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Biological nitrogen fixation occurs
in species of more than 100 genera distributed among several of the
major phylogenetic divisions of prokaryotes (Eubacteria and
Archaea) (25). Sequence and mutational analyses
of the genes necessary for nitrogen fixation (nif) in many
diazotrophs indicate that their products have common structures and
functions, while the degree of linkage and arrangement of specific
nif and associated genes vary considerably (5, 8,
17). In addition, nif genes and genes involved in
plant invasion and nitrogen fixation effectiveness, such as
nod and fix in species of
Rhizobiaceae, are often linked.
The identification of nitrogen-fixing bacteria with endophytic habitats
raises the possibility of a new classification of symbiosis
(3). The relationship of a proteobacterial 
group member,
Acetobacter diazotrophicus (syn. Gluconacetobacter
diazotrophicus), with sugarcane represents a promising model
system for the study of an association between a monocot and an
endophytic nitrogen-fixing bacterium (13, 22). The ability
of A. diazotrophicus to enhance sugarcane growth has been
documented, and while the benefit to plant growth might be due at least
in part to the transfer of bacterially fixed N, another plant
growth-stimulating factor(s) is indicated, possibly auxin production by
A. diazotrophicus (22). Because of its potential
agronomic use and unique status as the only diazotrophic species of
Acetobacter so far identified, it was of interest to isolate
and characterize genes that are involved in nitrogen fixation and regulation.
Identification of a major cluster of nif and associated
genes.
A genomic library of A. diazotrophicus
constructed in the wide-host-range cosmid pLAFR3 (Tetr)
(4) was transferred by conjugation from Escherichia
coli to several different nif mutants of
Azotobacter vinelandii (12). Two mutants that
yielded Nif+ Tetr transconjugants were DJ71
(nifV) and DJ35 (nifE), and several transconjugants of both strains carried the same cosmid, pAD71, the
22-kb insert fragment of which carries nifD through
mcpA (Fig. 1). One end of the
insert fragment contained sequences identical to a portion of the
nifHDK region cloned previously (M. Sevilla and C. Kennedy,
submitted for publication) and also to a portion of pAD101, another
cosmid from the pLAFR3 library, isolated by its ability to complement
nifA mutants of A. vinelandii (24). A
third cosmid, pAD102, which also complemented nifA
mutants, provided the region further upstream of nifA.
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FIG. 1.
Major nif gene map of A. diazotrophieus. Arrows indicate the positions and direction for
transcription initiation sites.   ,
 54-, NifA-dependent promoters. The thick lines
underneath represent the DNA fragments used as probes in Northern
analysis.
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Thirty-two open reading frames (ORFs) were identified in the>30
kbp of sequence obtained (Fig. 1). Gene characteristics,
potential regulatory sequences, and gene products are summarized
in Table 1. Genes not known to be
directly involved in nitrogenase structure, function, or regulation of
gene expression include mcpA, encoding a methyl-accepting
chemotaxis protein that responds to extracellular signals for
chemotactic responses (19), and modABCD, which
encode a high-affinity molybdate transport system in E. coli
(9), Staphylococcus carnosus (20), and
A. vinelandii (18). FixABCX may comprise an
electron transfer chain, and, while the fixABCX genes were
first identified in Rhizobium meliloti (6) and
subsequently in other diazotrophs, some or all are also present in the
genomes of E. coli (fixABCX) and
Mycobacterium tuberculosis (fixAB),
neither of which is a diazotroph. The Fix proteins in E. coli are required for carnitine breakdown, an anaerobic function
related to respiration (2), while their function in other
organisms is unknown (7). Also of unknown function but
similar to gene products in other diazotrophs are the ferredoxins FdxN
and FdxB and the products of ORFs 1, 4, and 6, named on the basis of
ORF numbers originally assigned. The only ORF without significant
similarity to known genes is the one between rpoN and
modC, orf11. The nif-fix and associated gene cluster characterized here for A. diazotrophicus is unique in representing the largest single
grouping of genes required for nitrogenase structure and function,
nif and fix gene regulation (nifA and
rpoN), and associated functions (molybdenum uptake and
electron transfer) found in any diazotroph so far studied.
Comparison of organization of nif genes and gene
products.
The individual A. diazotrophicus gene
products are generally most similar to those found in other group
proteobacteria, with 17 gene products being most like those in members
of the Rhizobiaceae and 9 gene products being most closely
related to Rhodobacter capsulatus proteins (Table 1). NifU
and NifS were most similar to the gene products of
Azotobacter species, members of the 
group of
proteobacteria. McpA was most similar to the mcpA gene
product of the unrelated Caulobacter crescentus (67% identity).
Although none of the individual gene products were most similar to
those of Azospirillum species, the overall organization of
genes in the A. diazotrophicus nif-fix cluster is most like that of Azospirillum brasilense. These are the only two
diazotrophs so far characterized that have an mcpA-like gene
associated with the nif-fix cluster (J. Frazzon and I. S. Schrank, personal communication; also this work). The McpA protein
is involved in chemotaxis in several organisms. Because both A. diazotrophicus and A. brasilense are found naturally
associated with monocot plants, it will be of interest to determine
whether McpA is responsible for chemotactic responses to plant exudates
for signaling or nutrition. An mcpA mutant strain of
A. diazotrophicus lost chemotaxis toward a wide range of
attractant stimuli (unpublished result).
Differences between the clusters are that nifA and
nifB of A. brasilense are not linked to the other
nif genes and nifQ, nifW, and fdxB are
absent from the latter (8, 15). nifY is not found
in the A. diazotrophicus cluster, but its requirement for nitrogen fixation is uncertain in other diazotrophs; it may be involved
in Fe-Mo cofactor insertion into nitrogenase enzyme or in fixed N
sensing (11, 23). One portion of the nif-fix
cluster of A. diazotrophicus is more like that of R. capsulatus (nifE to nifW) than to A. brasilense, although no ORF1-like gene is found in the latter. The
nifU gene in R. capsulatus is truncated compared
to most other nif genes, encoding only the C-terminal end of
NifU (16).
Transcriptional and translational organization of the cluster.
The transcriptional and translational organization of genes in the
nif-fix cluster of A. diazotrophicus shows
several interesting features. Northern analysis of mRNA was successful
in identifying the cotranscription of nifHDK, as occurs in
most other diazotrophs, according to size of transcripts hybridizing to
a nifD probe, and also of nifENX orf4 orfl fdxB
nifQ, indicated by hybridization of RNA to a nifE probe
(Fig. 1 and 2). The former was predicted by sequence analysis, which revealed N- and
NifA-binding sites upstream of nifH (Sevilla and Kennedy, submitted) required, respectively, for nif promoter
recognition and for nif gene transcriptional activation in
all other proteobacterial diazotrophs. A N
recognition sequences but no NifA recognition sequence was found upstream of nifE. Northern blots hybridized with
[32P]dCTP-labeled orf6, nifU, or
fixA probes showed only smears or broad bands after
autoradiography, suggesting rapid degradation of mRNA or a very low
level of transcription. The putative transcriptional organization of
the other genes was suggested by sequence analysis (Fig. 1). Locations
of possibly significant N, NifA recognition
sites upstream of genes and of potential transcription terminators
downstream of genes are given in Table 1.
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FIG. 2.
Northern analysis with probes for nifD (A)
and nifE (B). Probes were radiolabeleled with
[-32P]dCTP by random priming (Stratagene).  ,
cultures grown with low concentrations of fixed N (0.5 mM
NH4); +, cultures grown with high concentrations of fixed N
(10 mM NH4).
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An unusual degree of overlap between the 3' and 5' ends of adjacent
genes, indicating translational coupling, was found for nifN-nifX (30 bp). Other cases of overlap were found for
fixC-fixX (1 bp), rpoN-modC (3 bp),
modC-modB (3 bp), modB-modA (3 bp), nifZ-fixU (3 bp), and fixU-orfl (3 bp). Several
other cases of translational coupling are found between nif
and/or related genes in other diazotrophs, including one large overlap
of 34 bp between nifN and nifX in the archeal
diazotroph Methanococcus maripaludis and smaller
overlaps in M. maripaludis (nifD-nifK, 7 bp)
(14), A. vinelandii (orf8-nifW, 3 bp;
nifZ-nifM, 10 bp; nifM-orf9, 7 bp)
(12), and Klebsiella pneumoniae
(nifB-nifQ, 1 bp; nifL-nifA, 4 bp;
nifZ-nifM, 4 bp; and nifN-nifX, 14 bp)
(1). Of the A. diazotrophicus genes described
here, only nifN and modC had the translational
initiation codon GTG. This initiation codon also occurs in several
genes in various diazotrophs, for example, nifB and
nifX of Frankia alni and ORF1 of Plectonema
boryanum PCC 73110 (10, 21).
Characterization of genes related to nitrogen fixation and/or plant
colonization is important for elucidating these processes in endophytic
diazotrophs. This work examines a major cluster of genes that are
certainly or potentially important for the ability of A. diazotrophicus to fix nitrogen inside its plant host. This knowledge may be relevant for efforts to use this organism or others
like it, such as the endophyte Herbaspirillum
seropedicae, to better benefit growth of sugarcane and
possibly other monocot plants.
Nucleotide sequence accession number.
Sequences in previous
GenBank files have been compiled and combined with new data under
accession no. AF030414.
This work was supported by the National Science Foundation
(IBN-9728184) and by Deutsche (Germany), ME1254/3-1. S.L. was partially sponsored by Chunbuk National University in Korea (overseas scholarship).
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