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Journal of Bacteriology, August 1999, p. 5085-5089, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification and Characterization of
hupT, a Gene Involved in Negative Regulation of Hydrogen
Oxidation in Bradyrhizobium japonicum
C.
Van Soom,1
I.
Lerouge,1
J.
Vanderleyden,1,*
T.
Ruiz-Argüeso,2 and
J. M.
Palacios2
F. A. Janssens Laboratory of Genetics,
Katholieke Universiteit Leuven, B-3001 Heverlee,
Belgium,1 and Laboratorio de
Microbiología, Universidad Politécnica de Madrid,
Escuela Técnica Superior de Ingenieros Agrónomos, 28040 Madrid, Spain2
Received 5 March 1999/Accepted 27 May 1999
 |
ABSTRACT |
The Bradyrhizobium japonicum hupT gene was sequenced,
and its gene product was found to be homologous to NtrB-like histidine kinases. A hupT mutant expresses higher levels of
hydrogenase activity than the wild-type strain under
hydrogenase-inducing conditions (i.e., microaerobiosis plus hydrogen,
or symbiosis), whereas in noninduced hupT cells,
hupSL expression is derepressed but does not lead to
hydrogenase activity. We conclude that HupT is involved in the
repression of HupSL synthesis at the transcriptional level but that
enzymatic activation requires inducing conditions.
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TEXT |
A remarkable feature of hydrogen
oxidation in Bradyrhizobium japonicum is that induction of
the hydrogen uptake system is triggered when the bacterium shifts to
either of two extremely different lifestyles. Hydrogenase activity in
Hup+ strains develops during chemoautotrophic growth in a
microaerobic, hydrogen-containing environment and when the bacterium
differentiates into a symbiotic, nitrogen-fixing bacteroid (reviewed in
reference 20). Although the presence of the
substrate hydrogen appears to be the common denominator in both
lifestyles, it has become increasingly apparent that the regulatory
networks that govern the formation of the uptake hydrogenase are as
complex as the different lifestyles. The structural genes for the
B. japonicum hydrogenase enzyme are encoded by the
hupSL genes (28), and more than 20 other
hup-related genes with various functions in structure,
assembly, processing, maturation, nickel metabolism, and regulation
have been identified (3, 10-12, 23, 30, 32). In free-living
bradyrhizobia, hydrogenase expression is induced by hydrogen, low
oxygen concentrations, and trace amounts of nickel (14, 15,
29) and hupSL expression requires RpoN, integration host factor (2), and the DNA-binding transcriptional
activator HoxA (31). In bacteroids, HoxA is not essential
for transcription and it has been proposed that nitrogenase and
hydrogenase are coregulated through FixK2 (5).
The pathway through which these inducing signals are perceived and
transduced has not yet been elucidated in detail. A multicomponent
system including a pseudohydrogenase (HupUV/HoxBC) and a
repressor protein (HupT/HoxJ) has been proposed to be
involved in hydrogen sensing and signal transduction in Rhodobacter capsulatus (6, 8) and in
Alcaligenes hydrogenophilus (16, 17). The genes
encoding the pseudohydrogenase (HupUV) in B. japonicum have
been previously identified (3). Here, we present the
nucleotide sequence and mutant analysis of a regulatory gene,
hupT, showing that it has a repressor function in the
regulatory cascade leading to hup gene expression under both
free-living and symbiotic conditions. In addition, we studied the
expression of the gene encoding the transcriptional regulator HoxA and
demonstrated that hoxA is expressed under aerobic conditions
and that expression is positively autoregulated under
hydrogenase-inducing conditions (microaerobiosis plus hydrogen).
Bacterial strains, plasmids, and growth of cells.
B.
japonicum CB1809 (CSIRO, St. Lucia, Australia) is routinely grown
at 30°C in PSY medium (26). To measure hydrogenase activity under free-living conditions, cells were grown in minimal salts medium (described in reference 12),
supplemented with 0.05% sodium gluconate and 0.05% sodium glutamate,
under a gas atmosphere of 5% H2-5% CO2-1%
O2-89% N2 for 48 h. Then the cells were
lysed, and equal amounts of protein were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis for Western blotting
(1). To obtain soybean bacteroids, surface-sterilized soybean seeds (cultivar Williams) were inoculated with B. japonicum cultures grown on YMB (33) and plants were
grown in Leonard jar-type assemblies under bacteriologically controlled
conditions in a growth chamber (18 h of daylight [25°C]-6 h of
darkness [18°C] regime) as described previously (19).
After 24 days, bacteroids were isolated as previously described
(18) and hydrogenase activities of bacteroid suspensions
were determined amperometrically (27).
The hupT mutant FAJ1010 was constructed by replacing an
internal 900-bp NotI fragment of the B. japonicum
CB1809 genome with a tetracycline resistance gene cassette (Fig.
1). To this end, hupT was
cloned as a 4.8-kbp BamHI fragment in the conditionally lethal suicide vector pJQ200mp18 (24). After NotI
digestion, the ends were blunted with the Klenow fragment of DNA
polymerase and a 2-kb tetracycline resistance gene cartridge, isolated
from SmaI-digested pHP45-TcR (9), was ligated
into the coding sequence of hupT. This construct, pFAJ1029,
was mobilized to CB1809, and selection on PSY medium with 5% sucrose
and 100 µg of tetracycline/ml forced replacement of the wild-type
hupT copy with the truncated allele. The structure of the
mutated region in the hupT mutant, FAJ1010, was confirmed by
Southern hybridization (data not shown). To measure hupSL
expression, a promoterless gusA gene was inserted into the
hupS coding sequence in the wild-type and FAJ1010 strains as
follows: the hupS promoter region, encompassing the entire hupS gene and the 5' part of hupL on a 3-kb
BamHI fragment, was blunted with the Klenow fragment of DNA
polymerase and ligated in SmaI-digested pJQ200UC1.
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FIG. 1.
Physical and genetic map of the hup region of
B. japonicum. Horizontal arrows indicate the positions and
sizes of the B. japonicum hup (U to K
and T), hyp (A to E), and
hox (X and A) genes. The horizontal
line at the top of the figure represents the DNA region present in
cosmid pFAJ1005. Shown in details are the regions relevant to the
construction of the reporter and mutant strains, including the relevant
restriction sites (S = SacI, B = BamHI,
and N = NotI), as well as the locations of the
tetracycline interposon (TcR) in mutant FAJ1010 and of the
promoterless gusA-spectinomycin cassettes (SpR)
used to construct genomic reporter fusions in hupS (present
in strains FAJ1011 and FAJ1012) and hoxA (strain FAJ1005).
UAS, HoxA-binding site.
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A 4-kb
SacI fragment from pWM5 (
21), containing a
promoterless
gusA cassette preceded by a ribosome binding
site and a spectinomycin
resistance gene, was cloned in the
SacI site of
hupS, thus putting
the
gusA gene under the transcriptional control of the
hupS promoter
region. This construct, called pFAJ1050, was
mobilized to
B. japonicum,
and replacement of the wild-type
hupS copy on the chromosome was
forced by selecting
for double homologous recombination as described
above. The
resulting reporter strains, FAJ1011 (CB1809
hupS::
gusA)
and FAJ1012 (FAJ1010
hupS::
gusA), allow the measurement of
hupS expression in the presence and in the absence of
HupT, respectively
(Fig.
1). The structures of the mutated regions in
FAJ1011 and
FAJ1012 were confirmed as correct by means of
Southern hybridization
(data not
shown).
To induce cells to undergo
hupSL transcription, cells were
grown in HUM medium (
4), washed twice in modified minimal
salts
medium (as described above), and diluted in this medium to an
optical density of 0.1 at 600 nm. One-milliliter aliquots of these
cell
suspensions were transferred to 80-ml test tubes at 30°C
(time zero)
and cultured under air (aerobic growth conditions)
or under a defined
gas atmosphere (1% O
2-99% N
2 for microaerobic
growth conditions, 5% H
2-5% CO
2-1%
O
2-89% N
2 for inducing conditions).
The
tubes used for growth under a defined atmosphere were tightly
capped
with rubber stoppers and flushed twice a day with the gas
mixture.
-Glucuronidase activities of the cell suspensions were
quantified
after 48 h of growth by using the substrate
p-nitrophenyl-
-
D-glucuronide
(
13)
and expressed in Miller units (
22).
A chromosomal insertion of a promoterless
gusA cassette in
hoxA was obtained by insertion of the same 4-kb
gusA-Sp
r cassette described above into the
blunted
BamHI site of pFAJ1020
(
31) followed by
mobilization to
B. japonicum CB1809 and forced
double
homologous recombination. The structure of the mutated
region was
confirmed by Southern hybridization (data not shown),
and the resulting
hoxA mutant was called FAJ1005 (Fig.
1). An
intact
hoxA gene was supplied in
trans by introduction
of cosmid
pFAJ1005 (Fig.
1) (
32).
Sequencing of the B. japonicum hupT gene.
Double-stranded DNA sequencing was carried out on DNA located
downstream of the previously identified hoxA gene (31,
32), on both strands on overlapping fragments subcloned in pUC19,
with an automated sequencer (A.L.F.; Pharmacia Biotech). Nucleotide sequences were compiled and analyzed with PC/GENE software
(IntelliGenetics, Inc., Mountain View, Calif.). Analysis of codon
usage (25) showed a 1,368-bp open reading frame with a
codon profile characteristic for B. japonicum and
partially overlapping the 3' end of the hoxA gene, which
suggests translational coupling (see below). The deduced amino acid
sequence consists of 455 amino acid residues and encodes a putative
protein with a predicted molecular mass of 50,059 Da. When the amino
acid sequence was compared with sequences present in databases, the
highest degrees of similarity were found to be with the hoxJ
gene of A. hydrogenophilus and A. eutrophus (38.7 and 38.5% identity, respectively) (16, 17) and the
hupT gene of R. capsulatus (36% identity)
(8). These gene products are homologous to NtrB-like sensor
histidine kinases, and in the central and C-terminal regions,
functional domains for autophosphorylation, kinase or phosphatase
activity, and nucleotide binding are highly conserved. The N-terminal
parts of the proteins are much less conserved and are probably
involved in sensing a signal. In R. capsulatus, the
hupT gene product, together with the hupU and hupV gene products, is part of a repression system of
hydrogen uptake gene expression (6). In A. hydrogenophilus, HoxJ is proposed to be involved in signal
transduction from the hydrogen-sensing HoxBC complex to HoxA
(17).
hupT mutant analysis.
In FAJ1010, HupT is
truncated at amino acid 163 and thus lacks the conserved central
and C-terminal domains containing the typical histidine kinase motifs.
To study the function of hupT in B. japonicum,
we measured hydrogenase activity amperometrically both in
free-living and in symbiotic B. japonicum CB1809 (wild-type) and FAJ1010 (hupT mutant) cells. Table
1 shows that hydrogenase activity in the
hupT mutant is approximately twofold higher than that in the
wild type, both in free-living induced cells and in bacteroids. In
free-living cells grown aerobically in the presence or absence of
hydrogen or microaerobically in the absence of hydrogen, no hydrogenase
activity could be detected (Table 1), indicating that the simultaneous
presence of low oxygen concentrations and hydrogen is required to
obtain an active hydrogenase enzyme. When equal amounts of crude
protein of free-living induced cells were probed for the presence of
HupL protein by means of Western blotting with anti-HupL serum,
extracts of the hupT mutant clearly gave a stronger signal
than wild-type cells, indicating that relatively more hydrogenase
protein is formed in the absence of HupT (Fig. 2, lanes d and e). A similar increase in
HupL levels was observed in bacteroids of the HupT mutant (Fig. 2,
compare lanes f and g). These results are consistent with the observed
increase in hydrogenase activity associated with the absence of HupT
(Table 1). In protein extracts prepared from aerobically grown cells, no signal was present for wild-type cells but a clear signal could be
observed for the hupT mutant (Fig. 2, compare lanes a and
b). Since the hupT mutant grown aerobically had no
hydrogenase activity, the observed immunoreactive band presumably
corresponds to inactive (i.e., unprocessed) HupL protein. In fact, the
band observed in aerobic cells had a slower mobility (Fig. 2, compare
lanes b and c).
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TABLE 1.
Hydrogenase activity in B. japonicum wild-type
and hupT mutant strains grown under free-living and
symbiotic conditions
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FIG. 2.
Immunodetection of HupL in cell extracts. Western blots
containing crude cellular extracts were incubated with HupL antiserum
at a dilution of 1:1,000. Cell extracts were obtained from wild-type
CB1809 (lanes a, d, and f) or hupT mutant strain (lanes b,
c, e, and g) aerobically grown vegetative cells (panel A, lanes a and
b), induced vegetative cells (panel A, lane c, and panel B), or soybean
bacteroids (panel C).
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To study the effect of HupT on expression of the hydrogenase structural
genes under free-living conditions, a promoterless
gusA gene
was inserted in the
hupS coding sequence in the wild
type
and the
hupT mutant, as described above.
-Glucuronidase
activities of the reporter strains were measured after 48 h of
growth under aerobic, microaerobic, or inducing conditions (Table
2). In the wild-type FAJ1011 background,
hupSL expression is
blocked under aerobic and microaerobic
conditions and is induced
under conditions of microaerobiosis plus
hydrogen (inducing conditions).
When
hupT is knocked out (in
FAJ1012),
hupSL expression is derepressed
(although at low
levels) under aerobic and microaerobic conditions.
This low level of
expression does not increase over time (data
not shown) and is
consistent with the presence of HupL protein
in these cells (Fig.
2).
In induced cells, expression is highly
increased as a function of time
and is twice the expression level
seen in wild-type induced cells after
48 h (Table
2). These results
suggest that HupT somehow represses
hupSL expression under both
noninducing and inducing
conditions, since the level of
hupSL expression is
consistently higher in the absence of HupT. The
effect of HupT on
hydrogenase activity is observed only in induced
cells (free-living as
well as symbiotic).
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TABLE 2.
Effect of hupT on hupSL expression
in vegetative B. japonicum cells under aerobic,
microaerobic, or hydrogenase-inducing growth conditions
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We conclude from these results that HupT blocks transcription of the
hupSL promoter under conditions in which the expression
of a
hydrogenase enzyme would be futile (i.e., under aerobic conditions
or
in the absence of the substrate hydrogen) and somehow modulates
the
level of expression under inducing conditions. Under these
conditions,
HupT might be inactivated by a process dependent on
HupUV, as has
been proposed for
A. eutrophus
(
17). The observed
increase in
hupSL expression
in the absence of HupT may indicate
that the inactivation of HupT is
not complete under inducing
conditions.
The mechanism by which HupT affects transcription of the
hupSL promoter in free-living cells is likely mediated by
HoxA, since
it has been shown that under these conditions HoxA is
essential
for the induction of
hupSL expression and binds to
a defined region
of this promoter (
31). Therefore, we
propose that in free-living
B. japonicum cells, HupT
interacts with HoxA, modifying its transcription-inducing
activity.
HoxA is homologous to transcriptional activators of
the NtrC family,
whose transcriptional activities are dependent
on phosphorylation by
partner histidine kinases. Although the
phosphorylated form of an
NtrC-like regulator usually has transcriptional
activation activity, in
the case of
A. eutrophus it has been proposed
that HoxA is transcriptionally active in the
nonphosphorylated
form (
17). HupT
autophosphorylation has been demonstrated in
A. eutrophus (
17) and
R. capsulatus
(
7). The structural similarities
of
B. japonicum HupT and these homologous proteins, together with
the
observed effect of HupT on
hupSL expression, suggest that
in
B. japonicum HupT also modulates (represses) the
transcriptional
activity of HoxA via phosphorylation. Taking into
account these
observations, we hypothesize that the active form of
B. japonicum HoxA is the nonphosphorylated
form.
Expression of hoxA is positively autoregulated.
In
aerobically grown wild-type cells, there is no hupSL
transcription and, consequently, no hydrogenase activity. This lack of
hupSL transcription could be due to either the absence of
hoxA transcription under aerobic conditions or the
presence of inactive HoxA. To discriminate between these two
possibilities, we studied the expression pattern of hoxA
under aerobic and inducing conditions. The similar genetic
organizations of hypD, hypE, hoxX, and
hoxA suggest that the genes are translationally coupled
(32). Moreover, no obvious consensus promoter sequences
could be identified upstream of hoxA or hoxX,
except for one putative RpoN-dependent promoter 100 bp upstream of
hoxX (32). Since a reporter plasmid
containing 400 bp of DNA upstream of hoxX coupled to a
promoterless gusA gene failed to show any change in
expression level under inducing conditions compared to the level
observed during aerobiosis (results not shown), it was concluded that
this sequence does not correspond to a functional promoter. To monitor
hoxA expression driven from any promoter further upstream,
we constructed the reporter strain FAJ1005, in which the chromosomal
hoxA coding sequence is interrupted by a promoterless
gusA gene. An intact hoxA gene was supplied in trans on cosmid pFAJ1005, in which the insert
encompasses the hypABFCDE, hoxXA, and
hupT genes (Fig. 1). Expression was measured under aerobic
and inducing conditions, as shown in Table
3. Under aerobic conditions,
hoxA is expressed at a low basal level in the cells,
presumably from a housekeeping promoter. Expression is induced 10-fold
when cells are shifted to hydrogenase-inducing conditions, and this
expression is under positive autoregulatory control since no induction
can be seen in the absence of a functional hoxA gene. These
data strongly suggest that under aerobic conditions, HoxA is present in
the cells, although at a low level.
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TABLE 3.
hoxA expression in vegetative B. japonicum cells grown under aerobic or
hydrogenase-inducing conditions in the presence of HoxA
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In this view, the observed lack of
hupSL transcription in
wild-type cells under conditions of aerobiosis indicates that HoxA
is
present in the inactive form and is unable to activate transcription.
Since HoxA is the central transcriptional regulator of hydrogen
oxidation in free-living cells, the above-presented data suggest
that,
at least for this type of cells, HupT modulates the transcriptional
activity of HoxA, most probably through phosphorylation. Analogous
to
the situation in
A. eutrophus, we hypothesize that under
noninducing
conditions, HupT represses
hupSL transcription
by keeping the
HoxA pool in a phosphorylated state, for when
hupT is knocked
out,
hupSL is expressed. In the
absence of HupT, the available
HoxA would be nonphosphorylated, leading
to
hupSL transcription
and translation. It is clear that
levels of transcription of
hupSL and the content of HupL
protein are still lower in aerobically
grown cells than in induced
cells of the
hupT mutant. Full activation
of inactive
HoxA still seems to require the perception of inducing
conditions
by a not-yet-elucidated mechanism in which HupUV should
play a major
role.
Further research must focus on the identification of the additional
factors required to obtain an active
hydrogenase.
Nucleotide sequence accession number.
The nucleotide sequence
of the hupT gene has been deposited in the GenBank database
(accession no. AF132935).
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ACKNOWLEDGMENTS |
This work was supported by the Belgian Fund for Scientific Research
(FWO)
Flanders, by a postdoctoral fellowship to C.V.S., by a grant
from Spain's DGICYT (project PB95-0232) to T.R.-A., and by the IMPACT2
Project (Biotec Program; reference no. CT960027).
We thank C. Verreth and P. de Wilde for sequencing and L. Sayavedra-Soto for kindly providing the anti-HupL serum.
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FOOTNOTES |
*
Corresponding author. Mailing address: F. A. Janssens Laboratory of Genetics, Katholieke Universiteit Leuven,
Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium. Phone: 32-16-32 16 31. Fax: 32-16 32 19 66. E-mail:
Jozef.Vanderleyden{at}agr.kuleuven.ac.be.
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Journal of Bacteriology, August 1999, p. 5085-5089, Vol. 181, No. 16
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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