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Previous Article | Next Article 
J Bacteriol, July 1998, p. 3620-3628, Vol. 180, No. 14
F. A. Janssens Laboratory of Genetics,
K. U. Leuven, B-3001 Heverlee, Belgium
Received 23 February 1998/Accepted 17 May 1998
The Rhizobium etli rpoN1 gene, encoding the alternative
sigma factor Bacteria are able to activate or
switch off specific sets of genes as they face changing environmental
conditions. This can be achieved through the activities of RNA
polymerases containing alternate Rhizobium etli is the nodulating symbiont of the common bean
plant, Phaseolus vulgaris. It possesses at least two
different regulatory cascades of nitrogen fixation genes. The first
cascade is dependent on the transcriptional regulator nifA
and controls the expression of both nifHDK operons, the
third nifH gene, which is not linked to other nif
genes, and the production of the black pigment melanin (23, 24,
34). Although the R. etli nifA gene is
transcribed under aerobic and microaerobic conditions, NifA-dependent
gene activation is only operative at low oxygen tensions
(24). The second cascade is controlled by the R. etli fixLJ genes (6). Unlike the FixL protein from
Sinorhizobium meliloti (14), R. etli FixL is not a hemoprotein (7). The physiological
signal sensed by R. etli FixL is presently
unknown. We have recently identified the R. etli rpoN
gene (hereafter called rpoN1 [26]). Under
free-living conditions, rpoN1 controls growth on
C4-dicarboxylic acids and on several nitrogen sources. In
addition, inactivation of rpoN1 abolishes microaerobic
expression of nifH and the production of the black pigment
melanin under free-living conditions (26).
Here, we describe the isolation of a second rpoN gene of
R. etli CNPAF512, named rpoN2. This copy is
located near a cluster of nitrogen fixation genes. Mutant analysis
reveals that the rpoN1 and rpoN2 genes are both
active, but under different physiological conditions. The
rpoN1 gene is essential during free-living growth, while the
second copy, rpoN2, is required for symbiosis. Transcription of the R. etli rpoN2 gene is highly induced under
free-living microaerobic conditions by the RpoN1 and NifA proteins. In
contrast, expression of rpoN2 in bacteroids occurs
independently of the rpoN1 gene. We therefore propose that
the activity of rpoN2 is regulated, in addition to oxygen,
by a symbiosis-specific signal.
Strains, plasmids, and culture conditions.
The bacterial
strains and plasmids used in this work are listed in Table
1 or schematically presented in Fig.
1. Escherichia coli strains
were grown at 37°C in Luria-Bertani medium. R. etli strains were cultured in liquid TY (0.5% tryptone, 0.3% yeast extract, 7 mM CaCl2) medium at 30°C or maintained on
yeast-mannitol (YM) agar plates. Antibiotics were supplied to the
medium to maintain selection for plasmids or to select recombinant
strains at the following concentrations: 60 µg of nalidixic acid,
spectinomycin, and neomycin per ml; 30 µg of kanamycin and gentamicin
per ml; 100 µg of ampicillin per ml; and 1 µg (R. etli) or 10 µg (E. coli) of tetracycline per ml.
Growth tests of R. etli on plates were carried out with
acid minimal salts (AMS) medium (28) containing 1 mM
CaCl2 and the carbon and nitrogen sources indicated in the text. Plates were incubated at 30°C under aerobic or microaerobic conditions (0.5% oxygen), and colony size was monitored over a period
of 3 to 7 days. Triparental conjugations and site-directed mutagenesis
of R. etli were done as described previously
(6).
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Differential Regulation of Rhizobium etli rpoN2 Gene
Expression during Symbiosis and Free-Living Growth
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
54 (RpoN), was recently characterized and
shown to be involved in the assimilation of several nitrogen and carbon
sources during free-living aerobic growth (J. Michiels, T. Van Soom, I. D'hooghe, B. Dombrecht, T. Benhassine, P. de Wilde, and J. Vanderleyden, J. Bacteriol. 180:1729-1740, 1998). We identified a
second rpoN gene copy in R. etli,
rpoN2, encoding a 54.0-kDa protein which displays 59%
amino acid identity with the R. etli RpoN1 protein. The
rpoN2 gene is cotranscribed with a short open reading
frame, orf180, which codes for a protein with a size of
20.1 kDa that is homologous to several prokaryotic and eukaryotic
proteins of similar size. In contrast to the R. etli rpoN1
mutant strain, inactivation of the rpoN2 gene did not
produce any phenotypic defects during free-living growth. However,
symbiotic nitrogen fixation was reduced by approximately 90% in the
rpoN2 mutant, whereas wild-type levels of nitrogen fixation
were observed in the rpoN1 mutant strain. Nitrogen fixation
was completely abolished in the rpoN1 rpoN2 double mutant.
Expression of rpoN1 was negatively autoregulated during
aerobic growth and was reduced during microaerobiosis and symbiosis. In
contrast, rpoN2-gusA and orf180-gusA fusions were not expressed aerobically but were strongly induced at low oxygen
tensions or in bacteroids. Expression of rpoN2 and
orf180 was abolished in R. etli rpoN1 rpoN2 and
nifA mutants under all conditions tested. Under free-living
microaerobic conditions, transcription of rpoN2 and
orf180 required the RpoN1 protein. In symbiosis, expression
of rpoN2 and orf180 occurred independently of
the rpoN1 gene, suggesting the existence of an alternative symbiosis-specific mechanism of transcription activation.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
factors and their cognate
regulatory proteins. RNA polymerases containing the alternative sigma
factor 54 (N [RpoN]) recognize a
conserved sequence motif centered at 
24 and 12 nucleotides from the
transcriptional start site (5'-TGGCAC-N5-TTGCA/T-3' [see reference 9]). Transcription initiation
from these promoters depends on the presence of specific activators
which typically bind to DNA sequences located over 100 nucleotides
upstream from the transcriptional start site (4). The
activity of these activator proteins has been shown in several cases to
be regulated by phosphorylation in response to physiological signals.
In addition, evidence is accumulating that gene transcription by the
54 RNA polymerase holoenzyme not only is regulated by
the activator protein but also is regulated under certain conditions by
54 abundance (3). In Caulobacter
crescentus, transcription of rpoN is temporally
regulated during the cell cycle and is 10-fold induced at the
transition from swarmer to stalked cell (3). Upregulation of
rpoN coincides with the onset of stalk formation and occurs
before the 54-dependent increase in flagellar gene
expression. The rpoN gene from Rhodobacter
capsulatus is organized in a nifU2-rpoN
superoperon, and transcription of rpoN is under the control
of two promoters. One promoter upstream of
nifU2 is constitutively expressed with respect
to nitrogen; a secondary nitrogen-dependent promoter is autoactivated
by RpoN and NifA (5). Expression from the R. capsulatus rpoN secondary promoter is required for growth under certain stress conditions (5). Bradyrhizobium
japonicum is presently the only bacterium in which two
differentially regulated rpoN genes have been identified
(16). Expression of the first copy, rpoN1, is
activated in microaerobiosis by the FixLJ-FixK2 regulatory cascade,
while the rpoN2 gene is negatively autoregulated (9,
16). In Azorhizobium caulinodans, one of the two genes coding for 54, rpoF, is required for
high-level nifA expression (19, 33). Indirect genetic evidence suggests that Rhodobacter
sphaeroides (20) also contains a second
rpoN gene.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids
View larger version (8K):
[in a new window]
FIG. 1.
Physical map of the R. etli nif region
containing the rpoN2 gene. The physical map of the 2.8-kb
fragment that was sequenced is given below. The positions and
orientations of the different ORFs are presented below the restriction
maps. Triangles represent genomic insertions of the 
-Km interposon
or the gusA gene. B, BamHI; E, EcoRI;
H, HindIII; P, PstI; S, SalI; Sm,
SmaI; X, XbaI.
DNA methods. DNA preparation and recombinant DNA techniques were performed according to standard procedures (2, 30). DNA fragments were recovered from agarose gels with the Nucleotrap kit (Macherey-Nagel). Hybridizations were carried out with digoxigenin-labeled probes as described by the manufacturer (Boehringer Mannheim). To generate blunt ends to incompatible DNA fragments, DNA was incubated with Klenow or T4 DNA polymerases in the presence of the four deoxynucleoside triphosphates.
DNA sequencing. Automated DNA sequencing was performed with an A.L.F. sequencer (Pharmacia Biotech) with fluorescein-labeled oligonucleotide primers. Sequence data analysis was performed with the PC/Gene software package (IntelliGenetics, Inc.). Homology searches were carried out with the BLAST server at the National Center for Biotechnology Information.
PCR-mediated isolation of cosmid clones carrying the
R. etli rpoN2 gene.
A 0.6-kb fragment from
the R. etli rpoN2 gene was amplified with the
primers OJM081 (5'-CTGAGAATTCGACGCGGCTGACGGTGGATTCGTGC-3') and OJM082 (5'-CTGAGAATTCGATCCGCCGGGTGTTTTCGC-3') and genomic DNA from the R. etli rpoN1 mutant strain FAJ1154 as a
template (26). Amplification of DNA fragments by PCR was
performed as described previously (26), except that the
annealing temperature was 50°C. The primers were chosen in conserved
regions of rpoN genes and annealed at each site of the
PstI restriction site of the rpoN1 gene used to
insert the -Km interposon, thereby avoiding preferential
amplification of the rpoN1 gene. The 0.6-kb PCR product was
subsequently cloned into the pCR2.1 TA cloning vector (Invitrogen). The
nucleotide sequence of this fragment was different from that of
rpoN1, but was clearly homologous to the rpoN1
and rpoN genes from other bacterial species.
Expression analysis of gusA fusions and melanin
production.
Precultures were grown overnight in TY medium and
diluted 20-fold in AMS medium containing 10 mM NH4Cl and 10 mM succinate. Microaerobic induction of the different strains was
carried out overnight at 0.3% oxygen as described previously
(24). 
-Glucuronidase activity and melanin production were
quantified as described previously (26).
Plant culture and bacteroid isolation. Seeds of Phaseolus vulgaris cv. Limburgse vroege were sterilized, and seedlings were inoculated as previously described (25). Bean plants were grown in 250-ml cylindrical flasks in a Sanyo Gallenkamp Fitotron plant growth room with a 12-h photoperiod (day/night temperature, 22°C/18°C; day/night relative humidity, 65%/75%). Acetylene reduction activities (ARAs) were determined 4 weeks after inoculation on a Hewlett-Packard 5890A gas chromatograph equipped with a "PLOT fused silica" column. Ethylene production was quantified with propane as an internal standard.
To determine symbiotic expression of the different gusA fusion plasmids, bacteroids were isolated from the nodules of 4-week-old plants. For this, all of the nodules of one plant were collected and treated essentially as described by Leyva et al. (18). The nodules were crushed on ice in 15-ml plastic tubes in the presence of 0.5 g of polyvinylpolypyrrolidone and 2 ml of Mg-phosphate buffer (2.5 mM MgCl2, 50 mM potassium phosphate [pH 6.8]). Next, the paste was diluted with 10 ml of ice-cold Mg-phosphate buffer. The tubes were centrifuged at 121 × g for 1 min to remove plant cell material. This step was repeated with the supernatant. Bacteroids were finally precipitated at 4°C by centrifugation at 4,500 × g for 5 min and resuspended in 100 µl to 1 ml of Mg-phosphate buffer. Dilutions of the bacteroids were immediately assayed for glucuronidase activity.Construction of mutants.
To construct rpoN2 and
orf180 mutants, a 4.2-kb fragment containing the entire
rpoN2 gene, except the amino terminus, and the complete
orf180 gene was amplified by PCR with primers OJM104 (5'-CTGAGGATCCGCGGCCGCGCCTGTTTCCTTGAGCTTG-3') and OJM106
(5'-CT GAGGATCCGCGGCCGCTTTGTCCTGAATGTCAGTTC-3') annealing near
the 3' end of rpoN2 and approximately 2 kb upstream from
orf180, respectively. Both primers contain NotI
recognition sites at their 5' ends. The 4.2-kb DNA fragment was cloned
into the NotI site of pUC18Not (15). The
resulting plasmid, pFAJ1174, was digested with PstI (rpoN2) or EcoRI (orf180) and blunt
end ligated to the 1.8-kb BamHI fragment from pHP45-Km,
thereby inactivating the rpoN2 gene or orf180,
respectively. In both plasmids, the orientations of -Km and
rpoN2 or orf180 are opposed and the same,
respectively (Fig. 1). Finally, the 6-kb NotI fragments
from the resulting plasmids were cloned into the NotI site
of the Rhizobium suicide plasmid pJQ200-UC1, yielding
pFAJ1183 (rpoN2::Km) and pFAJ1184 (orf180::Km). These two mutations were finally
recombined into the genome of R. etli CNPAF512.
Insertion of the -Km cassette at the correct site was confirmed
by Southern hybridization with the appropriate DNA probes. The
R. etli rpoN2 mutant was named FAJ1169, and the
R. etli orf180 mutant was named FAJ1173.
-Sp cassette from pHP45-Sp,
thereby inactivating the gene. The construct carrying rpoN1
with the -Sp cassette in the opposite orientation (pFAJ1186) was
used to mutagenize FAJ1169. The resulting mutants were finally tested
for growth on AMS plates containing 10 g of mannitol and 2 g
of alanine per liter. rpoN1 mutants are unable to grow on
alanine (26). Appropriate insertion of the resistance cassette was verified by Southern hybridization. The R. etli
rpoN1 rpoN2 double mutant was named FAJ1170.
To confirm the results obtained with FAJ1170, we also constructed a
second, independent R. etli rpoN1 rpoN2 double mutant. R. etli CNPAF512 was mutagenized with plasmid pFAJ1186,
yielding the R. etli rpoN1 mutant FAJ1175. Next, the
rpoN2 internal 0.6-kb PCR fragment, amplified with primers
OJM081 and OJM082 (described above), was cloned into pCR2.1. The
resulting vector was digested with BamHI and ligated to the
3.8-kb BamHI fragment from pWM6, thereby inserting a
Nmr gene in a site flanking the 0.6-kb insert. The 4.4-kb
KpnI-NotI insert from this construct was finally
ligated into SmaI-digested pJQ200-UCI, after the ends of the
insert fragment had been blunted. This plasmid was used to mutagenize
R. etli FAJ1175. In the resulting R. etli
rpoN1 rpoN2 strain, FAJ1172, the rpoN2 gene is
disrupted by insertion of the entire plasmid into the gene.
Finally, to determine whether the R. etli rpoN2 and
fixLJ genes belong to the same regulatory cascade, a
R. etli fixL rpoN1 double mutant was generated. For
this, the R. etli fixL mutant RpFAJ1002 (6)
was mutagenized with pFAJ1186. The resulting strain was named FAJ1176.
Construction of rpoN2-gusA and orf180-gusA plasmid and chromosomally integrated fusions. To construct transcriptional rpoN2-gusA and orf180-gusA fusions, pFAJ1174 was digested with PstI (rpoN2) or EcoRI (orf180) and blunt end ligated to the 3.8-kb BamHI fragment from pWM6. The two 8-kb NotI fragments carrying the gusA gene insertion in the right orientation were cloned, after being blunted, into BamHI-digested pLAFR3 or into the NotI site of pJQ200-UC1. The resulting plasmids were named pFAJ1175 (pLAFR3-rpoN2-gusA), pFAJ1176 (pLAFR3-orf180-gusA), pFAJ1187 (pJQ200-UC1-rpoN2-gusA), and pFAJ1188 (pJQ200-UC1-orf180-gusA). To construct chromosomally integrated rpoN2-gusA and orf180-gusA fusions, pFAJ1187 and pFAJ1188 were integrated at the homologous site into the R. etli CNPAF512 genome, yielding CNPAF512::pFAJ1187 and CNPAF512::pFAJ1188. Promoter deletions of pFAJ1175 and pFAJ1176 were constructed as follows (also see Fig. 1). In pFAJ1180, the 6.3-kb SmaI fragment from pFAJ1175 was blunt end ligated into the BamHI site of pLAFR3. Similarly, to generate pFAJ1181, the 6.3-kb SmaI fragment from pFAJ1176 was blunt end ligated into the BamHI site of pLAFR3.
Nucleotide sequence accession number. The nucleotide sequence of the R. etli rpoN2 gene locus has been deposited in the DDBJ-EMBL-GenBank nucleotide sequence databases under accession no. AJ005696.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation and localization of the R. etli rpoN2
gene.
We have recently characterized the R. etli
rpoN1 gene (26). Mutant analysis indicated that
although the R. etli rpoN1 mutant had phenotypic
defects under free-living conditions (26), symbiotic nitrogen fixation was not affected, suggesting the existence of a
second rpoN copy. Therefore, based on the DNA sequence of
R. etli rpoN1, several PCR primers were designed
such that they annealed to conserved regions of rpoN
genes belonging to different organisms. The primers were chosen to
anneal at both sites of the rpoN1 -Km insertion, making
it possible to distinguish between PCR fragments originating from the
rpoN1 and rpoN2 genes, because PCR was performed with genomic DNA isolated from the R. etli rpoN1
mutant, FAJ1154. A 0.6-kb internal fragment of the rpoN2
gene was amplified with one of these primer combinations (see also
Materials and Methods). Based on the DNA sequence of this fragment, new
primers, specific for the rpoN2 gene, were designed and used
to screen a pLAFR1 genomic library of R. etli CNPAF512
(6), as detailed in Materials and Methods. Two overlapping
cosmid clones, pFAJ1172 and pFAJ1173, were isolated. Partial DNA
sequence analysis and restriction mapping of the DNA fragments common
to both clones confirmed the identity of rpoN2 and allowed
us to localize the rpoN2 gene approximately 3.5 kb from the
previously identified nifB-nifA-fixABCX cluster of nitrogen
fixation genes (23, 24). A physical map of this DNA region
is presented in Fig. 1.
Sequence analysis of R. etli rpoN2. To further characterize the rpoN homologous DNA region, the DNA sequence of a 2,739-bp fragment was established (Fig. 2). Two putative open reading frames (ORFs) were identified (Fig. 1 and 2). The first ORF codes for a protein that displays 59% amino acid identity with the previously characterized R. etli RpoN1 protein and was therefore named rpoN2. RpoN2 contains 483 amino acids and has a calculated molecular mass of 54,013 Da and a pI of 5.71, which is higher than that of RpoN1 (pI, 4.32). A putative ribosome-binding site (AGGAG) is located 13 bp upstream from the rpoN2 initiation codon. An alignment was made between R. etli RpoN1 and RpoN2 and the RpoN proteins from S. meliloti (1), B. japonicum (16), A. caulinodans (33), and Rhizobium sp. strain NGR234 (36). The three domains (as defined by van Slooten et al. [36]) of RpoN2 are amino-terminal glutamine-rich region I (M1 to E50), acidic region II (E50 to E118), and carboxy-terminal region III (E118 to G483). The latter domain contains a conserved helix-turn-helix motif involved in DNA binding and the RpoN box (36). All rhizobial RpoN proteins have a domain II of 98 to 110 amino acids, with two notable exceptions in which the acidic domain is considerably shorter, B. japonicum RpoN1 (62 amino acids [E50 to T112]) and R. etli RpoN2 (68 amino acids). Two hydrophobic heptad repeats have been proposed to form an intramolecular leucine zipper in the E. coli RpoN protein (31). Similar structures were found in rhizobial RpoN proteins and have the consensus L21-L28-L35-E42 (amino acid numbering refers to R. etli RpoN1) and (L/I)198-L205-V212-(L/I)219. An alignment of RpoN proteins indicated that while B. japonicum RpoN1 and RpoN2 are highly similar (87% amino acid identity), the RpoN proteins from R. etli are more divergent. The R. etli RpoN1 protein displays higher amino acid identity with the RpoN proteins of S. meliloti (68%) and Rhizobium sp. strain NGR234 (65%) compared to the R. etli RpoN2 protein. Also at the level of DNA sequence, the G+C contents of both R. etli rpoN genes differ (rpoN1, 64%; rpoN2, 56%). It is therefore likely that the rpoN2 gene did not arise by gene duplication but by lateral transfer.
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Sequence analysis of R. etli orf180.
Upstream from
the R. etli rpoN2 gene, a second ORF was identified
(Fig. 1 and 2). This ORF codes for a putative protein of 180 amino
acids with a predicted molecular mass of 20,180 Da and a pI of 7.82. orf180 is located 78 bp upstream from rpoN2 and might therefore be cotranscribed with the rpoN2 gene. A
putative Shine-Dalgarno sequence (AGGAG) is found 8 bp upstream from
orf180. In addition, putative regulatory sequences were
identified in the 5' region of orf180. A sequence
(TGGCA-N6-TTGCT [underlined in Fig. 2])
resembling the consensus of a 24/12 promoter (9) was
located 84 nucleotides upstream from the putative ATG initiation codon.
Approximately 270 bp upstream from this 24/12 type of promoter, a
possible NifA-binding (TGT-N10-ACA) site was
found. Homology searches in databases revealed homology between the
gene product of R. etli orf180 and a protein
encoded by the y4vd ORF located on the symbiotic
plasmid of Rhizobium sp. strain NGR234 (74% amino acid
identity [Q53212]), a protein from Haemophilus influenzae
(44% identity [P44758]), and a putative membrane protein from
Synechocystis sp. strain PCC6803 (60% identity [D90909]). An alignment of these proteins is shown in Fig.
3. In addition, lower homology was
detected with yeast peroxisomal proteins from Lipomyces
kononenkoae (26% identity [U11244]), Candida
boidinii (26% identity [P14292] and 25% identity [P14293]),
Schizosaccharomyces pombe (35% identity [G2598045]), and
an unknown protein from Arabidopsis thaliana (32% identity
[AC002292]). Homology is found over the entire protein length, except
for the amino terminus (the first 44 amino acids in the R. etli protein encoded by orf180). Amino acids conserved
between the prokaryotic proteins and the yeast peroxisomal proteins are
underlined in Fig. 3. The conserved amino acids are also found in the
A. thaliana protein, with the exception of the strongly
conserved PGAFTP(T/I/P)C motif, in which only the G and F residues are
maintained. The positive and negative charges in the protein encoded by
orf180 are evenly distributed throughout the protein, with
the exception of a stretch of 17 amino acids (overlined in Fig. 3)
predicted to form a putative transmembrane segment (17).
Interestingly, this hydrophobic segment is conserved among the
prokaryotic and eukaryotic (except A. thaliana) proteins and
contains the PGAFTP(T/I/P)C motif.
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Analysis of R. etli rpoN mutants. To further characterize the rpoN genes of R. etli, a phenotypical analysis of R. etli rpoN1 (FAJ1154), rpoN2 (FAJ1169), rpoN1 rpoN2 (FAJ1170 and FAJ1172), and rpoN1 fixL (FAJ1176) mutants was carried out under free-living and symbiotic conditions. First, growth of these mutants was tested under aerobic and microaerobic conditions on defined media containing ammonium, nitrate, and alanine as nitrogen sources (Table 2). These compounds were previously shown to reduce growth of an R. etli rpoN1 mutant during aerobic growth (26). In contrast to the wild type, growth of FAJ1154 was clearly reduced on alanine and mannitol both aerobically and microaerobically, while growth was not affected on the complex TY medium and on AMS minimal medium containing mannitol (carbon source) and glutamine (nitrogen source) (Table 2). Similar phenotypes were observed with the double mutants rpoN1 rpoN2 (FAJ1170 and FAJ1172) and rpoN1 fixL (FAJ1176). However, the R. etli rpoN2 mutant displayed wild-type growth on each of the different media tested under aerobic as well as microaerobic conditions. Growth phenotypes were different when nitrate or ammonium was used as an N source and mannitol was used as a C source (data not shown). Aerobic growth and microaerobic growth of the wild type and the rpoN2 mutant were indistinguishable when nitrate or ammonium was used as an N source. Also, the growth rates of the rpoN1, rpoN1 fixL, and rpoN double mutants were identical both aerobically and microaerobically on these media. Growth of these mutants was clearly reduced compared to that of the wild type during aerobic growth. Under microaerobic conditions, these mutants produced increased amounts of exopolysaccharides compared to the rpoN2 mutant and the wild-type strain. Excessive production of exopolysaccharides by the rpoN1 mutants may indicate a low intracellular concentration of fixed nitrogen, as can be expected from nitrate assimilatory mutants. These results indicate that under free-living conditions, the phenotypes of the R. etli wild type and the rpoN2 mutant are similar, and also the growth rates of the rpoN1 and rpoN double mutant strains are indistinguishable from each other on the different media tested.
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Analysis of an R. etli orf180 mutant.
Immediately upstream from R. etli rpoN2, an ORF coding
for a 180-amino-acid protein was identified. To determine its function, orf180 was inactivated by insertion of the -Km cassette
into the EcoRI restriction site of the gene, yielding strain
FAJ1173 (Fig. 1). This mutant was tested for growth on several nitrogen sources under aerobic and microaerobic conditions (Table 2). No
difference between the wild type and mutant FAJ1173 was observed on
these media. In addition, the symbiotic phenotype of this mutant strain
was determined (Table 3). The nodulation phenotypes of the wild type
and mutant were indistinguishable. However, nitrogen fixation activity
was strongly reduced compared with the wild-type level and was not
different from that of the R. etli rpoN2 mutant. The
phenotypes of the R. etli orf180 mutant and the
rpoN2 mutant, as shown in Tables 2 and 3, are identical.
Since the -Km interposon is known to create polar mutations, it
is possible that the observed phenotypes of FAJ1173 are due to absence
of transcription of the rpoN2 gene, which is located
downstream from orf180. This hypothesis was tested by
complementation of the R. etli orf180 mutant
strain, FAJ1173, with plasmid pFAJ1175 containing R. etli orf180 and an inactivated rpoN2 gene (Table
3). No complementation of the symbiotic phenotype of FAJ1173 was
observed, indicating that orf180 and rpoN2 are
probably transcribed from a promoter located upstream from
orf180.
Expression analysis of nifH-gusA and production of
melanin.
The effect of mutations introduced into the
rpoN1 and rpoN2 genes and orf180 on
the RpoN/NifA-dependent expression of nifH and production of
melanin was tested under free-living aerobic and microaerobic (0.3%
oxygen) conditions and in bacteroids (Table 4). Expression of nifH was
monitored by using the Azospirillum brasilense translational
nifH-gusA fusion plasmid, pFAJ21 (35). The
production of the black pigment melanin in R. etli was
previously shown to be dependent on the presence of NifA and RpoN1
(24, 26). From Table 4, it can be seen that as expected,
expression of the nifH gene and the synthesis of melanin are
abolished under aerobic conditions in all strains tested but are highly
induced under microaerobic conditions in the wild type. No expression of pFAJ21 under microaerobic conditions or production of melanin during
microaerobiosis or symbiosis is observed in the R. etli nifA mutant and in both rpoN1 rpoN2 double mutants.
However, levels of -glucuronidase activity of pFAJ21 and
melanin production during microaerobic growth were identical
in the rpoN2 mutant, the orf180 mutant, and the
wild type. These results indicate that rpoN1, but not
rpoN2, is required for free-living expression of the
A. brasilense nifH-gusA fusion and for the production of
melanin. During symbiosis, melanin production was abolished only in the nifA mutant and in the rpoN double mutant strain,
suggesting that both rpoN genes can substitute for each
other with respect to synthesis of melanin. Induction of pFAJ21 was
also assayed in complex TY medium and in AMS medium containing mannitol
as a carbon source and NH4Cl or nitrate as a nitrogen
source (data not shown). In contrast to the wild type, no activity of
the fusion could be observed in the rpoN1 mutant strain
under any of the growth conditions tested.
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Expression analysis of R. etli rpoN1, rpoN2, and orf180. From the data concerning growth and symbiotic phenotypes, expression of nifH, and production of melanin in single and double R. etli rpoN mutant strains, it appears that both rpoN genes are active under different physiological conditions. The rpoN1 gene is essential during free-living growth, and the presence of rpoN2 is required for an effective symbiosis. We tested whether these observations could be explained by differential expression of both genes. First, the expression of genomic gusA fusions, integrated at their homologous site, was monitored (Table 5). Expression of the R. etli rpoN1 gene was previously shown to occur independently of the nitrogen concentration (26). In addition, activity of a chromosomally integrated rpoN1-gusA fusion was shown to be negatively autoregulated. Here, we observed that the level of expression of rpoN1-gusA under free-living microaerobic and symbiotic conditions in a wild-type background was reduced approximately fourfold compared to free-living aerobic expression (Table 5). On the other hand, microaerobic expression of rpoN1-gusA in the rpoN1 mutant strain FAJ1156 was only slightly reduced (approximately 40%) compared to the aerobic expression level. In contrast to the expression of rpoN1-gusA, wild-type genomic orf180-gusA and rpoN2-gusA fusions were not expressed aerobically but were strongly induced during microaerobic growth and symbiosis (Table 5). From this expression analysis, it can be concluded that in the R. etli wild type, rpoN1 but not rpoN2 is expressed during aerobic growth, while the rpoN2 gene, but not rpoN1, is strongly transcribed in bacteroids.
|
24/12 promoter and
the NifA-binding site in the promoters of rpoN2 and
orf180 were not deleted in these plasmids. These fusion plasmids were pFAJ1180 (rpoN2) and pFAJ1181
(orf180). Similar expression patterns to those obtained with
pFAJ1175 and pFAJ1176 were observed in the different mutant
strains during free-living growth (Table 6).
|
| |
DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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We have isolated the second R. etli rpoN gene, rpoN2. This rpoN copy is clustered with nitrogen fixation genes and is located approximately 3.5 kb upstream from the R. etli fixA gene (23). Kullik et al. (16) have previously demonstrated that B. japonicum contains two differentially regulated rpoN genes. None of the genes maps closely to one of the previously identified nif gene regions. The B. japonicum rpoN2 gene, but not rpoN1, is flanked by ORFs that are also conserved in other species (21). In A. caulinodans, the rpoN gene is flanked by conserved ORFs (33). A second rpoN copy, named rpoF, was shown to control the expression of the regulatory nifA gene (19). On the basis of indirect evidence, a second rpoN gene has also been suggested to exist in the photosynthetic bacteria R. sphaeroides (20) and R. capsulatus (10). In R. sphaeroides, the identified rpoN gene is not linked to conserved ORFs but is adjacent to the 3' end of the nifUSVW gene cluster and is probably cotranscribed with the latter genes (20). Because the inactivation of the rpoN gene in R. sphaeroides does not impair diazotrophic growth, the existence of a second copy was postulated.
The R. etli rpoN2 gene product displays 59% amino acid identity with the RpoN1 protein. This is considerably lower than the RpoN proteins from B. japonicum, which are 87% identical to each other. Also, R. etli RpoN1 is more closely related to the RpoN proteins from S. meliloti and Rhizobium sp. strain NGR234 than to the R. etli RpoN2 protein. In addition, the G+C contents of the R. etli rpoN1 and rpoN2 genes differ considerably. It is therefore likely that the R. etli rpoN2 gene did not arise by duplication of a common ancestral rpoN gene, but may be the result of lateral transfer.
The protein encoded by orf180 is homologous to several
prokaryotic (Rhizobium sp. strain NGR234, H. influenzae, and Synechocystis sp. strain PCC6803)
and eukaryotic (yeast and A. thaliana) proteins. The H. influenzae protein possesses an additional C-terminal
glutaredoxin domain and is therefore assumed to participate in redox
reactions. The highest similarity is found with the gene product
of Rhizobium sp. strain NGR234 y4vd,
which maps on the symbiotic plasmid pNGR234a approximately 1.4 kb
upstream from fixA (11). The promoter region of
y4vd contains a consensus 24/12 promoter sequence and a
NifA-binding site, as observed in the R. etli orf180
promoter region. In C. boidinii, two PMP20-like peroxisomal
proteins are also homologous to the R. etli
orf180-encoded protein. These proteins are abundantly synthesized
when methanol is used as the sole carbon source and are
believed to be associated with the membrane of peroxisomes (13). The function of orf180 in R. etli is unclear, but because it is coregulated with nitrogen
fixation genes, it is tempting to speculate that it is involved in
processes related to symbiotic nitrogen fixation.
R. etli orf180 is probably cotranscribed with the rpoN2 gene. First, no recognizable terminator structure is found in the 78-bp intergenic region between orf180 and rpoN2. Second, under free-living conditions, expression of both genes is controlled by NifA and RpoN1, in agreement with the presence of RpoN- and NifA-binding sites in the promoter region of orf180. Finally, the reduced nitrogen fixation activity of an R. etli orf180 insertion mutant cannot be restored to the wild-type level by a plasmid carrying only orf180.
During aerobic growth, the R. etli rpoN1 gene was shown to be expressed at a constant level independently of the nitrogen source and was negatively autoregulated. Under these conditions, the rpoN2 gene is not expressed. The R. etli rpoN1 mutant strain displays growth defects on C4-dicarboxylic acids and on the nitrogen sources ammonium, nitrate, alanine, and serine (26).
When cells were cultured under free-living microaerobic conditions, expression of the R. etli rpoN1 gene was strongly decreased and transcription of rpoN2 was activated. The latter process was strictly dependent on the presence of RpoN1 and NifA proteins. Therefore, also under microaerobic conditions, no growth of the rpoN1 mutant strain was observed when alanine was used as a nitrogen source. Similarly, microaerobic production of melanin or expression of a nifH-gusA fusion plasmid was abolished in the rpoN1 mutant strain. Therefore, no active RpoN2 protein is formed under free-living aerobic or microaerobic conditions in the rpoN1 mutant. In B. japonicum, both rpoN genes are functional during free-living growth. The rpoN2 gene is required aerobically for growth on nitrate, while both copies are active under microaerobic conditions (16).
We have previously proposed that the consensus of a 24/12 promoter
overlapping the 10 promoter region and the transcription start site
of rpoN1 is responsible for the observed negative
autoregulation (26). RpoN1 may bind to this sequence and
thereby inhibit transcription initiation from the 35/10 promoter of
the rpoN1 gene. If active RpoN2 protein is formed during
microaerobiosis in the wild-type strain, this protein could account for
the strongly decreased expression level of rpoN1 by directly
interacting with the rpoN1 promoter. This is not the case in
B. japonicum, because repression of rpoN2
transcription has been specifically attributed to the presence of RpoN2
and not RpoN1 (16).
Microaerobic expression of rpoN genes has been reported in two other organisms. In B. japonicum, rpoN1 is activated by an Fnr-like protein, FixK2. The expression of the fixK2 gene is controlled by the oxygen sensor proteins FixLJ (9, 16). The R. capsulatus rpoN gene is organized in a nifU2-rpoN superoperon structure (5). A primary promoter upstream from rpoN is expressed constitutively. The secondary promoter, which is NifA and RpoN dependent, accounts for increased induction under low-nitrogen and low-oxygen conditions. R. etli rpoN2 constitutes the second example of a NifA-controlled rpoN gene.
Expression of the R. etli rpoN1 gene is shut off during symbiosis. The RpoN2 protein could account for this effect, because it is strongly expressed under these conditions. In addition, an R. etli rpoN2 mutant strain still produces wild-type levels of melanin and activates rpoN2-gusA and orf180-gusA fusions, indicating that in this mutant, the rpoN1 gene is no longer repressed. In contrast to rpoN1, transcription of rpoN2 is highly activated during symbiosis. Transcription activation of rpoN2 requires the NifA protein, but in contrast to free-living conditions, is not abolished in an R. etli rpoN1 mutant strain. Therefore, symbiotic expression of rpoN2 involves a symbiosis-specific mechanism, as suggested for other Rhizobium genes (37). It is possible that this mechanism accounts for a low basal level of expression of the rpoN2 gene. After initial transcription, autoactivation by NifA/RpoN2 may then amplify rpoN2 transcription. The mechanism involved is at present unclear, but it does not involve the R. etli fixLJ genes, because an rpoN1 fixL double mutant still fixes nitrogen.
Nitrogenase activity of nodules induced by the rpoN1 mutant
strain is not different from that of the wild type. On the other hand,
nitrogen fixation is reduced by approximately 90% in the rpoN2 mutant. At present, it is unclear whether the
difference between the symbiotic phenotypes of rpoN1 and
rpoN2 mutants is due to a difference in expression level of
the rpoN genes. The rpoN2 gene is highly induced
in bacteroids, while rpoN1 is negatively autoregulated and
can therefore not reach the high expression levels that may be required
during symbiosis to activate nitrogen fixation genes.
Alternatively, the RpoN1 and RpoN2 proteins may have distinct
affinities for different 24/12 promoters, depending on their DNA
sequence, as was previously reported for A. caulinodans (19).
Transcriptional regulation of sigma factors ensures that bacteria
express specific sets of genes during specific physiological conditions
(38). In recent years, it has become clear that expression levels of 54 may vary according to the physiological
need. Organisms seem to have evolved two different mechanisms. One
unique rpoN gene may be subject to different control
mechanisms. In C. crescentus, expression of the
rpoN gene is increased at the swarmer-to-stalked-cell transition, where 54 is required for stalk biosynthesis
and transcription of flagellar genes (3). Expression of the
rpoN gene from R. capsulatus is controlled
by two promoters (5). The first promoter is expressed constitutively and allows cells to grow under normal nitrogen-limiting conditions. The secondary promoter is autoactivated and is required for
growth on nitrogen-limiting high-salt or low-iron medium. A second
mechanism that bacteria use to cope with an increased demand of
54 is a differential regulation of two rpoN
gene copies. In B. japonicum, rpoN2 is
expressed under all conditions tested. In contrast, transcription of
rpoN1 is activated during microaerobiosis and symbiosis,
allowing rpoN2 to boost its expression during nitrogen
fixation (16). A. caulinodans also possesses two
genes coding for 54 (19, 33). Their
regulation has not yet been studied. Also in R. etli,
two differentially regulated rpoN genes were identified. The
first copy, rpoN1, is required for housekeeping functions and allows cells to grow on several nitrogen and carbon sources under
free-living conditions. The second copy is essential for symbiotic
nitrogen fixation.
| |
ACKNOWLEDGMENTS |
|---|
J.M. is a Postdoctoral Fellow of the Fund for Scientific
Research
Flanders. We acknowledge financial support from the Fund for
Scientific ResearchFlanders (FWO G.0220.97).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: F. A. Janssens Laboratory of Genetics, K. U. Leuven, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium. Phone: 32 16 321631. Fax: 32 16 321966. E-mail: jozef.vanderleyden{at}agr.kuleuven.ac.be.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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