(Received for publication, July 19, 1994; and in revised form, November 30, 1994)
From the
The two-component system sensor/response regulator pair, FixL/FixJ, controls the expression of Rhizobium meliloti nitrogen fixation (nif and fix) genes in response to changes in oxygen concentration. A truncated version of FixL, FixL*, is an oxygen-binding hemoprotein kinase that phosphorylates and dephosphorylates the nif and fix gene transcriptional activator, FixJ. Phosphorylation of FixJ is required for optimal transcriptional activation, and anaerobic conditions in vitro result in a substantial increase in the level of FixJ-phosphate. In this study, site-directed mutagenesis was carried out at histidine residues in FixL*. Mutant FixL* derivatives were purified and analyzed in vitro for their heme/oxygen binding properties and phosphorylation/dephosphorylation activities. Mutation of histidine 285, the putative autophosphorylation site, to glutamine results in the loss of FixL* phosphorylation activities. However, this mutant protein retains a substantial level of FixJ-phosphate dephosphorylation activity. Mutation of histidine 194 to asparagine results in the loss of heme binding and in the failure of FixL* to regulate its phosphorylation/dephosphorylation activities in response to changes in oxygen concentration. The FixL*H194N mutant protein also exhibits an increased FixJ phosphorylation activity under aerobic conditions. This study provides further evidence for the importance of the heme binding domain of FixL* in regulating FixJ phosphorylation and dephosphorylation activities in response to oxygen.
The soil bacterium, Rhizobium meliloti, fixes
dinitrogen (N) into ammonia when it is in symbiotic
association with its plant host, Medicago sativa (alfalfa).
There are at least 23 genes from R. meliloti required for
nitrogen fixation (nif and fix genes) as well as a
host of other both bacterial and plant genes that are required for the
nodulation process. The nitrogen fixation genes in R. meliloti are expressed relatively late in nodule development and the key
signal that induces the expression of most of the nif and fix genes is low oxygen concentration(1, 2) .
This is consistent with the fact that the nitrogenase enzyme is oxygen
labile, making it imperative for the bacterium to express the nitrogen
fixation machinery only when oxygen concentration is low. Two genes, fixL and fixJ, are responsible for sensing and
transducing the low oxygen signal which results in the activation of nif and fix gene expression(2, 3) .
The products of the fixL and fixJ genes are
homologous to the two-component regulatory system proteins (2) that are involved in signal transduction in
bacteria(4) . Genetic and biochemical evidence from many of
these systems supports a phosphotransfer model for signal
transduction(5, 6, 7, 8) . FixL is
an oxygen sensor that is homologous to the sensor class, and FixJ is a
transcriptional activator homologous to the response regulator class of
two-component system proteins. Genetic studies show that FixL responds
to low oxygen concentration by activating the transcriptional
activator, FixJ(9) . Activated FixJ then increases
transcription at the nifA and fixK promoters(9) , whose products are transcriptional
activators of other nif and fix genes(10, 11, 12) .
Our laboratory
has undertaken a biochemical approach to understanding the mechanism by
which the FixL protein senses oxygen and transduces this signal to
FixJ, ultimately resulting in the transcriptional activation of nif and fix genes (see (13) for review). Because
FixL is a transmembrane protein with four transmembrane
helices(14) , a soluble, truncated version of FixL, FixL*, was
constructed to facilitate purification and in vitro studies(15) . FixL* is an oxygen-binding hemoprotein and a
kinase capable of autophosphorylation as well as the phosphorylation of
the FixJ protein(15) . Analysis of purified deletion
derivatives established that FixL* consists of two separable,
functional domains; the N-terminal domain that functions in heme and
oxygen binding and the C-terminal domain that possesses
autophosphorylation and FixJ phosphorylation activities(16) .
The rate of FixL* autophosphorylation increases when reactions are
performed under anaerobic conditions relative to aerobic
conditions(16, 17, 18) , and this increased
activity requires the presence of the heme binding domain(16) .
The phosphorylation of FixJ occurs through direct phosphotransfer from
FixL*-phosphate to FixJ, and the rate of the phosphotransfer reaction
is not affected by oxygen concentration(17, 18) .
Additionally, FixL* possesses a FixJ-phosphate dephosphorylation
activity which decreases in response to anaerobic
conditions(17) . The FixJ dephosphorylation activity resides in
the C-terminal kinase domain. ()The net result of anaerobic
conditions in vitro is an increase in the level of
phosphorylated FixJ that is available to activate transcription from
the nifA and fixK promoters. Recently the entire
signal transduction pathway starting with anaerobiosis and ending with
transcriptional activation at the nifA and fixK promoters was reconstituted in vitro(19, 20) using purified truncated FixL
derivatives, purified FixJ, and purified RNA polymerase from Escherichia coli or R. meliloti. The FixLJ system
thus presents a unique system for understanding the molecular basis of
oxygen sensing in R. meliloti.
In this work, we analyze the effects of mutations of several histidine residues in FixL* on oxygen sensing and signal transduction in vitro. The histidine at position 285 in the kinase domain of FixL* was mutated, because it is a highly conserved histidine that is predicted to be the site of autophosphorylation based on sequence alignments with other two-component system sensors(5) . Consistent with this prediction, mutation of histidine 285 results in loss of autophosphorylation and FixJ phosphorylation activities. All three histidine residues that lie in the previously defined heme binding region of the protein (16) were also mutationally altered because of the similarity of the absorption spectra of FixL* and hemoglobin (15) and the likelihood that a histidine is involved in coordination of the heme iron. Histidine 194 is identified as the putative site of heme coordination. Finally, it is shown that mutations in the heme binding region have a significant impact on C-terminal kinase activity.
Mutagenesis was carried out by the method of
Kunkel(27) . Oligonucleotides used for the mutagenesis were
phosphorylated using T4 polynucleotide kinase purchased from U. S.
Biochemical Corp. Single-stranded DNA was prepared from E. coli strain RZ1032 bearing plasmid pEM13.8, pEM19.2, or pEM28.5 by
using the helper phage M13K07(28) . Ten pmol of each of the
phosphorylated oligonucleotides were annealed to 0.5 pmol of the
appropriate single-stranded DNA (prepared from strain RZ1032) in PE1
buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl, 50 mM NaCl, 1 mM dithiothreitol)(26) . Second strand synthesis and ligation
were performed using 0.5 mM fast protein liquid
chromatography-purified dNTPs (Pharmacia Biotech Inc.), 1 mM ATP, 2 units of T4 DNA ligase, and 25 units of exonuclease-free
Klenow (U. S. Biochemical Corp.) in PE1 buffer without NaCl. The
reaction was incubated overnight at 15 °C. The reaction mixture was
transformed into E. coli strain MC1061, and clones with the
appropriate restriction enzyme changes were sequenced. The entire
stretch of fixL DNA was sequenced to ensure that the sequences
were as expected and that there were no additional mutations. The
restriction fragments bearing the appropriate mutations were cloned
back into the pGG820 FixL* overexpression vector(15) . The
resulting pGG820 derivatives were named according to the position
number and identity of the amino acid change. Thus, pEMH138Q, pEMH192N,
pEMH194N, and pEMH285Q are plasmids expressing mutant FixL* proteins
that have a glutamine at position 138, asparagines at positions 192 or
194, and a glutamine at position 285, respectively.
Figure 1:
Schematic
structure of FixL and the truncated FixL derivative, FixL. Filled boxes indicate the four transmembrane (TM)
regions(14) . Stippled boxes indicate the heme and
oxygen binding region (16) , and the hatched boxes indicate the conserved histidine kinase
domain(2, 16) . The positions of the histidines that
were individually mutated, and the resulting amino acid replacements
are shown. Numbers refer to amino acid position based on the
first ATG in the open reading frame(2) . The mutant FixL*
proteins are designated according to the position and identity of the
amino acid change. Thus, FixL*H138Q is a FixL* protein with a histidine
to glutamine mutation at position 138. All of the FixL* derivatives
were produced in E. coli from the FixL* overexpression vector,
pGG820, and therefore all the FixL* derivatives have the same
N-terminal fusion to lacZ as does wild-type
FixL*(15) .
FixL*H138Q, FixL*H285Q, and FixL*H194N were purified to approximately 90% purity based on Coomassie Blue staining of SDS-PAGE gels. FixL*H138Q and FixL*H285Q are orange-red in color, like purified FixL*, indicating the presence of heme. The absorption spectra of FixL*H138Q and FixL*H285Q are very similar to those of FixL*, both in the presence (Fig. 2A) and absence (Fig. 2B) of oxygen. The absorption maxima for each peak for the mutant and wild-type proteins, both oxygenated and deoxygenated, are within 0.5 nm of one another (data not shown). This shows that changing either histidine 138 or 285 to glutamine has no major effect on the abilities of FixL* to interact with heme or to bind oxygen (although changes in oxygen binding affinity would not be addressed by this analysis).
Figure 2:
Absorption spectra and gel heme assays of
FixL* and FixL*histidine mutants. A, oxygenated spectra of
FixL* (thick solid line), FixLH138Q ( ), and
FixL
H285Q(- - - -). B,
deoxygenated spectra of FixL
(thick solid line),
FixL
H138Q ( ), and FixL
H285Q(- -
- -). Spectra were measured at 0.5-nm intervals using a
Hitachi U-2000 spectrophotometer. Proteins were in 20 mM Tris-HCl, pH 7.8, 125 mM NaCl, 20% (v/v) glycerol, 10
mM 2-mercaptoethanol. Deoxygenation was achieved by the
addition of a crystal of sodium dithionite to a 0.5-ml
sample.
The purified FixL*H194N protein is colorless and fails to show the
characteristic heme absorption spectrum of FixL*, FixL*H138Q, and
FixL*H285Q (data not shown). FixL*H194N also fails to show a
FixL*-specific heme staining band in crude extracts suggesting that the
lack of heme in FixL*H194N is not simply due to the loss of heme during
purification (Fig. 3, lane 2). The failure of
FixL*H194N to bind heme suggests that the histidine at position 194 is
involved in coordination of the heme iron. In hemoglobin, the fifth
coordination position of the heme iron is occupied by a histidine
residue, the so-called proximal histidine(33) , and mutation of
this histidine to glutamine in human -hemoglobin results in the
loss of heme binding(34) .
Figure 3: Nondenaturing gel heme assay of FixL* and FixL* mutant derivatives. Lanes 1-5 contain approximately 200 µg of total protein of the indicated crude extracts: lane 1, BL21 (pEMH192N); lane 2, BL21 (pEMH194N); lane 3, TG1 (pGG820); lane 4, BL21 (pUC9), lane 5, BL21 (pAF1). Lane 6 contains 3.6 µg of purified FixL*H285Q. Each strain used to make the crude extracts also carried the nifA::lacZ reporter plasmid pCHK57. Plasmid pAF1 overexpresses fixJ from the lac promoter in pUC9 (A. Lois, personal communication).
Although FixL*192N was not purified and examined further, analysis of crude extracts revealed that FixL*192N does indeed contain heme (Fig. 3, lane 1). This indicates that the histidine at position 192, despite its close proximity to the important histidine at position 194, is not likely to be involved in heme binding.
FixL*H194N was quantitated relative to the other FixL* derivatives on the basis of Coomassie Blue staining material on SDS-PAGE gels. On the basis of densitometric scans, we estimate that the variation in the amounts of the FixL* derivatives used in each of the assays was no more than 15%.
Fig. 4A shows the autophosphorylation activity of FixL* compared with that of FixL*H138Q in the presence and absence of oxygen. As reported previously, the FixL* protein shows approximately a 3-fold increase in the rate of autophosphorylation under anaerobic relative to aerobic conditions at 22 °C(16) . FixL*H138Q shows an elevated autophosphorylation rate in the presence of oxygen, a rate that is essentially equivalent to the rate of FixL* autophosphorylation in the absence of oxygen. Upon deoxygenation, the rate of FixL*H138Q autophosphorylation increases by about 2-fold; an oxygen response similar in magnitude to that of FixL*. Thus, the autophosphorylation activity of FixL*H138Q depends on oxygen concentration, consistent with the fact that H138Q retains oxygen binding ability. Furthermore, FixL*H138Q has even greater autophosphorylation activity than FixL* both in the presence and in the absence of oxygen.
Figure 4:
Autophosphorylation of FixL* and mutants. A, autophosphorylation of FixL* (circles) and
FixL*H138Q (squares). B, autophosphorylation of FixL* (circles) and FixL*H194N (triangles). Open
symbols indicate aerobic conditions and filled symbols anaerobic conditions. Each of the FixL* derivatives (1
µM) was incubated with [-
P]ATP
as described under ``Materials and Methods.'' Proteins were
analyzed by SDS-PAGE and autoradiography. Quantitation was by laser
densitometry.
Fig. 4B shows the autophosphorylation of FixL*H194N in comparison with FixL*. As would be expected for a heme-less FixL* protein, the autophosphorylation activity of FixL*H194N fails respond to oxygen. Since the absolute level of FixL*194N autophosphorylation is higher than wild-type FixL* in the presence of oxygen, this suggests that a major function of the oxygenated heme moiety is to repress the autophosphorylation activity of the C-terminal portion of the protein. Although the rate of FixL*H194N autophosphorylation was always higher than that of oxygenated FixL*, the values measured in different experiments were somewhat variable, ranging from 1.5 to 3 times that of FixL* in the presence of oxygen. This is in contrast to the very reproducible rates exhibited by FixL*H138Q and FixL*. A possible explanation for this is that FixL*H194N may be less stable than FixL*. It is well known that apomyoglobin and apohemoglobin denature much more readily than do their heme containing counterparts(36) .
Unlabeled FixJ was added to each P-labeled FixL* derivative and the rate of transfer from
FixL* to FixJ was monitored. The initial rates of phosphate loss and
FixJ-phosphate accumulation from FixL*H138Q and FixL*H194N correlate
well with each other and are equivalent to the rates observed for FixL* (Fig. 5, A and B). These results suggest that
the FixL*H138Q and the FixL*H194N mutations have no significant impact
on the initial rate of phosphotransfer from FixL* to FixJ. At later
time points, there is evidence of loss of transfer activity with a
subsequent gradual hydrolysis of phosphate from FixJ-phosphate.
FixJ-phosphate ultimately accumulates to somewhat higher levels for
FixL*H138Q (Fig. 5A) and FixL*H194N (Fig. 5B) than for FixL*, suggesting that there may
differences in the stability of FixJ-phosphate in the presence of the
different L* derivatives. The rates of phosphotransfer from the FixL*
derivatives to FixJ were unaffected by oxygen concentration (data not
shown) as is true for the wild-type FixL* protein(17) .
Figure 5: Transfer of phosphate from phosphorylated FixL*, FixL*H138Q and FixL*H194N. A, transfer of phosphate from FixL* (open circles) to FixJ (filled circles) and from FixL*H138Q (open squares) to FixJ (filled squares). B, transfer of phosphate from FixL* (open circles) to FixJ (filled circles) and from FixL*H194N (open triangles) to FixJ (filled triangles). Phosphorylated FixL*, FixL*H138Q, or FixL*H194N (0.6 µM) was incubated under aerobic conditions with FixJ (1.2 µM) at 22 °C. Proteins were analyzed by SDS-PAGE and autoradiography. Quantitation was by laser densitometry.
The FixJ-phosphate dephosphorylation activity of FixL* is shown in Fig. 6A. The dephosphorylation activity of FixL* decreases under aerobic conditions. In contrast to earlier results (17) , we have found that this decrease in phosphatase activity in response to anaerobiosis is of the same magnitude whether or not FixL* is preincubated with ATP (data not shown). In addition, the amount of the decrease is somewhat less than was reported previously (see ``Discussion'').
Figure 6:
Dephosphorylation of FixJ-phosphate by
FixL* and FixL* mutant derivatives. P-Labeled
FixJ-phosphate (0.45 µM) was incubated with FixL*,
FixL*H138Q, FixL*H194N, or FixL*H285Q (1 µM) under aerobic (open symbols) or anaerobic (filled symbols)
conditions as described under ``Materials and Methods.''
Dephosphorylation of FixJ-phosphate by FixL* (A, circles),
FixL* (B, circles) and FixL*H138Q (B, squares); FixL* (C, circles) and FixL*H194N (C, triangles); and FixL* (D, circles) and FixL
H285Q (D, diamonds)
is shown. FixJ-phosphate incubated under aerobic assay conditions
without a FixL* derivative is indicated by the
symbol. Proteins
were analyzed by SDS-PAGE, autoradiography, and laser
densitometry.
FixL* 138Q exhibits aerobic and anaerobic phosphatase activities equivalent to those of FixL* (Fig. 6B), demonstrating that the mutation at position 138 has no significant effect on phosphatase activity. Since the H138Q mutation substantially affects kinase activity (Fig. 4A); however, the data suggest that the kinase and phosphatase activities are to some extent independently regulated by the N-terminal heme binding domain of FixL*.
FixL*H194N and FixL* dephosphorylate FixJ-phosphate at equivalent rates under anaerobic conditions (Fig. 6C), but FixL*H194N consistently displays a reduced rate of FixJ-phosphate dephosphorylation under aerobic conditions. The rate of FixJ-phosphate dephosphorylation by FixL*H194N is essentially unaffected by oxygen concentration, i.e. the aerobic and anaerobic rates are similar. This is consistent with the fact that FixL*H194N lacks heme and suggests that an oxygenated heme domain could be required for full FixJ dephosphorylation activity.
The FixL*H285Q protein dephosphorylates FixJ-phosphate, although at a significantly reduced rate relative to that of FixL* (Fig. 6D), suggesting that the histidine at position 285 plays a role in, but is not absolutely required for, phosphatase activity. The FixL*H285Q phosphatase activity is still down-regulated in response to anaerobiosis, consistent with the ability of this protein to bind heme and oxygen.
Figure 7: Phosphorylation of FixJ by FixL* and FixL* mutant derivatives. FixL*, FixL*H138Q, or FixL*H194N (0.5 µM) was incubated with FixJ (1 µM) as described under ``Materials and Methods'' under aerobic (open symbols) or anaerobic (filled symbols) conditions. The amount of phosphorylated FixJ protein is plotted. A, phosphorylation of FixJ by FixL* (circles) and FixL*H138Q (squares). B, phosphorylation of FixJ by FixL* (circles) and FixL*H194N (triangles). Proteins were analyzed by SDS-PAGE and autoradiography, followed by laser densitometry.
Under aerobic conditions, FixL*H194N also shows an increased level of FixJ phosphorylation relative to that seen for FixL* (Fig. 7B). Although the results with FixL*H194N were again more variable than for the other FixL* derivatives, the level of FixJ phosphorylation by FixL*H194N under both aerobic and anaerobic conditions is always 4-7-fold higher than that seen for FixL* in the presence of oxygen. The increased level of FixJ phosphorylation by FixL*H194N under aerobic conditions is consistent with the increased autophosphorylation activity of FixL*H194N under aerobic conditions (Fig. 4B). The H285Q mutant does not show any FixJ phosphorylation activity (data not shown), consistent with its lack of autophosphorylation activity.
In this work, we have examined the effects of mutations of selected histidine residues on the heme binding properties as well as the in vitro phosphorylation and dephosphorylation activities of FixL* in the presence and absence of oxygen. Two mutations in the region of the protein previously demonstrated to bind heme, H138Q and H194N, increase FixL* autophosphorylation under aerobic conditions. Significantly, a change of histidine 194 to glutamine abrogates heme binding. Since this is the only histidine residue in the heme binding domain which, when mutated, results in the loss of heme binding, this histidine is likely to be the site of heme iron coordination. Finally, we showed that mutation of the highly conserved histidine residue at position 285 results in the loss of autophosphorylation and FixJ phosphorylation activities, suggesting that histidine 285 is the site of autophosphorylation. The H285Q mutant is also defective in FixJ dephosphorylation activity, but a significant level of activity remains, and this activity is regulated by oxygen.
The FixL*H194N data are consistent with a model in which the heme binding region of FixL* exerts a negative effect on the autophosphorylation activity of the C-terminal kinase domain, since the loss of heme in FixL*H194N results in rates of autophosphorylation and FixJ phosphorylation under aerobic and anaerobic conditions that are roughly equivalent to the rates of these reactions catalyzed by FixL* under anaerobic conditions. This increased in vitro activity of FixL*H194N in the presence of oxygen is consistent with data showing that deletion of the heme binding region (but not the transmembrane regions) results in elevated transcriptional activity at the nifA and fixK promoters under aerobic conditions in vivo in E. coli(38) .
The H138Q protein shows a significantly higher level of autophosphorylation activity than does FixL* under both repressed (aerobic) and derepressed (anaerobic) conditions. In this regard, it is similar to the FixL*362 mutant, an alanine-to-valine mutation at amino acid position 362(17) . However, FixL*H138Q does not show a reduction in phosphatase activity as does FixL*362(17) . The H138Q mutation may affect the conformation of the C-terminal kinase domain in such a way that it has increased access to the ATP substrate or that the active site is better positioned to carry out ATP hydrolysis. Alternatively, the H138Q mutation could alter the monomer-dimer equilibrium such that a greater proportion of dimers are formed. Gel filtration data are consistent with FixL* existing as a dimer(15) , and there is a growing body of evidence that two-component system sensors function as dimers with autophosphorylation occurring through a transphosphorylation between the subunits within a dimer(39, 40) .
It has been shown previously that anaerobiosis decreases the rate of FixJ-phosphate dephosphorylation by FixL*(17) . The FixJ dephosphorylation activity of FixL* was shown to be significantly inhibited under anaerobic conditions when FixL* was incubated with ATP (to generate FixL*-phosphate) prior to the addition of the FixJ-phosphate substrate. Anaerobic conditions had a less dramatic effect on reducing phosphatase activity when FixL* was in the unphosphorylated state (not preincubated with ATP)(17) . However, in this study we saw no significant difference in the phosphatase activities between unphosphorylated and phosphorylated FixL* either in the presence or absence of oxygen (data not shown). Similarly, we observed no effect of phosphorylation on the dephosphorylation activities of the FixL*H138Q, FixL*H194N, or FixL*H285Q mutant proteins (data not shown). The magnitude of the oxygen effect that we observed in this work was also less dramatic than that seen in the earlier study. The reason for these differences is presently unclear but might be due to differences in the FixL* and FixJ preparations used in each study. It is interesting that FixJ-phosphate dephosphorylation by FixL* appears to occur in at least two phases: an initial rapid phase followed by a slower phase (Fig. 6A). For FixL* and the FixL* histidine mutants, the decreased rate of dephosphorylation under anaerobic conditions is evident only in the later, slower phase of the reaction. In a previous study of phosphorylated FixL*, the effect of oxygen was evident at earlier times in the reaction(17) . More detailed kinetic experiments must be done in order to understand the biphasic nature of FixL* dephosphorylation of FixJ-phosphate and the effects of oxygen in the early, fast phase of the reaction.
The phosphatase activities of the histidine mutants demonstrate that mutations in both the heme binding region (H194N) and the conserved kinase domain (H285Q) can affect phosphatase function. The FixL*H194N mutant shows a reduction in aerobic, but not anaerobic, phosphatase activity that is consistent with the loss of oxygen sensing capability. This suggests that an oxygenated heme domain is required for full phosphatase activity. This situation is analogous to NtrB, whose NtrC-phosphate dephosphorylation activity is repressed (41, 42) in the absence of an activating signal from the PII protein. Mutation of the conserved, putative autophosphorylation site, histidine 285, reduces, but does not eliminate FixL* phosphatase activity. In addition, the phosphatase activity remaining is regulated by oxygen concentration. Work with other two-component system sensors has shown that mutations of the conserved histidine sometimes result in the complete loss of phosphatase activity. For example, a histidine-to-valine mutation at the conserved position 139 in NtrB results in a mutant protein that is devoid of phosphatase activity. However, an asparagine replacement at the same position retains phosphatase activity (as assayed by the ability of the mutant NtrB protein to negatively regulate transcription from the glnAp2 promoter)(43) . Such data together with the FixL*H285Q results reported here suggest that although the conserved histidine is not absolutely required for phosphatase activity, perturbations in structure around this histidine can significantly influence this activity.
Interestingly, the H138Q mutation has a significant effect on FixL* kinase activity but no apparent effect on phosphatase activity. This suggests that the kinase and phosphatase activities are to some degree independent. The independence of phosphatase and kinase activities has recently been demonstrated for NtrB by showing that it is possible to mutate one activity without altering the other(43) .
This study has focused on the effects of specific mutations on the in vitro activities of a soluble truncated derivative of FixL, FixL*. The in vitro response of FixL* to its oxygen ligand will facilitate the understanding of the molecular basis of oxygen sensing in R. meliloti as well as the understanding of signal transduction by two-component regulatory system proteins in general. It is important to keep in mind, however, that in vitro studies with FixL* have limitations. Although it has been demonstrated that the membrane spanning regions are not absolutely required for oxygen sensing and signal transduction in E. coli(38, 44) or R. meliloti(14) , maximal responsiveness to oxygen requires that the transmembrane segments be present(14, 44) . The membrane-spanning regions are therefore likely to play a role in FixL function. The membrane region of FixL may sense other signals related to low oxygen growth such as the redox state of the cell. There is precedent that a single sensor can respond to multiple input signals. VirA, which is required for virulence of Agrobacterium tumefaciens, responds in its periplasmic domain to sugars (45, 46) and in its cytoplasmic domain to phenolic compounds(47) . In addition, it has recently been suggested that the full-length FixL protein responds to ammonia in vivo(48) . Clearly, more work needs to be done with the full-length FixL protein before we understand the basis of signal transduction by FixL in R. meliloti.