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)

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

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.


This technique has been described by Berk and Sharp (21) and allows the physical mapping of RNA transcripts on a specific DNA fragment. The largest such region that is apparently devoid of essential nif functions is defined by a group of five Fix' insertions (nos. 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).9-kb fragments prior to hybridization also resulted in the protection of a 1. DNA fragments are end labeled and hybridized to RNA under conditions that allow RNA-DNA hybrids to form but prevent DNA-DNA hybridization (22. the plants grew no better than the uninoculated controls. and then immediately transferring it to 53-560C for hybridization. Three strains. 1). Fig. Acad. 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.4 M NaCI/50 mM Pipes. The digestion products were resolved by electrophoresis through alkaline agarose gels (24) and visualized by autoadiography. Nuclease S1 Mapping. 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. and 264 would be expected to define additional smaller regions between adjacent operons. When a RNA transcript that overlaps the labeled end of one strand of the fragment hybridizes to the 15-kb segment of DNA that includes the nitrogenase genes. pH 7/1 mM EDTA]. Proc.6kb Bgl II nitrogenase gene fragment (9. 1). Mutagenesis gave 70 independent Tn5 in- (Fig. 23). DNA used to probe RNA or Southern blots was labeled with 32P by nick-translation (18). Hybridization Probes. 2 ttl of 25% Ficoll/0. Even though plant growth was severely reduced.1-kb Bgl II fragment to the right of the nitrogenase genes. RNA Blot Analysis. and the sample was electrophoresed through a vertical 1% agarose/6% formaldehyde gel formed in 20 mM Mops. lanes c were also and d). These RNAs constitute the bulk of the RNA in such preparations and' always yield faint hybridization bands regardless of the probe used. 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. a fragment of 1. 1-kb HindIII'fragment with Bgl II into 1. The remainder of the gel was washed for 5 min with H20 and for 5 min with 3. the samples were diluted to 220 . Fix+ insertions 326. 215. When identical blots were probed with the labeled 8. denaturing the double-stranded probe by heating the mixture at 73°C for 10 min. 3.ul of hybridization buffer [80% formamide (Baker.3% bromphenol blue/0. distinguishable transcripts were notdiscussed above 2. Use of a 5'-end-labeled probe will map 5' ends of transcripts. The significance of the other bands is not clear. Since Tn5 insertions normally create polar mutations (25).000 Ci/mmol) and the Klenow fragment of DNA polymerase I. color.000 Ci/mmol.1 kb was protected (Fig. and nifKare within a single operon of about 6 kb in R. The suspension was cooled on ice. nitrogenase activity. Natl. 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-kb fragment (lanes c and . lanes a and b). Thirty strains. 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. nifD. nmliloti (9). The size of this protected fragment is the distance from the end of the RNA transcript to the end of the original fragment. and 220) mapped within 150 base pairs (bp) of each other. These blots were hybridized to nick-translated probes as described (9). 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. Nuclease Si Mapping Applied to the Nitrogenase Operon. 312.l of 20 mM 3-(N-morpholino)propanesulfonic acid (Mops).0 M NaCl/0. Many of the minor bands observed. 7-kb segment of the nif gene cluster. and 505) that span 1. formed small nodules that were detectably pink but had less than 10% of the nitrogenase activity of normal nodules. 1 Ci = 37 GBq) by using polynucleotide kinase. we used the nuclease S1 mapping technique to locate the ends of the RNA on the physical map.3006 Genetics: Corbin et al. formed small white nodules that completely lacked nitrogenase activity as measured by the acetylene reduction assay. and ability to support plant growth. They formed nodules that were indistinguishable from wild type on the basis of size. 218. Five micrograms of total nodule RNA was suspended in 20 A. RNA Blot Analysis Identifies the Nitrogenase Transcript. lanes a and b). The three nitrogenase structural genes-nifH. Hybridization to the labeled 3. RESULTS Tn5 Mutagenesis Completely Defines a Discrete nif Gene Cluster. Cleavage of this labeled 2. less abundant or more degraded than the nitrogenase transcript. while use of a 3'-end-labeled probe will map 3' ends. 292. In these cases. Sci. 219. USA 80 (1983) sertions distributed throughout a 35-kb segment of the genome that encompasses the nitrogenase structural genes (Fig. designated Fix+.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. These three Fix+ insertions (nos. nmliloti (10). leaf color was noticeably greener than uninoculated controls. Fix+ insertion 326.1 kb of DNA (Fig. pH 8/5 mM NaOAc/1 mM EDTA. 1 shows the fragments used in the analysis and summarizes the results. All 33 insertions that caused partial or complete inability to fix nitrogen mapped within a 14. 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. To more thoroughly characterize transcription from this operon.3 M Na citrate and transferred to nitrocellulose by standard blotting techniques (20). these Fix' insertions presumably define regions between operons. 2. 305. When the 2. appears to be an exception and is discussed in detail below.3% xylene cyanol was added. designated Fix-. In our experiments. 303. RNA blots of total nodule RNA were probed with two 32P-labeled DNA fragments that constitute a contiguous 11. The remaining 37 strains were designated Fix'. A fully protected fragment indicates that one strand of the entire fragment has been transcribed. 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. end-labeled DNA fragments were hybridized to total nodule RNA by suspending the nucleic acids in 20 . 304. therefore. clearly observed (Fig. 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. Twenty-eight insertions that mapped as far as 10 kb to the right or the left of this 14.1kb HindIII fragment was 5' end labeled and used in S1 mapping 15kb region yielded only Fix' phenotypes. One lane was stained with ethidium bromide to visualize the ribosomal RNAs that served as molecular size standards (19). Transcripts from this region are. 1). Fragments with 5' overhangs were 3'end labeled by using [a-32P]dNTPs (3.2. however.and 0. After 6-8 hr. recrystallized three times)/0.

9- 3. Fix.9 x . pneumoniae: region containing primarily nifH homology.3 .1- sgo 2. c and d. Lanes c and d: results of hybridization of the 32P-labeled 8.4 2.I 1.6 1. -43 5 30 I f23 4i 2. Bgi H. Nuclease S1 mapping of nif transcripts. a. putative nif promoter regions. Lanes a.6 2.8 I 1. RNA blot analysis of R. e. Nuclease S1 mapping analysis of the 5'a b c d e f g h i i 3.9-kb Xho I/EcoRI fiagment.0 RA_ RNA 1 P2 A2PI - 1 -~~~~ E.! 2. Xho I. Sci. 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.9 FIG. g. .0.and 6-wk nodules.7 0.8 I 3.6 3. meliloti nif region. Molecular size markers were ribosomal RNA molecules in the RNA preparation.7 2. Lanes b. 1.1-kb HindIII fragment dig with Bgl E. Proc. Sizes of fragments are indicated below in kb. this 5' end defines a b c d the promoter region. M. g and h. c.11. The horizontal lines represent the portions of DNA that were mapped for the respective enzymes.and 6-wk nodules. with transcription proceeding leftward into the nitrogenase genes.7 0. e and f.6 0. P1.0-kb Xho I fragment digested with EcoRI. 2. 3. Physical map of the R. Hindl. * a 2. h. In the absence of RNA processing. Fix' phenotype.2 a 21020 2 4 -25 2 7 i217 222fSztf-23 29231 221 -22 - Ni323gen4 se a Nitrogenase I*I a a 3. The bar above the 3. 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). Nat.7 aI I 4.0- 1.Genetics: Corbin et al.1-kb Bgi II fagment to RNA blots of total nodule RNA from 3. 3. Lanes a and b: results of hybridization of the 3P-labeled 3. niftranscripts deduced from S1 mapping experiments using 5'-end-labeled DNA fragments.5- 3. meliloti nif genes. d. R. Acad. dotted extensions ofthe bars show that transcription continues past arrows but a unique 3' end has not been mapped. P1 and P2. region containing primarily nifD homology.3 a b I a 1. and i show results of mock hybridization of probe without RNA or nuclease S1 treatment. 2.4 3. f. RNA 1 and RNA 2. E2. for the nitrogenase transcript (RNA 1). USA 80 (1983) -202 296 3007 -29S -2219 206 -26f6g gfi5 310 312 303 - 305 256n iEi292 8.3 a I I 4. their positions are marked in kb. Bm.6 . d). X. 1.4Xr 1. i andj. BamHI. e. Fix' phenotype..4 306I 17 t533B II i|| || 1 1128 2II30931 Bg H R Bm I * 3. Numbered circles designate locations of specific Tn5 insertions: o.0 0.8 8.9 12. As shown in Fig. RNA 2 kb FIG.1 I 4. FIG.phenotype. andj show protected fragments resulting from hybridization with RNA and treatment with nuclease Si.6-kb Bgi II fragment indicates the region that is homologous to the nitrogenase genes of K. 1. The arrow indicates the position of the prominent nitrogenase transcript.6-kb Bgi II nitrogenase gene fragment to RNA blots of total nodule RNA from 3. the 5' end of this transcript therefore maps approximately 100 bp to the left of Bgl II site a. EcoRI. H. a. parts of fragments protected from S1 digestion. as expected. Restriction enzyme map: Bg.1-kb HindIMl faent. 3.9 3.0-kb Xho I fragment. Lanes: a and b. 2.

(10). although. This operon opposite direction from the nitrogenase operon.0-kb HindIII fragment was used to map the 3' ends.Tn5 502 within the 2. However. It should be noted that Berg et al. The 1. Another possible reason for this nonpolarity could be that. 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). We have evidence from studies with two strains of R. 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. The very faint band resulting from S1 mapping experiments. 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. Mock hybridization without RNA did not protect fragments. 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. but the full extent of the nif cluster was not determined. with transcription proceeding leftward as expected (data not shown. pneumoniae.9 to 0.4-kb Bgl II fragments (9). 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. strong full-fragment protection was observed (data not shown). The data presented here extend the analysis of Ruvkun et al.4-kb fragment (lanes g and h).to 6-kb nitrogenase RNA in root nodules that is tran- as they are in K. 1).6 kb to the right of Xho I site a and is transcribed rightward at least as far as Xho I site b (Fig. but they -map 20-30 kb downstream from the nitrogenase operon (27). meliloti. We observed full-fragment protection and leftward polarity (data not shown. These genes are located within a 14. coli. 1). complementation analysis of Tn5-mutagenized R.9-kb EcoRI/Xho I fragment that overlaps this point yields only full-fragment protection (Fig.7-kb Xho I fragment was used to map the 3' end. as does the nitrogenase operon in K. we of another sponding region have detected transcription within this region in mature nodules by Southern blot hybridizations (unpublished data).9-kb region between RNA 1 and RNA 2 does not contain genes that are essential for nitrogen fixation. 1).9 kb of DNA. it seems unlikely that such genes are located in this cluster or within 10 kb on either side. Early nodulation genes have been identified in R. the Another nif operon is separated from nitrogenase operon is transcribed in the by 1. indicating that transcription from P1 does continue through the nitrogenase 6-wk nodules and that nitrogenase genes are expressed by transcription originating at P1. Analysis of the 5'-end-labeled 3. In the study of Ruvkun et al. Therefore. 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.4-kb fragment (Fig. approximately 3. there might be a weak secondary promoter in the intergenic region. indicating that the hybridization conditions were not permitting DNA-DNA probe rehybridization. (10) mapped eight strain Tn5R. a nitrogenase transcript of 5 to 6 kb could possibly encode other functions. and analysis of the 0. This identifies another transcript (RNA 2) that originates from a promoter (P2) approximately 0. we observed strong full-fragment protection relative to several smaller protected fragments (data not shown). If this . Other possible explanations for the Fix' phenotype of Tn5 326 are discussed below. Proc. The reason for this is unknown. several faint smaller bands were detected. Natl Acad. Fix+ of meliloti. DISCUSSION We have used Tn5 mutagenesis of a 35-kb segment of the R.7-kb fragments formed by cleavage of the original fragment with EcoRI also indicated protection of a 2. Very different results were obtained when the 4. RNA blot analysis failed to reveal any unique transcripts derived from the symbiotically essential region to the right of the nitrogenase operon (see above).7-kb Bgl II fragment. This suggests that detectable transcription from a promoter within the nifH-nifD intergenic region does not normally occur in 4.3. 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. we were surprised to find a Tn5 insertion between nifH and nifD that did not significantly alter symbiotic nitrogen fixation (Tn5 326).2. meliloti genome to identify the boundaries of a large cluster of nif genes. None of the 70 Tn5 insertions described in this paper interfered with the early stages of nodule formation. 15-kb region that is flanked on both sides by at least 10 kb of DNA that is symbiotically unessential. (10). when the 1.3008 Genetics: Corbin et al. In light of the above information supporting a single transcript for the nitrogenase genes.3 kb between Tn5 insertions 264 and 299.6 kb of DNA would be required to encode a nitrogenase complex of similar size in R. mappedhave Fix' Tn5 insertions in thisinsertionsand Ruvkun et in the correal.9 kb (data not shown). we were unable to identify a unique 3' end.7-kb Bgl II fragment or the 0. results summarized in Fig. 3. there was very little full-fragment protection and a large collection of faint bands ranging in size from 3.and 2. In addition. 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. We have identified by physical analysis a single 5. meliloti. However. USA 80 (1983) end-labeled 1. lanes e and f). In this case. 1). The 3.9-kb HindIII fragment was 3' end-labeled and used in S1 mapping experiments. Considering the large number of insertions analyzed.and 2. 3. Our data show that there exists at least one additional operon of up to 2. A unique 3' end was not detected. Based on the estimated sizes of nitrogenase polypeptides from other species of Rhizobium (29-31). although we did not detect a5' end in this region by nuclease S1 mapping. Three nif operons were identified in that work. results summarized in Fig. 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. also reported two scribed leftward from a promoter (P1) about 100 bp to the left of Bgl II site a (Fig.0-kb Xho I fragment indicated protection of a 2. 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.4-kb HindIII fragment was used in similar 5'-end mapping experiments to extend the analysis through the nitrogenase gene region. However. 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. Sci. (25) have reported occasional weak nonpolarity of Tn5 insertions in the lacZ gene of E. lanes i and j).the data suggest that transcription from P1 terminates somewhere near Fix' Tn5 13. when either the 2. We have five region. pneumoniae. Ruvkun et al. Identification of a Second nif Promoter Region. However. which was confined to a 12-kb region corresponding approximately to that bounded by our Fix' Tn5 insertions 13 and 215.

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

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