hydrogen independent expression of hupsl genes in thiocapsa roseopersicina bbs

Hydrogen independent expression of hupSL genesin Thiocapsa roseopersicina BBSAkos T. Kovacs1, Gabor Rakhely1, Judit Balogh1, Gergely Maroti1, Laurent Cournac2,Patrick Carrier2, Lıvia S. Meszaros1, Gilles Peltier2 and Kornel L. Kovacs1

1 Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, and Department of Biotechnology,

University of Szeged, Hungary

2 CEA Cadarache, DSV, Departement d’Ecophysiologie Vegetale et de Microbiologie, Laboratoire d’Ecophysiologie de la Photosynthese,

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. Kovacs, Department of Biotechnology,

University of Szeged, H-6726 Szeged,

Temesvari krt. 62, Hungary

Fax: +36 62 544352

Tel: +36 62 544351

E-mail: [email protected]

(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.

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 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. Rakhely and K. L. Kovacs,

unpublished data). A 708-bp DNA fragment contain-

ing the first 76 bp of the hupS coding 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

Transcription regulation of HupSL hydrogenase A. T. Kovacs 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.

Rakhely, A. T. Kovacs and K. L. Kovacs, 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.

Strain or plasmid Relevant genotype or phenotype Reference or source

Thiocapsa roseopersicina

GB11 hynSLD::Smr [11]

GB1121 hynSLD::Smr, hupSLD::Gm [11]

HRMG hupR::Emr, GB11 This work

RPON rpoN::Gmr, GB11 This work

HUVMG hupUVD, GB11 This work

HTMG hupTD, GB11 This work

Escherichia coli

S17-1(kpir) 294 (recA pro res mod) Tpr, Smr (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 lacIqZDM15 Tn10 (Tetr)]cStratagene

Plasmids

pGEM T-Easy Ampr, cloning vector, ColE1 Promega

pHUPU1 pGEM T-Easy, contains 272-bp fragment of hupU This work

pBluescript SK(+) Ampr, 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 Kmr, mob+, sacB+ [28]

pLO2 Kmr, mob+, sacB+ [29]

p34S-Gm Cloning vector carrying Gmr [36]

pRL271 Cloning vector carrying sacB, Emr (ermC), Cmr GenBank no. L05081

pHRIMER2 Kmr, 2833-bp region of hupR gene in pK18mobsacB carrying Emr cassette at BstXI site This work

pRPON2 Kmr, 1618-bp region of rpoN gene in pK18mobsacB carrying Gmr cassette at SmaI site This work

pHTD2 Kmr, in-frame up and downstream homologous regions of hupT in pK18mobsacB This work

pHUVD2 Kmr, in-frame up and downstream homologous regions of hupUV in pLO2 This work

pBBRMCS2 Kmr, mob+, broad host range vector [37]

pFLAC Gmr, mob+, pBBRMCS5 carrying the promoterless lacZ gene [15]

pHUPRIP Gmr, mob+, pFLAC carrying the promoter region of hupS gene fused to the lacZ gene This work

pBBRcrt Kmr, mob+, pBBRMCS2 carrying the promoter region of crtD gene This work

pTUVC1 Kmr, mob+, hupTUV genes cloned after the promoter region of crtD gene This work

pTUVC2 Kmr, mob+, hupT gene cloned after the promoter region of crtD gene This work

pMHE6crtKm Kmr, 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 H2 uptake and b-galactosidase activities in different strains grown in the absence or presence of hydrogen. ND, Not

detected; NA, not adaptable (antibiotic conflict).

Strain Inactivated genes

HupSL hydrogenase activitya LacZ activityb

– H2 + H2 – H2 + H2

GB11 DhynSL 100 ± 6.1 94.9 ± 15.4 57.7 ± 5.6 54.2 ± 7.9

GB1121 DhynSL, DhupSL 0 ± 0 0 ± 0 ND ND

RPON DhynSL, rpoN::Gmr 0 ± 0 0 ± 0 NA NA

HRMG DhynSL, hupR::Emr 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 H2. b Specific b-galactosidase activity (same strains containing pHUPRIP) given in micromoles of o-nitrophenol min)1ÆD �1650 .

A. T. Kovacs et al. Transcription regulation of HupSL hydrogenase


element (Fig. 1) [10]. Promoters harbouring )24 ⁄)12elements 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. Meszaros, G. Rakhely, H. P. Klenk and

K. L. Kovacs, 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

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

r54 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

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.



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

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 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 H2

(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.



HupT. 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 r54 promoter 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 H2 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

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. H2 uptake activities in complementation experiments. The

results are given in percentage compared to the T. roseopersicina

grown in the absence of H2.

Plasmid

Complementing

gene

HupSL hydrogenase activity

– H2 + H2

– – 100 ± 6.1 94.9 ± 15.4

pTrTUVC2 hupT 0 ± 0 0 ± 0

pTrTUVC1 hupTUV 95.1 ± 22.3 104.4 ± 11.6

pMHEUVC2 hupUV 93.5 ± 9.2 107.9 ± 21.6



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 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. eutropha lacking 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 hydro-

genase was repressed. Thus uncoupling of the hydro-

genase activity and the H2 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 H2 sensing

(HupUV) system are functional, yet the expression

of HupSL does not apparently depend on the pres-

ence or absence 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) erythromycin (50).

Conjugation

Conjugation was carried out as described previously [12].



Identification 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 recombination 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

ATCCGGAAAGGCTC-3¢) and )20 (5¢-GTAAAACGA

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



Dnase I at 37 �C 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 H2 and

HD formation, resulting from exchange between D2 and

protons of the medium, measured at 30 �C, were monitored

continuously by MS as described in detail previously

[31,32]. For each experiment 1.5 mL (D600 ¼ 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 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 Rozsa Verebely for excel-

lent technical assistance.

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hydrogen independent expression of hupsl genes in thiocapsa roseopersicina bbs

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