Laboratoire des Interactions Plantes-Microorganismes, UMR 2594 INRA-CNRS, Chemin de Borde-Rouge, BP 27, 31326 Castanet-Tolosan Cedex, France
Correspondence
Daniel Kahn
dkahn{at}toulouse.inra.fr
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ABSTRACT |
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Present address: Molecular Microbiology Laboratory, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Present address: Laboratoire de Microbiologie et de Génétique Moléculaires, UMR5100 CNRS-UPS, 118 route de Narbonne, 31062 Toulouse Cedex, France.
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INTRODUCTION |
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The FixLJ, FixK and NifA regulatory elements are conserved among rhizobia, but they differ in connectivity, targets and function. As a consequence, different Rhizobium species possess distinct, species-specific regulatory networks for symbiotic nitrogen fixation (Fischer, 1994). In S. meliloti the nifA gene is directly regulated by FixJ, in Azorhizobium caulinodans nifA is under the direct control of FixK, while in Bradyrhizobium japonicum nifA expression is fully independent of FixJ. Moreover, two copies of fixK are present in the latter species, with FixJ activating fixK2, which in turn activates fixK1 (Nellen-Anthamatten et al., 1998
). In Rhizobium etli, fixL is not even involved in the regulation of nitrogen fixation (D'Hooghe et al., 1995
, 1998
). Therefore, there appears to be a lot of plasticity in the regulatory pathways controlling nitrogen fixation in rhizobia, making generalization across species difficult.
So far, nifA and fixK have been the only genes known to be directly regulated by FixJ in S. meliloti (Fischer, 1994). The position of the FixLJ system at the top of the regulatory cascade led us to hypothesize the existence of other FixJ targets in the S. meliloti genome. Here we used an in vitro selection approach, genomic SELEX (Singer et al., 1997
), in order to systematically identify FixJ binding sites in the S. meliloti genome. Twenty-two FixJ binding sites were thus isolated. Functional analysis of five of these sites led to the identification of two novel FixJ-regulated genes. This FixJ-dependent expression appeared to be mediated by tandem duplication of the entire pfixK promoter region. A systematic search throughout the genome indicated that promoter duplication is a frequent event underlying the evolution of regulatory pathways in S. meliloti.
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METHODS |
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Construction of the uidA-sp cassette.
A 1·9 kb fragment containing the uidA reporter gene and a preceding ShineDalgarno sequence was extracted from pRG960SD-32 (Van den Eede et al., 1992) by digestion with BamHI and EcoRI, then inserted into pBlueScript-II KS+ (Promega) to generate pGMI2191. A truncated
sp cassette, lacking the T4 transcriptional terminator located upstream of aadA, was amplified by PCR using pHP45
(Prentki & Krisch, 1984
) as a template, with
4 and
5 as primers (Table 2
). To avoid the introduction of mutations, the amplification was carried out with the Pfu polymerase (2 min at 94 °C; 20 cycles of 30 s at 94 °C, 30 s at 60 °C, 4 min at 72 °C; 5 min at 72 °C). The PCR product was purified, digested with EcoRI and integrated into the EcoRI site of pGMI2191, leading to pGMI2192. In pGMI2192, aadA is positioned downstream and in the same orientation as uidA.
Construction of S. meliloti mutant strains.
Genomic fragments (200300 bp) corresponding to the beginning of each gene to be inactivated were amplified by PCR, using the gene-specific A and B primers (Table 2), 10 ng of total genomic DNA from Rm1021 and the Expand Long Template PCR System Kit (Boehringer Mannheim). Fragments corresponding to the end of each gene were similarly amplified using the gene-specific C and D primers (Table 2
). The full uidA-
sp cassette was also amplified from pGMI2192 with the Pfu polymerase and primers uidA-1 and
3. This cassette was then fused with the two amplified gene fragments by primerless elongation with the Expand Long Template PCR System Kit polymerase (2 min at 94 °C; 10 cycles of 30 s at 94 °C, 30 s at 60 °C, 3 min at 68 °C; 5 min at 68 °C). The gene-specific B and C primers were engineered so that they overlapped the uidA-
sp cassette, allowing for the integration of uidA-
sp between the ends of the target gene. In order to amplify the resulting 3·8 kb fusion product, 10 µl of the reaction mixture was added to a 50 µl PCR reaction mixture containing the gene-specific A and D primers. The long range PCR was performed using the same polymerase as above, with the following amplification conditions: 2 min at 94 °C; 10 cycles of 30 s at 94 °C, 30 s at 65 °C, 3 min at 68 °C; 15 cycles of 30 s at 94 °C, 30 s at 65 °C, 3 min plus 20 s/cycle at 68 °C; 7 min at 68 °C. The PCR product was digested with XbaI and cloned into the S. meliloti pJQ200KS suicide plasmid (Quandt & Hynes, 1993
). The functionality of the uidA and aadA genes was subsequently verified by introducing the resulting plasmid into DH5
and plating with X-Gluc and streptomycin. This plasmid was then introduced into Rm1021 by tri-parental mating, using GMI3442 as helper strain. E. coli strains were counter-selected on TY medium containing gentamicin, spectinomycin and streptomycin (400 µg ml1). GMI516, GMI518, GMI520, GMI522, GMI524 and GMI526 S. meliloti mutants strains were finally isolated as Spr, Sucr and Gms colonies. The GMI517, GMI519, GMI521, GMI523, GMI525, GMI527 and GMI528 fixJ strains were obtained by transducing the fixJ2·3 : : Tn5 mutation from GMI347 (David et al., 1988
) into GMI516, GMI518, GMI520, GMI522, GMI524, GMI52 and Rm1021, respectively, using bacteriophage N3 (Martin & Long, 1984
).
-Glucuronidase assays.
-Glucuronidase activity was assayed as described by Jefferson (1987)
. Mid-log culture cells (OD600=0·5) were harvested and resuspended in lysis buffer (50 mM sodium phosphate, pH 7·0, 10 mM
-mercaptoethanol, 10 mM EDTA, 0·1 % sodium lauryl sarcosine and 0·1 % Triton X-100). MUG, 4-methyl-umbelliferyl-
-D-glucuronide (1 mM; Sigma M-9130), was added to initiate the enzymic reaction. After 15 min incubation at 37 °C, the reaction was stopped by 0·2 M Na2CO3. Samples were kept on ice and protected from light until analysis. The concentration of methyl-umbelliferone (MU) was determined by fluorescence at 455 nm (excitation at 365 nm) on a Kontron SFM 25 spectrofluorimeter, using 10 µM MU (Sigma M-1508) as a standard.
RT-PCR.
RNA was extracted from a 15 ml culture grown at 30 °C under aerobic or microoxic conditions, as described by Cabanes et al. (2000). Briefly, cells were harvested at OD600=0·5, lysed for 10 min at 65 °C in 600 µl pre-warmed lysis solution [1·4 % SDS, 4 mM EDTA and 420 µg proteinase K ml1 (Boehringer Mannheim)]. Proteins were precipitated on ice by addition of 150 µl 5 M NaCl solution. The supernatant was recovered, and nucleic acids were precipitated with ethanol, resuspended and digested at 37 °C for 1 h with 80 U DNase I (FPLC pure, Amersham Pharmacia Biotech) in 0·1 M sodium acetate, pH 5, containing 5 mM MgSO4. After phenol/chloroform extraction, RNA was precipitated with ethanol, dissolved in DEPC-treated water and verified on a 1 % agarose gel.
First-strand cDNA synthesis was achieved in a 20 µl volume. RNA (100 ng) was incubated for 10 min at 70 °C in the presence of the appropriate reverse primer (500 nM final concentration), then quickly chilled on ice. Reaction buffer was added (50 mM Tris/HCl, pH 8·3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 500 µM each dNTP), the mix was kept at 42 °C for 2 min, and 200 U SuperScript II (Life Technologies) was added for cDNA synthesis (50 min at 42 °C). Reverse transcriptase was inactivated for 5 min at 95 °C. A portion (2 µl) of this cDNA was then introduced into a 50 µl PCR reaction mix (10 mM Tris/HCl, pH 8·3, 50 mM KCl, 1·5 mM MgCl2, 200 µM each dNTP, 0·02 U Taq polymerase µl1, 800 nM reverse and forward primers) and amplified as follows: 2 min at 94 °C; cycle of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C; 5 min at 72 °C. We used 25 cycles for hemA, fixK and proB2 and 40 cycles for smc03253. The resulting PCR products were analysed on a 2 % agarose gel. The following forward and reverse oligonucleotides were used: HemAS and HemAR for hemA; FixK-PF and FixK-PR for fixK; PROHYD1 and PROHYD2 for smc03253; ProB2-A and ProB2-B for proB2 (see Table 2).
Plant assays.
Seeds of Medicago sativa cv europe were surface sterilized and grown in tubes containing nitrogen-free Fahraeus medium slants (growth conditions: 22 °C, 60 % relative humidity, 16 h of light/8 h of darkness alternations). Three weeks after root inoculation with S. meliloti, nitrogen-fixing activity was assayed by acetylene reduction (Turner & Gibson, 1980).
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RESULTS |
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DNA fragments covering the entire genome were generated by random priming and size-selected as described by Singer et al. (1997). Genomic fragments ranging from 150 to 200 bp were incubated with the GSTFixJC fusion protein, and proteinDNA complexes were adsorbed on glutathioneSepharose beads. Bound DNA was eluted, amplified by PCR and proteinDNA complexes were selected again. After three selection cycles, the mix appeared highly enriched in FixJ binding fragments. These fragments were cloned and sequenced, generating 71 independent sequences. The sequences were compared with one another and clustered into 32 groups, corresponding to different regions in the genome. Among these, 22 regions showed affinity for FixJ
P, as determined by gel retardation assays (listed in Fig. 1
). Importantly, many of them were isolated on distinct overlapping fragments, indicating that they contain genuine FixJ binding sites, independently of the flanking sequences used in the SELEX procedure.
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FixJ-dependent regulation of smc03253 is mediated by duplication of the fixK promoter
The FixJ-regulated smc03253 gene is positioned in a particular region of the chromosome, proposed to have been acquired by horizontal transfer (Capela et al., 2001). smc03253 follows the two Ic and Id FixJ binding sites, which share extensive homology with the pfixK region located on pSymA (Fig. 3
). Besides the FixJ binding site of pfixK, the Ic and Id regions include 124 bp from the fixK coding region. These constitute the beginning of two short identical ORFs,
fixK1 and
fixK2. The corresponding protein sequences share homology with only the first 21 residues of FixK, which suggests that
fixK1 and
fixK2 are pseudogenes resulting from the duplication of the fixK promoter region. As in the pfixK region, a copy of the fixT gene, fixT3, precedes the Id FixJ binding site (Foussard et al., 1997
), with, however, a frameshift occurring 52 bp before the fixT3 stop codon. Moreover, only 12 bp are conserved between the upstream sequences of fixT1 and fixT3, suggesting that the two genes might be regulated differently. This is consistent with previously published genetic data, which indicate that fixT3 is not involved in the negative regulation of the FixLJ cascade (Foussard et al., 1997
). A 384 bp portion of the Id region, containing the FixJ binding site, the
fixK1 pseudogene and the 3' end of fixT3, is tandemly duplicated and forms the Ic site. The Ic and Id duplications differ by a single nucleotide, which is deleted in the Ic copy (Fig. 3
), indicating that this is a recent tandem duplication. We therefore propose that the chromosome acquired the Id region first by duplication of the pfixK region from pSymA, followed by tandem duplication to form the Ic site. Expression of smc03253 is thus regulated by FixJ via a functional duplication of the fixK promoter.
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In this way, 49 distinct families of duplicated promoters were identified. Twenty-three promoter duplications were found upstream of whole-gene duplications. These include promoter regions of fixN1/fixN2, fixT1/fixT2 and fixK1/fixK2, belonging to the fix-1/fix-2 duplicated clusters, respectively (Renalier et al., 1987). The three previously reported syrB-like genes, syrB1, syrB2 and sma0806 (Barnett et al., 2001
), also display strong similarity in their upstream regions. traA1/traA2, repC1/repC2 and groES1/groES2 are other examples of gene duplications that include upstream promoter regions (refer to http://sequence.toulouse.inra.fr/rhime/Promoter_Duplication for complete data). Furthermore, we identified 17 duplications of promoter regions located upstream of non-homologous genes, potentially bringing them under heterologous control (Table S1, available as supplementary data with the online version of this paper at http://mic.sgmjournals.org). These include the P3 copy of pnifH (cluster C02) (Better et al., 1983
), which is located 939 bp upstream of the original nifH promoter, and the pfixK-like site described above (cluster C16). Curiously, a duplication of the rpoN 5' region (rpoN') was found between P3 and the downstream sma0824 gene (cluster C21). Further analysis indicates that rpoN' results from a duplication of the full rpoN gene followed by a 1280 bp internal deletion (Fig. 4
). Indeed, sma0824 shares homology over 190 bp with the 3' end of rpoN, which is consistent with the strong similarity observed between the N-terminal end of SMa0824 and the C-terminal end of
54 (Barnett et al., 2001
). It thus appears that this region has been extensively rearranged. It does not contain essential symbiotic elements, as evidenced by Tn5 insertion analysis (Hirsch et al., 1983
). A third duplication of the nifH promoter region was identified 320 bp upstream of the nodD2 gene (cluster C02). However, the homology between the two sequences extends over 52 bp only, including the first 48 bp of nifH, but excluding promoter elements. The 6 to 89 region including the nifH promoter was found in the opposite orientation, suggesting that the nifH promoter was inverted after duplication. Finally, nine intergenic elements appeared to be duplicated within the coding regions of putative genes (Table S2, available as supplementary data with the online version of this paper at http://mic.sgmjournals.org).
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DISCUSSION |
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Non-functional FixJ binding sites?
Most of the FixJ binding sites isolated by genomic SELEX do not seem to be directly functional for transcriptional activation. Indeed 11 of the 22 newly identified FixJ targets are located downstream from or inside genes, making them unlikely candidates as regulatory targets. Furthermore, three of the five candidate sites chosen for further analysis were found not to mediate fixJ-dependent regulation of downstream genes. The determination of the function of these sites will require further experimentation. One hypothesis is that they might serve as reservoir sites facilitating FixJP diffusion and target location (Gowers & Halford, 2003
; von Hippel & Berg, 1989
). Indeed it has been shown that topologically linked non-specific proteinDNA interactions facilitate target location through 3D space (von Hippel & Berg, 1989
), accounting for the very fast forward rates typically found (Gowers & Halford, 2003
; von Hippel & Berg, 1989
). This hypothesis would also rationalize the strong bias in the distribution of non-functional FixJ sites, which places them in the vicinity of functional FixJ targets (Gowers & Halford, 2003
). The prevalence of seemingly non-functional FixJ sites in the S. meliloti genome can also be paralleled with earlier work by Berg and von Hippel, who predicted the existence of a large number of CRP pseudo-sites in the E. coli genome (Berg & von Hippel, 1988
).
Novel fixJ-regulated genes
In this work, two novel FixJ-regulated genes have been identified in S. meliloti: smc03253 and proB2. Like nifA and fixK, these genes are induced microaerobically under FixJ control. However, unlike nifA and fixK, they are not essential for symbiotic nitrogen fixation, and their role remains elusive. Both genes share homology with genes involved in proline metabolism. SMc03253 is homologous to L-proline cis-3-hydroxylase isolated from Streptomyces. This enzyme modifies free L-proline by stereospecific hydroxylation (Mori et al., 1997). ProB2 is homologous to the ProB
-glutamate kinase of E. coli, which acts at the first step in the proline biosynthetic pathway. However, proB2 is not the likely proB orthologue in S. meliloti. Indeed, the genome contains another copy, proB1, which displays a stronger similarity with proB and forms an operon with proA. Furthermore, proB2 was not required for proline prototrophy in S. meliloti (data not shown). We therefore suggest that proB2 and smc03253 are involved in a facultative proline-related pathway in bacteroids. In this context, it is also worth mentioning the importance of proline catabolism for symbiotic nitrogen fixation (King et al., 2000
; Jimenez-Zurdo et al., 1997
). The possible function of hydroxyproline in S. meliloti is unclear. In eukaryotes, hydroxyproline has been identified in collagen and in many plant cell-wall proteins in which L-proline is hydroxylated post-translationally (Adams & Frank, 1980
). In prokaryotes, hydroxyproline has been found in some peptide antibiotics such as etamycin (Sheehan et al., 1958
), plusbacin (Shoji et al., 1992
) and telomycin (Irreverre et al., 1962
). Detection of cis-3-proline hydroxylase activity in the Streptomyces canus strain producing telomycin suggests that hydroxyproline is incorporated as a free residue into peptide antibiotics (Mori et al., 1996
). By analogy, smc03253 might be involved in secondary metabolism in S. meliloti. Although we have shown that proB2 and smc03253 are not essential for symbiosis, we cannot rule out that they might provide an advantage to the bacterium in a natural environment, as was shown to be the case for the mos genes involved in rhizopine synthesis (Murphy et al., 1988
). Orthologues of smc03253 and proB2 can be found in the symbiosis island of Mesorhizobium loti strain MAFF303099: mlr6283 and mlr6298, respectively (Kaneko et al., 2000
). However, they are missing in the symbiosis island of another strain of M. loti, R7A (Sullivan et al., 2002
), and in the Bradyrhizobium japonicum genome (Kaneko et al., 2002
).
FixJ-independent microaerobic gene induction
During the course of this study we identified two microaerobically induced genes, smc03247 and sma1447, which unexpectedly escaped FixJ-dependent regulation, despite an adequate FixJ binding site. Other genes have been shown to be regulated by oxygen in a FixJ-independent fashion, such as the asnO gene, which encodes an asparagine synthetase homologue (Bergès et al., 2001), and the six loe-2, loe-3, loe-5, loe-6, loe-8 and loe-9 loci (Trzebiatowski et al., 2001
). Furthermore, microaerobic induction of nifA appears not to be solely regulated by the FixLJ system (Kahn & Ditta, 1991
). These observations point to the existence of other regulatory systems in S. meliloti that respond to low oxygen concentration.
Promoter duplication and genome adaptation
The FixJ-dependent expression of smc03253 is mediated by duplication of the fixK promoter region. This is not unprecedented; indeed, earlier studies have identified several duplications of the pnifH promoter, which controls expression of nitrogenase in S. meliloti. pnifH is a 54-dependent promoter which requires the NifA transcriptional regulator for activation. Four copies of pnifH (P1, P2, P3 and P4) have been identified in S. meliloti strain 102F34 (Better et al., 1983
). P1, P2 and P3 are located in the nif region of pSymA (Better et al., 1983
). P1 is the original nifH promoter, while P2 controls expression of the fixABCX genes involved in electron transport to nitrogenase (Earl et al., 1987
; Better et al., 1985
). The function of P3 remains unknown, as it is not essential for symbiotic nitrogen fixation (Hirsch et al., 1983
). Intriguingly, this region contains duplications of parts of the
54 gene rpoN, a gene required for pnifH activity. Another copy of pnifH has been identified upstream of the mos operon in S. meliloti strains L5-30 (Murphy et al., 1988
) and Rm220-3 (Rao et al., 1995
). mos genes are involved in rhizopine production in these strains, but are absent from strain Rm1021. Rhizopines have been proposed to be used as growth substrates by nodule-inducing bacteria, providing a selective advantage for the bacterial partner. However, they are not required for symbiotic nitrogen fixation. Two key symbiotic promoters, pnifH and pfixK, have thus been recruited for the control of other genes which are not essential for symbiosis. A systematic search for other such promoter duplications in the genome revealed the existence of 15 additional candidates, suggesting that promoter duplication is a common phenomenon that extends the regulatory repertoire of S. meliloti. It thus appears that bacteria can generate new regulatory pathways by the combinatorial assortment of pre-existing cis-regulatory elements.
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ACKNOWLEDGEMENTS |
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Received 4 February 2004;
revised 5 April 2004;
accepted 6 April 2004.
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