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J Bacteriol, June 1998, p. 3253-3256, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The FixK2 Protein Is Involved in
Regulation of Symbiotic Hydrogenase Expression in
Bradyrhizobium japonicum
Meredith C.
Durmowicz and
Robert J.
Maier*
Department of Biology, Johns Hopkins
University, Baltimore, Maryland 21218
Received 9 February 1998/Accepted 21 April 1998
 |
ABSTRACT |
The roles of the nitrogen fixation regulatory proteins NifA,
FixK1, and FixK2 in the symbiotic regulation of
hydrogenase structural gene expression in Bradyrhizobium
japonicum have been investigated. Bacteroids from FixJ and
FixK2 mutants have little or no hydrogenase activity, and
extracts from these mutant bacteroids contain no hydrogenase protein.
Bacteroids from a FixK1 mutant exhibit wild-type levels of
hydrogenase activity. In 
-galactosidase transcriptional assays with
NifA and FixK2 expression plasmids, the FixK2
protein induces transcription from the hup promoter to
levels similar to those induced by HoxA, the transcriptional activator
of free-living hydrogenase expression. The NifA protein does not
activate transcription at the hydrogenase promoter. Therefore,
FixK2 is involved in the transcriptional activation of
symbiotic hydrogenase expression. By using -galactosidase
transcriptional fusion constructs containing successive truncations of
the hup promoter, the region of the hup
promoter required for regulation by FixK2 was determined to be between 29 and 44 bp upstream of the transcription start site.
| |
TEXT |
The slow-growing symbiont of the
soybean plant, Bradyrhizobium japonicum, expresses a
hydrogen uptake hydrogenase that oxidizes hydrogen under both
free-living and symbiotic conditions. In the free-living state, the
expression of the NiFe hydrogenase is regulated at the transcriptional
level by hydrogen, oxygen, and nickel (21). These three
signals exert their effects within a 50-bp region of DNA located
between 99 and 149 bp upstream of the transcription start site of the
hydrogenase structural genes (22). In addition, the
hydrogenase promoter is 
54 dependent and requires
integration host factor for full induction (5).
The hoxA gene (32) is located approximately 12 kb
downstream of the hydrogenase structural genes, in a region of the
hydrogenase gene cluster previously shown to be necessary for
free-living hydrogenase activity (23). The hoxA
gene encodes a protein with extensive homology to transcriptional
activators of hydrogenase expression in several other organisms,
including HoxA in Alcaligenes eutrophus (12),
HupR1 in Rhodobacter capsulatus (30),
and HydG in Escherichia coli (31), all of which
are members of the NtrC-like family of response regulators
(17). Subsequent studies of the role of the HoxA protein in
the biosynthesis of hydrogenase by our group (11) and
another (33) have confirmed that HoxA is a transcriptional
activator of hydrogenase expression under free-living, microaerobic
conditions. Its cognate sensor protein is presently unknown. However,
bacteroids from nodules formed by B. japonicum HoxA mutants
exhibit wild-type levels of hydrogenase activity and extracts from
Hup+ bacteroids used in gel retardation assays do not
cause a shift of a fragment of the hydrogenase promoter containing the
50-bp regulatory region (11). Because the hydrogenase
promoter is 54 dependent, there must be some other,
symbiosis-specific, activator of hydrogenase expression that binds the
promoter at an alternative site.
The hydrogenase enzyme in the pea symbiont, Rhizobium
leguminosarum, is expressed solely in symbiotic conditions
(28). Interestingly, a defective hoxA gene that
has been inactivated by several frameshift and deletion mutations has
been reported for this organism (6). The hydrogenase
structural genes have been shown to be under the transcriptional
control of the nitrogen fixation regulatory protein NifA
(6). Several groups have suggested that symbiotic
hydrogenase expression in B. japonicum is also linked to the
regulation of nitrogen fixation (11, 33). In addition to
NifA, another candidate for a symbiotic regulator is the Fnr-like, DNA
binding protein FixK (2). In rhizobial species, FixK is part
of an oxygen-responsive regulatory cascade controlled by the FixL-FixJ
two-component system (13). B. japonicum contains
two homologs of the FixK protein (13). FixK2
activates the expression of several genes, including nitrate metabolism
genes, the fixNOQP operon, and fixK1,
in response to low oxygen levels (13). Mutants in this gene
are Nif
. The target or targets for FixK1 are
unknown (2). It is possible that one or both of the FixK
homologs may be involved in the regulation of other oxygen-responsive
genes, including hydrogenase (11, 13).
In this work, we show that mutants in the fixLJ-fixK
nitrogen fixation regulatory cascade are deficient in symbiotic
hydrogenase activity. Unlike in R. leguminosarum, NifA does
not seem to be involved in the symbiotic transcriptional activation of
B. japonicum hydrogenase expression. Instead, the
results here are consistent with FixK2 acting as a
symbiotic transcriptional activator. A possible FixK2
binding site is centered at 40 bp from the transcription start site.
Bacterial strains and plasmids.
All bacterial strains and
plasmids used in this work are listed in Table
1. B. japonicum JH
(16) is a derivative of USDA I-110 and is considered the
wild type. JH
A (11) is derived from strain JH and
contains an 886-bp in-frame genomic deletion that removes most of the
hoxA gene. Strains 7360 (1), 7454 (2),
and 9043 (13) are all derived from B. japonicum
110spc (29). Strains 7360 and 7454 contain an insertion of
the kanamycin resistance gene that disrupts the fixJ and
fixK1 genes, respectively. In strain 9043, the
fixK2 gene is replaced by the spectinomycin resistance gene. Escherichia coli ET8000 (24) is
a lac mutant strain that is used as a background strain in
-galactosidase transcriptional assays. Plasmid pRJ9044 (unpublished
data; a gift of H. Fischer) contains the B. japonicum
fixK2 gene on a 1.85-kb BamHI-SalI fragment cloned into pBluescript SK
under control of the lac promoter. Plasmid pMC71A
(7) contains the Klebsiella pneumoniae nifA gene
cloned into the multicopy vector pACYC184 (9) under control
of the promoter of the tetracycline resistance gene. Plasmid pSKA
contains the B. japonicum hoxA gene on a 1.5-kb KpnI-SpeI fragment cloned into pBluescript SK in
the same orientation as the vector lac promoter. Plasmid
pSY7 (21) is a hup-lacZ transcriptional
fusion construct derived from pGD499 (10) and contains
a 2.4-kb BamHI-PstI fragment of the hydrogenase
structural genes including 680 bp of the promoter region. The remaining
plasmids, pGHh1, pGHp1, pGR1, pGHf1, pGBs3, and pGNSdB (21,
22), are all derived from plasmid pSY7 and contain successive
deletions of the hup promoter region (listed in Table 1)
fused to a promoterless lacZ gene.
The hup phenotype of fixJ,
fixK1, and fixK2
regulatory mutants.
Free-living B. japonicum strains
were grown in modified Bergerson's medium (3) and
derepressed for hydrogenase activity by incubation for 18 to 20 h
in no-carbon medium (26) under standard conditions (4,
11) of 5 µM nickel and an atmosphere of 84% nitrogen, 10%
hydrogen, 5% carbon dioxide, and 1% oxygen. Whole bacteroids were
prepared as previously reported (11, 20) by crushing nodules
harvested from soybean plants inoculated with each B. japonicum strain and grown for 5 to 6 weeks as described previously (20, 25).
As shown in Table
2, all three of the
nitrogen fixation regulatory mutants (
fixJ,
fixK1, and
fixK2) are not
affected in hydrogenase
activity under free-living, microaerobic
conditions. The FixK
1 mutant is also Hup
+
in symbiosis (Fig.
1, lane 5). However,
bacteroids from the FixJ
and FixK
2 mutants exhibit little
or no hydrogenase activity and
extracts from these mutant bacteroids
contain little or no hydrogenase
protein (Fig.
1, lanes 3 and 4) as
detected by immunoblotting
with antibody to the large subunit of
hydrogenase (
15). NifA
mutants are severely affected in the
ability to form an effective
symbiosis and in nodule morphology
(
14). Therefore, a NifA mutant
could not be assayed for
symbiotic hydrogenase activity. The HoxA
mutant JH
A was assayed for
hydrogenase activity as a control
and was Hup
in
free-living conditions (Table
2) and Hup
+ in symbiosis
(Fig.
1, lane 2) as expected.
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FIG. 1.
Immunoblotting and hydrogenase activities of wild-type
and mutant bacteroid samples. The hydrogenase activity of whole
bacteroids was measured amperometrically (18, 34).
Activities are the averages ± standard deviations of six separate
determinations. Western blots of bacteroid extracts prepared as
previously described (11) were probed with antibody against
the large subunit of B. japonicum hydrogenase. Lane 1, JH;
lane 2, JHA; lane 3, 9043; lane 4, 7360; lane 5, 7454.
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|
Since the FixJ and FixK
2 mutants are defective in nitrogen
fixation, the possibility exists that the Hup
phenotype
observed in bacteroids from these strains is due to
an indirect effect
of a lack of hydrogen (a known requirement
for
hup
transcription) produced by the nitrogenase enzyme rather
than the
absence of either FixJ or FixK
2. To investigate this
possibility, bacteroids from a
B. japonicum mutant strain
harboring
a Tn
5 insertion in the nitrogenase structural gene
nifD were assayed
for hydrogenase activity. Bacteroids from
this
nif mutant are
also Hup
(data not shown).
However, the data do not rule out a role for
FixK
2 in the
symbiotic regulation of hydrogenase genes. It is
possible and, in fact,
probable that, as in free-living conditions,
symbiotic hydrogenase
expression is regulated by multiple signals.
Presumably, hydrogen acts
as an environmental signal in addition
to oxygen and the positive
signals for
hup transcription are passed
on by
as-yet-unidentified components to the oxygen-responsive
FixLJ-FixK
regulatory cascade, which then acts on the hydrogenase
genes.
Transcriptional control of the hup promoter by
FixK2.
To demonstrate a role for the FixK2
protein in the symbiotic regulation of hydrogenase biosynthesis and to
rule out the NifA protein as a symbiotic transcriptional activator,
-galactosidase transcriptional assays (27) were done in
the heterologous background of the lac mutant E. coli strain ET8000 (24). Plasmid pSY7, containing the
hup promoter fused to a promoterless lacZ gene, was cotransformed into ET8000 with each of the following plasmids: pRJ9044 and pSKA, which constitutively express B. japonicum
FixK2 and HoxA, respectively, from the lac
promoter of pBluescript SK, and pMC71A, which constitutively expresses
K. pneumoniae NifA from the tetracycline resistance gene
promoter of pACYC184.
Both the HoxA and FixK
2 proteins induce expression from the
hydrogenase promoter to levels 6- to 14-fold above the background
levels represented by plasmid pSY7 and plasmid pSY7 cotransformed
with
pBluescript KS (Table
3). Expression of
the
hup promoter
was not activated by the NifA protein. In
addition, the
B. japonicum hydrogenase promoter was provided
on a multicopy plasmid in a
K. pneumoniae wild-type strain
and the activity of the nitrogenase
enzyme was measured by acetylene
reduction (
19). It has been
shown that multiple copies of a
NifA binding sequence will reduce
nitrogenase activity as measured by
acetylene reduction due to
the titration of NifA (
6,
8). The
hup promoter plasmid reduced
levels of acetylene reduction
to the same extent as a control
vector with no insert, while a plasmid
containing the
B. japonicum nifH promoter region, which is
known to bind NifA, eliminated
acetylene reduction activity (data not
shown). These data indicate
that FixK
2, and not NifA, has a
role in the transcriptional regulation
of the
B. japonicum
hydrogenase structural genes. This is in contrast
to the situation in
R. leguminosarum, in which NifA has been shown
to bind and
regulate the hydrogenase promoter (
6).
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TABLE 3.
Transcriptional activity of the hup promoter
measured in a lac mutant E. coli strain (ET8000)
carrying various plasmidsa
|
|
Localization of a potential FixK2 binding site in the
hydrogenase promoter region.
A visual inspection of the
hydrogenase promoter region revealed the presence of two potential
binding sites for FixK2 (Fig. 2A). Both sites are about 50% identical
to the FixK consensus binding sequence (Fig. 2B) (13). The
sites are located between 213 and 228 bp and between 32 and 47 bp
upstream of the transcription start site. When compared to the
placement of FixK binding sites upstream of genes known to be regulated
by FixK (13), either site upstream of the hydrogenase
structural genes is equally likely to be the actual binding area.
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FIG. 2.
Sequence of the upstream region of the hydrogenase
structural gene operon and FixK consensus binding sequence. (A) Two
potential FixK binding sites extending from  213 to 228 and from
32 to 47 bp upstream of the transcription start site of the
hupSL operon (marked with an asterisk) are underlined. (B)
Comparison of the hydrogenase promoter region with the FixK consensus
binding sequence. Identical residues in each potential binding site are
in boldface type.
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|
To determine the region of the hydrogenase promoter necessary for
regulation by FixK
2, plasmid pRJ9044 was cotransformed into
strain ET8000 with various plasmids containing successive truncations
of the hydrogenase promoter region fused to a promoterless
lacZ gene (Table
1) (
21,
22) and
-galactosidase activity was
measured (see Table
4). Induction of the
hydrogenase promoter
by the FixK
2 protein is unaffected
when the promoter region is
truncated to 99 bp upstream of the
transcription start site as
in plasmid pGR1 but is reduced to just
twice background levels
when the promoter region is truncated to 44 bp
upstream of the
hydrogenase structural genes as in plasmid pGHf1 (Table
4). Hydrogenase
promoter activity is
reduced even further, to background levels,
when the upstream region is
truncated to 29 bp (i.e., plasmid
pGBs3). A deletion of the promoter
region between 29 and 64 bp
upstream of the transcription start site in
plasmid pGNSdB also
abolishes induction of the
hup promoter
by FixK
2 (Table
4). In
plasmids pGBs3 and pGNSdB, the
integration host factor binding
site shown to be necessary for full
induction of the hydrogenase
promoter under free-living conditions
(
5) is not present. However,
in previous studies with
pGNSdB,
hup promoter activity was only
reduced to 50% of
the activity observed with full-length promoter
constructs
(
5). No
hup promoter activity was measured with
either plasmid pGBs3 or pGNSdB in our experiment. Therefore, the
reduction in
hup promoter activity is due to a lack of
binding
of a factor (FixK
2) other than integration host
factor. The partial
reduction of hydrogenase promoter activity with a
44-bp upstream
region indicates that sequences within the putative FixK
binding
site closest to the transcription start site are important for
activation of
hup transcription by FixK
2.
Taken together, these data show that in contrast to
R. leguminosarum, NifA is not a regulator of symbiotic hydrogenase
expression
in
B. japonicum. Instead, the Fnr-like DNA
binding protein FixK
2 is involved in the symbiotic
transcriptional activation of the
hydrogenase genes in this organism.
The possibility exists that
FixK
2 exerts its effect on
hydrogenase expression in an indirect
fashion, through some
as-yet-unidentified component(s). However,
the
-galactosidase
transcriptional assays with the FixK
2 expression
plasmid
provide strong evidence for the FixK
2 protein being at
least one point at which the regulation of symbiotic hydrogenase
expression and the regulation of nitrogen fixation merge.
| |
ACKNOWLEDGMENTS |
We thank Hans-Martin Fischer for providing several B. japonicum mutant strains and plasmid pRJ9044. We are grateful to
Mike Merrick for his generous gifts of plasmid pMC71A and strain ET8000 and for his helpful suggestions. We also thank Sue Maier for her assistance with plants and Jon Olson for helpful discussions.
This work was supported by Department of Energy grant
DEFG02-89-ER14011.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Johns Hopkins University, Baltimore, MD 21218. Phone: (410) 516-7218. Fax: (410) 516-5213. E-mail:
Maier_rj{at}jhuvms.hcf.jhu.edu.
| |
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J Bacteriol, June 1998, p. 3253-3256, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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