Proc. Nati Acad. Sci. USA Vol. 80, pp.

3005-3009, May 1983 Genetics

Organization and expression of Rhizobium meliloti nitrogen fixation genes(nif gene/Tn5 mutagenesis/nuclease SI mapping)

DAVID CORBIN, LESLIE BARRAN*, AND GARY DITTA
Department of Biology, University of California at San Diego, La Jolla, California 92093

Communicated by Donald R. Helinski, February 9, 1983 ABSTRACT The boundaries of a nif gene cluster in Rhizobium meliloti were determined by Tn5 mutagenesis. These genes are clustered within a 14- to 15-kilobase (kb) region that includes the nitrogenase structural genes. Sequences within 10 kb on either side of this region are devoid of symbiotically essential -gene function. RNA blot analysis identified a 5- to 6-kb transcript that corresponds to the nitrogenase structural gene operon. The 5' end of this transcript and its polarity were determined by nuclease Si mapping. The 5' end of another nif transcript was also identified by nuclease SI mapping. The promoter regions for these two nif transcripts control transcription in divergent directions and are separated by 1.9 kb of symbiotically unessential DNA. One Tn5 insertion within the nitrogenase operon did not create a polar mutation. The implications of this finding and the overall emerging picture of the genetic organization of this nif region are discussed.
genes other than nitrogenase have not been determined in Rhi-

zobium. In this report, we describe experiments in which transposon mutagenesis was used to define the limits of this cluster of Rhizobium meliloti nif genes. We show that the region is limited to a 14- to 15-kb segment of the genome.surrounding the nitrogenase genes. In addition, we have defined two-nif promoter regions by mapping the 5' ends of two divergently transcribed RNAs. One of these promoters controls the synthesis of a 5- to 6-kb transcript that covers the entire nitrogenase structural gene
region.

Gram-negative soil bacteria of the genus Rhizobium have the unique ability to form nitrogen-fixing nodules on the roots of leguminous plants. This is a complex process involving recognition and invasion of the appropriate legume by the bacteria, stimulation of plant cell division, multiplication and differentiation of bacteria within root cortical cells into morphologically altered forms called bacteroids, and the reduction of atmospheric nitrogen to ammonia, which is then assimilated by the plant (1, 2). Mutants of Rhizobium that are defective at various stages of symbiotic development may be phenotypically categorized as either Nod- or Fix- (3, 4). Nod- mutants are blocked prior to nodule meristem induction so that nodules are not formed. Fixmutants form visible nodules, but these nodules do not fix enough nitrogen to support normal plant growth. In some rhizobia, Nod and Fix genes are closely linked and reside on large indigenous plasmids (5-8). Fix genes have been shown to be clustered around a region of DNA that encodes the three structural polypeptides of the key nitrogen fixation enzyme, nitrogenase (9, 10). Preliminary characterization of this symbiotically essential gene cluster has suggested that there are several operons (9, 10) and at least some of the genes are transcribed only during symbiosis (9). This situation is similar to that found in the well-studied nitrogen-fixing enteric bacterium Klebsiella pneumoniae in which 17 nif genes, including nitrogenase, are specifically expressed under nitrogen-limited anaerobic growth (11). These genes are tightly clustered in seven or eight operons along a 24-kilobase (kb) segment of the genome and encode enzymes involved in the biosynthesis and processing of nitrogenase peptides, synthesis of an iron/molybdenum cofactor necessary for nitrogenase activity, transport of electrons to nitrogenase, and regulation of the operons (see ref. 12 for review). Biochemical functions for nif
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MATERIALS AND METHODS Strains and Plasmids. R. meliloti 102F34 was provided by Nitragin, Milwaukee, WI. The Escherichia coli strains used were HB101 (pro leu thi lacy endoI recA hsdR hsdM Strr) and HB101: :Tn5. Recombinant plasmids from our R. meliloti gene bank carry large segments of the nif region cloned into the broad host range cloning vector pRK290 (9, 13). Cloned sequences flanking those previously obtained were identified by overlapping homology. Tn5 Mutagenesis. Tn5 insertions into recombinant plasmids were obtained as described (14) except that HB101: :Tn5 was the source of the transposon. Mutated sequences were recombined into the Rhizobium genome by a marker-exchange procedure (15) as described (9). The fidelity of the exchange events was confirmed by probing Southern blots of restriction enzyme digests of the mutated genomic DNAs with cloned homologous DNA lacking Tn5. In every case, the restriction pattern was altered as expected for a Tn5 insertion at the mapped position. The symbiotic phenotype of each mutated Rhizobium strain was determined by monitoring plant growth and acetylene reduction (16) as described (9). Nodule RNA Isolation. Nodules were obtained from alfalfa roots (Medicago sativa cv. Moapa) 4-6 wk after germination, and 500 mg was pulverized in liquid nitrogen with a mortar and pestle. The resulting powder was transferred to 5 ml of 0.5 M mannitol/20 mM NaOAc, pH 5.5/1 mM EDTA containing 0.1% diethylpyrocarbonate. This suspension was homogenized at low speed at 40C in a Potter-Elvehjem tissue grinder. The homogenate was immediately poured into 10 ml of 0.5 M mannitol/20 mM sodium acetate, pH 5.5/1 mM EDTA/3% NaDodSO4 that had been preheated to 1000C. The mixture was heated at 100'C for 2-5 min, until the lysate became nonviscous. It was then extracted with phenol and CHCl3/isoamyl alcohol (24: 1), and RNA was precipitated as described (9). Residual DNA was removed by passing the preparation through
Abbreviations: kb, kilobase(s); Mops, 3-(N-morpholino)propanesulfonic acid; bp, base pair(s). * Present address: Chemical and Biological Research Inst., Agriculture Canada, Ottawa, Ont., KlAOC6.

3005

DNA used to probe RNA or Southern blots was labeled with 32P by nick-translation (18). The significance of the other bands is not clear. USA 80 (1983) sertions distributed throughout a 35-kb segment of the genome that encompasses the nitrogenase structural genes (Fig. end-labeled DNA fragments were hybridized to total nodule RNA by suspending the nucleic acids in 20 .to 15kb region yielded only Fix' phenotypes.l of 20 mM 3-(N-morpholino)propanesulfonic acid (Mops). One lane was stained with ethidium bromide to visualize the ribosomal RNAs that served as molecular size standards (19). we used the nuclease S1 mapping technique to locate the ends of the RNA on the physical map. distinguishable transcripts were notdiscussed above 2. The size of this protected fragment is the distance from the end of the RNA transcript to the end of the original fragment. Hybridization Probes. a fragment of 1.000 Ci/mmol.9-kb fragments prior to hybridization also resulted in the protection of a 1. Three strains. 215. 10) revealed a prominent transcript of 5-6 kb that we presume is the major nitrogenase transcript and a number of less intense minor bands (Fig. recrystallized three times)/0. The remainder of the gel was washed for 5 min with H20 and for 5 min with 3. pH 8/5 mM NaOAc/1 mM EDTA. 305. lanes a and b).1-kb Bgl II fragment to the right of the nitrogenase genes. nitrogenase activity. and 505) that span 1. Nuclease S1 Mapping.000 Ci/mmol) and the Klenow fragment of DNA polymerase I.ul of hybridization buffer [80% formamide (Baker. 1 shows the fragments used in the analysis and summarizes the results. and 264 would be expected to define additional smaller regions between adjacent operons. These three Fix+ insertions (nos. Nuclease Si Mapping Applied to the Nitrogenase Operon. nifD. and 220) mapped within 150 base pairs (bp) of each other.to 15-kb segment of DNA that includes the nitrogenase genes. leaf color was noticeably greener than uninoculated controls. Cleavage of this labeled 2.and 0. When the 2. The largest such region that is apparently devoid of essential nif functions is defined by a group of five Fix' insertions (nos. Twenty-eight insertions that mapped as far as 10 kb to the right or the left of this 14. Hybridization to the labeled 3. 1 Ci = 37 GBq) by using polynucleotide kinase. Fix+ insertions 326. formed small nodules that were detectably pink but had less than 10% of the nitrogenase activity of normal nodules. RNA blots of total nodule RNA were probed with two 32P-labeled DNA fragments that constitute a contiguous 11. pH 8/5 mM NaOAc/1 mM EDTA/ 50% formamide (deionized with Amberlite MB-1 ion exchange resin)/6% formaldehyde and heated at 60'C for 5 min. None of the insertions were in genes involved in early stages of root infection or nodule formation since all 70 Tn5-containing strains induced the formation of nodules on alfalfa roots. lanes c were also and d). When a RNA transcript that overlaps the labeled end of one strand of the fragment hybridizes to the probe. RESULTS Tn5 Mutagenesis Completely Defines a Discrete nif Gene Cluster.1 kb of DNA (Fig. The remaining 37 strains were designated Fix'. They formed nodules that were indistinguishable from wild type on the basis of size. Fragments to be 5'-end labeled were treated with calf intestinal alkaline phosphatase to remove 5' phosphate residues and then labeled with [y-32P]ATP (5. and ability to support plant growth. formed small white nodules that completely lacked nitrogenase activity as measured by the acetylene reduction assay.1kb HindIII fragment was 5' end labeled and used in S1 mapping experiments. 32P-Labeled bacteriophage A DNA digested with Cla I or HindIII and bacteriophage 4X174 replicative form DNA digested with Hae III served as molecular size standards. 1). 3. Nine Fix+ Tn5 insertions mapped within the nif gene cluster a nitrocellulose column (9) or by pelleting it through a CsCl solution (17). We used site-directed Tn5 mutagenesis (15) to determine the filll extent of a previously identified cluster of nif genes in R. 2. Fig.6kb Bgl II nitrogenase gene fragment (9. 303. A fully protected fragment indicates that one strand of the entire fragment has been transcribed.3% xylene cyanol was added. color. All 33 insertions that caused partial or complete inability to fix nitrogen mapped within a 14. When identical blots were probed with the labeled 8. denaturing the double-stranded probe by heating the mixture at 73°C for 10 min. Sci. The digestion products were resolved by electrophoresis through alkaline agarose gels (24) and visualized by autoadiography. while use of a 3'-end-labeled probe will map 3' ends. 1). Transcripts from this region are. The suspension was cooled on ice.0 M NaCl/0. and then immediately transferring it to 53-560C for hybridization.3% bromphenol blue/0. 292. 23).4 M NaCI/50 mM Pipes. Many of the minor bands observed. lanes a and b). After 6-8 hr. clearly observed (Fig. Even though plant growth was severely reduced. less abundant or more degraded than the nitrogenase transcript. These blots were hybridized to nick-translated probes as described (9). these Fix' insertions presumably define regions between operons. Five micrograms of total nodule RNA was suspended in 20 A.2. Use of a 5'-end-labeled probe will map 5' ends of transcripts. Fragments with 5' overhangs were 3'end labeled by using [a-32P]dNTPs (3. 1-kb fragment (lanes c and . Thirty strains. the samples were diluted to 220 . nmliloti (9). 219. and nifKare within a single operon of about 6 kb in R.ul by addition of ice-cold S1 buffer and digested with 30 units of nuclease S1 (Bethesda Research Laboratories) for 30 min at 37°C. 7-kb segment of the nif gene cluster. RNA Blot Analysis Identifies the Nitrogenase Transcript. 1-kb HindIII'fragment with Bgl II into 1. These RNAs constitute the bulk of the RNA in such preparations and' always yield faint hybridization bands regardless of the probe used. In these cases. 304. The mutagenized strains were classified into three categories based on the appearance of the nodules and their ability to reduce acetylene and support plant growth. Acad. The direction of transcription of the fragment can be determined by cleaving the original labeled fragment into two distinguishable end-labeled fragments before hybridization and then determining which labeled end is protected. 312. and the sample was electrophoresed through a vertical 1% agarose/6% formaldehyde gel formed in 20 mM Mops. designated Fix-. however. pH 7/1 mM EDTA]. The three nitrogenase structural genes-nifH. 1). Natl. therefore. that portion of the fragment hybridized will be resistant to the single-strand-specific nuclease Si and yield a radioactive "protected" fragment after Si digestion. This technique has been described by Berk and Sharp (21) and allows the physical mapping of RNA transcripts on a specific DNA fragment. nmliloti (10).1 kb was protected (Fig.3006 Genetics: Corbin et al. Mutagenesis gave 70 independent Tn5 in- (Fig. To more thoroughly characterize transcription from this operon.3 M Na citrate and transferred to nitrocellulose by standard blotting techniques (20). Since Tn5 insertions normally create polar mutations (25). 218. appears to be an exception and is discussed in detail below. the plants grew no better than the uninoculated controls. In our experiments. Fix+ insertion 326. Four of the latter bands corresponded in po- sition to the plant and bacterial ribosomal RNAs that were visible in the ethidium bromide-stained gel (data not shown). RNA Blot Analysis. 2 ttl of 25% Ficoll/0. DNA fragments are end labeled and hybridized to RNA under conditions that allow RNA-DNA hybrids to form but prevent DNA-DNA hybridization (22. designated Fix+. Proc.

e and f. * a 2. Fix' phenotype. As shown in Fig.6 1. pneumoniae: region containing primarily nifH homology. The presence of a Fix' Tn5 insertion (Tn5 326) 1 kb downstream from the nitrogenase promoter raises the question of whether or not there could be another promoter immediately downstream of this insertion that maps within the nifH-nifD intergenic region (26). parts of fragments protected from S1 digestion. X. f. meliloti nif genes. Nuclease S1 mapping of nif transcripts.6-kb Bgi II nitrogenase gene fragment to RNA blots of total nodule RNA from 3.1-kb HindIMl faent. meliloti nif region. with transcription proceeding leftward into the nitrogenase genes.4Xr 1.4 2. R. Fix.0 0.1-kb Bgi II fagment to RNA blots of total nodule RNA from 3.7 2.4 3.9 x . c. a.2 a 21020 2 4 -25 2 7 i217 222fSztf-23 29231 221 -22 - Ni323gen4 se a Nitrogenase I*I a a 3.3 . In the absence of RNA processing. putative nif promoter regions.8 I 3. Physical map of the R.phenotype.9 FIG.6-kb Bgi II fragment indicates the region that is homologous to the nitrogenase genes of K. M. c and d. -43 5 30 I f23 4i 2. 3. d). USA 80 (1983) -202 296 3007 -29S -2219 206 -26f6g gfi5 310 312 303 - 305 256n iEi292 8. The bar above the 3.1-kb HindIII fragment dig with Bgl E.6 2. Hindl. 2. 1. for the nitrogenase transcript (RNA 1).4 306I 17 t533B II i|| || 1 1128 2II30931 Bg H R Bm I * 3. i andj. Lanes a.6 0. Lanes b.0 RA_ RNA 1 P2 A2PI - 1 -~~~~ E. P1. h. 2.0-kb Xho I fragment. the 5' end of this transcript therefore maps approximately 100 bp to the left of Bgl II site a.7 aI I 4. Numbered circles designate locations of specific Tn5 insertions: o.and 6-wk nodules. 3. e. a.7 0. region containing primarily nifD homology. their positions are marked in kb. Lanes: a and b.Genetics: Corbin et al.0. Restriction enzyme map: Bg.9 3. Bars designated 5' below the restriction enzyme map correspond to restriction enzyme fragments used for 5' nuclease S1 mapping: pointed ends indicate direction of transcription. dotted extensions ofthe bars show that transcription continues past arrows but a unique 3' end has not been mapped. P1 and P2. H. g and h. Fix' phenotype. Proc. Xho I. Nat.9 12.9- 3. 2. . 1.I 1.6 3. BamHI. The arrow indicates the position of the prominent nitrogenase transcript.1- sgo 2. andj show protected fragments resulting from hybridization with RNA and treatment with nuclease Si.! 2.8 8.3 a I I 4. Bgi H. FIG. Bm. RNA blot analysis of R.0- 1.3 a b I a 1. RNA 2 kb FIG.7 0. E2. and i show results of mock hybridization of probe without RNA or nuclease S1 treatment.6 .8 I 1. d. 1.11. g. niftranscripts deduced from S1 mapping experiments using 5'-end-labeled DNA fragments.and 6-wk nodules. Molecular size markers were ribosomal RNA molecules in the RNA preparation. RNA 1 and RNA 2. this 5' end defines a b c d the promoter region.. The horizontal lines represent the portions of DNA that were mapped for the respective enzymes.9-kb Xho I/EcoRI fiagment. Acad.0-kb Xho I fragment digested with EcoRI. 3. EcoRI. Lanes c and d: results of hybridization of the 32P-labeled 8. Lanes a and b: results of hybridization of the 3P-labeled 3. e.5- 3. Sizes of fragments are indicated below in kb. Sci. Nuclease S1 mapping analysis of the 5'a b c d e f g h i i 3.1 I 4. as expected.

when either the 2. USA 80 (1983) end-labeled 1.to 6-kb nitrogenase RNA in root nodules that is tran- as they are in K. strong full-fragment protection was observed (data not shown). The 1.4-kb fragment (lanes g and h). results summarized in Fig.7-kb Bgl II fragment or the 0. but they -map 20-30 kb downstream from the nitrogenase operon (27). Identification of a Second nif Promoter Region. indicating that transcription from P1 does continue through the nitrogenase genes. We have five region. meliloti. (10). we were unable to identify a unique 3' end.and 2.7-kb Bgl II fragment. However. Ruvkun et al. The 3. as does the nitrogenase operon in K.3008 Genetics: Corbin et al. (10). These genes are located within a 14.9 kb of DNA.2. The very faint band resulting from S1 mapping experiments. We have evidence from studies with two strains of R. there was very little full-fragment protection and a large collection of faint bands ranging in size from 3. However. mappedhave Fix' Tn5 insertions in thisinsertionsand Ruvkun et in the correal. also reported two scribed leftward from a promoter (P1) about 100 bp to the left of Bgl II site a (Fig. lanes i and j). Genetic data of Ruvkun and Ausubel (10) suggest that the right-hand boundary of this operon maps near the site of insertion of Fix' Tn5 292. (10) mapped eight strain Tn5R. A unique 3' end was not detected.4-kb Bgl II fragments (9).6 kb of DNA would be required to encode a nitrogenase complex of similar size in R. The reason for this is unknown.9-kb region between RNA 1 and RNA 2 does not contain genes that are essential for nitrogen fixation. None of the 70 Tn5 insertions described in this paper interfered with the early stages of nodule formation. In light of the above information supporting a single transcript for the nitrogenase genes. pneumoniae. complementation analysis of Tn5-mutagenized R. 1). and analysis of the 0.and 2.7-kb fragments formed by cleavage of the original fragment with EcoRI also indicated protection of a 2. Three nif operons were identified in that work. In addition. we observed strong full-fragment protection relative to several smaller protected fragments (data not shown).4-kb HindIII fragment was used in similar 5'-end mapping experiments to extend the analysis through the nitrogenase gene region. Fix+ of meliloti. Therefore. In the study of Ruvkun et al.0-kb HindIII fragment was used to map the 3' ends. meliloti indicated that the nitrogenase operon is about 6 kb in size and partial DNA sequence analysis indicated that the genes are transcribed in the order nif HDK Tn5 insertions near the nifH-nifD intergenic region that were weakly nonpolar.9 kb (data not shown). RNA blot analysis failed to reveal any unique transcripts derived from the symbiotically essential region to the right of the nitrogenase operon (see above). 1). However. which was confined to a 12-kb region corresponding approximately to that bounded by our Fix' Tn5 insertions 13 and 215. we were surprised to find a Tn5 insertion between nifH and nifD that did not significantly alter symbiotic nitrogen fixation (Tn5 326). as well as the inability to detect another prominent transcript by RNA blot analysis suggests that the abundance of intact RNA from this operon is lower than that of the nitrogenase operon. several faint smaller bands were detected. Very different results were obtained when the 4. This identifies another transcript (RNA 2) that originates from a promoter (P2) approximately 0. The size of the nitrogenase transcript obtained from RNA blots suggests that the 3' end of the transcript should be between Fix' Tn5 13 and Fix. Based on the estimated sizes of nitrogenase polypeptides from other species of Rhizobium (29-31).4-kb fragment (Fig.6 kb to the right of Xho I site a and is transcribed rightward at least as far as Xho I site b (Fig. the Another nif operon is separated from nitrogenase operon is transcribed in the by 1.3 kb between Tn5 insertions 264 and 299. We have not been able to detect unique 3' ends for either RNA 1 or RNA 2 although genetic studies (10) have established the approximate boundaries of each operon. we of another sponding region have detected transcription within this region in mature nodules by Southern blot hybridizations (unpublished data). meliloti that Tn5 insertions near the site of insertion of Tn5 326 can result in high levels of expression of a promoterless chloramphenicol acetyltransferase gene cartridge (28) inserted into the HindIII site near the nifH/nifD junction (unpublished data). Another possible reason for this nonpolarity could be that. coli. but the full extent of the nif cluster was not determined.to 15-kb region that is flanked on both sides by at least 10 kb of DNA that is symbiotically unessential. Natl Acad. Early nodulation genes have been identified in R. One possible reason for the strong nonpolarity of this particular Tn5 insertion is that under certain circumstances Tn5 is capable of promoting transcription of adjacent sequences. with transcription proceeding leftward as expected (data not shown.7-kb Xho I fragment was used to map the 3' end. Analysis of the 5'-end-labeled 3. We have now applied nuclease S1 mapping to this region and found that this approach is sensitive enough to detect the 5' end of at least one transcript. In this case. Other possible explanations for the Fix' phenotype of Tn5 326 are discussed below. we had previously shown that this region is transcribed in mature nodules by hybridizing total RNA labeled in vitro to Southern blots of the 8. indicating that the hybridization conditions were not permitting DNA-DNA probe rehybridization. Decreasing the RNA concentration in the hybridization mixture resulted in a concomitant decrease in intensity of all bands with no effect on the relative intensities. The data presented here extend the analysis of Ruvkun et al. Sci. We observed full-fragment protection and leftward polarity (data not shown. there might be a weak secondary promoter in the intergenic region.9 to 0.9-kb EcoRI/Xho I fragment that overlaps this point yields only full-fragment protection (Fig.the data suggest that transcription from P1 terminates somewhere near Fix' Tn5 13.3. although. This indicates that the hybridizations were in excess DNA and that detection of a unique 3' end was not prevented by displacement of the 3' ends from the probe by hybridization with a less-abundant read-through transcript. It should be noted that Berg et al. This suggests that detectable transcription from a promoter within the nifH-nifD intergenic region does not normally occur in 4. it seems unlikely that such genes are located in this cluster or within 10 kb on either side. If this . results summarized in Fig. lanes e and f).Tn5 502 within the 2. 1). when the 1. Mock hybridization without RNA did not protect fragments. 3. We have identified by physical analysis a single 5. meliloti genome to identify the boundaries of a large cluster of nif genes. Therefore. DISCUSSION We have used Tn5 mutagenesis of a 35-kb segment of the R. approximately 3. although we did not detect a5' end in this region by nuclease S1 mapping.0-kb Xho I fragment indicated protection of a 2. 1). This operon opposite direction from the nitrogenase operon. (25) have reported occasional weak nonpolarity of Tn5 insertions in the lacZ gene of E.to 6-wk nodules and that nitrogenase genes are expressed by transcription originating at P1. pneumoniae. meliloti. Considering the large number of insertions analyzed. 3. Proc. However. Our data show that there exists at least one additional operon of up to 2. a nitrogenase transcript of 5 to 6 kb could possibly encode other functions.9-kb HindIII fragment was 3' end-labeled and used in S1 mapping experiments.

. M. MacNeil. 20. 9 kb of which appears to be unessential for nitrogen fixation. Cannon. R. 5. 257. eds. L. Loenig. the nif region of R. M. & Studier. A. Leong. 7. F. 326-333. Sci. (1982)1 Bacteriol 149. A. 21. C. & Atherly. H. 5. G. W. Biophys. 22. A. Kassavetis. (1980) Mol Gen. 15. Berk. S. Newton. Kiss. (1976) Proc. J. R.. W. V. Newcomb. Finally. & Ausubel. 503-517. Ditta. all nif operons are transcribed from one strand of DNA (11. provide renewed transcription from their termini as hypothesized above. P. B.. E. Rigby. D. W.. A. Giles. & Ausubel. (1979) Biochim. P. B. W. (University Park Press. G. 1539-1552. U. E. 30. (1981) Mol. Hombrecher. G.. J. White. & Orme-Johnson. A. 348357. J. L. B. C. F. Natl. D. Sundaresan. (1980) in Nitrogen Fixation 2. S. Acta 371. F. R. Genet. E. M. M.. S. Long. J. Brewin. J. pneumoniae nif region span a 24-kb segment of DNA (32). 18. C. G. K. then some of the genes must be located in another region of the genome. 13. M. L. If genes analogous in function to all of the nif genes of Klebsiella are present in Rhizobium. Biol 98. 133-136. W. (1981)J Bacteriol 145. McDonell. N. G. & Geiduschek. 1129-1136. E. S. (University Park Press. Roberts. W. C. Long.. (1979) Mol Gen. Beringer. 16.. 6. W. 119-146. A. Burns.... & Orme-Johnson. M. E. Genet. (1981) Annu. Zhu. Gen. This leaves a maximum 12 to 13 kb of indispensible nif function in this region. 721-732. & Kondorosi. 85-93. The most striking difference at this time is that. G. (1977) Cell 12. M.. pneumoniae. Riedel. S. R. F. Acta 565. J.. J. P. 500-508. Prakash. W. 33). Thomas. pp. J. Simon. Buchanan-Wollaston. R. 4. Scott. 9. 1. V. V. 185-190. We are indebted to Dr. A. New York). W. E. 33. (1977)J Mol.. We gratefully acknowledge the excellent technical assistance of Marty Yanofsky and Patty Tooker. B. M. Natl. J. 28. F. (1982)J Biol Chem. M. R. W. Newton. & Sharp. M. & Dilworth. & Davis. & Johnston. R. B.. Rev. 32. F. M. Baltimore). F. 207235. Whiting. (1982) Gene 20. J. Banfalvi. (1975) 1 Mol. 184. Vincent. E. R. R. Acad. Genet. F. either it must be a negative one or the Tn5 insertions within the region. M.. Corbin. E. K. S. N. eds. I. A.. Dieckmann. A. A. Torok. 12. G. Ruvkun. & Ausubel. Baltimore).. A. Dusha. 365-378. 2. 9. while disrupting normal patterns of transcription. either it is unrelated to nitrogen fixation or it is possibly a negative regulatory element of the Rhizobium nif system. 355-365. Now that the size of the R. it is interesting to compare certain features with those of the K. fewer genes may be required in Rhizobium to elaborate a functional nitrogenase complex during symbiosis. Hardy. 178. P. Casey.. & Helinski. & Brill. eds. 318-325.. (1981) Mol Gen. & Russell. D. W. 103-129. meliloti nif gene cluster has been determined. 337-351. Hennecke. Berg. 19. 485-488. (1981) Mol Gen.. & Ausubel. R. USA 73. Ruvkun. 303-314. F. Weiss. Ausubel. 24. P. 23. Z. R. W. Ditta. 59-66. If transcription alone plays a regulatory role. (1968) J Mol Biol 38. K. & Davidson. 182. (1980) in Nitrogen Fixation 1. R. W. 26. in K. Helinski in whose laboratory this study was conducted. (1974) J Biol Chem. C. 439-446. T. 47-81. Donald R. 5711-5723. Rosenberg. 149. J. (Academic. Rhodes.. I. 107-114. A. (1980)1J Bacteriol 142. (1980) Proc. 110. & Brill. R.. E. Israel. 11. G. 29. C. while the 17 closely spaced genes of the K. E. Sakanyan. 249. W. & Lim. M. Evans. Acad. E. This work was supported by National Science Foundation Grant PCM 82-04730. T. 237-251.. Biol. Janssen. J. pp. Close. J. & Holsten. G.. (1981) Nucleic Acids Res. (1977)J Mol. & Casse-Delbart. Biophys. Southern. Riedel. Buikema. D. & Rodriquez. (1982) Cell 29. D. D. & Nuti.. 8588. J.. P. 31. & Helinski. 2294-2298. B. 14.Genetics: Corbin et al. A.. & Cannon. Biol 113. & Crossland. G. 3.. R. M. 174. 7347-7351. 1. 551-559. meliloti occupies only 14 to 15 kb. (1982)J Bacteriol. D. (1973) Soil Biol Biochem. Boistard. S. Ruvkun. . 27. D. On the other hand. (1977) Nucleic Acids Res. & Johnston. USA 80 (1983) 3009 region encodes a protein. J. Corbin. G.. 184. & Kondorosi. Ditta. P. Hirsch. 247-298. Genet. Microbiol 35. Koncz. W. Nati Acad. & Berg. M. Sci. H. 17. (1982) EMBOJ 1. (1981) Nature (London) 289. 4. L. (1982) Nature (London) 298. 221-228. The findings of two promoters that control divergent transcription in Rhizobium indicates substantial differences in the genetic organization of nif functions in these two nitrogen-fixing organisms. J. 8724-8730. 114-122. P. Proc. Howard. S. H. D. Schilperoort. & Helinski. F. Meade.. Genet. H. Denarie. H. H. pp. D. A. (1974) Biochim. 10.. pneumoniae nif gene cluster. Brewin. L. 25. Stanfield. Brown. R. S. M. G. Sci USA 77. (1981)J Bacteriol 145. A W. (1981) in Biology of the Rhizobiaceae. 8. & Ausubel.