Directed transposon Tn5 mutagenesis and complementation analysis of rhizobium meliloti symbiotic nitrogen fixation genes

Cell, Vol. 29, 551-559,

June 1982,

Copyright

0 1982

by MIT

Directed Transposon Tn5 Mutagenesis and Complementation Analysis of Rhizobium meliloti Symbiotic Nitrogen Fixation Genes Gary B. Ruvkun, Venkatesan Sundaresan Frederick M. Ausubel Department of Cellular and Developmental Harvard University Cambridge, Massachusetts 02138

and Biology

Summary An 18 kb region adjacent to and surrounding the genes for nitrogenase (nif) was cloned from the genome of the symbiotic nitrogen-fixing species Rhizobium meliloti. A total of 31 Tn5 insertions in the nif region were constructed and assayed for their effect on symbiotic nitrogen fixation (Fix phenotype). Fix- insertions were found in two clusters, one 6.3 kb and a second at least 5.0 kb, separated by a 1.6 kb region not containing essential symbiotic genes. The locations of at least three transcription units containing Fix genes were deduced from complementation analysis between genomic nif: :TnS insertions and nif: :Tn5 insertions on mobilizable cloning vectors. The locations of R. meliloti genes nifH, nifD and nifK, which code for the single subunit of the nitrogenase Fe protein and for the two subunits of the nitrogenase MoFe protein respectively, were determined by DNA hybridization to cloned Klebsiella pneumoniae nif genes and by comparison of partial R. meliloti DNA sequences with K. pneumoniae nif gene sequences. R. meliloti nifH, D and K are located in the 6.3 kb Fix-::TnB cluster and are transcribed in the order nifH, nifD, nifK, which is the same order as in K. pneumoniae. Introduction Bacteria in the genus Rhizobium normally fix nitrogen only in symbiotic association with their legume host. The establishment of symbiosis begins with penetration of root hairs by Rhizobium and culminates in the differentiation of the bacteria into nitrogen-fixing “bacteroids” within a highly specialized macroscopic structure, the root nodule, formed by interaction of the bacteria and plant (Vincent, 1980). Bacteria invade nodule cells via infection threads (cellulose tubes formed by the plant), which grow into the interior of the nodule. Within the mature nodule, some plant cells are packed with nitrogen-fixing bacteroids. In several Rhizobium species, symbiotic genes have been defined by the isolation of nodulation-defective (Nod-) and nitrogen-fixation-defective (Fix-) mutants (Buchanan-Wollaston et al., 1980; Meade et al., 1982; Scott et al., 1982). We define the Nod- phenotype as failure of the bacteria to penetrate the root and signal the plant to initiate divisions in root cortical cells. In the Fix- phenotype, bacteria invade the root via infection threads and cause the plant to differentiate a morphologically identifiable nodule; however, the bac-

teria either fail to differentiate into bacteroids or differentiate into bacteroids that fail to fix nitrogen. A specific subclass of Fix- mutants are those containing defects in nitrogen fixation (nif) genes, genes that code for nitrogenase and for ancillary enzymes directly involved in the nitrogen-fixation reaction. In several Rhizobium species, genes that code for nitrogenase are located on large indigenous plasmids (Nuti et al., 1979; Ruvkun and Ausubel, 1980; Prakash et al., 1981). In addition, Rhizobium genes required for bacterial-plant recognition (nod genes) are located on plasmids (Johnston et al., 1978; Zurkowski and Lorkiewicz, 1979; Beynon et al., 1980; Brewin et al., 1980; Hirsch et al., 1980). Moreover, in at least some Rhizobium species, nif and nod genes are linked on the same large plasmid (Banfalvi et al., 1981; Rosenberg et al., 1981; Long and Ausubel, 1982; A. W. B. Johnston, personal communication) and are preferentially transcribed in root nodules (Krol et al., 1980). These observations suggest that symbiotic genes are clustered on plasmids and are coordinately regulated. We previously showed that the K. pneumoniae DNA sequences coding for two nitrogenase genes (nifD and nifH) share DNA homology with 13 widely divergent nitrogen-fixing species, including R. meliloti (Rm), in which Rm nifH and part of Rm nifD are located on a 3.9 kb Eco RI fragment (Ruvkun and Ausubel, 1980). We used this interspecies homology to clone the 3.9 kb R. meliloti Eco RI fragment, and we verified that this fragment contains essential symbiotic nitrogen fixation genes by replacing the wild-type 3.9 kb fragment in the R. meliloti genome with the cloned fragment altered by transposon Tn5 mutagenesis (Ruvkun and Ausubel, 1981). In this paper, we report the construction and genetic analysis of a variety of recombinant plasmids that are adjacent to or overlap the 3.9 kb Eco RI fragment containing Rm nifH. In particular, using Tn5 mutagenesis, we construct a correlated physical-genetic map that spans 14 kb surrounding Rm nifH and Rm nifD. We find that the region examined contains at least two clusters of symbiotic genes (approximately 6.3 kb and at least 5.0 kb); Tn5 insertions in these clusters result in a Fix- phenotype. Using a mobilizable cloning vector to carry out complementation analyses between pairs of appropriate nif::Tn5 insertions, we deduce that that 6.3 kb cluster of symbiotic genes is a single transcription unit containing the genes Rm nifH, Rm nifD and Rm nifK, transcribed in that order. Results Construction of Plasmids carrying R. meliloti Symbiotic Genes In a previous study (Ruvkun and Ausubel, 1980), we utilized cloned K. pneumoniae nitrogenase genes as hybridization probes to identify a 3.9 kb R. meliloti

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Eco RI fragment partially homologous to the K. pneumoniae nifH and nifD genes. This interspecies homology was used in a colony hybridization procedure (Hanahan and Meselson, 1980) to clone a region of the R. meliloti genome (in plasmid pRmR1) that contains the 3.9 kb Eco RI fragment. Plasmid pRmR1 was derived from a partial Eco RI digest of total R. meliloti DNA ligated to Eco RI-digested pBR322. It contains five Eco RI fragments, ranging in size from 1.2 kb to

Figure

I.

nif Region

Restriction

Map of R. meliloti

The map shows the location and associated phenotype of nif::Tn5 insertions 7. L5 and 22 and restriction sites used in the construction of the plasmids shown. Table 1 lists the plasmid vectors used and nif region restriction sites used in plasmid constructions and insertion mutations in each plasmid. To clone each of the nif: :Tn5 mutations such that they were contained on nif region Barn HI fragments, total genomic DNA was isolated from each of the three strains (see Table I) and cleaved with Barn HI. (The Barn HI sites in the nif region [I 3 kb apart] flank Tn5 insertions 7. 22 and LS.) Barn HI-digested DNA, isolated from strains containing each of these Tn5 insertions, was ligated into the Barn HI site of plasmid pBR322, yielding plasmids pRmR8L2 derived from genomic Tn5 insertion 7, pRmRT2 from Tn5 insertion 22, and pRmRQ42 from Tn5 insertion L5. To clone the nif region Cla I fragment with nif::Tn5 insertions, DNA isolated from R. meliloti strains containing Tn5 insertions 22 and 7 was digested with Cla I and ligated into the Cla I site of cosmid pHC79 (Collins and Hohn, 1978). The ligation mixtures were packaged in vitro into A phage heads (Hahn, 1975); E. coli strain HBlOl was infected with these phage particles and Ap’Nm’ colonies were selected. Cosmid DNA was prepared from the resulting colonies, digested with Cla I, and a 23-25 kb Cla I fragment was found in cosmids derived from each nit :Tn5 plasmid. Plasmid pRmR8LP contains a 9.8 kb Barn HI fragment insert that includes the Nm’ gene of Tn5 plus the nif region of R. meliloti from the site of Tn5 insertion 7 to Barn HI site (a). In addition, plasmid pRmR8LP contains a 1.0 kb Barn HI fragment from the R. meliloti genome that is not adjacent to the 13 kb nif region Barn HI fragment. Fortuitously, this 1 .O kb Barn HI fragment contains an Xho I site that was used to construct a derivative of pRmR8LP that does not contain the Nm’ gene. This was accomplished by digestion of pRmRBL2 with Xho I, which deletes the Nm’gene of Tn5 but leaves the rest of the nif region intact. Ligation of the resulting fragments into the Sal I site of pBR322 and selection for Ap’Tc*Nm’ colonies, yielded plasmid pRmRQ8. which contains the region from the insertion site of nit :Tn5 insertion 7 to Barn HI site (a) plus 0.5 kb of Tn5 DNA from the end of the Tn5 inverted repeat plus 0.4 kb of R. meliloti DNA of unknown origin. C: Cla I. B: Barn HI. R: Eco RI. X: Xho 1. Bg: Bgl II.

5.0 kb, in addition to the 3.9 kb Eco RI fragment homologous to K. pneumoniae nitrogenase genes. Analysis of R. meliloti DNA by the Southern gel transfer and hybridization procedure (Southern, 19751, with 32P-labeled restriction fragments purified from pRmR1 used as hybridization probes, showed that the 5.0 kb Eco RI fragment is adjacent to the 3.9 kb Eco RI fragment on the R. meliloti genome (data not shown). To facilitate the analysis of these two Eco RI fragments, the 3.9 kb and the 5.0 kb fragments were recloned individually in the vector pACYCl84 to yield plasmids pRmR2 and pRmR3, respectively. A restriction map of a portion of the R. meliloti genome containing the 3.9 and 5.0 kb Eco RI fragments is shown in Figure 1. To clone additional fragments that partially overlap and extend the region cloned to the left and right of pRmR2 and pRmR3, we made use of transposon Tn5 insertions in the R. meliloti genome (Ruvkun and Ausubel, 1981). The placement of Tn5 in the R meliloti nif region “marks” restriction fragments from that region with the neomycin resistance gene of Tn5. Therefore, by use of the appropriate restriction enzyme, recombinant plasmids containing R. meliloti nif DNA adjacent to Tn5 were isolated by genetic selection in E. coli for the Tn5 neomycin resistance gene. Two different restriction enzymes were used to clone Table

I.

R. meliloti Strains

and Recombinant

Plasmids

R. meliloti Strains Strains

Used

nif Region

Genotype

Phenotype

Rml021

Fix+

Rml491

nif::Tn5

insertion

7

Fix

Rm1431

nif::Tn5

insertion

22

Fix+

RM1480

nif::Tn5

insertion

L5

Fix+

Recombinant

Plasmids Size of Insertion

nif Region Tn5 Insertions Ineluded

Plasmids Used

Vector

Restriction Enzvme Used

pRmR2

pACYCl84

EGO RI

3.9 kb

pRmR3

pACYCl84

Eco RI

5.0 kb

pRmR8LP

pBR322

Barn HI

9.8 kb

nif::Tn5

7

pRmRT2

pBR322

Barn HI

10.8 kb

nif::Tn5

22

pRmRQ42

pBR322

Barn HI

8.8 kb

nif::Tn5

L5

pRmRO8

pBR322

Xho I-Sal

8.0 kb

nif::Tn5

7 7

I

pRmR298L2

pRK290

Barn HI-Bgl

II

9.8 kb

nif::Tnd

pRmR29T2

pRK290

Barn HI-Eel

II

10.6 kb

nif::Tn5

22

pRmR29042

pRK290

Barn HI-Egl

II

8.6 kb

nif::Tnd

L5

pRmRD5

pHC79

Cla I

25.0 kb

nif::Tn5

7

pRmRE2

pHC79

Cla I

25.0 kb

nif::Tn5

22

pRmR29D5

pRK294

Cla I

25.0 kb

nif::Tn5

7

pRmR29E2

pRK294

Cla I

25.0 kb

nif::Tn5

22

Genetics 553

of Symbiotic

Nitrogen

Fixation

nif region fragments in this way: Cla I, which does not cut within Tn5, and Barn HI, which cuts once in Tn5 adjacent to the neomycin resistance gene (Jorgensen et al., 1979). Cla I and Barn HI cut infrequently in the nif region to generate 18 kb and 13 kb fragments, respectively, which contain R. meliloti nifH and nifD (see Figure 1). Three different nif::Tn5 insertions were used to construct recombinant plasmids as described in Figure 1: Fix+: :Tn5 insertion 22, which maps 100 bp to the right of Xho I site(b); Fix-: :Tn5 insertion 7, located in the R. meliloti nifH coding region 800 bp to the left of Tn5 insertion 22; and Fix+: :Tn5 insertion L5, which maps 200 bp to the left of Xho I site (b) (see Figure 1). Plasmids pRmR8L2 and pRmRT2 both include nif region DNA to the left of the Tn5 insertions 7 and 22 respectively, extending to Barn HI site (a). Plasmid pRmRQ42 contains the nif region to the right of Tn5 insertion L5 extending to Barn HI site (b). (The orientations of Tn5 insertions 7 and 22 are opposite to that of Tn5 insertion L5.) Recombinant plasmids containing the same nit :Tn5 mutations as above, but extending further to the left and right, were also constructed. Based on restriction maps of the R. meliloti nif region, we expected Cla Idigested DNA from the nif::Tn5 strains to contain a 23-25 kb Cla I fragment, “marked” by the Nm’ gene of Tn5. Cosmid pRmRE2, which contains nif::Tn5 insertion 22, and cosmid pRmRD5, which contains nif::Tn5 insertion 7 on the analogous R. meliloti nif region Cla I restriction fragment, were constructed as described in Figure 1. Plasmid pRmR8L2 contains nif region DNA to the left of the 3.9 kb Eco RI fragment, which had not yet been surveyed by Tn5 mutagenesis for symbiotic genes. Before the nif region cloned on pRmR8L2 could be used as a target for Tn5 mutagenesis, it was necessary to construct, as described in Figure 1, a derivative plasmid, pRmRQ6, with the Nm’ gene of Tn5 inactivated. Localization of Rhizobium nif Genes by Interspecies DNA Homology As described above, at least two K. pneumoniae nitrogenase genes, nifH and nifD, are partially conserved between K. pneumoniae and R. meliloti (Ruvkun and Ausubel, 1980). Hybridization of probes carrying K. pneumoniae nifD or nifH genes to various restriction digests of plasmids pRmR8L2 and pRmRT2 showed that homology to nifD is localized to the left of Hind Ill site (b) (see Figure 2) and to the right of Bgl II site (a), and that homology to nifH is located to the right of Hind Ill site (b) and to the left of Xho I site (b) genes. To map the location and direction of transcription of the presumptive R. meliloti nifH and nifD genes more precisely, we used the method of Maxam and Gilbert (1980) to determine the DNA sequence of both strands of the region between Bgl II site (b) and Xho

I site (a) (see Figure 2). which, based on the interspeties DNA hybridization data, was expected to contain the N-terminal coding region of Rm nifH. We also determined the DNA sequence of one strand extending from Hind Ill site (b) towards the left, which was expected to include the N-terminal coding region of Rm nifD (see Figure 2). Some of the sequence obtained is compared in Figure 2 to the corresponding DNA sequences of K. pneumoniae nifH and nifD (Scott et al., 1981; Sundaresan and Ausubel, 1981). On the basis of these DNA sequencing studies, we conclude that the K. pneumoniae and R. meliloti nifH genes share 61% DNA homology and 72% amino acid homology in the 360 bp region analyzed with several runs of exact amino acid homology, as illustrated in Figure 2. If we assume that the location of the Nterminal amino acid is the same in R. meliloti and K. pneumoniae, the R. meliloti nifH coding region starts 130 bp to the left of the Bgl II site and translation proceeds from right to left. T&ok and Kondorosi (1981) have determined the entire DNA sequence of the nifH gene from a different strain of R. meliloti. Their sequence matches exactly our partial sequence in the protein-coding regions; they find that the Rm nifH gene is 890 bp in length and ends 200 bp to the right of Hind Ill site (b) (see Figure 2). Two regions of significant homology were detected between the K. pneumoniae nifD gene and R. meliloti sequences distal to Rm nifH. One, with 50% amino acid homology, was located 40 bp to the left of Hind Ill site (b) (Figure 2) and corresponds to K. pneumoniae nifD amino acids 28 to 36, and the second, with 87% amino acid homology, was located 120 bp to the left of Hind Ill site (b) and corresponds to K. pneu-

Figure 2. Location Genome

of Genes

nifH.

MD

and nifK

in the R. meliloti

(Top) Restriction map of nifff. nifD and nifK genes. (Bottom) An example of interspecies ONA sequence homology in nif/-/ and nifD genes used to map these genes in R. meliloti. Amino acid residue numbers are derived from published K. pneumoniae DNA sequence (Sundaresan and Ausubel. 1981; Scott et al., 1981). Arrows 1, 2 and 3: region from which DNA sequences shown below were derived. The probability of random occurrence of these DNA homologies are: homology (1 ), less than 1 O-“; homology (2). 10-s; and homology (3), 1 O-‘. R: Eco RI. H: Hind III. Bg: Bgl II. X: Xho I.

Cell 554

moniae nifD amino acids 56 to 62. If the N-terminal amino acid is at the same relative location in R. meliloti and K. pneumoniae, the coding sequence of R. meliloti nifD starts approximately 50 bp to the right of the Hind Ill site (b), or about 100 bp after the Ft. meliloti nifH termination codon (T&ok and Kondorosi, 1981). With an assumed molecular weight of 55 kd for the nifD gene product (Isreal et al., 1974; Whiting and Dilworth, 19741, the coding sequence should end approximately 500 bp to the left of Eco RI site (a). Based on analogy to the nif gene organization in K. pneumoniae, we expected to find the R. meliloti gene equivalent to nifK to the left of Rm nifD, and we expected it to be approximately 1.5 kb long, ending near Hind Ill site (a) (Figure 2). We previously reported no detectable R. meliloti DNA sequence homology to K. pneumoniae nifK DNA (Ruvkun and Ausubel, 1980). However, upon hybridization of a 32P-labeled 2.5 kb Eco RI-Hind Ill K. pneumoniae restriction fragment containing K. pneumoniae nifKYE to Eco RI-digested plasmid pRmR8L2 DNA fractionated on a 1.8% agarose gel and transferred to nitrocellulose, a hybridization band at 1.7 kb was detected. Based on the location of the 1.7 kb fragment (shown in Figure 2) in the R. meliloti nif region, the fragment most likely contains homology to K. pneumoniae nifK. Hybridization of other cloned K. pneumoniae nif gene probes to the R. meliloti nif region recombinant plasmids showed no detectable homology. Tn5 Mutagenesis and Gene Replacement To construct a fine-scale genetic map of the symbiotic genes in the R. meliloti nif region cloned on plasmids pRmR2, pRmR3 and pRmRQ6, these plasmids were mutagenized with Tn-5 as described by Ruvkun and Ausubel (1981). The Tn5 insertion site in each of the resulting plasmids was mapped with restriction enzymes Barn HI or Hind Ill, and each R. meliloti Eco RI fragment in plasmids pRmR2, pRMR3 and pRmRQ6 containing a Tn5 insertion was recloned into the broad host-range cloning vector pRK290 (Ditta et al., 1980). Plasmid pRK290 is 20 kb, confers Tc’, contains Eco RI and Bgl II cloning sites and can be mobilized into and replicates stably within R. meliloti. Each pRK290 recombinant plasmid containing a Tn5-mutagenized R. meliloti fragment was conjugated into the Fix+ R. meliloti strain 1021, and R. meliloti transconjugants were selected in which the wild-type R. meliloti sequences were replaced by the homologous sequences containing Tn5 (see Experimental Procedures for details). The location of each Tn5 insertion in the resulting strains was mapped by the Southern gel transfer and hybridization technique (Southern, 1975) with use of Xho I-, Hind Ill- or Bgl II-digested total DNA isolated from each strain and 32P-labeled pRmR2 or pRmR3 DNA as a hybridization probe (data not shown). Finally, each R. meliloti strain containing a single genomically mapped Tn5 insertion

was inoculated onto at least three separate sterile alfalfa seedlings and after 4 weeks a nitrogenase assay was performed on each plant (Ruvkun and Ausubel, 1981). A total of 31 Tn5 insertions distributed over 14 kb of the R. meliloti nif region were analyzed by the above method and the results are summarized in Figure 3. The analysis revealed two clusters of Fix-: :Tn5 insertions of 6.3 f 0.2 kb and at least 5.0 kb, separated by a 1.6 f 0.2 kb cluster of Fix+ Tn5 insertions between Bgl II site (b) and Eco RI site (b). None of these Tn5 insertions resulted in any defect in ability to form nodules or any gross abnormality in nodule morphology. All of the strains tested (except two) formed nodules with either the wild-type level of acetylene reduction (500 nmole ethylene per 24 hr; Fix+) or less than 1 nmole of ethylene per 24 hr (Fix-). (Tn5 insertions P20 and Pl 1, located between Rm nifD and Rm nifH [see below] displayed a low level [5 f 2 nmole ethylene produced per 24 hr] of nitrogenase activity.) The 6.3 kb cluster of Fix-::Tn5 insertion mutations was bounded on the left by Fix+::Tn5 insertion A52 and on the right by Fix+::Tn5 insertion L5 (see Figure 3). Because the reliability of the Fix+ phenotype of Tn5 insertion A52 was important to the conclusion that this insertion defined the boundary of a symbiotic gene cluster, Tn5 insertion A52 was recombined into the R. meliloti genome in five independent experiments. In all five cases, a Fix+ phenotype was obtained, and in all five cases the Tn5 insertion was shown to map in the same position. The locations of the R. meliloti DNA sequences homologous to K. pneumoniae nif genes correlate well with the Tn5 mutagenesis results: all mutations in the nif region to the left of the Bgl II site (b) and to the right of Tn5 insertion A52, a region that includes R. meliloti nifH and nifD, result in a Fix- phenotype. Tn5 insertions to the left of the Rm nifD gene also result in a Fix- phenotype. Based on analogy to the nif gene organization in K. pneumoniae and the interspecies hybridization of a DNA fragment containing the K. pneumoniae nifK genes, we tentatively conclude that the R. meliloti equivalent to nifK is located in the 2.5 kb to the left of Rm nifD and to the right of Tn5 insertion A52. The direction of transcription of R. meliloti nifH and R. meliloti nifD can be inferred to be from right to left (Figure 2) on the basis of the amino acid homology to K. pneumoniae nifH and nifD. The promoter for this transcription unit was expected to be located within 400 bp to the right of the nifH putative initiation codon because of the location of Fix+: :Tn5 insertion L5. The DNA sequence 5’ to the R. meliloti nifH putative initiation codon was scanned for canonical promoter-like sequences (Pribnow, 1975; Rosenberg and Court, 1979) and for homology to the K. pneumoniae nif promoter region (Scott et al., 1981; Sundaresan and Ausubel, 1981). No significant homology to the K.

Genetics 555

of Symbiotic

Nitrogen

Nit phenotype

Fixation

-

A

,A,

I

pRmR297

pRmR29E2

I

pRmR29042 Transcription

units

f

I Figure

3. Physical

Genetic

-I

--

Map of Ft. meliloti nif Region

(Above restriction map) Locations of all Tn5 insertions and their associated phenotypes ([+I Fix+::Tn5. [-] Fix-::TnS) (Below restriction map) nif region included on each plasmid used in the complementation analysis and the location of nif::Tn5 insertions they contain. Above each plasmid map is the result (+ or -) of complementation analysis of that plasmid with the different genomic nif::Tn5 insertion numbered above the restriction map. The genomic insertion ISFtml is a Fix- mutation caused by the insertion of an Ft. meliloti insertion element ISRml (Ruvkun et al., 1982). (Bottom) Transcription unit map of the R. meliloti nif reaion deduced from complementation analysis and DNA sequence analysis. Arrow: direction of transcription of the nifHD”K” transcription unit. C: Cla I. B: Barn HI. R: Eco RI. H: Hind Ill. X: Xho I. Bg: Bgl II.

pneumoniae nifH promoter region was found. A potential Shine-Dalgarno sequence (AGGA; Shine and Dalgarno, 1974) was found at -6 to -10 bp, as reported by T&ok and Kondorosi (19811, but no canonical promoter sequence of statistical significance was found in the region from -125 to + 1 bp, numbering from the putative translation initiation codon (data not shown). Complementation Analysis of nif: :Tn5 Insertions To determine whether the genes in 6.3 kb and 5.0 kb Fix-::TnS clusters are organized into operons, we performed complementation analysis between selected pairs of nif::Tn5 insertions. Tn5 normally causes polar mutations (Berg, 1977); therefore, complementation analysis between different nit :Tn5 insertions can establish the boundaries of transcription units, but not the boundaries of genes within operons. The strategy we adopted to perform complementation analysis was to: first, construct several mobilizable plasmids, each containing a different nif::Tn5 insertion on a large Barn HI or Cla I nif region fragment; second, conjugate these recombinant nif region plasmids into a variety of nif::Tn5 R. meliloti strains; and finally, assay the resulting merodiploid R. meliloti strains for ability to fix nitrogen after establishment of symbiosis. Because this analysis was performed in a Ret+ R. meliloti strain, it was necessary to be able to distinguish complementation from marker rescue in the merodiploid strains.

For reasons described in detail below, Tn5 insertions 22, 7 and L5 were chosen for placement on plasmids that could be used in the complementation analysis. The cloning of Barn HI and Cla I fragments containing nit :Tn5 insertions 22, 7 and L5 in plasmids pBR322 or pHC79 is described in Figure 1 legend. Because pHC79 and pBR322 do not replicate in R. meliloti, it was necessary to reclone each nif region fragment onto the broad host-range vector pRK290 (Ditta et al., 1980) for mobilization into R. meliloti. The Barn HI fragments from plasmids pRmR8L2, pRmRT2 and pRmRQ42 were ligated into the pRK290 Bgl II site. The Cla I restriction fragments containing nit :Tn5 insertions from cosmids pRmRE2 and pRmRD5 were recloned in pRK294, a derivative of pRK290, containing a Cla I cloning site (see Experimental Procedures). Two additional plasmids, pRmR297 and pRmR2922, containing Tn5 insertions 7 and 22 respectively, cloned on the 3.9 kb nif region Eco RI fragment inserted into the Eco RI site of pRK290, were also constructed. Each of the pRK290-derived plasmids described above (see Table 1 and Figure 1) containing nif region Cla I, Barn HI or Eco RI restriction fragments with one of the three nif::Tn5 insertions, were transferred by conjugation into 18 different nit :Tn5 R. meliloti strains for complementation analysis. Each R. meliloti strain containing a genomic nit :Tn5 insertion and a different plasmid-borne nif: :Tn5 insertion was inoculated onto alfalfa seedlings and after 3-4 weeks, whole plants

Cdl 556

were assayed for the ability to reduce acetylene (Ruvkun and Ausubel, 1981). The results of this complementation analysis are summarized in Figure 3. Plasmid pRmR29E2, containing Fix+: :Tn5 insertion 22 on the Cla I fragment, which spans the entire nif all region surveyed, complemented genomic Fix-: :Tn5 insertions. Similarly, plasmid pRmR29T2, which begins at Fix+: :Tn5 insertion 22 and spans the 6.3 kb Fix-::Tn5 cluster shown in Figure 3, complemented all Fix-::Tn5 mutations in the 6.3 kb cluster. For both pRmR29E2 and pRmR29T2, the level of acetylene reduction by alfalfa nodules containing merodiploid R. meliloti strains was about 10% that of wildtype strains. This level of acetylene reduction could be due to instability of the pRK290 recombinant plasmids in bacteria in nodules (S. R. Long, unpublished observations). In contrast, plasmid pRmR298L2, which has the same left (Figure 3) Barn HI site end point as pRMR29T2, but which lacks the N-terminal region of Rm nifH, did not complement any Fix-: :Tn5 insertions in the 6.3 kb cluster. A similar result was obtained with plasmid pRmR29D5, which contains Fix-::Tn5 insertion 7 (located in Rm nifH) on the cloned Cla I fragment. These data are consistent with the interpretation that plasmid pRmR29T2 contains an intact 6.3 kb transcription unit that begins to the left of Tn5 insertion 22 and ends between Tn5 insertion A5-2 and A4-3. The fact that plasmids pRmR298L2 and pRmR2922 do not complement any Fix-::Tn5 mutations tested suggests that they do not contain any complete transcription units in which we have constructed genomic nit :Tn5 insertions. The trivial explanation that the lack of complementation with plasmids pRMR2922, pRMR298L2 and pRMR29D5 was due to segregation of these plasmids in the nodule was ruled out by extraction of DNA from the mature nodules and by showing, with Southern blot analysis, that the plasmidborne nif sequences were present (data not shown). The orientation of insertion of the nif region Barn HI fragments in the vector pRK290 was determined for plasmids pRmR29T2 and pRmR298L2 and was found to be the same. In addition, plasmids with the nif region Barn HI fragments inverted in the vector relative to pRmR298L2 and pRmR29T2 yielded the same complementation results (data not shown). These results argue that the observed complementation of nif::Tn5 mutations by pRmR29T2 was not due to transcription from a vector promoter. Therefore, we conclude that the R. meliloti nif region Barn HI fragment on pRmR29T2 contains the promoter for the nifHD“K” transcription unit. The genomic Tn5 insertion A.52, which overlaps pRmR29T2, yields a Fix’ phenotype. We conclude that this insertion is distal to the end of the nifHD”K” transcription unit, a result consistent with the conclusion that pRmR29T2 contains the complete transcription unit. This transcription unit is 6.3 f 0.2 kb long

and contains Rm nifH, Rm nifD, Rm nifK and coding capacity for another protein up to 30 kd in size. It is unlikely that the Nif+ phenotypes observed in merodiploid strains with genomic Fix-: :Tn5 insertions and plasmids pRmR29T2 or pRmR29E2 are due to recombinational rescue, for the following reasons. First, plasmid pRmR298L2 did not “complement” any Fix-::Tn5 mutations, even though it should be capable of recombinational rescue of nif::Tn5 mutations that it overlaps. Second, plasmid pRmR2922, which contains the nifH promoter but not genes distal to Rm nifD, also did not complement any Fix-::TnS mutations that it overlaps. Finally, plasmid pRmR29D5, which contains Fix-: :Tn5 insertion 7 on the nif region Cla I fragment, did not complement any Fix-::Tn5 insertions to the left of Tn5 insertion 7, but overlaps all genomic nit :Tn5 insertions tested. To characterize the transcription unit organization of the region to the right of the nif pHD “K” transcription unit, plasmids pRmR29Q42, pRmR29D5 and pRmR29E2 were conjugated into R. meliloti strains containing nif::Tn5 insertions to the right of Tn5 insertion 22. We found that plasmid pRmR29Q42 complemented to wild-type levels all mutations it overlaps. If we assume, as above, that recombinational rescue is not responsible for this observation, these data suggest that plasmid pRmRQ42 contains at least one intact transcription unit and that all Tn5 insertions constructed that it overlaps are included in this transcription unit. Fix-::Tn5 mutation 2239 is in a different transcription unit from that contained in plasmid pRmR29Q42, because pRmR29042 does not overlap this Tn5 insertion, but complements Tn5 insertions between Eco RI site (b) and Barn HI site (b). Fix-: :Tn5 insertion 2239 is complemented by plasmids pRmR29D5 and pRmR29E2, suggesting that these plasmids contain an additional intact transcription unit to the right of Barn HI site (b). Discussion Transposon Tn5 mutagenesis of 14 kb of the R. meliloti genome adjacent to the genes for nitrogenase resulted in the isolation of 31 Fix-: :Tn5 mutations. All of the Tn5 insertions constructed (except two; see below) yielded either a completely wild-type or a completely Fix- phenotype, indicating that, in general, Tn5 insertion results in a nonleaky Fix- mutation. Tn5 insertions P20 and Pl 1 were located in the intergenic region between Rm nifH and Rm nifD (based on DNA sequence analysis) and, therefore, did not insertionally inactivate either gene. The low level of acetylene reduction in these strains most likely results from incomplete polarity of these Tn5 insertions on Rm nifD. The locations of Rm nifD and Rm nifH relative to mapped restriction sites were determined from DNA sequence comparisons with K. pneumoniae nifH and

Genetics 557

of Symbiotic

Nitrogen

Fixation

nifD and were correlated exactly with the locations of Fix-: :Tn5 insertions in this region (see Figure 3). The direction of transcription of Rm nifD and Rm nifH is from right to left in Figure 3, because the “bottom” DNA strand translated into an amino acid sequence homologous to the K. pneumoniae nitrogenase Fe and MoFe proteins. Based on these results, we expected the promoter for Rm nifH to be located in the 400 bp region between the initiation codon of Rm nifH and Fix+: :Tn5 insertion L5. However, a search of the DNA sequence 5’ to Rm nifH up to Bgl II site (b) revealed no evidence of statistically significant canonical promoter sequences (Rosenberg and Court, 1979). This result is not surprising, because the canonical Pribnow box was derived by comparison of DNA sequences of promoters primarily from E. coli genes, which may not be generally used by all procaryotic species. The transcription initiation point of nifH has been determined to be 70 bp upstream from the translation initiation codon by the Sl-nuclease mapping method of Berk and Sharp (1978) using RNA isolated from nodule bacteroids (V. Sundaresan, J. D. G. Jones and F. M. Ausubel, in preparation). The data in Figure 3 show that not only is the DNA sequence of the genes nifH, nifD and nifK conserved between R. meliloti and K. pneumoniae, but their transcriptional organization is also the same. In contrast, in Anabaena 7120, the nif gene sequences of nifH and nifD are separated by 11 kb from nifK and, therefore, are probably in different transcription units (Mazur et al., 1980; Rice, 1981). The Rm nifHDK transcription unit also has space for a gene coding for a protein of molecular weight 30 kd. In K. pneumoniae there is a protein of 24 kd coded for by nifY located distal to nifK in the same transcription unit (Puhler and Klipp, 1980). The transcription unit to the right of the .nifHDK transciption unit was defined by plasmid pRmR29Q42, which complements all genomic nit :Tn5 insertions to the left of Barn HI site (b) and to the right of Eco RI site (b). Therefore, this plasmid contains at least one intact transcription unit, at most 4.2 kb long, bounded on the left by Fix+: :Tn5 insertion VII2 and on the right by Barn HI site (b). The direction of transcription of this region is not known. Because Tn5 insertion 2239, located 0.5 kb to the right of Barn HI site (b), is not polar on the region to the left of Barn HI site(b), this insertion defines another transcription unit. This Fix-: :Tn5 mutation is complemented by plasmids pRmR29E2 and pRmR29D5, which extend to the right to the Cla I site located 3.7 kb to the right of Barn HI site(b). Therefore, this region of 3.7 kb contains at least one additional intact nif transcription unit. The fact that plasmid pRmR29E2 can complement all Fix-: :Tn5 mutations tested shows that none are dominant Fix- mutations. It is not known whether the 1.6 kb Fix+: :Tn5 cluster contains any genes at all. All of the strains containing

Tn5 insertions in this region are prototrophic. It is possible that the 1.6 kb region could contain genes involved in regulation of symbiotic nitrogen fixationthat is, a gene for a repressor of nif gene expression. None of the nit :Tn5 insertions showed any defects in ability to form nodules nor any gross defects in nodule morphology. There is evidence that both nitrogenase and nodulation genes exist on the same large indigenous plasmid in R. meliloti. Banfalvi et al. (1981) and Rosenberg et al. (1981) have reported a spontaneous 80 kb deletion of a 500 kb “mega” plasmid in R. meliloti strain 41 that results in simultaneous loss of ability to nodulate alfalfa and loss of DNA hybridization to nif plasmids pRmR2 and pRmR3. R. meliloti strain 1021 also contains the 500 kb megaplasmid on which the nif genes are located (W. J. Buikema, W. Szeto and S. R. Long, personal communication). After random Tn5 mutagenesis of strain 1021, a nodulationdefective mutant was isolated that has recently been shown to contain a single Tn5 insertion into an Eco RI fragment located 23 kb to the left (as in Figure 3) of Rm nifH (Long and Ausubel, 1982). Experimental

Procedures

Bacterial Strains and Media R. meliloti strains used in this study are listed in Table 1. The medium, LB (1% Bacto-tryptone. 0.5% yeast extract and 0.5% pH 7.2) was used for growing both E. coli and Ft. meliloti strains was solidified with 1.5% agar. Alfalfa seedlings (variety Iroquois) grown on a nitrogen-free medium, as described by Ruvkun Ausubel (1981). E. coli strains HBlOl and MM294 have also described by Ruvkun and Ausubel (1981).

same NaCI. and were and been

Construction of Plasmids and Tn5 Mutagenesis Procedures for isolating plasmid DNA, for digesting DNA with restriction endonucleases, for ligating DNA with phage T4 DNA ligase and for transforming E. coli strain HBlOl with plasmid DNA have been described by Ruvkun and Ausubel(1980.1981). Restriction enzymes were purchased from Bethesda Research Laboratories and were used according to the manufacturer’s instructions. Multicopy recombinant plasmids in E. coli strain HBlOl were mutagenized with X::TnS as described by Ruvkun and Ausubel (1981). To transfer a Tn5 containing R. meliloti Eco RI fragment from pACYC184 to pRK290. the pACYCl84derived plasmids were digested with Eco RI and ligated to EGO RI-digested pRK290, as described by Ruvkun and Ausubel(1981). Plasmid pRK294 was constructed by ligation of Barn HI-digested pACYCl84 (Chang and Cohen, 1978) into Bgl II-digested pRK290; transformation of this mixture into E. coli; mobilization to another E. coli strain: and selection for Tc’ (10 gg/ml and Cm’ (20 as/ml) colonies. The resulting plasmid pRK294 contains the single Cla I site of pACYCl84. To select strains in which Tn5 (carried on a pRK29Oderived plasmid) had recombined into the R. meliloti genome by homologous recombination, R. meliloti strains containing pRK290-nif::TnS plasmids were conjugated with E. coli strain 2124, which contains the IncPGm’Spc’ plasmid pPH1 JI (Hirsch, 1978). Because pPH1 JI and pRK290 are incompatible, selection for Sm’ (200 pg/ml) Gm’ (70 rg/ ml) Nm4 (20 pa/ml) R. meliloti transconjugants yields strains in which Tn5 had recombined (or transposed) into the R. meliloti genome. To identify strains in which Tn5 had recombined into the genome as the result of a double homologous recombination event, Gm’ Nm’ Tc’ transconjugants were purified, total DNA was isolated from these strains and the DNA was subjected to hybridization analysis with use of the Southern gel transfer technique (Southern. 1975). 32P-labeled

Cell 558

DNA probes were scribed by Ruvkun

prepared by a nick translation and Ausubel. 1981).

procedure

as de-

Complementation Analysis Merodiploid R. melliloti strains were constructed by conjugating pRK290-derived plasmids carrying Tn5 insertions from E. coli strain MM294 to R. meliloti Fix- mutant strains as described above. Str’ (200 pg/ml) Tc’ (5 cg/ml) Nm’ (20 pg/ml) transconjugants were purified and eight individual colonies from each cross were inoculated into 3-7 day old alfalfa seedlings in 18 X 150 mm test tubes. Afler 3-4 weeks of growth at 27’C in a 12 hr daylight regime, nitrogenase assays on whole plants in test tubes were performed as described by Ruvkun and Ausubel(1981). DNA Sequencing Plasmid pRmR2 was digested with Xho I and the 730 bp Xho I fragment was eluted from a 5% acrylamide gel and labeled at the 3’ end with ‘*P-a-NTPs with DNA polymerase I Klenow fragment or at the 5’ end with 32P~ATP and T4 kinase. After DNA strand separation by acrylamide electrophoresis. the DNA sequence of each strand was determined by the technique of Maxam and Gilbert (1980). The Hind Ill site(b). Sal I site (a) and Bgl II site (b) of pRmR2 were also labeled at the 3’ ends with 32Pw-NTP with the Klenow fragment of DNA polymerase I. The DNA sequence was determined on all of these fragments after each fragment was recut with the appropriate restriction enzyme and separated by electrophoresis. Sequence comparisons were carried out on the Stanford SUMEX:AIM facility by using the SEQ program of D. Brutlag. P. Friedland and J. Clayton. Acknowledgments The authors would like to thank V. Corbin for the construction of plasmid pRmRQ6 and the restriction map of pRmR8L2, R. Hyde for preparation of the manuscript, the late F. Lang, to whom this paper is dedicated, for suggestions on reading DNA sequencing films, the Stanford SUMEX facility for DNA sequence analysis computer programs and A. Friedman for packaging cosmid clones. This work was supported by a National Science Foundation grant and a United States Department of Agriculture grant awarded to F. M. A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

25, 1982;

revised

March

mutants in Rhizobium leguminosarum by insertion of transposon into a transmissable plasmid. Mol. Gen. Genet. 178, 185-l 90.

Tn5

Chang, A. C. Y. and Cohen, S. N. (1978). Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from P15A cryptic miniplasmid. J. Bacterial. 134, 1141-l 156. Collins, J. and Hohn. B. (1978). Cosmids: a type of plasmid gene cloning vector that is packageable in vitro in bacteriophage lambda heads. Proc. Nat. Acad. Sci. USA 75, 4242-4245. Ditta, G., Stanfield. S.. Corbin, 0. and Helinski. D. (1980). Broad host range DNA cloning sytem for gram negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Nat. Acad. Sci. USA 77, 7347-7351. Hanahan. D.. and Meselson. M. (1960). colony density. Gene 10, 63-67. Hirsch,

P. R. (1978).

Ph.D. thesis,

University

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screening

at high

of East Anglia. England.

Hirsch, P. R., van Montagu. M.. Johnston, A. W. B.. Brewin. N. J. and Schell. J. (1980). Physical identification of bacteriochinogenic, nodulation. and other plasmids in strains of Rhizobium leguminosarum. J. Gen. Microbial. 120, 403-412. Hohn. B. (1975). DNA as substrate for packaging lambda in vitro. J. Mol. Biol. 98, 93-108.

into bacteriophage

Isreal. D. W.. Howard, R. L.. Evans, H. J. and Russel. S. A. (1974). Purification and characterization of the molybdenum-iron protein component of nitrogenasa from soybean bacteroids. J. Biol. Chem. 249, 500-508. Johnston, A. W. B., Beynon. J. L., Buchanan-Wollaston, A. V.. Setchell. S. M.. Hirsch, P. R. and Beringer. J. E. (1978). High frequency transfer of nodulating ability between strains and species of Rhizobium. Nature 276, 634-636. Jorgenson, restriction resistance.

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Krol, A. J. M., Hontelez, J. G. J.. van den Boa.. R. C. and van Kammen. A. (1980). Expression of large plasmids in the endosymbiotic form of Rhizobium leguminosarum. Nucl. Acids Res. 8, 4337-4347. Long, S. R.. and Ausubel. F. M. (1982). Cloning of Rhizobium nodulation genes by direct complementation of Nod- mutants. in press. Maxam. A. and Gilbert, base specific chemical

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