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 Rhizo-
bium 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 func-
tion. RNA blot analysis identified a 5- to 6-kb transcript that cor-
responds 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 mu-
tation. The implications of this finding and the overall emerging
picture of the genetic organization of this nif region are discussed.
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 rec-
ognition and invasion of the appropriate legume by the bac-
teria, stimulation of plant cell division, multiplication and dif-
ferentiation of bacteria within root cortical cells into mor-
phologically 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. Fix-
mutants 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 sit-
uation is similar to that found in the well-studied nitrogen-fix-
ing 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) seg-
ment of the genome and encode enzymes involved in the bio-
synthesis and processing of nitrogenase peptides, synthesis of
an iron/molybdenum cofactor necessary for nitrogenase activ-
ity, transport of electrons to nitrogenase, and regulation of the
operons (see ref. 12 for review). Biochemical functions for nif
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertise-
ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3005
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 Rhi-
zobium meliloti nif genes. We show that the region is limited
to a 14- to 15-kb segment of the genome.surrounding the ni-
trogenase 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.
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 overlap-
ping 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 recom-
bined into the Rhizobium genome by a marker-exchange pro-
cedure (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 re-
duction (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 ho-
mogenate was immediately poured into 10 ml of 0.5 M man-
nitol/20 mM sodium acetate, pH 5.5/1 mM EDTA/3% Na-
DodSO4 that had been preheated to 1000C. The mixture was
heated at 100'C for 2-5 min, until the lysate became nonvis-
cous. It was then extracted with phenol and CHCl3/isoamyl
alcohol (24: 1), and RNA was precipitated as described (9). Re-
sidual 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.
3006 Genetics: Corbin et al.
a nitrocellulose column (9) or by pelleting it through a CsCl so-
lution (17).
Hybridization Probes. DNA used to probe RNA or Southern
blots was labeled with 32P by nick-translation (18). 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 (5,000 Ci/mmol; 1 Ci = 37 GBq) by using
polynucleotide kinase. Fragments with 5' overhangs were 3'-
end labeled by using [a-32P]dNTPs (3,000 Ci/mmol) and the
Klenow fragment of DNA polymerase I.
RNA Blot Analysis. Five micrograms of total nodule RNA
was suspended in 20 A.l of 20 mM 3-(N-morpholino)pro-
panesulfonic acid (Mops), 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. The sus-
pension was cooled on ice, 2 ttl of 25% Ficoll/0.3% brom-
phenol blue/0.3% xylene cyanol was added, and the sample
was electrophoresed through a vertical 1% agarose/6% form-
aldehyde gel formed in 20 mM Mops, pH 8/5 mM NaOAc/1
mM EDTA. One lane was stained with ethidium bromide to
visualize the ribosomal RNAs that served as molecular size
standards (19). The remainder of the gel was washed for 5 min
with H20 and for 5 min with 3.0 M NaCl/0.3 M Na citrate and
transferred to nitrocellulose by standard blotting techniques (20).
These blots were hybridized to nick-translated probes as de-
scribed (9).
Nuclease S1 Mapping. This technique has been described
by Berk and Sharp (21) and allows the physical mapping of RNA
transcripts on a specific DNA fragment. DNA fragments are
end labeled and hybridized to RNA under conditions that allow
RNA-DNA hybrids to form but prevent DNA-DNA hybridiza-
tion (22, 23). When a RNA transcript that overlaps the labeled
end of one strand of the fragment hybridizes to the probe, that
portion of the fragment hybridized will be resistant to the sin-
gle-strand-specific nuclease Si and yield a radioactive "pro-
tected" fragment after Si digestion. The size of this protected
fragment is the distance from the end of the RNA transcript to
the end of the original fragment. Use of a 5'-end-labeled probe
will map 5' ends of transcripts, while use of a 3'-end-labeled
probe will map 3' ends. A fully protected fragment indicates
that one strand of the entire fragment has been transcribed.
The direction of transcription of the fragment can be deter-
mined by cleaving the original labeled fragment into two dis-
tinguishable end-labeled fragments before hybridization and
then determining which labeled end is protected.
In our experiments, end-labeled DNA fragments were hy-
bridized to total nodule RNA by suspending the nucleic acids
in 20 ,ul of hybridization buffer [80% formamide (Baker; re-
crystallized three times)/0.4 M NaCI/50 mM Pipes, pH 7/1
mM EDTA], denaturing the double-stranded probe by heating
the mixture at 73°C for 10 min, and then immediately trans-
ferring it to 53-560C for hybridization. After 6-8 hr, the sam-
ples were diluted to 220 ,ul by addition of ice-cold S1 buffer and
digested with 30 units of nuclease S1 (Bethesda Research Lab-
oratories) for 30 min at 37°C. The digestion products were re-
solved by electrophoresis through alkaline agarose gels (24) and
visualized by autoadiography. 32P-Labeled bacteriophage A DNA
digested with Cla I or HindIII and bacteriophage 4X174 rep-
licative form DNA digested with Hae III served as molecular
size standards.
RESULTS
Tn5 Mutagenesis Completely Defines a Discrete nif Gene
Cluster. We used site-directed Tn5 mutagenesis (15) to deter-
mine the filll extent of a previously identified cluster of nif genes
in R. nmliloti (9). Mutagenesis gave 70 independent Tn5 in-
Proc. Natl. Acad. Sci. USA 80 (1983)
sertions distributed throughout a 35-kb segment of the genome
that encompasses the nitrogenase structural genes (Fig. 1). 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. The muta-
genized strains were classified into three categories based on
the appearance of the nodules and their ability to reduce acet-
ylene and support plant growth. Thirty strains, designated Fix-,
formed small white nodules that completely lacked nitrogenase
activity as measured by the acetylene reduction assay. In these
cases, the plants grew no better than the uninoculated controls.
Three strains, designated Fix+, formed small nodules that were
detectably pink but had less than 10% of the nitrogenase ac-
tivity of normal nodules. Even though plant growth was se-
verely reduced, leaf color was noticeably greener than uninoc-
ulated controls. These three Fix+ insertions (nos. 218, 219, and
220) mapped within 150 base pairs (bp) of each other. The re-
maining 37 strains were designated Fix'. They formed nodules
that were indistinguishable from wild type on the basis of size,
color, nitrogenase activity, and ability to support plant growth.
All 33 insertions that caused partial or complete inability to fix
nitrogen mapped within a 14- to 15-kb segment of DNA that
includes the nitrogenase genes. Twenty-eight insertions that
mapped as far as 10 kb to the right or the left of this 14- to 15-
kb region yielded only Fix' phenotypes.
Nine Fix+ Tn5 insertions mapped within the nif gene cluster
(Fig. 1). Since Tn5 insertions normally create polar mutations
(25), these Fix' insertions presumably define regions between
operons. The largest such region that is apparently devoid of
essential nif functions is defined by a group of five Fix' in-
sertions (nos. 312, 303, 305, 304, and 505) that span 1.1 kb of
DNA (Fig. 1). Fix+ insertions 326, 292, 215, and 264 would be
expected to define additional smaller regions between adjacent
operons. Fix+ insertion 326, however, appears to be an excep-
tion and is discussed in detail below.
RNA Blot Analysis Identifies the Nitrogenase Transcript.
RNA blots of total nodule RNA were probed with two 32P-la-
beled DNA fragments that constitute a contiguous 11. 7-kb seg-
ment of the nif gene cluster. Hybridization to the labeled 3.6-
kb Bgl II nitrogenase gene fragment (9, 10) revealed a prom-
inent transcript of 5-6 kb that we presume is the major nitro-
genase transcript and a number of less intense minor bands (Fig.
2, lanes a and b). Four of the latter bands corresponded in po-
sition to the plant and bacterial ribosomal RNAs that were vis-
ible in the ethidium bromide-stained gel (data not shown). These
RNAs constitute the bulk of the RNA in such preparations and'
always yield faint hybridization bands regardless of the probe
used. The significance of the other bands is not clear.
When identical blots were probed with the labeled 8.1-kb
Bgl II fragment to the right of the nitrogenase genes, clearly
observed (Fig. 2, lanes c
distinguishable transcripts were notdiscussed
and d). Many of the minor bands above were also
observed. Transcripts from this region are, therefore, less
abundant or more degraded than the nitrogenase transcript.
Nuclease Si Mapping Applied to the Nitrogenase Operon.
The three nitrogenase structural genes-nifH, nifD, and nifK-
are within a single operon of about 6 kb in R. nmliloti (10). To
more thoroughly characterize transcription from this operon,
we used the nuclease S1 mapping technique to locate the ends
of the RNA on the physical map. Fig. 1 shows the fragments
used in the analysis and summarizes the results. When the 2.1-
kb HindIII fragment was 5' end labeled and used in S1 map-
ping experiments, a fragment of 1.1 kb2. was protected (Fig. 3,
lanes a and b). Cleavage of this labeled 1-kb HindIII'fragment
with Bgl II into 1.2- and 0.9-kb fragments prior to hybridization
also resulted in the protection of a 1. 1-kb fragment (lanes c and
Bg
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R
Bm
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I f23
Genetics: Corbin et al.

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. 310

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-

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Nitrogenase

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.
2.1

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296
-29S
21020

256n
iEi292
8.2

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FIG. 1. Physical map of the R. meliloti nif region. Restriction enzyme map: Bg, Bgi H; H, Hindl; R, EcoRI; Bm, BamHI; X, Xho I. Sizes of
a

fragments are indicated below in kb. The horizontal lines represent the portions of DNA that were mapped for the respective enzymes. Numbered
1.6

circles designate locations of specific Tn5 insertions: o, Fix' phenotype; e, Fix- phenotype; a, Fix' phenotype. The bar above the 3.6-kb Bgi II
fragment indicates the region that is homologous to the nitrogenase genes of K. pneumoniae: region containing primarily nifH homology; E2,
3.8

E,
Proc. Nat. Acad. Sci. USA 80 (1983)

2.6
-2219

2 4 -25 2 7 -23 29231

i217 222fSztf
2.4

I
221 -22

4.9
-

1 kb
3007

region containing primarily nifD homology. 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; M, parts of fragments protected from S1 digestion; RNA 1 and RNA
2, niftranscripts deduced from S1 mapping experiments using 5'-end-labeled DNA fragments; dotted extensions ofthe bars show that transcription
continues past arrows but a unique 3' end has not been mapped. P1 and P2, putative nif promoter regions.

d). As shown in Fig. 1, the 5' end of this transcript therefore the promoter region, P1, for the nitrogenase transcript (RNA 1).
maps approximately 100 bp to the left of Bgl II site a, with tran- The presence of a Fix' Tn5 insertion (Tn5 326) 1 kb down-
scription proceeding leftward into the nitrogenase genes, as ex- stream from the nitrogenase promoter raises the question of
pected. In the absence of RNA processing, this 5' end defines whether or not there could be another promoter immediately
downstream of this insertion that maps within the nifH-nifD
a b c d intergenic region (26). Nuclease S1 mapping analysis of the 5'-
a b c d e f g h i i

3.9-
3.0-
3.0- ! 1.1- sgo 2.4Xr 1.9-
2.1-
1.5-

FIG. 2. RNA blot analysis of R. meliloti nif genes. Lanes a and b:
results of hybridization of the 3P-labeled 3.6-kb Bgi II nitrogenase gene
fragment to RNA blots of total nodule RNA from 3- and 6-wk nodules.
Lanes c and d: results of hybridization of the 32P-labeled 8.1-kb Bgi II
fagment to RNA blots of total nodule RNA from 3- and 6-wk nodules.
Molecular size markers were ribosomal RNA molecules in the RNA
preparation; their positions are marked in kb. The arrow indicates the
position of the prominent nitrogenase transcript.
FIG. 3. Nuclease S1 mapping of nif transcripts. Lanes: a and b,
2.1-kb HindIMl faent; c and d, 2.1-kb HindIII fragment dig with
Bgl E; e and f, 3.0-kb Xho I fragment; g and h, 3.0-kb Xho I fragment
digested with EcoRI; i andj, 1.9-kb Xho I/EcoRI fiagment. Lanes a, c,
e, g, and i show results of mock hybridization of probe without RNA or
nuclease S1 treatment. Lanes b, d, f, h, andj show protected fragments
resulting from hybridization with RNA and treatment with nuclease
Si.
3008 Genetics: Corbin et al.
end-labeled 1.9-kb EcoRI/Xho I fragment that overlaps this
point yields only full-fragment protection (Fig. 3, lanes i and
j), with transcription proceeding leftward as expected (data not
shown; results summarized in Fig. 1). This suggests that de-
tectable transcription from a promoter within the nifH-nifD
intergenic region does not normally occur in 4- to 6-wk nodules
and that nitrogenase genes are expressed by transcription orig-
inating at P1. Other possible explanations for the Fix' phe-
notype of Tn5 326 are discussed below.
The 3.4-kb HindIII fragment was used in similar 5'-end
mapping experiments to extend the analysis through the nitro-
genase gene region. We observed full-fragment protection and
leftward polarity (data not shown; results summarized in Fig.
1), indicating that transcription from P1 does continue through
the nitrogenase genes.
The size of the nitrogenase transcript obtained from RNA
blots suggests that the 3' end of the transcript should be be-
tween Fix' Tn5 13 and Fix- Tn5 502 within the 2.7-kb Bgl II
fragment. However, when either the 2.7-kb Bgl II fragment or
the 0.9-kb HindIII fragment was 3' end-labeled and used in S1
mapping experiments, strong full-fragment protection was ob-
served (data not shown). In addition, several faint smaller bands
were detected. Decreasing the RNA concentration in the hy-
bridization mixture resulted in a concomitant decrease in in-
tensity of all bands with no effect on the relative intensities.
This indicates that the hybridizations were in excess DNA and
that detection of a unique 3' end was not prevented by dis-
placement of the 3' ends from the probe by hybridization with
a less-abundant read-through transcript. Mock hybridization
without RNA did not protect fragments, indicating that the hy-
bridization conditions were not permitting DNA-DNA probe
rehybridization. Very different results were obtained when the
4.0-kb HindIII fragment was used to map the 3' ends. In this
case, there was very little full-fragment protection and a large
collection of faint bands ranging in size from 3.9 to 0.9 kb (data
not shown). Therefore, although- the data suggest that tran-
scription from P1 terminates somewhere near Fix' Tn5 13, we
were unable to identify a unique 3' end.
Identification of a Second nif Promoter Region. RNA blot
analysis failed to reveal any unique transcripts derived from the
symbiotically essential region to the right of the nitrogenase
operon (see above). However, we had previously shown that
this region is transcribed in mature nodules by hybridizing total
RNA labeled in vitro to Southern blots of the 8.2- and 2.4-kb
Bgl II fragments (9). 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. Analysis of the
5'-end-labeled 3.0-kb Xho I fragment indicated protection of
a 2.4-kb fragment (Fig. 3, lanes e and f), and analysis of the
0.3- and 2.7-kb fragments formed by cleavage of the original
fragment with EcoRI also indicated protection of a 2.4-kb frag-
ment (lanes g and h). This identifies another transcript (RNA
2) that originates from a promoter (P2) approximately 0.6 kb to
the right of Xho I site a and is transcribed rightward at least as
far as Xho I site b (Fig. 1). 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. However, when the
1.7-kb Xho I fragment was used to map the 3' end, we observed
strong full-fragment protection relative to several smaller pro-
tected fragments (data not shown). A unique 3' end was not
detected.
DISCUSSION
We have used Tn5 mutagenesis of a 35-kb segment of the R.
meliloti genome to identify the boundaries of a large cluster of
nif genes. These genes are located within a 14- to 15-kb region
Proc. Natl Acad. Sci. USA 80 (1983)
that is flanked on both sides by at least 10 kb of DNA that is
symbiotically unessential. The data presented here extend the
analysis of Ruvkun et al. (10), which was confined to a 12-kb
region corresponding approximately to that bounded by our Fix'
Tn5 insertions 13 and 215. Three nif operons were identified
in that work, but the full extent of the nif cluster was not de-
termined. Our data show that there exists at least one addi-
tional operon of up to 2.3 kb between Tn5 insertions 264 and
299.
None of the 70 Tn5 insertions described in this paper in-
terfered with the early stages of nodule formation. Considering
the large number of insertions analyzed, 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. meli-
loti, but they -map 20-30 kb downstream from the nitrogenase
operon (27).
In the study of Ruvkun et al. (10), complementation analysis
of Tn5-mutagenized R. 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
as they are in K. pneumoniae. Ruvkun et al. also reported two
Tn5 insertions near the nifH-nifD intergenic region that were
weakly nonpolar. We have identified by physical analysis a sin-
gle 5- to 6-kb nitrogenase RNA in root nodules that is tran-
scribed leftward from a promoter (P1) about 100 bp to the left
of Bgl II site a (Fig. 1). In light of the above information sup-
porting a single transcript for the nitrogenase genes, we were
surprised to find a Tn5 insertion between nifH and nifD that
did not significantly alter symbiotic nitrogen fixation (Tn5 326).
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. We have evi-
dence from studies with two strains of R. meliloti that Tn5 in-
sertions near the site of insertion of Tn5 326 can result in high
levels of expression of a promoterless chloramphenicol acetyl-
transferase gene cartridge (28) inserted into the HindIII site
near the nifH/nifD junction (unpublished data). Another pos-
sible reason for this nonpolarity could be that, although we did
not detect a5' end in this region by nuclease S1 mapping, there
might be a weak secondary promoter in the intergenic region.
It should be noted that Berg et al. (25) have reported occasional
weak nonpolarity of Tn5 insertions in the lacZ gene of E. coli.
Based on the estimated sizes of nitrogenase polypeptides from
other species of Rhizobium (29-31), approximately 3.6 kb of
DNA would be required to encode a nitrogenase complex of
similar size in R. meliloti. Therefore, a nitrogenase transcript
of 5 to 6 kb could possibly encode other functions, as does the
nitrogenase operon in K. pneumoniae.
Another nif operon is separated from the nitrogenase operon
by 1.9 kb of DNA. This operon is transcribed in the opposite
direction from the nitrogenase operon. The very faint band re-
sulting from S1 mapping experiments, 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.
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. The reason for this
is unknown.
The 1.9-kb region between RNA 1 and RNA 2 does not con-
tain genes that are essential for nitrogen fixation. We have
five Fix' Tn5 insertions in this region, and Ruvkun et
mappedhave
al. (10) mapped eight Fix+ Tn5R.insertions in the corre-
sponding region of another strain of meliloti. However, we
have detected transcription within this region in mature nod-
ules by Southern blot hybridizations (unpublished data). If this
 Genetics: Corbin et al.
region encodes a protein, either it is unrelated to nitrogen fix-
ation or it is possibly a negative regulatory element of the Rhi-
zobium nif system. If transcription alone plays a regulatory role,
either it must be a negative one or the Tn5 insertions within the
region, while disrupting normal patterns of transcription, pro-
vide renewed transcription from their termini as hypothesized
above.
Now that the size of the R. meliloti nif gene cluster has been
determined, it is interesting to compare certain features with
those of the K. pneumoniae nif gene cluster. The most striking
difference at this time is that, while the 17 closely spaced genes
of the K. pneumoniae nif region span a 24-kb segment of DNA
(32), the nif region of R. meliloti occupies only 14 to 15 kb, 1. 9
kb of which appears to be unessential for nitrogen fixation. This
leaves a maximum 12 to 13 kb of indispensible nif function in
this region. If genes analogous in function to all of the nif genes
of Klebsiella are present in Rhizobium, then some of the genes
must be located in another region of the genome. On the other
hand, fewer genes may be required in Rhizobium to elaborate
a functional nitrogenase complex during symbiosis. Finally, in
K. pneumoniae, all nif operons are transcribed from one strand
of DNA (11, 33). The findings of two promoters that control
divergent transcription in Rhizobium indicates substantial dif-
ferences in the genetic organization of nif functions in these two
nitrogen-fixing organisms.
We are indebted to Dr. Donald R. Helinski in whose laboratory this
study was conducted. We gratefully acknowledge the excellent tech-
nical assistance of Marty Yanofsky and Patty Tooker. This work was
supported by National Science Foundation Grant PCM 82-04730.
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