Hydrogen independent expression of hupSL genes
in Thiocapsa roseopersicina BBS
A
´
kos T. Kova
´
cs
1
,Ga
´
bor Ra
´
khely
1
, Judit Balogh
1
, Gergely Maro
´
ti
1
, Laurent Cournac
2
,
Patrick Carrier
2
,Lı
´
via S. Me
´
sza
´

eutropha [2]. In the presence of H
2
, 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 H
2
.
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

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.
FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS 4807
hydrogenases 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 H
2
-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
a
2
b
2
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 H
2
to HupUV before or during the incubation with HupT
rendered the complex unstable [6]. The transmission of

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 H
2
[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]-H
2
ases [16]. Many
members of this group are expressed only in the pres-

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
Transcription regulation of HupSL hydrogenase A
´
. T. Kova
´
cs et al.
4808 FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS
anaerobic conditions as the presence of trace amount
of oxygen abolished HupSL activity (J. Balogh, G.
Ra
´
khely, A
´
. T. Kova

(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 (Tet
r
)]
c
Stratagene
Plasmids
pGEM T-Easy Amp
r
, cloning vector, ColE1 Promega
pHUPU1 pGEM T-Easy, contains 272-bp fragment of hupU This work
pBluescript SK(+) Amp
r
, cloning vector, ColE1 Stratagene
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]
pK18mobsacB Km
r
, mob
+
, sacB
+
[28]
pLO2 Km
r
, mob

, mob
+
, broad host range vector [37]
pFLAC Gm
r
, mob
+
, pBBRMCS5 carrying the promoterless lacZ gene [15]
pHUPRIP Gm
r
, mob
+
, pFLAC carrying the promoter region of hupS gene fused to the lacZ gene This work
pBBRcrt Km
r
, mob
+
, pBBRMCS2 carrying the promoter region of crtD gene This work
pTUV
C
1Km
r
, mob
+
, hupTUV genes cloned after the promoter region of crtD gene This work
pTUV
C
2Km
r
, mob

GB1121 DhynSL, DhupSL 0 ± 0 0 ± 0 ND ND
RPON DhynSL, rpoN::Gm
r
0 ± 0 0 ± 0 NA NA
HRMG DhynSL, hupR::Em
r
0 ± 0 0 ± 0 7.5 ± 1.6 5.9 ± 1.1
HTMG DhynSL, DhupT 106.9 ± 24.1 112.8 ± 14.2 48.3 ± 8.7 59.1 ± 5.9
HUVMG DhynSL, DhupUV 89.5 ± 17.9 102.3 ± 9.9 58.9 ± 8.2 63.2 ± 4.8
a
Relative hydrogenase activities in the membrane fraction given in percentage compared to the T. roseopersicina GB11 strain grown in the
absence of H
2
.
b
Specific b-galactosidase activity (same strains containing pHUPRIP) given in micromoles of o-nitrophenol min
)1
ÆD
À1
650
.
A
´
. T. Kova
´
cs et al. Transcription regulation of HupSL hydrogenase
FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS 4809
element (Fig. 1) [10]. Promoters harbouring )24 ⁄ )12
elements require the sigma factor RpoN (r
54

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
hupDHIR genes. 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
r
54
interaction 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

Transcription regulation of HupSL hydrogenase A
´
. T. Kova
´
cs et al.
4810 FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS
Isolation 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

2), hupUV genes
(pMHEUVC2) or hupTUV genes (pTrTUV
C
1) 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 hydrogenase
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

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 O
2
[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 (pTrTUV
C
1) showed
0.19 ± 0.06 lmolÆL
)1
Æmin
)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
)1
Æmin
)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-

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
r
70
-dependent in R. capsulatus [1]. This is in line with
the observation that the putative r
54
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 H
2
insensitive 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

2
– – 100 ± 6.1 94.9 ± 15.4
pTrTUV
C
2 hupT 0±0 0±0
pTrTUV
C
1 hupTUV 95.1 ± 22.3 104.4 ± 11.6
pMHEUVC2 hupUV 93.5 ± 9.2 107.9 ± 21.6
Transcription regulation of HupSL hydrogenase A
´
. T. Kova
´
cs et al.
4812 FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS
take 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 so-called RH
STOP
mutant protein of
Ra. eutropha lacking a C-terminal peptide of 55 amino
acids in HoxB lost its H
2
-sensing ability but still cata-
lysed the H
2
oxidation [7]. In this case the RH
STOP
was incapable of forming the (ab)
2
dimeric heterodi-
mer and the complex with HoxJ kinase, therefore the
expression of the membrane bound HoxKG hydro-
genase was repressed. Thus uncoupling of the hydro-
genase activity and the H
2
sensing 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 H
2
sensing
(HupUV) system are functional, yet the expression

4
Cl [27].
Sodium acetate (2 gÆL
)1
)ord-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 g Æ L
)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) erythromycin (50).
Conjugation
Conjugation was carried out as described previously [12].
A
´
. T. Kova
´

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-

1 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 (pTrTUV
C
2) 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
Transcription regulation of HupSL hydrogenase A
´
. T. Kova
´
cs et al.
4814 FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS
Dnase I at 37 °C for 60 min in a total volume of 40 lL
[40 mm Tris ⁄ HCl pH 7.5, 20 mm MgCl
2
,20mm CaCl
2
,4U
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 lLH

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 (i.
nih.nlm.gov). Multiple alignments were performed with the
clustal x program.
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, Birmingham, UK) for many helpful discussions.
We gratefully acknowledge Ro
´
zsa Verebe
´
ly for excel-
lent technical assistance.
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