2. Nodulation behavior of B. japonicum llOspc4 (wild type) and several deletion mutants on soybean and other host plants

Delay of Avg nodule no. per plant" on:Fix nodulationB. japonicum strain Extent of deletion phenotype' on G. max M.(dpoGays) G. max V. unguiculata V. radiata atropurpureum

llOspc4 + 0 20.3 ± 6.6 38.1 ± 11.8 43.3 + 12.9 21.1 ± 6.1AE1-7d1 From left of RSa4 +C 2-4 21.0 ± 12.1 <id <d <id

to beginning ofcluster I

AEl-7d1(pL30-LLB) Same as in AE1-7dl; NTe 0 NT 36.7 ± 10.3 37.0 ± 18.7 14.5 ± 3.4strain carriescomplementingcosmid

AE1-8d1 From left of RSa4 - 4-6 4.3 ± 2.9 <id <id <idto right to RSa3

lAA3-4 From RSa8 to RSa3 - 2-3 13.0 ± 5.4 NT NT NTAF4-4 From RSa8 to - 1 17.1 ± 4.8 NT NT NT

RSa12AA3-R8 From RSa8 to RSal - 1 18.9 ± 5.8 20.3 + 3.5 21.6 + 8.0 8.9 + 2.0AD3-R2 From RSa4 to RSal - 2-4 7.9 ± 4.3 <id <id <idA4199 17 kb on the right of + 0 NT NT NT NT

RSa3a +, Fix' phenotype; -, Fix- phenotype.b Numbers are mean values from at least six plants.c Only 25 to 30% of wild-type fixation activity.d Less than 20% of the plants were nodulated; when nodulated, the plants had one to three nodules.eNT, not tested.

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NOVEL nod REGION OF B. JAPONICUM 57

20kb

Wild- type

pL38-12 F

nod-1 nif-clusterl

c4 a9,,,,,,,,,,,

E E RSx3 E EI I~~~~~~~~

X x ,,

1kb_---

A4199

FIG. 1. Map of the unstable DNA region surrounding cluster I in B. japonicum and construction of deletion mutant A4199. In the upperpart, genomic DNA is indicated by a horizontal line which is interrupted by slashes where physical distances are unknown. U, Positions ofRSa copies. The location of cluster I and of two nod regions is shown. In the lower part, all restriction sites for EcoRI (E) and the relevantsites of XhoI (X) are shown. r0, 2.35-kb XhoI fragment of Tn5 carrying the gene for neomycin phosphotransferase II (nptII [14]); - - -, thetwo sections of B. japonicum DNA which allowed marker replacement via homologous recombination (see Materials and Methods).

moles of C2H4 produced per hour per gram (dry weight) ofnodule.

RESULTSDeletion mutants. From the collection of available B.

japonicum mutants with deletions around nif cluster I (10),we chose six strains for this study (Table 2). The seventhstain, A4199, was constructed by marker replacement muta-genesis (see Materials and Methods (Fig. 1) for the followingreason. Strain AE1-8d1, carrying the largest deletion, wasfound to have one deletion endpoint close to the right ofRSa3 (10). To determine whether symbiotically essentialgenes are located in the region next to this deletion endpoint,cosmid pL38-12F was used to construct a 17.5-kb deletion,resulting in strain A4199 (Fig. 1). Taken together, two of themutants had retained the cluster I region (AE1-7dl andA4199), whereas the other five mutants had lost cluster I plusdifferent amounts of flanking DNA.

Prototrophy of the deletion mutants. The strain with thelargest deletion (AE1-8dl) and strain A4199 were both able togrow on minimal HMS-arabinose medium (3) like the wildtype, whereas only abortive growth was visible with ahistidine-requiring auxotroph (strain USDA I11O-FNhis4,kindly provided by D. Kuykendall), which was included as acontrol. Hence, the whole region depicted in Fig. 1 does notseem to contain any genes essential for prototrophic growthof B. japonicum on laboratory media.

Fix phenotypes of the deletion mutants. All deletion strainswere able to nodulate soybean (see below). Five strains withdeletions affecting cluster I were unable to reduce acetylenesymbiotically (Fix-; Table 2). Strain A4199 was Fix', indi-cating that there are no nif or fix genes within the deletedDNA (Fig. 1; Table 2). Strain AE1-7dl showed only 25 to30% of the wild-type Fix activity. Similar reduced valueswere also obtained for its parent strain, El, which carries aTn5 insertion in the region upstream of the nifD promoter (9;not shown). Since strain AE1-7dl has the right deletionendpoint within TnS DNA of the parental El strain (15), weconclude that there are no additional nif or fix genes in the

DNA segment which is missing in AE1-7dl. This conclusionis in contradiction to the data of Noti et al. (22), whosuggested the presence of a fix region approximately 9 kbupstream of nilj), on the basis of the phenotypes of Tn5mutants.

Nodulation phenotypes of the deletion mutants. All deletionmutants nodulated soybean; however, they exhibited dif-ferent degrees of defectiveness in nodule formation. Thephenotypes of the strains were studied by determining thetime of the first appearance of root nodules and the totalnodule number per plant at a given time after infection. Wealso used competition assays to distinguish the strains.Comparison of the deletion mutants revealed a correlation

between nodulation behavior and the amount of deletedDNA (Table 2). The most defective phenotype was observedin the strain with the largest deletion, AE1-8dl, whichinduced nodules with a delay of 4 to 6 days. Furthermore,these nodules were found only in small numbers at thesecondary roots, along with a varying number of small,nondifferentiated outgrowths. However, the few larger nod-ules were similar to wild-type nodules, i.e., they possessed apink central zone, and the infected nodule cells containedwell-differentiated bacteroids (as revealed by electron-mi-croscopic examination; D. Studer, personal communica-tion). After reisolating bacteria from such nodules andretesting them on plants, we again found that the bacteriafrom the nodules produced the same irregular nodulationphenotype as the original AE1-8d1 mutants did. This showedthat the nodules were not produced by any kinds of geneticsegregants. Strains with deletions smaller than that in AEl-8dl exhibited a less abnormal nodulation (Table 2), yet theydid have significant differences from the wild type (seebelow). No difference from wild-type-induced nodulationcould be observed with strain A4199.

Phenotypic parameters such as nodulation delay and nod-ule number were found to show considerable variation. Toconfirm our data by independent experimental means, wedeveloped a simplified competition assay. The principle wasto mix a Fix- mutant (strain B) with a Fix' reference strain

nod-2

CL1 cX12 3

I r) ,

X X

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58 HAHN AND HENNECKE

(AE1-7dl) in a certain ratio (see Materials and Methods).The competitiveness of strain B was measured, in mixed-infected nodules, by its capability to suppress the fixationactivity obtained with the reference strain. Significant differ-ences were found between some of the deletion mutants (seebelow) (Fig. 2). Similar differences were observed when theinoculation ratio between strain AE1-7d1 and strain B was

1:10 (not shown).Preliminary localization of two nod regions. The results

described above strongly indicated that in the deletionmutants, genes had been lost which were required for rapidand competitive nodulation of soybean. It was possible tomap these presumptive nod genes by correlating the pheno-types with the amount of deleted DNA of different strains.This comparison was facilitated because mutants had beenchosen which carried deletions with identical endpoints onone side but different endpoints on the other side: these werestrains AD3-R2 and AA3-R8 (common endpoint at RSal) andstrains AA3-4, AF4-4, and AA3-R8 (common endpoint atRSa8 [Table 2]) (10). One nod region, called nod-i, was thuspreliminarily mapped between RSa4 and RSa8, on the basisof a comparison of strains AD3-R2 and AA3-R8 (Table 2).These mutants differed significantly in nodulation delay,nodule number, and competitiveness (Table 2; Fig. 2).Another presumptive nod region, called nod-2, was locatedbetween RSa12 and RSa3 (Table 2). Of the three strainshaving the common left deletion endpoint in RSa8, strainAA3-4 was phenotypically more defective than strains AF4-4and AA3-R8; the last two did not differ significantly fromeach other (Table 2; Fig. 2). The difference between AA3-4and the two other strains was most evident in mixed-infection experiments, in which it was clearly the worstcompetitor (Fig. 2).Mutants AF4-4 and AA3-R8, which both lacked cluster I

but still possessed nod-i and nod-2, were also found to besomewhat impaired in nodulation and competitiveness com-

pared with the wild-type strain or the nifD::Tn5 mutant A3(Table 2; Fig. 2). Possible explanations for this effect are

given in the Discussion.Inability of mutants lacking nod-i to nodulate other host

plants. B. japonicum is able to infect not only its original

427

'ac

l.2

46-La ! 50

% Go_=16

o l

Strain B: A3 AD3-R2 MA3-R8 AF4-4 &A3-4

FIG. 2. Competitiveness of B. japonicum mutants against strainAE1-7d1. The vertical bars represent mean values obtained from atleast five plants each. Each test was done twice. Standard deviationsare indicated. The Fix activity of plants infected with AE1-7d1 alone(100%) was determined as 37.6 ,umol of ethylene h-' g (dry weight)of nodule-l.

host, soybean, but also several other legumes includingcowpea, mung bean, and siratro (16). When these plantswere inoculated with the wild-type strain, they formed pinknodules which were able to reduce acetylene (with siratro,the Fix phenotype was variable). In contrast, all mutantswhich had lost the nod-i region (strains AE1-8d1, AE1-7dl,and AD3-R2) were found to be almost completely Nod- on

these hosts (Table 2).Cloning and mapping of nod-i. In a previous work, we

described the isolation of pLAFR1 cosmids containing RSacopies which surround cluster I in the B. japonicum genome(10) (Fig. 1). Since pLAFR1 can be mobilized and is able toreplicate in a wide range of gram-negative bacteria includingB. japonicum, we attempted to clone nod-i by means ofcomplementation. Cosmids carrying RSao4 or RSot8 plusvarious amounts of flanking DNA were mobilized intodeletion mutant AE1-7d1 by using the triparental matingsystem described by Ditta et al. (5). Tcr transconjugantswere used as inoculum for infection of cowpea, mung bean,and siratro. One cosmid, pL30-11B, was found to restorecompletely the ability of AE1-7d1 to nodulate these plants(Table 1). Furthermore, strain AE1-7d1 carrying pL30-11Binduced nodules on soybean without the delay that wascharacteristic for strain AE1-7d1 (not shown). To confirmthat pL30-11B was responsible for the complementation,bacteria were isolated from siratro nodules infected withAE1-7d1 containing pL30-11B. Of the colonies obtained,2.5% were Tcr, indicating a significant loss of the cosmidduring passage of the bacteria through the nodules. Whenthese colonies were again used for infection of siratro, onlythe Tcr isolates were able to nodulate.A physical map of the B. japonicum DNA cloned in

pL30-11B is shown in Fig. 3. The insert is 21.6 kb long. Tworepeated sequences, called RSE4 and RSot8, have beenlocalized on this DNA (10). By comparing the restrictionsites of RSe4 with those from another cloned RSe copy(RSe1 [10]), the unit length of RSe was estimated to be atleast 1.9 kb. The genomic orientation of the pL30-11B DNArelative to RSa4 and to cluster I has been determinedpreviously (10). Since nod-i has been preliminarily mappedbetween RSa4 and RSa8, it was expected to be located inthe region on the left of RSa8 (Fig. 3).To more precisely localize the region which was able to

complement the nodulation defect of strain AE1-7dl, Tn5-containing derivatives and subclones of pL30-11B wereconstructed (see Materials and Methods). After transfer ofthese clones into AE1-7d1, the transconjugants were testedfor their ability to nodulate siratro.Of 16 Tn5 insertions isolated, 8 were found to abolish

complementation completely (TnS-8 to TnS-14) or partially(TnS-IS; Fig. 3). These insertions were mapped in a region of3 kb. Outside this region, complementation was not affectedby TnS insertions. Among the subclones of pL30-11B tested,only those which carried the whole DNA region between thesites of the insertions Tn5-7 and TnS-16 (pRJ4350 andpRJ4354) were able to complement (Fig. 3). Two in vitroconstructed insertions in plasmid pRJ4350 both abolishedcomplementation (Fig. 3), which was consistent with thephenotype of the TnS insertions. Taken together, theseresults show that the nod-i region as defined by its ability torestore nodulation of strain AE1-7d1 on siratro could bedelimited to a DNA region of 3.1 to 3.5 kb (Fig. 3). The sameregion was also found to fully restore the nodulation abilityon cowpea, mung bean and soybean (not shown).Lack of hybridization of nod-i with known host-specific

nodulation (hsn) genes. The data have shown that the nod-i

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NOVEL nod REGION OF B. JAPONICUM 59

RS-4MOM

nod-1 RSc8- _ - to nifO

+ pL30-11B

- pRJ4327

- pRJ4348

+ pRJ4350

+ pRJ4354

E B BC B E C BgE C C B CI a

I axi £ £8 inaa1 a

1 2 3 45 6 78 9-14 15160 *- 0 *0 0 Q

E H HE.-w-

E

(B) B H (B)L-

a bo o

(B) (B)

FIG. 3. Mapping of the nod-I region. , Cloned B. japonicum DNA is indicated by horizontal lines, ---, deleted DNA. The bars on topshow the location of nod-i and of two repeated sequences. For nod-I, the black part of the bar marks the minimal size and the whole barrepresents the maximal size. For RSE4, the minimal size is indicated. The ability of various plasmids to restore wild-type nodulation of strainAE1-7d1 is indicated on the left(+, 10 to 20 nodules per plant on siratro; -, no nodules). The effects of TnS insertions (A) or in vitro insertions(A) are shown by circles (0, full complementation; 0, no complementation; OP, partial complementation, i.e., nodules are formed with a delayof ca. 10 days). Restriction sites are shown for EcoRI (E), BgII (Bg), BamHI (B), ClaI (C), and Hindlll (H). Sites in parentheses were lostduring cloning.

region was more essential for nodulation of siratro, mung

bean, and cowpea than for nodulation of soybean. Wetherefore tested whether nod-i was homologous to nodgenes from other sources which had been shown to encodehost-specific functions. No hybridization of a nod-i DNAprobe (see Materials and Methods) was found with a clonecarrying hsnA, hsnB, hsnC, and hsnD from Rhizobiummeliloti (13) and with cosmids carrying three unlinked hsnregions from Rhizobium sp. strain NGR234 (19; data notshown).The nod-i probe was also hybridized with total DNA of

Bradyrhizobium strains known to nodulate siratro and of thefast-growing soybean symbiont Rhizobium fredii; in ourassays the latter strain was also found to be Nod' on siratro.Only the Bradyrhizobium strains exhibited strong hybridiza-tion bands (Fig. 4). After longer exposure of the autoradio-graph, a weak band of 4.0 kb was seen with wild-type R.fredii and a sym-plasmid-deleted derivative thereof (notshown).

DISCUSSIONIn B. japonicum, the DNA region surrounding cluster I

was found to have several unusual features. More than 230kb can be deleted spontaneously without obvious effects onfree-living growth; another 10 to 15 kb was shown to benonessential by use of the newly constructed deletion instrain A4199. Many copies of repeated sequences are accu-mulated in this region (10). In addition to the nifandfix genesin cluster I, the deletable region was now shown to containgenes involved in nodulation. A preliminary mapping ofthese genes was performed by phenotypic comparison ofseveral deletion mutants. That this approach was valid was

proved by the subsequent cloning of nod-l.

A novel mixed-infection assay was used to characterizethe mutants and allowed us to compare their competitivebehavior. Although the assay did not include the determina-tion of nodule occupancy, it was sensitive and reproduciblydistinguished between strains carrying or lacking certaingenomic regions. In all cases, a correlation was foundbetween nodulation delay and reduction in competitiveness.This is in agreement with data obtained by Bohlool et al. (1),who were able to shift the relative competitiveness of twostrains by changing the timing of infection. It thus appears asif all deletion mutants are, to a different degree, defective inthe early infection process.A region called nod-2 was identified somewhere between

RSal2 and RSM3. It must therefore be located in a DNAsegment which has previously been shown, by mapping, tobe at least 84 kb long (10). Interestingly, within the deletableregion, two DNA fragments were identified between RSal2and RSa3 which showed hybridization to a probe containingthe nod box, a highly conserved sequence which has beenfound in front of nod genes in many rhizobia (25; M.Gottfert, personal communication).

Deletion mutants lacking the DNA between RSa9 andRSal but still possessing nod-i and nod-2 were also weaklydefective in nodulation (data not shown) (Table 2; Fig. 2). Aninterpretation of this phenomenon is difficult, since in suchmutants all nifandfix genes of cluster I are deleted. The lossof either one or more of the known cluster I genes or ofadditional, as yet unidentified genes could be responsible forthe observed phenotype. It should be emphasized, however,that the defect cannot be caused by the lack of N2 fixationper se, because the nifD::TnS mutant A3 was reported to besimilar to the wild type with respect to nodulation andcompetitiveness (12).

Bg E BgI I

E

E

1kb

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60 HAHN AND HENNECKE

21.7-

9.6-

6.6 -

5.0 -4.3-35.- ,

23-

FIG. 4. Presence of nod-i in other Bradyrhizobium strains. Theautoradiograph was obtained from a filter membrane carryingEcoRI-digested total DNA after hybridization with a nod-i probe(see Materials and Methods). DNA of the following strains wasloaded: B. japonicum 110spc4 (lane 1); B. japonicum 61-A-76 (lane2); Bradyrhizobium sp. (Parasponia) strain ANU289 (lane 3); Brady-rhizobium sp. (Crotolaria) strain 32H1 (lane 4); R. fredii USDA191str (lane 5); and R. fredii USDA 191C3 (lane 6). In the leftmargin, the sizes of molecular weight standards are indicated.

The nod-i region was cloned by complementation of strainAE1-7d1 with cosmid pL30-11B, which fully restored thenodulation ability on all hosts. During symbiosis, the cosmidwas lost by more than 95% of the bacteria. This confirmedthat nod-i is required only in the early stages of the symbi-osis. nod-i was found to span a contiguous stretch of 3.1 to3.5 kb. Any insertion within this region completely abolishedits complementing activity, except for one insertion at theright end (TnS-IS), which only partially affected complemen-tation. One can expect, therefore, that the genes in nod-i are

coding for similar or cooperating functions. Which kind offunctions they encode, however, is unknown. They are

required for normal nodulation of soybean, but are almostindispensable for nodulation of other hosts. Thus, they seemto contribute to the host range of B. japonicum.Although being linked, by deletions, to cluster I, nod-i is

located at least 25 kb upstream of nifD (10, 22). Similarly tocluster I, nod-i was found to be close to a copy of RSot.Since strain AE1-7d1 is Fix' and its nodulation ability can becompletely restored by nod-i DNA, probably no symbioticgenes other than those of the nod-i region are present in thedeleted DNA.

Hybridization experiments revealed that nod-i was alsopresent in other Bradyrhizobium strains. The results did notconclusively indicate, however, whether a region homolo-gous to nod-i also existed (maybe on the chromosome) in R.fredii. No hybridization of nod-i was found with cloned hsngenes from R. meliloti and Rhizobium sp. strain NGR234(13, 19). Furthermore, we could not detect homology to a B.

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japonicum hsn locus described recently (21), nor to a frag-ment carrying B. japonicum nodD (not shown). Thus, nod-iappears to represent a novel nodulation region.

ACKNOWLEDGMENTSWe thank D. Studer and M. Gottfert for communicating unpub-

lished data, H. Dreyer for help with the competition experiments, S.Hitz for expert technical assistance, and H. Paul for typing themanuscript. We are also grateful to A. Kondorosi, W. J. Broughton,P. Gresshoff, G. Ditta, D. Kuykendall, and E. Appelbaum forproviding strains or seeds.

Financial support for this work was provided by the FederalInstitute of Technology, Zurich, Switzerland, and by LubrizolGenetics Inc./Agrigenetics Research Associates Ltd.

LITERATURE CITED1. Bohlool, B. B., R. Kosslak, and R. Woolfenden. 1984. The

ecology of Rhizobium in the rhizosphere: survival, growth andcompetition, p. 287-293. In C. Veeger and W. E. Newton (ed.),Advances in nitrogen fixation research. Nijhoff/Junk Publishers,The Hague, The Netherlands.

2. Casadaban, M. J., A. Martinez-Arias, S. K. Shapira, and J.Chou. 1983. P-Galactosidase gene fusions for analyzing geneexpression in Escherichia coli and yeast. Methods Enzymol.100:293-308.

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