Previous Article | Next Article 
Journal of Bacteriology, July 2000, p. 3632-3637, Vol. 182, No. 13
Laboratoire de Biologie Moléculaire des
Relations Plantes-Microorganismes, UMR 215, CNRS-INRA, 31326 Castanet-Tolosan Cedex, France
Received 3 January 2000/Accepted 28 March 2000
RNA fingerprinting by arbitrarily primed PCR was used to isolate
Sinorhizobium meliloti genes regulated during the symbiotic interaction with alfalfa (Medicago sativa). Sixteen partial
cDNAs were isolated whose corresponding genes were differentially
expressed between symbiotic and free-living conditions. Thirteen
sequences corresponded to genes up-regulated during symbiosis, whereas
three were instead repressed during establishment of the symbiotic
interaction. Seven cDNAs corresponded to known or predicted
nif and fix genes. Four presented high sequence
similarity with genes not yet identified in S. meliloti,
including genes encoding a component of the pyruvate dehydrogenase
complex, a cell surface protein component, a copper transporter, and an
argininosuccinate lyase. Finally, five cDNAs did not exhibit any
similarity with sequences present in databases. A detailed expression
analysis of the nine non-nif-fix genes provided evidence
for an unexpected variety of regulatory patterns, most of which have
not been described so far.
Rhizobia are taxonomically diverse
members of the We reasoned that bacterial genes whose expression is differentially
regulated in nodules as compared to under free-living conditions could
be of special relevance for the symbiotic interaction. With the aim of
identifying such genes, we employed the RNA fingerprinting by
arbitrarily primed PCR (RAP) technique (30), a method
closely related to the differential display technique, which has been widely used in eukaryotes. RAP uses an arbitrary primer at a low annealing temperature for synthesizing the first strand of cDNA by
reverse transcription (16), thus accommodating the lack of polyadenylation of bacterial RNAs. In this work, we compared the RNA
fingerprints of bacterial cells in the nitrogen-fixing symbiotic state
(bacteroids) to those of free-living cells grown under oxic or
microoxic conditions in rich medium.
This work has led to the identification of a set of Sinorhizobium
meliloti genes whose expression is either positively or negatively
regulated during symbiosis. Sequence analysis of the corresponding
expressed sequence tags (ESTs) pointed to new functions that could be
important for the establishment of the symbiotic interaction.
Furthermore, a detailed expression analysis of nine genes by reverse
transcription (RT)-PCR showed the existence of a variety of regulatory
patterns, most of which have not been described so far.
Microbiological material and techniques.
All S. meliloti strains used in this study were isogenic with GMI211
(lac Smr) (7). GMI5601 (lac
nifAZ239::Tn5) and GMI5704 (lac
fixJ2.3::Tn5) were constructed by David et
al. (7). GMI942 was constructed by Foussard et al.
(12). S. meliloti strains were grown at 30°C in
TY complex medium (25) or in defined M9 medium
(17) supplemented with 0.3 mM CaCl2, 1 mM
MgSO4, and 2 µM biotin. Sterilized carbon sources were
added at 20 mM final concentration. Microoxic conditions were achieved
either as described by de Philip et al. (8) (2% oxygen for
4 h) or using the stoppered-tube assay (STA) described by Ditta et
al. (9) (<1% oxygen for 16 h).
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Identification of Sinorhizobium meliloti
Genes Regulated during Symbiosis
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
subdivision of the proteobacteria that induce the
formation of specialized organs, called nodules, on the roots of
leguminous plants. During nodule formation, bacteria invade the plant
cells and differentiate into nitrogen-fixing bacteroids that export
fixed nitrogen into the plant cells. Nodules provide the endosymbiotic
bacteria with a microenvironment suitable for nitrogen (N2)
fixation, including a very low ambient O2 concentration
compatible with nitrogenase activity. Many plant and bacterial genes
contribute to the development of a nitrogen-fixing nodule, only a few
of which are known. The best-characterized bacterial symbiotic genes
include those involved in nitrogenase synthesis and activity (the
nif and fixABCX genes) and two operons involved
in the synthesis of a respiratory oxidase with high affinity for oxygen
(fixNOQP and fixGHIS). Remarkably, these two sets
of genes belong to the same regulon, coordinated by the FixLJ
two-component regulatory system (7). FixL is an oxygen-sensitive sensor histidine kinase that phosphorylates the FixJ
transcription regulator in response to the dramatic drop in oxygen
concentration that takes place inside the nodule. Phosphorylated FixJ
activates the transcription of two intermediary regulatory genes,
nifA and fixK, that control expression of the
nif and fixNOQP genes, respectively.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
strains were involved. Nodules were ground in
liquid nitrogen with a mortar and pestle, and the powder was suspended
in 2 ml of 50 mM Tris-hydrochloride (pH 7.6)-500 mM mannitol. Debris
were sedimented by centrifugation for 5 min at 800 × g. Bacteroids were pelleted by centrifugation of the supernatant
for 5 min at 12,000 × g, and stored at 
80°C if necessary.
RNA preparation. The bacterial pellet from a 25-ml culture (optical density at 600 nm = 0.5) or from 0.25 g of nodules was resuspended and incubated for 10 min at 65°C in 2 ml of prewarmed lysis solution (1.4% sodium dodecyl sulfate, 4 mM EDTA, 75 µg of proteinase K). Proteins were precipitated by adding 1 ml of NaCl (5 M) at 4°C. Nucleic acids were precipitated from the supernatant by addition of 1 volume of isopropanol, and the pellet was resuspended in nuclease-free water. DNA was eliminated by the addition of 7.5 U of fast protein liquid chromatography-purified RNase-free DNase I (Amersham-Pharmacia Biotech, Little Chalfont, Buckinghamshire, England). RNAs were further extracted with 1 vol of a 24:24:1 mix of phenol-chloroform-isoamylalcohol and 1 vol of a 24:1 mix of chloroform-isoamylalcohol and then precipitated with 90% ethanol. The RNA pellet was washed with 70% ethanol and resuspended in nuclease-free water. RNAs were quantitated by measuring the absorbancy at 260 nm. Absence of contaminating DNA in the preparation was ensured by PCR amplification, with each specific primer pair.
RAP. The arbitrary primers used originated from an Arabidopsis thaliana primer library. They were 10 to 18 nucleotides in length, with a mean GC content of 50%.
First-strand cDNA synthesis took place in a 16.5-µl reaction volume. One microliter of total bacterial RNA (50 ng), 7.1 µl of diethyl pyrocarbonate-treated H2O, and 1 µl (50 ng) of an arbitrary primer were heated at 70°C for 10 min and quickly chilled on ice. After brief centrifugation, 3.4 µl of the following 5× buffer (250 mM Tris-HCl [pH 8.3], 375 mM KCl, 15 mM MgCl2), 2 µl of 0.1 M dithiothreitol (DTT) and 1 µl of a 25 mM deoxynucleoside triphosphate mix were added and incubated at 42°C for 2 min. After addition of 1 µl (200 U) of SuperScript II reverse transcriptase (Life Technologies, Rockville, Md.) and thorough mixing by pipetting up and down, the reaction tube was incubated for 50 min at 42°C. Reverse transcriptase was inactivated by heating at 95°C for 5 min as we observed that heating at 70°C did not fully inactivate the enzyme. For PCR amplification, 2.5 µl of 10× PCR buffer (200 mM Tris-HCl [pH 8.4], 500 mM KCl), 1 µl of 50 mM MgCl2, 1 µl of deoxynucleoside triphosphate mix (25 mM), 1 µl of reverse primer (50 ng), 1 µl of sense primer (50 ng), 0.2 µl of [-33P]dCTP (10 µCi/µl; 2,000 Ci/mmol), and 0.5 µl (2.5 U) of Taq DNA polymerase were added to the
first-strand synthesis reaction mix. After gentle mixing, the reaction
mixture was layered with 1 drop of silicone oil and submitted to a
first, low-stringency, PCR cycle (94°C for 5 min, 40°C for 5 min,
and 72°C for 5 min) and then 30 high-stringency cycles (94°C for 1 min, 60°C for 1 min, 72°C for 1 min) and 1 cycle at 72°C for 10 min. Two microliters of 80% formamide with bromophenol blue and xylene
cyanol dyes was added to 2.5 µl of each PCR sample. The samples were
heated to 95°C for 3 min and electrophoresed on a standard
acrylamide-urea sequencing gel. After autoradiography, the RAP products
of interest were eluted from the polyacrylamide sequencing gel by
incubating the gel band in Tris-HCl (10 mM)-EDTA (0.1 mM) solution at
65°C for 2 h. After centrifugation, 50 µl of eluate was
reamplified by PCR using the primers and the stringent PCR conditions
described above, omitting the radioisotope. Products were
electrophoresed on a 2% agarose gel, and bands of the correct size
were extracted from the agarose gel with the Jetsorb kit (Genomed,
Beverly Hills, Calif.). The products were directly cloned into the
pGEMT vector (Promega, Madison, Wis.), or blunt-ended and cloned by
standard methods (26) into an EcoRV digested
pBluescript II vector (Stratagene, La Jolla, Calif.).
RT-PCR expression analyses. Sequence data from fixN, nifH, hemA, and from each differentially expressed RAP product were used to design specific primers. Primer sequences are available at http://www.toulouse.inra.fr/lbmrpm/eng/hp_jb.htm. The reactions were performed as described above for the RAP procedure, except that (i) Moloney murine leukemia virus reverse transcriptase was used, (ii) radioisotope was omitted, and (iii) the first low-stringency PCR cycle was omitted and replaced by 94°C for 3 min. RT-PCR products were electrophoresed on a 2% agarose gel, blotted onto a nylon membrane (Biodyne A Transfer membrane; Pall, East Hills, N.Y.) and probed with a 32P-labeled DNA probe, generated by PCR using the same set of primers. Washing of membranes was carried out in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 sodium citrate)-0.1% sodium dodecyl sulfate at 42°C for 30 min.
DNA sequence analysis. Clones were sequenced using an ABI PRISM Dye terminator cycle-sequencing ready reaction kit (Perkin-Elmer, Oak Brook, Ill.) on an ABI 373 automated sequencer (ABI, Columbia, Md.). Database searches were conducted through the National Center for Biotechnology Information Web page using the BLAST2 package program (1). The sequences were aligned using the Multalin program and the modified blosum62 table (5). Homologous domain searches were conducted using the ProDom database (6).
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
ESTs detection by RAP. The RAP technique (30) uses arbitrary-primed RT of RNA molecules followed by PCR (RT-PCR) to highlight differences in the composition of two different RNA populations. Briefly, a first strand of cDNA is made by RT of a subset of RNA molecules using an arbitrary primer hybridizing under poorly stringent conditions. The second cDNA strand is synthesized from a second arbitrary primer at low stringency in the presence of Taq polymerase. The resulting cDNA products are then amplified by PCR using the same pair of primers, at high stringency with simultaneous radiolabeling. The PCR products are separated by polyacrylamide-urea gel electrophoresis and visualized by autoradiography. The cDNAs of interest can then be eluted from the gel and cloned for further analysis. The original RAP technique was slightly modified here in order to achieve RT, second-strand synthesis, and PCR amplification in one tube and to minimize the amount of RNA material needed (see Materials and Methods for details). The arbitrary primers used originated from an A. thaliana primer library that was available in the laboratory. Primers were 10 to 18 nucleotides in length, with a mean GC content of 50%.
The RAP procedure was applied to total RNAs extracted from S. meliloti bacteria grown in rich medium under oxic, mild (2% oxygen), or severe (<1% oxygen) microoxic conditions and from symbiotic bacteroids isolated from nodules 3 weeks after inoculation of alfalfa. Different control reactions were performed: (i) a reaction without RNA to eliminate bands due to primer self-amplification, (ii) a reaction without reverse transcriptase to detect DNA contamination of the RNA preparations, and (iii) a reaction on RNAs isolated from plant roots, to identify bands due to plant RNA contamination of the bacteroid preparation. RAP reactions were performed at least two times independently to ensure that the fingerprints were reproducible. In total, 50 different pairs of primers were tested that gave rise to 32 bands reproducibly enhanced in symbiotic bacteroids (see examples in Fig. 1). These differential bands were sliced out of the gel, and the corresponding cDNA molecules were eluted. The released DNA molecules were reamplified by PCR using the same pair of arbitrary primers as originally used, and the products were either directly cloned into the pGEMT vector (Promega), or blunt ended and cloned into an EcoRV-digested pBluescript II vector (Stratagene).
|
ESTs sequence analysis. Out of 90 ESTs clones sequenced, 72 were unique. Forty-nine of them were rejected as they corresponded to bacterial rRNAs or plant RNAs.
Of the 23 remaining clones, 4 corresponded to already-known S. meliloti nifB, nifH, fixB, and fixC genes. In addition, three of the clones were predicted to encode proteins homologous to Nif proteins from other rhizobium species (Table 1). We identified these genes as the likely nifD, nifE, and nifK orthologues of S. meliloti. nifHDKE and fixABCX expression in S. meliloti strain 2011 was indeed known from previous work (29) to be specifically induced under symbiotic conditions as compared to free-living microoxic conditions (see also Discussion).
|
-proteobacteria Zymomonas mobilis and Rickettsia prowazekii and from
mitochondria. The PDH complex consists of three enzymes: PDH (E1p),
dihydrolipoamide acetyltransferase (E2p), and lipoamide dehydrogenase
(E3) (22). The prediction that RAP3 encoded the E1p
component of the complex was validated by further sequence analysis
(3). The PDH complex catalyzes the synthesis of acetyl
coenzyme A and has indeed been postulated to be a key enzyme of carbon
metabolism in bacteroids.
The RAP5 protein showed limited but very high sequence similarity (66%
identity over 33 amino acids) with argininosuccinate lyases from
Haemophilus influenzae, Escherichia coli, and
Z. mobilis (AAD19417). Argininosuccinate lyase, encoded by
the argH gene in E. coli, contributes to the last
step of arginine biosynthesis. No other potential homologs to RAP5
besides argininosuccinate lyases were detected using the BLASTX 2.0.10 program (Expect = 1). Hence we identified RAP5 as the possible
argH orthologue.
RAP6 is predicted to encode a protein with similarity to the
wall-associated protein WAPA from Bacillus subtilis (Q07833) (11) and to the Rhs proteins from E. coli
(14) (Table 1). The sequence similarity was observed within
a domain (domain PD004656 [ProDom release 99.2]) only detected so far
in WAPA and Rhs proteins. These proteins have been proposed to be
ligand-binding proteins at the surface of the bacterial cell.
The RAP8 translation product showed similarity with CopB of
Xanthomonas campestris and Pseudomonas syringae
and with PcoB of E. coli (Table 1). The sequence similarity
was observed on a relatively large portion of the protein (160 amino
acids) and specifically with CopB of X. campestris and
P. syringae (P12375) and PcoB of E. coli. Again,
the region of similarity lies within a domain (domain PD024328 [ProDom
release 99.2]) that is unique to these proteins. The cop
operon from P. syringae encodes two periplasmic proteins,
CopA and CopC; an outer membrane protein, CopB; and a probable inner
membrane protein, CopD. Together, these proteins contribute to copper
resistance in sequestering and compartmentalizing copper in the
periplasm and outer membrane (4, 18).
Expression analysis of the RAP genes by RT-PCR. Detailed expression analysis of the RAP genes, except those corresponding to nif and fix genes, was performed by RT-PCR on different total RNA preparations. Using specific primers designed from the DNA sequence of the different clones, RT-PCR products were obtained and subjected to Southern hybridization. Specific radiolabeled probes were generated by PCR amplification of the corresponding clones. As a control, the constitutively expressed S. meliloti hemA gene (2) was in the same way subjected to the RT-PCR procedure using hemA-specific primers. No RT-PCR product was detected in reaction mixture controls that contained no reverse transcriptase, thus excluding a contamination of the RNA preparations by DNA. RT-PCRs were performed at least two times independently to assess reproducibility.
To validate the RT-PCR procedure under our experimental conditions, the expression profiles of the nifH and fixN genes were determined by this technique. nifH and fixN are two S. meliloti symbiotic genes whose regulation of expression has been characterized in detail using gene fusions. nifH and fixN expression is regulated by the nifA and fixK genes, respectively, in response to low oxygen signalling mediated by the FixLJ system (7). Results obtained by RT-PCR (Fig. 2) were in full accordance with previous data. Expression of fixN was indeed detected under both microoxic and symbiotic conditions (Fig. 2A), whereas nifH expression was almost exclusively detected under symbiotic conditions, as reported before (26).
|
Expression analysis of the symbiotically regulated genes in nif and fix mutants of S. meliloti. To test whether expression of the newly isolated genes was controlled by the fixLJ, fixK, nifA regulatory pathway, RT-PCRs were performed on RNAs isolated from selected mutants of S. meliloti. RNAs were extracted from bacteroids of the wild type (GMI211), fixK (GMI942), nifA (GMI5601), and fixJ (GMI5704) S. meliloti strains (Fig. 2B). Because non-nitrogen-fixing nodules senesce rapidly, RNAs were extracted from mutant and wild-type bacteroids only 2 weeks after inoculation of alfalfa.
RT-PCRs showed that fixN expression depended both on fixJ and fixK, whereas nifH expression was abolished in both a fixJ and a nifA mutant (Fig. 2B). These data are fully consistent with those described before, using gene fusions (6, 26). No effect of the fixJ, fixK, and nifA mutations was detected on RAP3 symbiotic expression. RAP3 is thus a clear example of a gene whose expression is induced in bacteroids independently of oxygen and of the fixLJ regulatory cascade and whose expression does not depend on the nitrogen fixation status of the nodule. RAP1 and RAP2 genes showed reduced expression in a fixJ mutant (Fig. 2B). In addition, RAP1 expression was reduced in a fixK mutant, whereas RAP2 expression was reduced in a nifA mutant. These results suggest a positive, albeit partial, control of RAP1 and RAP2 expression by fixK and nifA, respectively. By contrast, expression of RAP4, -5, and -6 was markedly decreased in all three nif and fix mutants tested. We hypothesized that this could be a consequence of the Fix
status of these nodules. Further support for this hypothesis came from
the observation that RAP4 symbiotic expression was also greatly
decreased in a nifH mutant (data not shown). This feature thus clearly distinguishes RAP4, -5, and -6 from the known
nif and fix genes whose expression is not
affected by the Fix status of the nodule (see
nifH and fixN in Fig. 2B).
The RAP8, RAP9, and RAP10 genes were clearly derepressed in the
fixK mutant, thus suggesting their symbiotic repression is mediated by the FixK transcriptional regulator.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Detection of differential transcription by RAP. Genetics is the method of choice for identifying bacterial genes involved in complex, symbiotic or pathogenic, interactions. Because some of these genes can be essential for free-living bacteria and because not all of them display a clear phenotype upon inactivation, molecular techniques are also of interest. As far as the rhizobium-legume symbiosis is concerned, the in vivo expression technology strategy, originally developed in plant and animal pathogens (21), has been recently used to identify bacterial genes expressed within the nodule (20). Also, a transcriptional survey of the recently sequenced 500-kb symbiotic plasmid of Rhizobium sp. strain NGR234 (13) was performed by reverse-Northern blotting (23). Here we report on another approach, based on the RAP molecular technology (30) in an effort to identify new bacterial genes regulated during symbiosis. The use of both the in vivo expression technology and RAP approaches is based on the hypothesis that genes specifically expressed in bacteroids could be of special importance for establishment of the symbiosis. The RAP approach allowed us to isolate 13 ESTs corresponding to S. meliloti genes activated in bacteroids. Seven of these 13 ESTs corresponded to known or predicted nif and fix genes of S. meliloti, including nifB, nifD, nifE, nifH, nifK, fixB, and fixC. These genes are under the control of the NifA transcriptional activator. It is known from previous work that expression of a nifH-lacZ fusion, although it depends on the fixLJ-nifA regulatory cascade, cannot be obtained under free-living microoxic conditions in strain 2011, which we used in this work (28). The reason for this lack of nifH expression in pure culture is not completely clear. We previously proposed that NifA synthesis or activity could be limiting under nonsymbiotic conditions in strain 2011 (28, 29). In the present work, we have confirmed using RT-PCR that nifH expression was indeed restricted to nodules (Fig. 2A). Thus, isolation of nifA-dependent genes in the course of the RAP screening procedure is consistent with the literature.
We observed a high proportion of contaminants and false positives in our screening, many of which were of ribosomal origin, as already described (19). We also isolated sequences of plant origin probably because abundant plant mRNAs copurified with bacteroids. Three products were isolated that corresponded to genes actually repressed under symbiotic conditions, i.e., that had a pattern of expression opposite to what we expected. The likely reason that explains isolation of these sequences is the following. As described above, a band that appears differential on a gel (Fig. 1) actually consists of several molecular species, of which relative abundance may vary. We suspect that the RAP8, -9, and -10 sequences were isolated, for they corresponded to poorly abundant but readily clonable contaminant species. Nevertheless, these genes were retained for further analysis as their characterization might reveal genetic and physiological regulations operating during symbiosis. In addition, it is known in other rhizobia that turning some genes off, such as nod genes in R. leguminosarum bv. viciae, is essential for normal establishment of the symbiotic interaction (15). Thus, the characterization of these repressed genes is of interest as it could shed light on critical changes occurring during bacteroid differentiation.New symbiotically regulated S. meliloti genes.
Identification of orthologues from limited sequence data is of course
difficult. The forthcoming availability of the complete sequence of the
genome of S. meliloti
(http://sequence.toulouse.inra.fr/meliloti.html) will
help solve this problem. However, the high similarity observed over the
full length of the RAP sequences suggests that the predictions we have
made in this work are reasonably safe. For example, further sequence
analysis of RAP3 indeed confirmed that it encodes the -subunit of
the PDH complex of S. meliloti (3). It has been proposed that PDH contributes, together with malic enzymes, to the
synthesis of acetyl coenzyme A from malate and succinate, which are the
restricted carbon sources of bacteroids (10).
Regulation of the new S. meliloti genes. Symbiotic expression of nine RAP genes was investigated in the known regulatory mutants, fixJ, fixK, and nifA by RT-PCR. As a control, the profile of expression of nifH and fixN, two S. meliloti symbiotic genes whose expression has been studied in great detail with gene fusions, was determined in the RT-PCR assay. Data obtained using these two techniques were in full agreement, thus validating the RT-PCR procedure. The RT-PCR assay, however, should be considered only as a semiquantitative assay under our experimental conditions. Nevertheless, we are confident that major and reproducibly observed changes reported here are indeed meaningful.
Most of the selected RAP genes exhibited original profiles of expression. As for RAP1 and RAP2, RAP3 expression is induced in the symbiotic stage as compared to that under free-living conditions. Symbiotic expression of RAP3 did not depend on the fixLJK-nifA regulatory pathway. Moreover, we have found recently that expression of the corresponding pdhA gene is not triggered in minimal M9 medium using either mannitol or succinate as a carbon source (3). Instead high expression of pdhA (RAP3) was observed in the presence of pyruvate, likely acting as a coinducer (3). Although RAP1, -2, and -3 all displayed initially the same pattern of expression (Fig. 2A), several lines of evidence indicate that RAP1 and RAP2 are under a different genetic control than RAP3. First, RAP1 and RAP2 expression is not influenced by pyruvate. Second, whereas RAP3 expression is completely independent of the fixLJ regulatory operon, both RAP1 and RAP2 expression was partly but reproducibly weakened in a fixJ background. Moreover, RAP1 expression was lowered in a fixK background as compared to the wild type but was unaffected by a nifA mutation. Conversely, RAP2 expression was weakened in a nifA background and unaffected in a fixK mutation. This suggests that RAP1 and RAP2 might be under fixK and nifA control, respectively. fixK clearly acts also as a repressor. In the case of RAP8, RAP9, and RAP10, whose expression is repressed in bacteroids, strong derepression was observed in a fixK mutant. Thus, fixK represses directly or indirectly expression of these genes. However, for RAP9 and -10, no significant derepression was observed in a fixJ mutant in spite of the fact that fixJ positively regulates fixK expression (2). One possibility to rationalize this observation would be that fixJ positively regulates RAP9 and RAP10 expression. Such a situation has been already reported for nifA and fixK, which are indeed both under positive control by fixJ and negative control by fixK. Finally, RAP4, -5, and -6 clearly differed from known nif and fix genes since their symbiotic expression was reduced in Fix mutants, including a nifH mutant in the
case of RAP4. We thus propose that symbiotic expression of these genes
depends not only on oxygen but also on the nitrogen fixation status of
the nodule.
Detailed studies are now required to assess the validity of the
hypotheses made above and to clarify the underlying genetic pathways.
The present work is highly complementary to the S. meliloti genome sequencing project which is under way. Knowledge of the complete
genome sequence will help in characterizing further the genes described
here. Inversely, the present work will contribute to functional
genetics as it will point to regions of the genome which might be of
special relevance for symbiosis.
| |
ACKNOWLEDGMENTS |
|---|
We thank C. Labeur and L. Mène-Saffrane for their contribution to this work, L. Sauviac for automatic cycle sequencing, and J. Gouzy for expert help in sequence analysis. J. V. Cullimore and F. Ampe are gratefully acknowledged for critical reading of the manuscript.
This work was supported in part by a grant from the European Union in the frame of the BIOTECH programme (FIXNET, BIOT4-CT97-2319). D.C. was supported by a doctoral fellowship from the French Ministère de l'Enseignement Supérieur et de la Recherche and by the FIXNET program.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Laboratoire de Biologie Moléculaire des Relations Plantes-Microorganismes, UMR 215, CNRS-INRA BP27, 31326 Castanet-Tolosan Cedex, France. Phone: (33) 5 61 28 50 54. Fax: (33) 5 61 28 50 61. E-mail: jbatut{at}toulouse.inra.fr.
Present address: Unité des Interactions Bacteries-Cellules,
Institut Pasteur, 75015 Paris, France.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Altschul, S. F.,
T. L. Madden,
A. A. Schaffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. J. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res.
25:3389-3402 |
| 2. | Batut, J., M. L. Daveran-Mingot, M. David, J. Jacobs, A. M. Garnerone, and D. Kahn. 1989. fixK, a gene homologous with fnr and crp from Escherichia coli, regulates nitrogen fixation genes both positively and negatively in Rhizobium meliloti. EMBO J. 8:1279-1286[Medline]. |
| 3. | Cabanes, D., P. Boistard, and J. Batut. Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti. Mol. Plant-Microbe Interact., in press. |
| 4. | Cooksey, D. A. 1993. Copper uptake and resistance in bacteria. Mol. Microbiol. 7:1-5[CrossRef][Medline]. |
| 5. |
Corpet, F.
1988.
Multiple sequence alignment with hierarchical clustering.
Nucleic Acids Res.
16:10881-10890 |
| 6. |
Corpet, F.,
J. Gouzy, and D. Kahn.
1999.
Recent improvements of the ProDom database of protein domain families.
Nucleic Acids Res.
27:263-267 |
| 7. | David, M., M. L. Daveran, J. Batut, A. Dedieu, O. Domergue, J. Ghai, C. Hertig, P. Boistard, and D. Kahn. 1988. Cascade regulation of nif gene expression in Rhizobium meliloti. Cell 54:671-683[CrossRef][Medline]. |
| 8. |
de Philip, P.,
J. Batut, and P. Boistard.
1990.
Rhizobium meliloti Fix L is an oxygen sensor and regulates R. meliloti nifA and fixK genes differently in Escherichia coli.
J. Bacteriol.
172:4255-4262 |
| 9. |
Ditta, G.,
E. Virts,
A. Palomares, and C. H. Kim.
1987.
The nifA gene of Rhizobium meliloti is oxygen regulated.
J. Bacteriol.
169:3217-3223 |
| 10. | Driscoll, B. T., and T. M. Finan. 1993. NAD(+)-dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation. Mol. Microbiol. 7:865-873[CrossRef][Medline]. |
| 11. | Foster, S. J. 1993. Molecular analysis of three major wall-associated proteins of Bacillus subtilis 168: evidence for processing of the product of a gene encoding a 258 kDa precursor two-domain ligand-binding protein. Mol. Microbiol. 8:299-310[CrossRef][Medline]. |
| 12. | Foussard, M., A. M. Garnerone, F. Ni, E. Soupene, P. Boistard, and J. Batut. 1997. Negative autoregulation of the Rhizobium meliloti fixK gene is indirect and requires a newly identified regulator, FixT. Mol. Microbiol. 25:27-37[CrossRef][Medline]. |
| 13. | Freiberg, C., R. Fellay, A. Bairoch, W. J. Broughton, A. Rosenthal, and X. Perret. 1997. Molecular basis of symbiosis between Rhizobium and legumes. Nature 387:394-401[CrossRef][Medline]. |
| 14. | Hill, C. W., C. H. Sandt, and D. A. Vlazny. 1994. Rhs elements of Escherichia coli: a family of genetic composites each encoding a large mosaic protein. Mol. Microbiol. 12:865-871[CrossRef][Medline]. |
| 15. |
Knight, C. D.,
L. Rossen,
J. G. Robertson,
B. Wells, and J. A. Downie.
1986.
Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.
J. Bacteriol.
166:552-558 |
| 16. |
Liang, P., and A. B. Pardee.
1992.
Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction.
Science
257:967-971 |
| 17. | Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 18. |
Mills, S. D.,
C. A. Jasalavich, and D. A. Cooksey.
1993.
A two-component regulatory system required for copper-inducible expression of the copper resistance operon of Pseudomonas syringae.
J. Bacteriol.
175:1656-1664 |
| 19. | Nagel, A., J. T. Fleming, and G. S. Sayler. 1999. Reduction of false positives in prokaryotic mRNA differential display. BioTechniques 26:641-643[Medline], 648. |
| 20. | Oke, V., and S. R. Long. 1999. Bacterial genes induced within the nodule during the Rhizobium-legume symbiosis. Mol. Microbiol. 32:837-849[CrossRef][Medline]. |
| 21. | Osbourn, A. E., C. E. Barber, and M. J. Daniels. 1987. Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid. EMBO J. 6:23-28[Medline]. |
| 22. | Patel, M. S., and T. E. Roche. 1990. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J. 4:3224-3233[Abstract]. |
| 23. | Perret, X., C. Freiberg, A. Rosenthal, W. J. Broughton, and R. Fellay. 1999. High-resolution transcriptional analysis of the symbiotic plasmid of Rhizobium sp. NGR234. Mol. Microbiol. 32:415-425[CrossRef][Medline]. |
| 24. | Preisig, O., R. Zufferey, and H. Hennecke. 1996. The Bradyrhizobium japonicum fixGHIS genes are required for the formation of the high-affinity cbb3-type cytochrome oxidase. Arch. Microbiol. 165:297-305[CrossRef][Medline]. |
| 25. | Rosenberg, C., P. Boistard, J. Denarie, and F. Casse-Delbart. 1981. Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol. Gen. Genet. 184:326-333[Medline]. |
| 26. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. |
| 27. | Shoham, N. G., T. Arad, R. Rosin-Abersfeld, P. Mashiah, A. Gazit, and A. Yaniv. 1996. Differential display assay and analysis. BioTechniques 20:182-184[Medline]. |
| 28. | Soupene, E. 1995. Régulation symbiotique de l'expression de gènes impliqués dans la fixation de l'azote lors de l'interaction Rhizobium meliloti- Medicago sativa: rôle de l'oxygène. This. Université Paul Sabatier, Toulouse, France. |
| 29. |
Soupene, E.,
M. Foussard,
P. Boistard,
G. Truchet, and J. Batut.
1995.
Oxygen as a key developmental regulator of Rhizobium meliloti N2- fixation gene expression within the alfalfa root nodule.
Proc. Natl. Acad. Sci. USA
92:3759-3763 |
| 30. |
Welsh, J.,
K. Chada,
S. S. Dalal,
R. Cheng,
D. Ralph, and M. McClelland.
1992.
Arbitrarily primed PCR fingerprinting of RNA.
Nucleic Acids Res.
20:4965-4970 |
Journal of Bacteriology, July 2000, p. 3632-3637, Vol. 182, No. 13
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Gurich, N., Gonzalez, J. E.
(2009). Role of Quorum Sensing in Sinorhizobium meliloti-Alfalfa Symbiosis. J. Bacteriol.
191: 4372-4382
[Abstract] [Full Text] -
Sauviac, L., Philippe, H., Phok, K., Bruand, C.
(2007). An Extracytoplasmic Function Sigma Factor Acts as a General Stress Response Regulator in Sinorhizobium meliloti. J. Bacteriol.
189: 4204-4216
[Abstract] [Full Text] -
Bontemps, C., Golfier, G., Gris-Liebe, C., Carrere, S., Talini, L., Boivin-Masson, C.
(2005). Microarray-Based Detection and Typing of the Rhizobium Nodulation Gene nodC: Potential of DNA Arrays To Diagnose Biological Functions of Interest. Appl. Environ. Microbiol.
71: 8042-8048
[Abstract] [Full Text] -
House, B. L., Mortimer, M. W., Kahn, M. L.
(2004). New Recombination Methods for Sinorhizobium meliloti Genetics. Appl. Environ. Microbiol.
70: 2806-2815
[Abstract] [Full Text] -
Berges, H., Lauber, E., Liebe, C., Batut, J., Kahn, D., de Bruijn, F. J., Ampe, F.
(2003). Development of Sinorhizobium meliloti Pilot Macroarrays for Transcriptome Analysis. Appl. Environ. Microbiol.
69: 1214-1219
[Abstract] [Full Text] -
Peck, M. C., Gaal, T., Fisher, R. F., Gourse, R. L., Long, S. R.
(2002). The RNA Polymerase {alpha} Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro. J. Bacteriol.
184: 3808-3814
[Abstract] [Full Text] -
Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., Molteni, E., Huffman, D. L., O'Halloran, T. V.
(2002). Metallochaperones and Metal-Transporting ATPases: A Comparative Analysis of Sequences and Structures. Genome Res
12: 255-271
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»