The hupT gene product, expressed from a plasmid, repressed HupSL synthesis as expected while introduction of act-ively expressed hupTUV genes together derepressed the HupSL activity in T
Trang 1in Thiocapsa roseopersicina BBS
A´ kos T Kova´cs1
, Ga´bor Ra´khely1, Judit Balogh1, Gergely Maro´ti1, Laurent Cournac2, Patrick Carrier2, Lı´via S Me´sza´ros1, Gilles Peltier2and Korne´l L Kova´cs1
1 Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, and Department of Biotechnology,
University of Szeged, Hungary
2 CEA Cadarache, DSV, De´partement d’Ecophysiologie Ve´ge´tale et de Microbiologie, Laboratoire d’Ecophysiologie de la Photosynthe`se, CNRS CEA, Saint Paul-Lez Durance, France
The presence of the substrate molecule of hydrogenases,
H2, triggers the expression of some hydrogenases through
a hydrogen-sensing regulatory hydrogenase (HupUV⁄
HoxBC) and a two-component signal transduction
system (HupT⁄ HoxJ and HupR ⁄ HoxA) as described
mainly in Rhodobacter capsulatus [1] and Ralstonia
eutropha [2] In the presence of H2, the expression of
the membrane bound HupSL (in R capsulatus) or
HoxKG (in Ra eutropha) and soluble HoxFUYH
(in Ra eutropha) hydrogenases is initiated, while the
gene products are not formed in the absence of H2 HupUV and⁄ or HoxBC are members of the regulatory [NiFe] hydrogenases (RH) [3] They show a predicted structure that is similar to the typical [NiFe] hydro-genases, possessing the small and the large subunits and the well known [NiFe] active site with two CN and one CO ligand [4] RH is a soluble protein in line with the absence of an N-terminal translocation signal sequence on the small subunit polypeptide Interestingly, the large subunit proteins of the sensor
Keywords
hydrogen sensor; [NiFe] hydrogenase;
transcriptional regulation; Thiocapsa
roseopersicina
Correspondence
K L Kova´cs, Department of Biotechnology,
University of Szeged, H-6726 Szeged,
Temesva´ri krt 62, Hungary
Fax: +36 62 544352
Tel: +36 62 544351
E-mail: kornel@brc.hu
(Received 16 June 2005, accepted 3 August
2005)
doi:10.1111/j.1742-4658.2005.04896.x
The expression of many membrane bound [NiFe] hydrogenases is regulated
by their substrate molecule, hydrogen The HupSL hydrogenase, encoded
in the hupSLCDHIR operon, probably plays a role in hydrogen recycling
in the phototrophic purple bacterium, Thiocapsa roseopersicina BBS RpoN, coding for sigma factor 54, was shown to be important for expres-sion, suggesting a regulated biosynthsis from the hup gene cluster The response regulator gene, hupR, has been identified in the hup operon and expression of hupSL was reduced in a chromosomal hupR mutant, which indicated that HupR was implicated in the activation process The hupT and hupUV genes were isolated, and show similarity to the histidine kinase element of the H2-driven signal transduction system and to the regulatory hydrogenases of Ralstonia eutropha and Rhodobacter capsulatus, respect-ively Although the genes of the entire H2 sensing and regulation system were present, the expression of the hupSL genes was not affected by the presence or absence of H2 Using reverse transcription PCR, we could not detect any mRNA specific to the hupTUV genes in cells grown under diverse conditions The hupT and hupUV mutant strains had the same phe-notype as the wild-type strains The hupT gene product, expressed from a plasmid, repressed HupSL synthesis as expected while introduction of act-ively expressed hupTUV genes together derepressed the HupSL activity in
T roseopersicina The gene product of hupUV behaves similarly to other regulatory hydrogenases and shows H–D exchange activity
Abbreviations
IHF, integration host factor; RH, regulatory hydrogenase; RT, reverse transcription.
Trang 2hydrogenases terminate at a histidine residue and lack
the commonly occurring C-terminal extension that is
proteolytically processed during the last step of
post-translational maturation in energy transducing [NiFe]
hydrogenases Some of the pleiotropic accessory
pro-teins (Hyp) are required for the proper assembly of
the H2-activating [NiFe] site in RH [5] The catalytic
activity of RH is low, but the activity is insensitive to
oxygen [4] It has been purified as a tetramer with an
a2b2 structure This tetramer forms a complex with
the HupT⁄ HoxJ kinase in vitro [4] The role of the
N-terminal part of the kinase, containing a PAS
domain, was established in signal transduction
between the RH and the kinase [6,7] Addition of H2
to HupUV before or during the incubation with HupT
rendered the complex unstable [6] The transmission of
H2-induced changes from the RH to the histidine
kin-ase in vivo inhibits phosphorylation of the response
regulator Therefore the DNA-binding positive
regula-tor remains unphosphorylated and binds to its target
site and activates the expression of the hupSL (hoxKG
and hoxFUYH) hydrogenase genes In the absence of
molecular hydrogen the kinase phosphorylates the
HupR⁄ HoxA regulator, which therefore looses its
activity and stops the transcription of the hydrogenase
structural genes [1] The main difference in the signal
transduction between R capsulatus and Ra eutropha
is displayed by the phenotype of hupT⁄ hoxJ and
hupUV⁄ hoxBC mutants, respectively R capsulatus
hupT and hupUV mutants show a high level of
hy-drogenase activity in the absence of H2 Thus both the
HupT and the HupUV proteins exert a negative
con-trol on hydrogenase gene expression [8] Phenotypic
analysis of Ra eutropha hoxJ and hoxBC mutants
revealed that the H2 sensing HoxBC protein
counter-acts the negative role of the HoxJ kinase [4]
Thiocapsa roseopersicina BBS is a purple sulphur
photosynthetic c proteobacterium belonging to the
Chromatiaceae family Two sets of genes coding for
membrane bound [NiFe] hydrogenases) the
hynS-isp1-isp2-hynL (formerly hydS and hydL) [9] and
hupSLCDHIR [10] – and a third, soluble hydrogenase
(hoxEFUYH) [11], together with other components
that are necessary for hydrogenase maturation [12,13]
were cloned and characterized Thiocapsa
roseoper-sicina provides an attractive model system for
com-parative studies of the structure–function–stability
relationships of different hydrogenase isoenzymes [14]
Transcriptional regulation of the T roseopersicina hyn
operon was demonstrated recently The expression of
the hyn genes was induced under anaerobic conditions
by an FNR homologue, FnrT, and it was unaffected
by H2[15]
We now report that transcription of T roseopersicina hupSL hydrogenase genes is regulated through an RpoN dependent promoter The elements (hupR, hupTUV) of a typical signal transduction system are present and HupR is functionally active The hupT and hupUV genes are apparently intact, yet the hydrogen sensing system is not functional in T roseopersicina BBS
Results
Hydrogen independent hupSL expression The HupSL enzyme of T roseopersicina is a member
of the Group 1 uptake [NiFe]-H2ases [16] Many members of this group are expressed only in the pres-ence of hydrogen In order to study directly the H2 dependent expression of hupSL the T roseopersicina GB11 strain was used because it lacks the other mem-brane associated [NiFe] hydrogenase, HynSL, which would interfere with the HupSL specific hydrogenase assay of the membrane fraction Deletion of the hynSL genes did not affect the activity of HupSL hydrogenase [11] Mutation in the structural genes of both membrane bound hydrogenases resulted in the loss of all membrane bound hydrogenase activity [11,12] (Table 1) Unexpectedly, the hydrogenase activity measurements indicated a constant level of HupSL activity, irrespective of the presence of hydro-gen (Table 2) The effect of H2 on the expression of HupSL hydrogenase was examined under conditions where nitrogenase was fully repressed and the HoxYH soluble hydrogenase did not produce detectable amount of H2 (G Ra´khely and K L Kova´cs, unpublished data) A 708-bp DNA fragment contain-ing the first 76 bp of the hupS codcontain-ing sequence, together with upstream sequences, was cloned into the broad host-range lacZ expression vector, pFLAC, to create an in-frame hupS::lacZ gene fusion The result-ing recombinant plasmid, pHUPRIP was introduced into T roseopersicina and b-galactosidase activities were measured during growth under various condi-tions The measurements revealed similar expression when cells were propagated in the absence or presence
of hydrogen (Table 2) Hydrogenase activity of HupSL could not be detected in Ni-free conditions; however, the b-galactosidase activities were unchaged (55.6 ± 6.2 Miller units in Ni-free conditions and 57.7 ± 5.6 Miller units in the presence of 5 lmolÆl)1 Ni) This suggests that Ni is important only for the maturation of the HupSL hydrogenase enzyme but not for the expression of hupSL genes During the experiments, cultures were grown under strictly
Trang 3anaerobic conditions as the presence of trace amount
of oxygen abolished HupSL activity (J Balogh, G
Ra´khely, A´ T Kova´cs and K L Kova´cs,
unpub-lished data)
Activation is dependent on RpoN Inspection of the upstream sequence region of hupS gene revealed a typical )24 ⁄ )12 promoter sequence
Table 1 Strains and plasmids.
Thiocapsa roseopersicina
Escherichia coli
S17-1(kpir) 294 (recA pro res mod) Tp r , Sm r (pRP4-2-Tc::Mu-Km::Tn7), kpir [35]
XL1-Blue MRF¢ D(mcrA)183, D(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1,
recA1, gyrA96, relA1 lac [F¢ proAB lacI q
ZDM15 Tn10 (Tetr)]c
Stratagene Plasmids
pTUV2 8576-bp HindIII fragment that contains the hupTUV operon in pBluescript SK (+) This work
pAK35 4568-bp SphI fragment that contains the hupCDHI and hupR genes in pUC18 [10]
pKK23 3313-bp PstI fragment that contains the upstream region of hupS gene in pUC18 [10]
pHRIMER2 Km r , 2833-bp region of hupR gene in pK18mobsacB carrying Em r cassette at BstXI site This work
pRPON2 Km r , 1618-bp region of rpoN gene in pK18mobsacB carrying Gm r cassette at SmaI site This work
pHTD2 Km r , in-frame up and downstream homologous regions of hupT in pK18mobsacB This work
pHUVD2 Km r , in-frame up and downstream homologous regions of hupUV in pLO2 This work
pHUPRIP Gm r , mob + , pFLAC carrying the promoter region of hupS gene fused to the lacZ gene This work
pTUVC1 Kmr, mob+, hupTUV genes cloned after the promoter region of crtD gene This work
pTUV C 2 Km r , mob + , hupT gene cloned after the promoter region of crtD gene This work
pMHE6crtKm Km r , mob + , expression vector containing the promoter region of crtD gene [30]
pMHEUVC2 Kmr, mob+, hupUV gene cloned after the promoter region of crtD gene This work
Table 2 HupSL specific H 2 uptake and b-galactosidase activities in different strains grown in the absence or presence of hydrogen ND, Not detected; NA, not adaptable (antibiotic conflict).
HupSL hydrogenase activity a LacZ activity b
a
Relative hydrogenase activities in the membrane fraction given in percentage compared to the T roseopersicina GB11 strain grown in the absence of H2 b Specific b-galactosidase activity (same strains containing pHUPRIP) given in micromoles of o-nitrophenol min)1ÆD 1
650
Trang 4element (Fig 1) [10] Promoters harbouring )24 ⁄ )12
elements require the sigma factor RpoN (r54)
Fur-ther upstream from the r54 element, an integration
host factor (IHF) box was recognized The role of
IHF in transcriptional regulation will be the subject
of future studies The rpoN gene was detected as
part of the ongoing genome project of T
roseopersi-cina (L S Me´sza´ros, G Ra´khely, H P Klenk and
K L Kova´cs, unpublished data) The sequence of
the rpoN gene was deposited in the GeneBank
(accession number: AY837592) The rpoN gene was
disrupted with a gentamycin cassette to generate
plasmid pRPON2 that was conjugated into T
roseo-persicina Double recombinant colonies were isolated
to yield the rpoN mutant, RPON Southern blot
ana-lyses on genomic DNA confirmed the inactivation of
the chromosomal rpoN gene in the expected way
(data not shown) The RPON strain was unable to
grow in the absence of ammonium as a nitrogen
source indicating that the N2 fixing ability was
impaired as well Results in Table 2 show that
HupSL activity was also lost in the RPON mutant
b-galactosidase activities were not measured as the
pHUPRIP vector contains a gentamycin resistance
marker and the T roseopersicina RPON strain is also
resistant to gentamycin
HupR activates hupSL transcription
The hupSLC structural genes are clustered with the
hupDHIRgenes blastp and clustal analyses
sugges-ted that the putative HupR protein belonged to the
family of response regulators The translated HupR
from T roseopersicina showed similarity to HoxA of
Ra eutropha(53% identity and 66% similarity) and to HupR (45% identity and 61% similarity) of R capsul-atus In addition, the putative T roseopersicina HupR possesses a helix-turn-helix DNA binding motif (resi-dues 434–474, with E-value of 5.4e-12) in its C-ter-minal domain The HupR architecture was determined using the SMART database, revealing that T roseo-persicina HupR contained a response regulator receiver domain (residues 6–125, with E-value of 6.4e-29) and a
r54interaction domain (residues 165–386, with E-value
of 1.2e-140)
The presence of the hupR gene in T roseopersicina
is in apparent contradiction with the absence of a hydrogen-dependent regulation of HupSL expres-sion In order to examine in detail the role of hupR
in T roseopersicina an interposon mutant strain (HRMG) was constructed The mutation in hupR affected the expression of HupSL hydrogenase dras-tically: no hydrogenase activity could be measured in the membrane fraction of T roseopersicina HRMG under any conditions compared to the wild-type GB11 strain (Table 2) The hydrogenase activity of HoxYH proteins in the soluble fraction was unaffec-ted in the T roseopersicina HRMG strain (data not shown) Plasmid pHUPRIP carrying the hupS::lacZ fusion was conjugated into the wild-type and HRMG mutant T roseopersicina strains; transconjugants were grown in the absence and presence of hydrogen and assayed for b-galactosidase activity Results in Table 2 show that the expression of hupS::lacZ is dramatically decreased in the hupR mutant independently from the presence of hydrogen Thus HupR is necessary for HupSL expression, but it is not sufficient for the
H2-dependent regulation
Fig 1 Structure of the hup operon and reg-ulatory region The 120-bp region upstream from hupS is presented Hypothetical )24 ⁄ )12 region and IHF site (on the bottom strand) are boxed and compared to the con-sensus RpoN and IHF sites A vertical line denotes residue identity Start codon of hupS is underlined and the first two amino acids of HupS are indicated.
Trang 5Isolation of the hydrogen sensor and sensor
kinase coding genes
Multiple alignments were performed with the known
HupUV⁄ HoxBC protein sequences and the conserved
regions were selected Because these proteins resemble
the regular [NiFe] hydrogenases, extreme care was
taken to avoid regions which were conserved also in
the nonregulatory hydrogenases Finally, a 272-bp
fragment of the hupU gene was successfully amplified,
cloned and sequenced This fragment was used to
iso-late an 8570-bp fragment carrying the hupT, hupU,
and hupV genes (Fig 2) and flanking sequences
(Gen-Bank accession number: AY837591) The hupT and
hupUV genes encode putative proteins that are most
similar to HupT and HupUV of Azorhizobium
caulino-dans(65% similarity and 53% identity for HupT, 78%
similarity and 68% identity for HupU, 68% similarity
and 56% identity for HupV [17]) Downstream from
the hupV gene parA and orf154 were identified The
predicted parA gene product showed similarity to the
partition protein A (57% similarity to ParA of
Actino-bacillus actinomycetemcomitans) and Orf154 showed
68% similarity to a hypothetical protein of
Synecho-cystis sp PC6803 Upstream from the hupT gene a
truncated orf, similar to nifS gene, was identified that
lacks translational signal elements Additionally, there
were numerous stop codons preceding this truncated
orf
Total RNA was isolated from cells grown under
var-ious conditions (Fig 3) and reverse transcription
(RT)-PCR was used to search for the hupTUV transcript
No mRNA corresponding to the hupTUV genes was
found (Fig 3) The quality of the RNA was checked
and found satisfactory using primers specific for the
coding region of Hyn hydrogenase (Fig 3B) The
results suggest that the transcript level of the hupTUV
genes is below the detection limit or is missing in
T roseopersicina
Mutagenesis and homologous expression of the
hupT and hupTUV genes
In-frame deletion mutagenesis was used to characterize
the hupT and hupUV deficient phenotype The
exten-sively truncated hupT derivative was cloned into
T roseopersicina, resulting in HTMG Similarly, the HUVMG strain contained a 64-amino acid fragment
of hupUV Both the hupT and the hupUV mutant strains had comparable HupSL hydrogenase activities
to the control GB11 strain (Table 2) We also assayed b-galactosidase activity in wild-type, HTMG, and HUVMG T roseopersicina strains carrying pHUPRIP Neither the hupT nor the hupUV mutation changed the expression of hupS::lacZ (Table 2)
The hupT gene (pTrTUVC2), hupUV genes (pMHEUVC2) or hupTUV genes (pTrTUVC1) were cloned behind the promoter of the crtD gene and expressed under anaerobic, phototrophic conditions Plasmids were transformed into T roseopersicina, and the transformants were grown in the presence or absence of hydrogen and assayed for HupSL hydroge-nase activity Table 3 shows that HupSL hydrogehydroge-nase activity was lost in the strain, which expressed the hupT gene The HupT expressed from a plasmid thus apparently performs the expected repressor function of
Fig 2 Identified hupTUV genes Restriction
sites used during construction of in-frame
deletion vectors are indicated The
sequence has been deposited with
Gene-Bank Accession Number AY837591.
A
B
Fig 3 RT-PCR analysis of T roseopersicina hupTUV expression Primers TUVo24 and TUVo13 were used to detect mRNA corres-ponding to hupU (A) Primers otsh11 and otsh14 were used to detect mRNA corresponding to hynS (B) and used to verify the quality of RNA prepared PCR products were analysed on agarose gel Samples were loaded as follows: cells were grown in Pfennig’s mineral medium (lanes 1, 2), and supplemented with sodium-acet-ate (lanes 3, 4), D -glucose (lanes 5, 6), grown in the presence of H 2 (lanes 7, 8), or ammonium chloride was omitted (lane 9, 10) In samples loaded in lanes 1, 3, 5, 7 and 9, reverse transcription was carried out before the PCR; in lanes 2, 4, 6, 8 and 10, reverse transcription was omitted M, Marker; C, control PCR made on genomic DNA Selected marker bands are indicated.
Trang 6HupT Production of HupTUV from a similar plasmid
construction, however, did not alter the HupSL
hydrogenase activity, i.e HupSL was not regulated by
H2 (Table 3) The HupSL activity was also unaltered
in strains expressing the hupUV genes only (Table 3,
pMHEUVC2) RT-PCR revealed the presence of hupT
and hupUV specific mRNA in strains expressing the
corresponding genes from the promoter of crtD gene
(data not shown), but not in strains without plasmid
b-Galactosidase activities were not measured as the
pHUPRIP vector contains the same origin of
replica-tion as pTrTUVC1, pTrTUVC2 and pMHEUVC2
The enzyme activities of the RH proteins measured
with various redox dyes showed very low activity
com-pared to those of energy conserving [NiFe]
hydro-genases [4] Therefore we tested the activity of the
T roseopersicina HupUV using the H–D exchange
reaction H–D exchange, catalysed by the energy
con-serving hydrogenases and by the RH, can be
distin-guished on the basis of their different response to O2
[18] Strains lacking the HupUV expression plasmids
had no detectable H–D exchange activity in the
pres-ence of oxygen, while those expressing the HupTUV
from the promoter of crtD (pTrTUVC1) showed
0.19 ± 0.06 lmolÆL)1Æmin)1 activity In comparison,
the H–D exchange activity of the soluble HoxEFUYH
hydrogenase, measured in the absence of oxygen, was
23.5 ± 2.1 lmolÆL)1Æmin)1 The H–D exchange
activ-ity of the soluble hydrogenase was sensitive to oxygen
as described earlier for other hydrogenases
Discussion
In a few organisms, e.g methanogens, whose
metabo-lism is strictly linked to H2, hydrogenases are
synthes-ized constitutively [19] In most other cases the
expression of hydrogenases is regulated by various
environmental signals The signal may be anaerobicity
[20], Ni [21], or hydrogen itself The signal
transduc-tion pathway that responds specifically to H2 has been
studied in detail in Ra eutropha [2,3,7], R capsulatus
[1,6] and in Bradyrhizobium japonicum [21,22] The pathway comprises HupUV (regulatory hydrogenase), HupT (kinase), and HupR (response regulator) in
R capsulatus
The genes coding for the membrane bound HupSL hydrogenase were cloned and sequenced in T roseo-persicina [10] The presence of HupR response regula-tor downstream from the hupSLCDHI genes prompted
us to assume that hydrogen-dependent regulation may function in T roseopersicina by analogy to R capsula-tus and Ra eutropha The regulation of the T roseo-persicina HupSL hydrogenase was followed by hydrogenase activity measurements and it was found that hydrogen did not affect HupSL activity This puz-zling observation could not explain the presence of the hupR gene and the r54promoter element The r54 spe-cific binding site in the hupS upstream region was investigated Indeed, the expression of T roseopersicina HupSL hydrogenase depended on the presence of func-tional RpoN protein The expression of hydrogenase was also RpoN-dependent in Ra eutropha [23] and
in B japonicum [24], while hupSL transcription is
r70-dependent in R capsulatus [1] This is in line with the observation that the putative r54 interaction sites within the HupR⁄ HoxA proteins are well conserved in
T roseopersicina, Ra eutropha and B japonicum, but not in R capsulatus [1]
The remote possibility of the inactive hupR gene was considered The functional role of HupR was therefore tested by creating a T roseopersicina hupR mutant strain Results obtained with this mutant provided straightforward evidence that HupR was essential for the hupSL transcription under all conditions investi-gated The H2insensitive HupSL expression was there-fore not due to an aborted hupR The promoter region
of the T roseopersicina hupSL genes did not reveal any unusual feature that could be responsible for the lack
of response to the environmental signal, hydrogen
If the presence of HupR and its effect on HupSL expression is a sign for the biosynthesis of the enzyme being under the H2 control, the other elements of the signal transduction cascade should be present in
T roseopersicina The clustered hupTUV genes were identified, cloned, sequenced, and analysed The trun-cated HupT and HupUV proteins were most similar
to the corresponding proteins of Azorhizobium cauli-nodans [17] The physiological role of HupT and HupUV in the regulation of HupSL was tested by creating hupT and hupUV deletion mutants in T roseo-persicina Hydrogenase activity measurements showed that deletion of hupT or hupUV genes did not change the level of HupSL hydrogenase activity, suggesting that the putative HupT and HupUV proteins do not
Table 3 H2uptake activities in complementation experiments The
results are given in percentage compared to the T roseopersicina
grown in the absence of H 2
Plasmid
Complementing
gene
HupSL hydrogenase activity
Trang 7take part in a hydrogen sensing function and do not
regulate the HupSL formation under the growth
con-ditions examined A possible explanation of these
data may implicate the apparently truncated nifS,
located immediately upstream from the hupT gene
This flawed gene residue may hamper the
transcrip-tion of the hupTUV genes due to a polar effect The
lack of expression of the HupTUV would explain the
hydrogen independent activity profiles To confirm
this idea, RT–PCR experiments were carried out to
test the presence or absence of the hupTUV message
RT–PCR experiments showed that no mRNA
corres-ponding to hupU gene was detected in cells grown
under various conditions It was therefore concluded
that the hupTUV gene cluster is cryptic in T
roseo-persicina The question remained whether a point
mutation in the hupTUV genes or the upstream
trun-cated nif gene is responsible for the failed
transcrip-tional regulation?
Multiple alignment of T roseopersicina HupT
pro-tein with other kinases revealed the presence of H, N,
G1, F and G2 motifs in the C-terminal region, those
necessary for kinase function Introduction of the hupT
gene behind the promoter region of crtD gene
repressed HupSL expression in T roseopersicina
sug-gesting that HupT can fulfil its function if expressed
behind a heterologous promoter Thus HupT is more
similar in function to the HoxJ protein of Ra
eutro-pha, i.e it represses transcription of the hupSL
Thio-capsa roseopersicina HupUV resembles typical features
of [NiFe] hydrogenases Introduction of hupTUV genes
cloned behind the promoter region of crtD gene
restored the expression of HupSL hydrogenase
How-ever, the expression of HupSL hydrogenase was
unal-tered by the presence of H2 These results suggest that
HupUV, expressed from a strong T roseopersicina
promoter, interacts with HupT and alters its
phos-phorylation state, but the HupUV cannot change the
interaction with HupT depending on the presence of
hydrogen Remarkably, HupUV, expressed from a
plasmid, clearly displayed catalytic activity in the H–D
exchange activity assay When expressed, the HupUV
regulatory hydrogenase is therefore active in T
roseo-persicina The so-called RHSTOP mutant protein of
Ra eutrophalacking a C-terminal peptide of 55 amino
acids in HoxB lost its H2-sensing ability but still
cata-lysed the H2 oxidation [7] In this case the RHSTOP
was incapable of forming the (ab)2 dimeric
heterodi-mer and the complex with HoxJ kinase, therefore the
expression of the membrane bound HoxKG
genase was repressed Thus uncoupling of the
hydro-genase activity and the H2sensing ability of HupUV is
conceivable
In summary, it can be concluded, that the expres-sion of the hupTUV genes from a broad host range vector could partially restore the signal transduction cascade, although irrespective of the presence of hydrogen Each of the elements of the known signal transduction (HupR and HupT) and H2 sensing (HupUV) system are functional, yet the expression
of HupSL does not apparently depend on the pres-ence or abspres-ence of H2 in the environment The lack
of functionally active hupTUV on the chromosome is
a likely reason for the constitutive expression of the hupSL genes in the wild type strain At this point one cannot exclude the possibility that additional genetic elements are also involved in the assumed
H2 dependent regulation of HupSL biosynthesis Impaired regulatory mechanisms, caused by point mutations, have been described previously in several cases In Ra eutropha H16, a mutation of HoxJ kin-ase resulted in the loss of HoxJ protein function and constitutive expression of hydrogenase genes [25] In Rhodopseudomonas palustris CGA009, the photosys-tem is synthesized in the dark due to a single point mutation in the helix–turn–helix DNA binding motif
of PpsR, rendering it inactive [26] Comparison of HupSL regulations and the functional roles of HupTUV in other T roseopersicina strains would provide further insight into the understanding of the loss of HupSL hydrogenase regulation
Experimental procedures
Bacterial strains and plasmids Strains and plasmids are listed in Table 1 T roseopersicina strains were grown in liquid cultures for 3–4 days in Pfen-nig’s mineral medium supplemented with 0.1% NH4Cl [27] Sodium acetate (2 gÆL)1) or d-glucose (5 gÆL)1) was added when needed NiCl was omitted only if indicated, otherwise
5 lmolÆL)1 was used Plates were solidified with 7 gÆL)1 Phytagel (Sigma, St Louis, MO, USA); when selecting for transconjugants plates were incubated for 2 weeks in anaer-obic jars using the GasPack (BBL, Kansas City, MI, USA)
or AnaeroCult (Merck, Rahway, NJ, USA) systems Escherichia coli strains were maintained on Luria–Bertani agar Antibiotics were used in the following concentrations (lgÆmL)1): for E coli: streptomycin (50), ampicillin (100), kanamycin (50), gentamycin (20), erythromycin (50); for
T roseopersicina: streptomycin (5), kanamycin (20), genta-mycin (5) erythrogenta-mycin (50)
Conjugation Conjugation was carried out as described previously [12]
Trang 8Identification of the hupU gene
A multiple alignment of the known HupU protein sequences
was performed and conserved domains were selected for
designing PCR primers PCR was carried out using
the primers: hupUo1 (5¢-AACGAGTTCTAIGAITAIAAG
GCN-3¢) and hupUo2 (5¢-GCIACGTTCCTIGCCTTNG
GCATRTC-3¢) (where R is A or G) on T roseopersicina
genomic DNA The isolated PCR product of the correct
size (272 bp) was cloned into pGEM T-Easy (Promega,
Madison, WI, USA; resulting in pHUPU1) and sequenced
Cloning of hupTUV genes from T roseopersicina
Southern analysis was performed with the NotI fragment of
pHUPU1 as a probe A HindIII partial genomic library
was created in pBluescript SK+ and pTUV2 was identified
by colony hybridization The insert of the pTUV2 plasmid
was subcloned and sequenced on both strands by primer
walking The 8576-bp sequence was deposited in the
Gene-Bank under the accession number AY837591
Site-directed mutagenesis of hupR, rpoN, hupT
and hupUV genes
The in-frame deletion vector constructs derived from the
pK18mobsacB [28] or pLO2 [29] vectors For insertion
mut-agenesis of the hupR gene, the 2833-bp ApaI (truncated)–
SphI fragment of pAK35 [10] was inserted into the EcoRV–
SphI site of pLO2, resulting in pHRIMER1 After digesting
the pHRIMER1 with BstXI and polishing, the truncated
SalI–EcoRI fragment (918 bp) of pRL271 (GenBank
acces-sion number L05081) containing the erythromycin
resist-ance gene was inserted (pHRIMER2)
For insertion mutagenesis of the rpoN gene, the 1618 bp
PCR fragment obtained with primers rpoN1 (5¢-GCTGC
ATCTCGACGATCTTC-3¢) and rpoN2 (5¢-ATCGCTTGC
GCTGAGCCTCT-3¢) from rpoN (GenBank Accession
Number AY837592) was inserted into the SmaI site of
pK18mobsacB, resulting in pRPON1 After digesting the
pRPON1 with SmaI, the SmaI fragment (855 bp) of
p34S-Gm (GenBank accession number AF062079) containing the
gentamycin resistance gene was inserted (pRPON2)
For removal of the hupT gene, the truncated 1379-bp
ApaI fragment of pTUV2 was inserted into the BamHI
digested and polished pK18mobsacB vector, resulting in
pHTD1 The 1311-bp SacI fragment of pTUV2 was
inser-ted into the SalI site of pHTD1 vector after polishing the
noncompatible ends, resulting in pHTD2
For removal of the hupU and hupV gene, the 1794-bp
BamHI fragment of pTUV2 (upstream region of the hupU)
was inserted into the 5924-bp BamHI vector fragment of
pTUV2 (containing the downstream region of the hupV),
resulting in pHUVD1 The 4534-bp KpnI–XbaI fragment of
the pHUVD1 was inserted into the SacI–XbaI site of pLO2 vector after polishing the noncompatible ends, resulting in pHUVD2
The pHRIMER2, pRPON2, pHTD2 and pHUVD2 con-structs were transformed into E coli S-17(kpir), then conju-gated into T roseopersicina GB11 resulting HRMG (hupR::Er), RPON (rpoN::Gm), HTMG (DhupT) and HUVMG?(DhupUV), respectively When creating the hupR::Er or rpoN::Gm strain, the selection for the recombi-nation was based on the erythromycin or gentamycin resist-ance and then the double recombinant clones, that were resistant to erythromycin or gentamycin and sensitive to kanamycin, were selected In the case of in-frame deletion
of hupT or hupUV genes, selection for the first recombina-tion event was based on kanamycin resistance The selec-tion for the second recombinaselec-tion was based on the sacB positive selection system [13] The mutant clones were veri-fied by PCR and⁄ or Southern blotting
Construction of hupS::lacZ fusion plasmid The PCR fragment obtained with ohup4 (5¢-CTCGAA
CGGCCAGT-3¢) primers on pKK23 [10] was digested with PstI and cloned into the XbaI (polished)-PstI site of pFLAC [15] resulting pHUPRIP1
Construction of hupTUV expressing plasmids The hupTUV and hupT genes of T roseopersicina were cloned downstream from the crtD promoter region of
T roseopersicina as follows: the promoter region of the crtD gene from T roseopersicina was isolated from pRcrt4
as an XhoI–BamHI fragment and after polishing the ends it was cloned to the SspI site of pBBRMCS2 resulting pBBRcrt The hupTUV genes were cloned as a HindIII– BglII(polished) fragment from pTUV2 into the HindIII– BstXI (polished) sites of pBBRcrt yielding pTrTUVC1 To express the hupT gene only the hupUV genes were deleted from pTrTUVC1 by replacing the EcoRI–StuI (polished) fragment (containing the 3¢ region of hupT and the hupUV genes) with the EcoRI–BamHI (polished) fragment of pTUV2 This construct (pTrTUVC2) restored the whole hupT gene, but lacked the hupUV genes The NdeI-HindIII digested TUVo31 (5¢-ACATATGAACCTGTTATGGCTC CAG-3¢)–TUVo28 (5¢-AAGCTTGTGGACCGTGCAGAC CAT-3¢) PCR fragment was cloned into the corresponding sites of pMHE6crtKm [30] resulting in pMHEUVC2
Isolation of total RNA and RT-PCR analysis RNA was isolated from cells using the TRI reagent (Sigma,
St Louis, MO, USA), following the manufacturer’s recom-mendations Isolated total RNA was treated with RNase-free
Trang 9Dnase I at 37C for 60 min in a total volume of 40 lL
[40 mm Tris⁄ HCl pH 7.5, 20 mm MgCl2, 20 mm CaCl2, 4 U
of RNase-free DNase I (Promega, Madison, WI, USA)]
prior to RT-PCR After phenol⁄ chloroform extraction and
ethanol precipitation, the RNA was dissolved in 20 lL H2O
RT–PCR was carried out as described previously [12] The
TUVo24 primer (5¢-GAGGTTGGTGGCCAGTTC-3¢) was
used for the reverse transcription and PCR The TUVo13
(5¢-AACGCCGTGTCGGACCATGT-3¢) served as the other
primer in PCR Using these primers a 592-bp fragment was
expected The quality of the RNA prepared was assayed with
primers specific for the hynS gene: otsh14 (5¢-GAT
CGCGATATTGAACATC-3¢) was used in the reverse
tran-scription and otsh11 (5¢-CTGCCCGAGCTTGACGC-3¢)
served as other primer in PCR Using these primers a 512-bp
fragment was expected
Enzyme assays
Hydrogenase uptake activities of membrane fractions were
determined using benzyl viologen [13] The rates of H2and
HD formation, resulting from exchange between D2 and
protons of the medium, measured at 30C, were monitored
continuously by MS as described in detail previously
[31,32] For each experiment 1.5 mL (D600¼ 0.464 ± 0.034
of 10-times diluted cultures) culture was used Hydrogenase
activity based on the rates of H2 and HD formation was
calculated as described by Cournac et al [33] The
b-galac-tosidase activity of the toluene-permeabilized cell extracts
was assayed as described earlier for T roseopersicina
[27,34] Cells were assayed at the late logarithmic growth
state One Miller unit corresponds to 1 lmol of
o-nitrophe-nyl-b-galactoside (Sigma-Aldrich) hydrolysed per minute
normalized to the optical density at 650 nm for T
roseo-persicina
Bioinformatics tools
Protein sequence comparisons in the various databases were
done with the blast (p, x) programs (http://www.ncbi
nih.nlm.gov) Multiple alignments were performed with the
clustal xprogram
Acknowledgements
Supported by Hungarian Ministry of Education
(OMFB-00768⁄ 03) and the European Commission
(QLK5-1999-01267 and NEST STRP SOLAR-H,
con-tract 516510) We thank Dr Annette Colbeau and Dr
Sylvie Elsen (DBMS, CEA-CENG, Grenoble, France)
and Dr Douglas F Browning (University of
Biming-ham, BirmingBiming-ham, UK) for many helpful discussions
We gratefully acknowledge Ro´zsa Verebe´ly for
excel-lent technical assistance
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