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J Biol Chem, Vol. 274, Issue 45, 32500-32506, November 5, 1999
From the Laboratoire de Biologie Moléculaire des Relations
Plantes-Microorganismes, CNRS-INRA BP27,
Castanet-Tolosan Cedex, 31326 France
Nitrogen fixation in symbiotic rhizobia is
subject to multiple levels of gene regulation. In Sinorhizobium
meliloti, the alfalfa symbiont, the FixLJ two-component
regulatory system plays a major role in inducing nitrogen fixation and
respiration gene expression in response to the low ambient
O2 concentration of the nodule. Here we report on the mode
of action of the FixT protein, a recently identified repressor of
nitrogen fixation gene expression in S. meliloti. First, we
provide evidence that FixT prevents transcription of the intermediate
key regulatory genes nifA and fixK by
counteracting the activity of the FixLJ two-component system under
otherwise inducing microoxic conditions. Second, we demonstrate that
FixT acts as an inhibitor of the sensor hemoprotein kinase FixL,
preventing the production or the accumulation of its phosphorylated
form. FixT is thus a new example of a regulatory protein that blocks signal transduction in two-component systems at the level of the sensor kinase.
In legumes, nitrogen fixation takes place in a specialized organ,
the nodule, that rhizobia elicit on the roots of their host plants.
Within the nodule, rhizobia acquire the capacity to reduce N2 to ammonia that is assimilated by the plant cells. In
the Rhizobium-legume symbiosis, as in all nitrogen fixing
systems that have been studied so far, nitrogen fixation is tightly
regulated, primarily but not uniquely, at the transcriptional level. In
Sinorhizobium meliloti (formerly Rhizobium
meliloti), the alfalfa symbiont, both positive and negative
control of nitrogen fixation gene expression have been described,
although positive control is by far the best understood. Positive
control is exerted by a two-component regulatory system, FixLJ, that
senses the microoxic conditions that prevail inside the nodule (1, 2).
Indeed, the legume nodule is a microoxic environment whose dissolved
O2 concentration is in the 10-30 nM range.
Under microoxic (or anoxic) conditions, the sensor hemoprotein kinase
FixL autophosphorylates from ATP on a conserved histidine residue
(His285) (3) and subsequently transfers its phosphate to
one of the conserved aspartate residues (Asp54) of the
regulator protein FixJ (4). Phosphorylation of FixJ dramatically
enhances its affinity for the promoters of two intermediate regulatory
genes, nifA and fixK, that control expression of
genes involved in the biosynthesis of nitrogenase and of an oxidase complex with high affinity for oxygen, respectively (5-7).
Early observations indicated that expression of nifA or
fixK was enhanced in a fixK mutant (8). More
recently, it was demonstrated that repression by fixK
actually involved activation of a repressor gene, called
fixT (9), located downstream of the fixLJ operon (Fig. 1A). We now provide evidence that the FixT protein
prevents transcription of nifA and fixK by
counteracting the activity of the master FixLJ two-component system
under microoxic conditions. We further demonstrate that FixT behaves as
an inhibitor of the FixL sensor kinase, preventing synthesis or
accumulation of its phosphorylated form. Biological implications of
this finding are discussed with respect to the regulation of symbiotic
nitrogen fixation as well as in terms of signal processing by
two-component regulatory systems in general.
Bacterial Strains--
GMI939, -940, and -941 are derivatives of
GMI211 (lac Smr), constructed as described in
Ref. 9. Briefly, GMI939 was derived from GMI211 by site-directed
inactivation of the Northern Blot Analysis of fixK Expression--
RNAs were
extracted from 30-ml cultures (A600 = 0.4) in M9
medium of cells incubated under microaerobic conditions (2% oxygen) for 4 h (10). Bacterial cells were collected by centrifugation and
incubated for 10 min at 65 °C in 2 ml of prewarmed lysis solution (1.4% SDS, 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 the addition of
1 volume of isopropyl alcohol, and the pellet was resuspended in
nuclease-free water. DNA was eliminated by the addition of 7.5 units of
fast protein liquid chromatography-pure RNase-free DNase I (Amersham
Pharmacia Biotech). RNAs were further extracted with
phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol and
then precipitated with ethanol. The RNA pellet was washed with 70%
ethanol and resuspended in nuclease-free water. RNAs were denaturated
for 10 min at 75 °C before loading on a 2.2 M
formaldehyde, 1.5% agarose gel. RNAs were electrotransferred to a
Hybond N nylon membrane (Amersham Pharmacia Biotech) and probed with a
32P-labeled BglII-EcoRV
fixK internal fragment. Equal loading of the different lanes
in Fig. 1 was ensured by hybridization of the membrane with a
hemA probe. For the purpose of quantification, a range of
appropriate dilutions of the various RNA preparations was transferred
to a Nylon membrane, and the hybridization signals were quantified on a
phosphorus screen apparatus (Fuji).
Reverse
Transcriptase-PCR1 Analysis
of nifA Expression--
Microaerobic induction of nifA was
achieved using the stoppered tube assay procedure (11). RNAs were
purified as described above and quantitated by measuring the absorbance
at 260 nm. The absence of contaminating DNA in the preparation was
ensured by PCR amplification in the absence of reverse transcriptase.
The following oligonucleotides were used to evaluate nifA
and hemA gene expression: 5'-ATT AGC TTC GCA AAG CA
(nifA reverse primer), 5'-CAG CAA GAA CAA CAG AA
(nifA sense primer), 5'-GTC GAT CGC GTT CTT (hemA
reverse primer), and 5'-TGG ATG GGC TGC ATC A (hemA sense primer).
First-strand cDNA synthesis took place in a 17-µl reaction
volume. 1 µl of total bacterial RNA (100 ng), 7.1 µl of diethyl pyrocarbonate-treated H2O, and 1 µl (50 ng) of the
nifA or hemA reverse primer were heated at
70 °C for 10 min and quickly chilled on ice. After a brief
centrifugation, 3.4 µl of 5× buffer (250 mM Tris-HCl pH
8.3, 375 mM KCl, 15 mM MgCl2), 2 µl of 0.1 M dithiothreitol, and 1 µl of a 25 mM dNTP solution were added and incubated at 42 °C for 2 min. After the addition of 1 µl (200 units) of Moloney murine
leukemia virus reverse transcriptase (Life Technologies, Inc.) 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, since we have 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 dNTP mix (25 mM), 1 µl of
reverse primer (50 ng), 1 µl of sense primer (50 ng), 0.5 µl (2.5 units) of Taq DNA polymerase (Life Technologies, Inc.) were
added to the first strand synthesis reaction mix. After gentle mixing,
the reaction mixture was layered with one drop of silicone oil, heated
to 94 °C for 3 min, and submitted to 25 cycles of the following
sequence (94 °C for 30 s, 50 °C for 30 s, 72 °C for
1 min) and one cycle at 72 °C for 10 min. Reverse transcriptase-PCR
products were electrophoresed on a 2% agarose gel, blotted onto a
nylon membrane (Biodyne A Transfer membrane; Pall), and probed with a
32P-labeled probe internal to nifA or
hemA genes.
Purification of FixT--
FixT was essentially purified by
affinity chromatography of a fusion protein between FixT and MalE, the
maltose-binding protein (Biolabs). DNA corresponding to the putative
fixT open reading frame was generated by PCR
(ExpandTM Long Template PCR System; Roche Molecular
Biochemicals) using the following two oligonucleotides:
5'-CCCAAGCTTGTTGGATGGGAAAACC and
5'-CCGGAATTCACTAAGGGAAATTCA as primers. These primer
oligonucleotides carried HindIII and EcoRI sites
(underlined) that were used to clone the fragment into a pBluescript
KS+ vector (Stratagene) for verification of the sequence generated by
PCR. The EcoRI-HindIII fragment was subsequently
recloned between the EcoRI and XmnI sites of
pMal-c2. The resulting plasmid (pMF116) was transformed into the TB1
strain of Escherichia coli.
MalE-FixT purification was done essentially as described by Biolabs
Inc. 3 liters of complete LB medium supplemented with 0.2% glucose and
ampicillin (50 µg/ml) were inoculated with 30 ml of an overnight
culture of TB1(pMF116). When the A600 of the culture reached about 0.5, isopropyl-1-thio-
Part of the above MalE-FixT protein (20 mg) was incubated with Xa
factor protease (200 µg, Roche Molecular Biochemicals) for 2 h
at room temperature. The protein mix was then sieved twice on a
Sephacryl S200-HR column (Amersham Pharmacia Biotech). Fractions containing pure FixT were pooled, concentrated, and stored at Phosphorylation Assays of FixL and FixJ and Transcription
Assays--
The FixL protein used in this work, FixL122, is a
truncation of the full-length FixL protein that lacks the N-terminal
part that normally anchors the protein to the membrane (12). FixL122 and FixJ were purified as described previously (5).
Phosphorylation assays were essentially as described (5) except that
[
Transcription assays were essentially as described (5). The assay
monitors the production of a 370-nucleotide-long RNA from supercoiled
pJMR300 plasmid, a pTE103 derivative that contains the 5'-end of the
fixK gene bracketed between two strong terminators of
transcription. All reactions were performed at 28 °C. Three tubes
containing FixJ (6 µM) and FixL122 (6 µM)
proteins were incubated under anoxic conditions in the presence of 0.4 mM ATP for 20 min. In two of the tubes, FixT (9 µM) or buffer (control) was added prior to ATP addition.
In the third tube, FixT was added only after 10 min. Formation of
closed complexes was allowed for 10 min after the addition of E. coli RNA polymerase-
In Fig. 2B, FixT was added after phosphorylation of FixJ by
acetylphosphate for 1 h at 25 °C (5). The transcription assay was then run in the presence of FixT as described above. Alternatively, the FixJ-P preparation was run over a CentriconTM 3000 microconcentrator (Amicon) in order to eliminate acetylphosphate before
adding FixT.
Purification of FixL-Phosphate--
600 µl of FixL122 protein
(6 µM) were allowed to autophosphorylate for 60 min under
standard conditions (see above). The reaction mix was loaded on top of
a G25 Sephadex sieving column. Fractions containing phosphorylated
(radioactive) FixL122 were individually screened by TLC
(polyethyleneimine-cellulose, Schleicher & Schuell) for the presence of
contaminating ATP, ADP, and Pi using 0.75 M
potassium dihydrogenophosphate (pH 3.5) as a buffer. Pure fractions were pooled, concentrated at 4 °C, and used immediately.
Phosphotransfer Assays--
Purified FixL122-phosphate (6 µM; see above) and FixJ (6 µM) were
incubated at 28 °C in standard phosphorylation buffer under aerobic
conditions. FixT action was tested by preincubating FixT and
FixL122-phosphate before the FixJ addition (t = 0).
Phosphotransfer was allowed to proceed for 5, 10, or 15 mn before the
addition of SDS-PAGE loading buffer.
Design and Purification of the FixLC Protein--
The C-terminal
part of FixL (from amino acid position 266 to the end of the protein,
position 505) was cloned into a pMalc2 vector to obtain a fusion
protein between FixLC and MalE. DNA fragment corresponding to FixLC was
purified from pDPD147 plasmid (12). pDPD147 was first restricted by
NheI, treated with Klenow polymerase, and restricted by
BamHI. The fragment of interest was purified on the gel and
cloned into XmnI- and BamHI-restricted pMalc2
vector. The resulting plasmid (pAM266L) was transformed into the TB1
strain of E. coli, and the fixLC insert was
checked by sequencing. MalE-FixLC purification was done as described
above for FixT. Fractions containing the MalE-FixLC fusion protein were pooled and treated with Xa factor protease. The cleaved preparation was
concentrated and stored at Phosphorylation Assays for KinA, DegS, and NtrB
Proteins--
Purified proteins were kindly provided by Dr. Tarek
Mzadek (Institut Pasteur, Paris, France). Standard phosphorylation
assays of these proteins were as described by Dahl et al.
(13). Briefly, phosphorylation took place at room temperature in a
buffer (100 mM Tris-HCl, pH 8.0, 200 mM KCl, 4 mM CaCl2, 4 mM MgCl2,
0.5 mM dithiothreitol, 0.1 mM EDTA, 5% (v/v)
glycerol). Phosphorylation was initiated by adding 5 µCi of
[ fixT Lowers fixK and nifA Transcript Levels in
Vivo--
fixT is a duplicated gene on the pSym-a
megaplasmid of S. meliloti. One copy of fixT,
that we shall call fixT1, lies between the
regulatory fixLJ and fixK genes (Fig.
1A) and is transcribed from a
fixK-dependent promoter (9). The second copy of
fixT, fixT2, is located ~260 kilobase
pairs from fixT1 in a reiterated region that
includes a second copy of fixK as well as of the
fixNOQP operon (14). nifA instead is unlinked to
fixT genes.
Northern blot analysis of RNAs extracted from fixT S. meliloti mutant strains and congenic controls incubated under
microoxic conditions showed that the levels of fixK
transcript were enhanced in fixT mutants (Fig.
1B). Selective inactivation of fixT1 (9)
led to a 10-fold increase in the level of fixK RNA as
compared with the wild-type GMI211 strain (lane
4). Similarly, a strain deleted for fixT2
as well as for the adjacent fixK and fixN genes
displayed an increase in the level of fixK RNA
(lane 2) as compared with GMI211. For a reason
that we ignore, this effect was, however, smaller than the one observed
with the fixT1 mutant. The combination of the two
sets of mutations in the same strain (GMI941, lane
3) resulted in an ~50-60-fold increase in the level of
fixK transcript as compared with the wild-type control strain.
We examined the effect of fixT on nifA expression
by reverse transcriptase-PCR, since we could not obtain consistent
Northern blot hybridization patterns with a nifA probe.
Wild-type (GMI211) and fixT (GMI941) S. meliloti
cells were incubated under nearly anoxic conditions in order to
maximize nifA expression (11). Comparison of the
nifA-specific amplification products from extracted RNAs
showed higher levels of nifA transcript in the double
fixT mutant as compared with the wild-type strain (Fig.
1C, lanes 1 and 2).
Expression of the oxygen-insensitive S. meliloti hemA gene
(8) was unaffected by mutations in fixT (Fig. 1C,
lanes 3 and 4).
These data suggested that fixT affected the synthesis or the
stability of fixK and nifA transcripts. We took
advantage of the availability of an in vitro transcription
assay for these genes to discriminate between these possibilities.
Purified FixT Protein Blocks fixK Transcription in
Vitro--
Previous genetic evidence indicated that fixT
encoded a repressor protein (9). The FixT protein, encoded by
fixT1, was overproduced in Escherichia
coli as a fusion protein with MalE, the maltose-binding protein,
and purified by affinity chromatography over an amylose column (see
"Experimental Procedures"). In the experiments described below,
either the MalE-FixT protein or the cleaved and purified FixT protein
has been used and has led to similar results.
A transcription assay for fixK was previously set up (5)
that relies on the ability of phosphorylated FixJ protein to drive fixK transcription in vitro, at the genuine
promoter, in the presence of E. coli
The addition of highly purified MalE-FixT protein at the onset of FixJ
phosphorylation (step 1 above) resulted in a dramatic decrease in the
level of fixK mRNA synthesized (Fig.
2A, lane 3). The effect of MalE-FixT on fixK transcription
was specific, since all other transcripts, which originate from
transcription of the vector template DNA genes by E. coli
holoenzyme, were unaffected. Thus, FixT specifically blocked initiation
of transcription of fixK or elongation of the nascent
fixK RNA. The inhibitory effect of FixT was also observed
when FixT was added 10 min after FixJ phosphorylation was started (Fig.
2A, lane 4; see "Discussion").
Interestingly, no inhibition of fixK transcription was
observed when FixJ was phosphorylated from acetylphosphate (Fig.
2B), a low molecular weight phosphodonor (5, 15), instead of
being phosphorylated from FixL-phosphate. Specifically, FixT had no effect on FixJ-driven transcription if FixT was added to FixJ at the
end (Fig. 2B) or at the beginning of FixJ incubation with acetylphosphate (data not shown). Furthermore, FixT did not inhibit the
transcriptional activity of a FixJ-phosphate preparation from which
acetylphosphate had been eliminated by molecular sieving (data not
shown). This demonstrated first that FixT did not directly dephosphorylate FixJ-phosphate (since the transcription assay relies on
phosphorylated FixJ) and, second, that FixT did not inhibit
FixJ-phosphate transcriptional activity per se. Rather, these data suggested an implication of FixL in inhibition by FixT.
FixT Inhibits FixL-Phosphate Synthesis--
When purified FixL is
incubated in the presence of
When purified FixT protein was preincubated with FixL prior to the
addition of
We also ruled out the possibility of an ATPase activity associated with
FixT, since no Pi production or depletion of the ATP pool
could be detected on TLC plates after extended incubation of FixT with
Both explanations are consistent with the further observation that
adding FixT to FixL 60 min after the addition of ATP (i.e. when the phosphorylation reaction FixL + ATP FixT Promotes FixJ-Phosphate Dephosphorylation in the Presence of
FixL--
When FixL and FixJ are incubated together in the presence of
Evidence for a Stoichiometric Interaction between FixT and
FixL--
Early trials led us to use FixT at a concentration of 9 µM and FixL at 6 µM (monomers) in the
experiments reported above. We subsequently investigated how the
activity of FixT varied with its concentration. In a first set of
experiments, FixL concentration was kept at 6 µM, and
FixT was titrated down from 9 to 0.1 µM. Half-maximum
inhibition of FixL autophosphorylation was observed when FixT was
~1.2 µM, i.e. at a 1:5 ratio between FixT
and FixL (Fig. 6). Complete inhibition
was observed at ~3 µM FixT, i.e. at a 1:2
ratio. Similar data were obtained at another concentration of
FixL (2 µM monomer) (Fig. 6).
These data are consistent with the action of FixT on FixL being
stoichiometric rather than catalytic. Based on previous observations that FixL is a dimer in solution (5, 18) and assuming that FixT is a
monomer, full inhibition of FixL autophosphorylation would occur upon
mixing one monomer of FixT with one dimer of FixL.
FixT Targets the Carboxyl-terminal Kinase Domain of FixL--
FixL
is a modular protein (12, 19, 20) made up of three domains: 1) a
N-terminal membrane-anchoring domain, which is not essential for
activity or for oxygen sensing in vitro (this domain is
missing in the soluble FixL122 protein used in this work), 2) a central
oxygen-sensing heme-containing domain whose structure has been solved
recently (20); and 3) a C-terminal kinase domain homologous to the
kinase domain of sensor regulatory proteins. This isolated domain,
previously called FixLC, is capable of constitutive autophosphorylation
independently of oxygen, although with a lower efficiency than the
full-length protein (19). We examined whether this isolated kinase
domain was prone to inhibition by FixT.
A FixLC protein was purified that consisted of the last 240 amino acids
fused to MalE (see "Experimental Procedures"). FixT significantly
inhibited phosphorylation of FixLC, although inhibition was not
complete (Fig. 7A,
lane 4), even at a molar ratio of 6 between FixT
and FixLC (data not shown). We conclude that the C-terminal kinase
domain of FixL is the primary target for FixT, although we do not
exclude the possibility that the central oxygen-sensing domain of FixL
may contribute to the interaction with FixT.
FixT Is Not Active on the Heterologous NtrB, DegS, and KinA Sensor
Kinases--
Since the C-terminal kinase domain of FixL is conserved
between the sensor proteins of the two-component family, it was of interest to test whether other targets could be identified for FixT.
Three purified kinases were kindly provided by Dr. T. Mzadek: the NtrB
protein from Salmonella typhimurium and the DegS and KinA
sensor proteins from Bacillus subtilis. Two different
phosphorylation conditions were tested. In a first set of experiments,
KinA, NtrB, and DegS were phosphorylated in the presence of 2.5 µM [ Mode of Action of FixT--
fixT was originally
identified by genetic studies that demonstrated its ability to repress
expression of the regulatory genes fixK and nifA
in S. meliloti (9). However, the mode of action of
fixT was completely unknown. Using in vitro
methods, we have now demonstrated that FixT counteracts the activity of
the FixLJ two-component system that mediates nifA and
fixK expression in response to oxygen in S. meliloti. Specifically, we provide evidence that FixT acts by
inhibiting primarily FixL-phosphate synthesis.
How FixT achieves its effect is not completely clear yet. When added
prior to ATP, FixT completely prevented incorporation of phosphate into
FixL (Fig. 3A). This is consistent with FixT blocking
autophosphorylation of the FixL protein from ATP. For example, FixT may
prevent access of ATP to the nucleotide binding pocket of FixL or slow
down histidyl-phosphate formation. By contrast, FixT had no detectable
ATPase activity (data not shown) and thus certainly does not act by
depleting the pool of ATP. Alternatively, FixT may favor rapid
dephosphorylation of neosynthesized FixL-phosphate. We have presented
evidence that FixT is not a phosphatase for FixL-P (Fig. 3C)
and does not phosphorylate itself from FixL-P (data not shown). By
contrast, we do not exclude the possibility that FixT may enhance the
rate of dephosphorylation of FixL-phosphate in the presence of ADP
(FixL-P + ADP
FixT does not resemble any known protein, nor does it have any
prominent motif that could give indications regarding its mode of
action. However, we presently have three clues: 1) FixT acts directly
on FixL; 2) FixT targets the C-terminal domain of FixL; 3) the
interaction between the two proteins is likely to be stoichiometric. Our working model is that FixT makes a protein-protein interaction with
the carboxyl-terminal kinase domain of FixL that may either inhibit
autophosphorylation of FixL or favor the reverse dephosphorylation reaction.
No other effect of FixT was observed besides its effect on FixL
phosphorylation. First, FixT did not detectably impair phosphotransfer between FixL-phosphate and FixJ (data not shown). Second, FixT had no
effect on the stability of FixJ-phosphate (in the absence of FixL), as
assayed by transcriptional activity (Fig. 2B). Note that
direct assessment of FixJ-phosphate dephosphorylation by FixT was not
appropriate because of the very low specific activity of
acetyl-phosphate that can be achieved (5). Instead, transcription is an
indirect but reliable assay for FixJ-phosphate dephosphorylation. Thus,
the primary, if not unique, role of FixT is to inhibit FixL phosphorylation.
The reported effect of FixT on FixL-phosphate synthesis fully accounts
for both the FixT-induced FixJ-phosphate dephosphorylation in the
presence of FixL (Fig. 5) and the inhibition of fixK
transcription (Fig. 2A). Even under low oxygen conditions,
phospho-FixJ is continuously and rapidly hydrolyzed because of the
phosphatase activity associated with unphosphorylated FixL (16). In the
presence of FixT, the FixL-phosphate pool drops with the consequence
that phospho-FixJ cannot be replenished and thus disappears rapidly.
FixJ-phosphate dephosphorylation in turn results in a dramatic (more
than 100-fold) decrease of its capacity to bind and activate the
nifA and fixK promoters (7). The comparison of
the kinetics of disappearance of FixJ-phosphate and of inhibition of
fixK transcription is consistent with this analysis
(compare, for example, Fig. 5, lane 8, and Fig.
2A, lane 4).
Modulators of Two-component Systems--
Two-component systems are
of paramount importance in bacteria, where they probably constitute the
most widespread device for signal transduction. E. coli and
B. subtilis each use up to 30 different two-component
systems to cope with environmental changes (21, 22). Some two-component
systems look very simple; in the FixLJ system, for example, a single
protein, FixL, is able to sense its cognate signal (molecular oxygen)
and to transduce it to the regulator protein, FixJ, by phosphotransfer.
Moreover, both FixL and FixJ proteins have a modular structure (12, 19, 23). As a result, a combination of four domains suffices for oxygen
signal transduction. Many two-component systems are more complex,
involving more proteins than a simple sensor and regulator pair. Many
of these proteins are phosphatases. For example, in B. subtilis, RAP phosphatases ensure dephosphorylation of the SpoOF-phosphate regulator protein (24). The SixA protein from E. coli is a phosphohistidine phosphatase that promotes
dephosphorylation of the sensor ArcB protein (25). The PII protein in
enteric ntr circuits is not a phosphatase per se
but stimulates the phosphatase activity associated with the NtrB sensor
kinase (26).
A more relevant comparison for FixT is KipI, for which the name
antikinase was coined in bacteria. kipI was originally
identified during the course of the B. subtilis genome
sequencing project (27). Subsequently, KipI was shown to block
sporulation of B. subtilis when overproduced in the absence
of another protein, KipA, with which KipI normally forms a complex
(28). KipI and FixT resemble each other by their inhibitory effect on
the (auto)phosphorylation of their cognate sensor kinases, KinA and
FixL, respectively. However, FixT and KipI display no amino acid
sequence similarity to each other, and, thus, whether they are related
in structure or mode of action is not known.
Biological Role of fixT in S. meliloti--
The primary role of
FixLJ in S. meliloti is to control, via nifA and
fixK, nif and fix gene expression in
response to oxygen availability. The present data reinforce the pivotal
role of the FixLJ system in S. meliloti, as an integration
site for both positive regulation by oxygen and negative regulation by
FixT. Typically, molecular devices such as the KipI, FixT, or the above
mentioned phosphatases may allow two-component systems to integrate
multiple environmental regulatory signals as demonstrated for the RAP
phosphatases of B. subtilis (24, 28).
Since FixT interacts with FixL, one obvious possibility would be that
FixT contributes to oxygen control. Available data indicate that FixL
is sufficient for oxygen regulation of nifA and
fixK expression both ex planta and in
vitro and that fixT does not grossly affect oxygen
regulation of the system (9). Furthermore, the central oxygen-sensing
domain of FixL does not seem to be the primary target for FixT, and no
difference in FixT activity was observed in vitro under
anoxic versus oxic conditions (data not shown). Still, FixT
may contribute to a fine tuning of the FixLJ response to oxygen,
ensuring, for instance, that the FixL protein, which has a very low
affinity for oxygen (29), does not become prematurely activated in
response to a moderate or transient oxygen deprivation. Alternatively,
FixT may prevent activation of the FixLJ system by low oxygen under
conditions that would be otherwise antithetical to symbiotic nitrogen
fixation such as, possibly, low energy charge, low carbon, or high nitrogen.
We are most grateful to Dr. T. Mzadek for the
kind gift of purified kinases, Prof. S. Kustu for stimulating
discussions and encouragement, Prof. S. R. Long for helpful
comments on the manuscript, and Dr. C. Gough for correcting the
English. Colleagues at the Institut National des Sciences
Appliquées, Toulouse are gratefully acknowledged for generous
access to anaerobic facilities.
*
This work was supported in part by European Union Biotech
Program Grant BIOT4-CT97-2319 and by the Action Incitative
Programmée-Institut National de la Recherche Agronomigue
Microbiologie program.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 33 5 61 28 50 54;
Fax: 33 5 61 28 50 61; E-mail: jbatut@toulouse.inra.fr.
The abbreviations used are:
PCR, polymerase
chain reaction;
PAGE, polyacrylamide gel electrophoresis.
Inhibition of the FixL Sensor Kinase by the FixT Protein in
Sinorhizobium meliloti*
,,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
10 promoter region of the
fixT1 gene. GMI940 carries an ~5-kilobase pair
deletion that removes the fixT2 gene as well as the
adjacent fixK and fixNOQP genes. GMI941 is a
derivative of GMI940, in which the 5-kilobase pair deletion has been
transduced into GMI939, using a phleomycin resistance marker associated
with the deletion (9).
-D-galactopyranoside (2 mM)
was added, and the culture was incubated for an additional 2 h at
30 °C. Cells were collected by centrifugation, washed, resuspended
in C buffer (20 mM Tris-HCl, pH 7.4, 200 mM
NaCl, 1 mM EDTA, 1 mM dithiothreitol), and
finally disrupted by sonication. The crude cell extract (15 ml) was
clarified by centrifugation, diluted to 200 ml with C buffer, and
loaded on top of a 40-ml amylose-agarose column (Biolabs). After
extensive washing (10 volumes) of the column with C buffer, bound
MalE-FixT protein was eluted with C buffer supplemented with 10 mM maltose. Fractions containing MalE-FixT were screened by
SDS-PAGE using an Amersham Pharmacia Biotech Phast Gel System
apparatus. Fractions containing pure MalE-FixT were pooled and
concentrated by centrifugation over Centripep 10 devices (Amicon).
Fractions were stored at 80 °C in the presence of 5% glycerol
before use. Quantification of the protein was done by Coomassie Blue
staining of protein separated on a SDS-acrylamide gel as compared with
standards. The purity of the preparation was estimated to be 80%.
80 °C in the presence of 5% glycerol. The purity of this FixT preparation was estimated to be 90% from inspection of a Coomassie Blue-stained SDS-polyacrylamide gel.
-33P]ATP (0.4 mM) was used as labeling
source (final specific activity ~5000 dpm/pmol). All reactions
were at 28 °C. Phosphorylation reactions were loaded on SDS-10%
polyacrylamide gels. When indicated, reactions were performed under an
anaerobic hood facility. Dried gels were autoradiographed at room
temperature. When required, quantification was obtained by liquid
scintillation counting of excised band gels.
70 holoenzyme (600 nM) and 12.5 nM supercoiled DNA template. This step was also performed
under anoxic conditions, since closed complexes are unstable and
dissociate in the absence of phosphorylated FixJ protein. Isomerization
of closed complexes into open complexes and elongation of RNAs was
initiated by adding UTP and GTP (0.4 mM) and 0.1 mM [
-32P]CTP (2 Ci/mmol). Heparin was also
added, so that the assay was a single-round transcription assay.
80 °C in the presence of 5% glycerol. Its purity was estimated to be ~50%.
-33P]ATP and 2.5 µM ATP. Reactions were
stopped in SDS-loading buffer after 5 min of phosphorylation. FixT
action was tested after preincubating FixT with KinA, NtrB, or DegS for
5 min prior to ATP addition. Alternatively, KinA, DegS, and NtrB were
phosphorylated under the conditions described above for FixL.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, genetic map of the fixLJ
cluster in S. meliloti. The black bar
delineates the duplicated part of the cluster. B, Northern
blot analysis of fixK expression in GMI939
(fixT1) (lane 1), GMI940
( 
fixT2) (lane 2), GMI941
(fixT1fixT2) (lane
3), and GMI211 (wild type) (lane 4).
C, reverse transcriptase-PCR analysis of nifA
(lanes 1 and 2) and hemA
(lanes 3 and 4) expression in GMI211
(wild type) (lanes 1 and 3) and GMI941
(fixT1fixT2) (lanes
2 and 4).
70-RNA polymerase
holoenzyme. The assay is designed in such a way that transcription
strictly depends upon phosphorylation of FixJ. It consists of three
steps (see "Experimental Procedures" for details). 1) Pure FixJ
protein is phosphorylated by incubation with pure FixL protein and ATP
for 20 min. The FixL protein that is used, FixL122, is a truncated,
soluble, oxygen-responsive form of the full-length FixL protein (5,
12). 2) Closed complexes are allowed to form for 10 min by the addition
of E. coli RNA polymerase-70 holoenzyme and a cloned
fixK DNA transcription template to phosphorylated FixJ. 3)
Isomerization of closed complexes and elongation of transcription is
started upon the addition of the three missing nucleotides. The first
two steps are performed under anoxic conditions, since they are readily
reversible in air.
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Fig. 2.
Effect of the addition of purified MalE-FixT
protein to an in vitro transcription assay of
fixK. A, transcription was driven by FixJ (6 µM) phosphorylated in the presence of FixL122 (6 µM) and ATP (0.4 mM). The small
arrowhead points to the position of fixK
transcript. Lane 1, negative control, no FixJ.
Lane 2, positive control, no FixT. MalE-FixT (9 µM) was added at the start of (t = 0 min,
lane 3) or during (t = 10 min,
lane 4) the phosphorylation step. See
"Experimental Procedures" for details. B, transcription
assay of fixK as driven by FixJ (2 µM)
phosphorylated from acetylphosphate (20 mM).
Lane 1, positive control, no FixT. Three
different concentrations of FixT were tested: 2, 5, and 10 µM (lanes 2, 3, and
4, respectively).
-labeled [33P]ATP, the
amount of (auto)phosphorylated FixL protein rises linearly over at
least the first 15 min of the reaction (16) (Fig.
3B). Subsequently, the amount
of FixL-phosphate levels off because of the accumulation of both
FixL-phosphate and ADP that promotes the reverse reaction,
i.e. FixL-P + ADP 
FixL + ATP (16).
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Fig. 3.
Effect of FixT on FixL-phosphate
synthesis. A, early kinetics of FixL
autophosphorylation in the presence (black bar)
or absence (open bar) of FixT. FixL122 (6 µM) alone or FixL122 and FixT (9 µM)
together were preincubated for 5 min before the phosphorylation
reaction was started (t = 0) by the addition of
[ -33P]ATP (0.4 mM). Samples were then
taken at regular intervals for SDS-PAGE analysis. Lanes
1 and 5, 1 min; lanes 2 and
6, 2 min; lanes 3 and 7, 5 min; lanes 4 and 8, 10 min.
B, FixL122 (6 µM) was allowed to
autophosphorylate for 5 min in the presence of
[-33P]ATP (0.4 mM) at 28 °C under
aerobic conditions. After 5 min, purified FixT protein (9 µM) (closed circles) or buffer
(open triangles) was added. Samples were taken at
10 and 15 min. Radioactivity in FixL122 was measured by liquid
scintillation counting of excised gel bands. C,
nucleotide-free FixL-phosphate (6 µM) was incubated with
buffer (odd lanes) or FixT protein (9 µM) (even lanes) for 15 min
(lanes 1 and 2), 30 min
(lanes 3 and 4), or 60 min
(lanes 5 and 6) at 28 °C.
-labeled ATP (400 µM), no FixL-phosphate
accumulation could be detected after 10 min (Fig. 3A) or
after 60 min of incubation (data not shown). In a second set of
experiments (Fig. 3B), FixL was incubated with -labeled
ATP for 5 min prior to the addition of FixT, thus allowing a detectable
amount of FixL-phosphate to be synthesized. Upon the addition of FixT,
the level of phosphorylated FixL stabilized but did not decrease, thus
excluding a prominent phosphatase activity associated with FixT. This
was confirmed by purifying FixL-phosphate from ATP (and ADP)
nucleotides in order to prevent further FixL phosphorylation. It was
then observed that the addition of FixT (Fig. 3C) or FixT
plus ATP (data not shown) did not enhance the rate of FixL-P
dephosphorylation. This is evidence that FixT is not a phosphatase.
-labeled ATP, even in the presence of FixL (data not shown). We were
also unable to detect any (reasonably stable) phosphorylation of FixT
after it was incubated with either radiolabeled ATP or FixL-phosphate
and separated by SDS-PAGE under neutral pH conditions (data not shown).
Thus, the model we presently favor is that FixT either blocks
autophosphorylation of FixL from ATP (FixL + ATP FixL-P + ADP) or
enhances the rate of the reverse reaction (i.e. FixL-P + ADP FixL + ATP).
FixL-P + ADP had reached equilibrium) resulted in the disappearance of most of FixL-phosphate within 15 min (Fig.
4).
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Fig. 4.
FixT action results in a depletion of the
FixL-phosphate pool. FixL122 (6 µM) was incubated
with [ -33P]ATP (0.4 mM) at 28 °C under
aerobic conditions. After a 65-min incubation (lane
1), the mix was split into two tubes to which either buffer
(; lanes 3, 5, and 7) or
MalE-FixT protein (+; 9 µM) was added (lanes
4, 6, and 8). Samples were withdrawn
at 70 min (lanes 3 and 4), 75 min
(lanes 5 and 6), and 80 min
(lanes 7 and 8) for SDS-PAGE analysis.
Lane 2, positive control. In this tube, MalE-FixT
protein was present throughout the phosphorylation reaction of
FixL122.
-labeled ATP under anoxic conditions, the phosphate first enters FixL and then readily transfers to FixJ so that only FixJ-phosphate accumulates (Fig. 5, lanes
1-3). The level of phosphorylated FixJ levels off after a
30-40-min incubation in our assay conditions because of the
phosphatase activity associated with FixL (3, 17). When FixT was added
after a 60-min incubation, the level of phosphorylated FixJ decreased
dramatically within 15 min (Fig. 5). FixT thus promoted
dephosphorylation of FixJ-phosphate. As pointed out above (Fig.
2B), this was not due to a direct dephosphorylation of
FixJ-phosphate by FixT. Rather, FixJ-phosphate disappearance was a
consequence of the block in FixL-phosphate synthesis that prevented
replenishing of the FixJ-phosphate pool (see "Discussion"). In
addition, we observed that FixT did not affect phosphotransfer between
FixL-phosphate and FixJ as monitored with purified (sieved), nucleotide-free, FixL-phosphate (data not shown).
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Fig. 5.
FixT promotes FixJ-phosphate
dephosphorylation in the presence of FixL. A mix of FixL122 (6 µM monomer), FixJ (6 µM), and
[ -33P]ATP (0.4 mM) was incubated at
28 °C under anoxic conditions. After a 65-min incubation
(arrow), the mix was split into two reaction tubes to which
either buffer (open bar) or purified MalE-FixT
protein (9 µM) was added (black
bar). Incubation was prolonged, and samples were withdrawn
at 70, 75, and 80 min for the purpose of SDS-PAGE analysis.
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Fig. 6.
Stoichiometry of the interaction between FixL
and FixT. FixL122 (6 µM (closed
circles) or 2 µM (open
triangles)) was incubated with [ -33P]ATP
(0.4 mM) in the presence of various concentrations of
cleaved FixT protein. Reactions were stopped after a 10-min incubation,
and samples were analyzed by SDS-PAGE. Quantification was by liquid
scintillation counting of the bands of gel containing FixL.
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Fig. 7.
FixT activity on FixLC and other
kinases. A, FixL122 (lanes 1 and
2) and FixLC (lanes 3 and
4) at 6 µM were phosphorylated from
[ -33P]ATP (0.4 mM) for 15 min in the
absence (lanes 1 and 3) or presence
(lanes 2 and 4) of cleaved FixT
protein (9 µM). Open triangles mark
the position of the FixL122 and FixLC proteins. B, FixT
activity was tested against 3 µM KinA (lanes
1 and 2), NtrB (lanes 3 and
4), and DegS (lanes 5 and
6). Lanes 1, 3, and
5, without FixT. Lanes 2,
4, and 6, with FixT (9 µM). See
"Experimental Procedures" for details.
-33P]ATP, as described before (13).
No effect of FixT on the phosphorylation of the heterologous kinases
was observed (Fig. 7B). In a second set of experiments, we
assayed phosphorylation of NtrB, DegS, and KinA under the conditions
used for FixL (i.e. 0.4 mM ATP) and, again,
found no effect of FixT (data not shown). Similar data were obtained
with higher concentrations of FixT (27 µM) (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FixL + ATP), i.e. may favor the reverse
reaction of autophosphorylation.
ACKNOWLEDGEMENTS
FOOTNOTES
Recipients of a fellowship from the Ministère de
l'Enseignement Supérieur et de la Recherche, France.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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