A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti
© Bergès et al, licensee BioMed Central Ltd. 2001
Received: 4 April 2001
Accepted: 22 May 2001
Published: 22 May 2001
Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown.
We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis .
Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.
Sinorhizobium meliloti forms N2-fixing nodules on the roots of alfalfa (Medicago sativa) and closely related plants. Expression of nitrogen fixation genes is under both positive and negative control. This regulation depends on a regulatory cascade, on top of which the two-component regulatory system FixLJ activates expression of nitrogen fixation genes in response to microoxic conditions , such as those that prevail inside the nodule . Under microoxic conditions, the sensor histidine kinase FixL autophosphorylates and transfers its phosphate to the FixJ transcriptional regulator protein . Phosphorylated FixJ then activates transcription of two intermediate regulatory genes, nifA and fixK, that both encode transcriptional regulators . NifA mediates activation of nif genes involved in nitrogenase biosynthesis whereas FixK, a member of the Crp/Fnr family, activates expression of genes involved in the synthesis of a respiratory oxidase complex [4, 5]. fixK is also indirectly responsible for negative regulation of the cascade since it controls expression of a gene, fixT, that negatively affects expression of FixLJ dependent genes (see Figure 6). We have shown recently that the FixT protein negatively affects the expression of nifA and fixK by inhibiting phosphorylation of the sensor hemoprotein kinase FixL and, by consequence, phosphorylation of FixJ . Whether FixT serves a mere homeostatic function in S. meliloti (the level of FixT protein feed-back controlling activity of the FixLJ system) or whether FixT allows integration of a physiological signal by the FixLJ system was so far unknown. We addressed this question by looking for S. meliloti mutants in which the FixT protein would not be active in repression.
Here we report the isolation of a S. meliloti mutant strain that phenotypically escapes the repressor activity exerted by FixT. The mutation lies in a gene named asnO encoding a protein homologous to glutamine-amidotranferases. We discuss the significance of this finding with respect to the regulation of symbiotic nitrogen fixation.
Results and Discussion
Isolation of a S. meliloti mutant strain escaping repression by FixT
Bacterial strains and plasmids used
SmR Nod+ Fix+
GMI211 asnO::Tn5 SmR NmR
GMI211 fixJ2.3::Tn5 SmR NmR BleoR
asnA31 asnB32 thi
Pro-82 thi-1 hsdR17 supE44 endA1
endA1 hsdR17 (rk-mk+) supE44 thi-1 recA1 gyrA relA1Δ (lacIZYA-
argF)U169 deoR (φ 80dlacΔ (lacZ)M15)
pGD926 (IncP broad host range vector) derivative carrying a fixK-
lacZ fusion with a mutation in the fixT promoter. TcR
pGMI41211 (Inc-P1 broad host range vector) derivative carrying a
nifA-lacZ fusion. TcR
Same as pMF10 but with fixT in reverse orientation. GmR
pBBR1-MCS3 derivative, carrying an HindIII fragment containing
the asnO gene and the SMb20482 orf.
pBBR1-MCS3 derivative, expressing asnO under the control of the
pUC18 digested with EcoRI, carrying the S. meliloti EcoRI
fragment containing the asn O region with the Tn5 insert. ApR,
pBeloBAC11 containing a 100 kb S. meliloti genome fragment
carrying the asn-like region of pSymB.
PRK600::Tn5, Cmr, Nm-Kmr
In the GMI401 mutant strain, the level of fixK gene expression in the absence of pMF10 was the same as in the wild-type strain (Figure 1A; compare lane 1 to lane 4 and lane 5). This result demonstrated that activation of fixK gene expression by the FixLJ two-component system was not affected in the mutant strain, but rather that the phenotype was genuinely due to decreased repression by fixT. Western-Blot analysis using an antibody directed against the FixT protein indicated that the level of FixT protein was the same in the GMI211(pMF10) wild-type strain and in the GMI401(pMF10) mutant strain (data not shown).
Similar results have been obtained using a nifA-lacZ fusion (pCHK57 plasmid). The inhibition of nifA-lacZ expression by overexpressed fixT that was observed in a GMI211 wild-type strain was not observed anymore in the GMI401 mutant strain (data not shown).
Altogether, these results tend to indicate that, in the GMI401 mutant strain, the absence of repression of fixK and nifA by fixT was due to a reduced inhibition of the FixLJ system by the FixT protein.
The GMI401 mutant strain produced nitrogen fixing nodules on M. sativa, as did the GMI211 wild-type strain (data not shown). Whereas the GMI211 parent strain expressing fixT constitutively produced non N2-fixing nodules (Figure 1B), the GMI401 mutant strain was able to induce effective N2-fixing nodules on alfalfa (Medicago sativa cv. gemini) thus enabling the plants to grow in the absence of combined nitrogen. Hence, the gene that modulates repression by FixT activity ex planta is also active in planta.
The Tn5-insertion maps in an asparagine synthetase-like gene
The asnO gene complements the mutant phenotype of GMI401
asnO inactivation in GMI401 does not lead to auxotrophy for asparagine
Because of the homology of the S. meliloti asn O gene product with known asparagine synthetases, we tested whether the asnO gene was involved in asparagine biosynthesis, by three complementary approaches (data not shown).
First, we observed that the mutant strain GMI401 and the isogenic GMI211 wild-type strain, grew at equal and similar rates in minimal medium with or without asparagine.
Second, we tested the ability of pBasn2 to restore prototrophy of a E. coli asparagine auxotroph, a double asnB/asnA mutant strain (Table 1). In asparagine-supplemented minimal medium, all strains grew at equal rates. However, in the absence of added asparagine, the S. meliloti asnO gene under the control of the plac promoter was unable to complement the asparagine auxotrophy of the asnA/asnB E. coli mutant strain.
Third, addition of asparagine at different concentrations in minimal medium, did not lead to complementation of the S. meliloti GMI401 mutant phenotype, i.e did not restore repression of fixK gene expression by overexpressed fixT. This observation suggested that the mutant phenotype is not dependent on the level of asparagine, and hence, that the asnO gene does not mediate asparagine synthesis.
The reactions catalyzed by asparagine synthetases involve two different family of proteins depending on whether glutamine or ammonia is used as a nitrogen source. Members of the AsnA family, that are found in procaryotes, only use ammonia as the amino group donor [11, 12]. The AsnB family, which is found in both procaryotes and eucaryotes, preferentially uses glutamine as nitrogen source [13, 14]. E. coli possesses an asnA and an asnB gene  whereas three asparagine synthetase genes, asnB, asnH and asnO, which all belong to the AsnB family, have been characterized in B. subtilis. No member of the AsnA family has been found in B. subtilis . Complete genome analysis indicated that S. meliloti carries no asnA-like gene and two asnB-type genes (see http://sequence.toulouse.inra.fr/meliloti.html) one of which is asn O and a second gene that was named asnB. Sequence analysis is consistent with the possibility that the asnB gene of S. meliloti might be the ortholog of the biosynthetic asnB gene of E. coli and B. subtilis whereas the asn O gene mutated in GMI401 may play a regulatory function, in relationship with nitrogen fixation and microoxic respiration (Figure 3). Similarly, the asnO gene of B. subtilis has a regulatory role in sporulation , whereas the ltsA gene of Corynebacterium glutamicum is involved in cell wall formation .
asnO gene expression is induced in microoxic conditions
FixT is an intriguing protein as it has not been described so far in any other bacterium besides S. meliloti. Furthermore its mode of action is original, as it has the capacity to block phosphorylation, and hence activity, of the FixL sensor histidine kinase. They are only a few examples of such anti-kinase proteins in the literature. Lastly, FixT primary sequence did not provide clues to its function. There is thus a great deal of interest in determining the biological role of fixT in S. meliloti.
Further work is required to elucidate the relationship between fixT, fixL and asnO. We propose as a working model that the absence of AsnO may result in an imbalance in the pool of a metabolite (e.g a substrate or a product of AsnO), that would affect the intrinsic repressing activity of FixT or, equally, the interaction between FixT and FixL. Identification of the reaction catalyzed by AsnO and further elucidation of the mode of action of the FixT protein should shed light to this model. Because glutamine, a likely by-product of nitrogen fixation in symbiotic rhizobia, is a predicted substrate of the AsnO protein, it is tempting to speculate that asnO and fixT may provide a link between the nitrogen status of bacteria -or of the plant cell- and nitrogen fixation activity and reducing power generation. Possibly, such a genetic device may connect the nitrogen needs of the plant to the nitrogen fixation activity of the microsymbiont.
Materials and Methods
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are listed in Table 1. S. meliloti GMI211, the symbiotically effective parent strain used in this study is a lac Smr, Nod+ Fix+ derivative of S meliloti RCR2011 . Tn5 transposon mutagenesis of GMI211 was performed using the pRK602 plasmid (Table 1, ). In order to isolate random Tn5 insertions in the S. meliloti genome, we conjugated E. coli MM294(pRK602) donor strain with a S. meliloti GMI211(pMF10) recipient strains, and screened for streptomycin (100 μg/ml) and neomycin (100 μg/ml) resistant transconjugants.
S. meliloti strains were grown at 30°C in TY complex medium or in defined M9 medium  supplemented with 0.3 mM CaCl2, 1 mM MgSO4 and 2 μM biotin. Sterilised carbon sources were added at 20 mM final concentrations. Microoxic conditions were achieved as described by de Philip et al  (2% oxygen for 4 h). For testing induction of fixK-LacZ expression by microoxic conditions, S. meliloti strains were previously grown to OD600 = 0.3 in M9 medium. β-Galactosidase assays were performed as described by de Philip et al. .
DNA techniques were carried out as described by Sambrook et al. . S. meliloti genomic DNA was prepared as described by Chen and Kuo . For Southern-Blot analysis, 1 μg of genomic DNA was digested at 37°C for 4 hours with the appropriate enzymes. DNA fragments were separated on a 0.8% agarose gel, transferred onto a nylon membrane (BiodyneA transfer membrane, Pall, East Hills, NY), and hybridized with a Tn5 32P-labelled probe.
asnO cloning and sequencing
A genomic fragment of S. meliloti GMI401 carrying the Tn5 insertion was cloned from a EcoRI-digested genome fragment into a pUC18 plasmid, resulting in the pUC23 plasmid (see Table 1). However, the presence of a Eco RI restriction site in the asnO gene (see Figure 2) did not permit to obtain the entire sequence of the gene from this construct. Therefore, a library of 96 BACs covering the S. meliloti genome , was screened to isolate the full-length asnO gene. PCR screening of the BAC library was performed using the oligonucleotide primers ASN+54 and ASN-9 shown in Figure 2. By this method, we could detect the presence of the asnO gene on BAC37, that was assigned to the pSymB megaplasmid . Complete sequence of the asnO region was determined by a primer-walking approach on the BAC37 and pBH1 plasmid (see Table 1), which was obtained by subcloning a HindIII fragment of BAC37 in pBBR1-MCS3 . Sequencing was performed on a ABI373 automated sequencer (ABI, Columbia, MD), using the ABI PRISM Dye terminator cycle sequencing ready kit (Perkin-Elmer, Oak, Brook, IL).
Putative open reading frame were predicted using the FrameD program  used for S. meliloti whole genome analysis (http://sequence.toulouse.inra.fr/meliloti.html). Protein analysis was done with the NCBI web page using the BLAST2 package program  against the NCBI-nr and Swiss-Prot databases. Homologous domains searches were driven using the ProDom database .
Medicago sativa cv. Gemini seedlings were aseptically grown on agar slants made up with nitrogen-free Fahraeus medium. Three-day-old plants were inoculated with the different S. meliloti strains and grown for 3 weeks before observation.
The bacteria from a 25 ml culture at 0.4 OD600nm were harvested and RNA prepared with the Qiagen RNeasy kit as described by the manufacturer. DNA was eliminated by addition of 7.5 Units of FPLC-Pure RNase-free DNaseI (Amersham-Pharmacia Biotech). RNA was further extracted with phenol-chloroform and then precipitated with ethanol. After washing with 70% ethanol, the pellet was resuspended in nuclease-free water. RNA was quantified by absorbance measurements at 260 nm. Absence of DNA contamination was verified by PCR amplification.
RT-PCR reactions were performed according to the manufacturer, using the SuperScript™ One-Step RT-PCR System (GibcoBRL).
The following oligonucleotides were used for reverse transcription of the messenger RNA and amplification of the product to evaluate gene expression :
For the hemA gene: hemAr (reverse primer) : 5'-GTCGATCGCGTTCTT-3'; hemAf (foward primer) : 5'-TGGATGGGCTGCATCA-3'
For the asnO gene: RTA1 (reverse primer) : 5'-TGCGTATTCTCGACCTG-3'; RTA2 (foward primer) : 5'-TCGCGAAAATTGTAGATG-3'
For the fixK gene: KPR (reverse primer) : 5'- CCGATTACCAGAAGATGC-3'; KPF (foward primer) : 5'-TATCTACCGCCTCCTTTC-3'
RT-PCR products were electrophoresed on a 2% agarose gel, blotted onto a nylon membrane and probed with a 32P-labeled DNA probe prepared from the gene of interest. Washing was done with 0.1XSSC, 0.1%SDS at 42°C during 30 min.
We thank Dr T. Hirasawa for generous gift of E. coli strain ER, Dr Delphine Capela for access to the S. meliloti BAC genomic library, Laurent Sauviac for automatic cycle sequencing, Isabelle Gairin for informatic technical assistance, Jérôme Gouzy and Frédéric Ampe for help in sequence analysis. We are grateful to Dr Julie Cullimore and Dr Eliane Meilhoc for critical reading of the manuscript. HB and part of this work were supported by a grant from the European Union in the frame of the BIOTECH programme (FIXNET, BIOT4-CT97-2319).
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