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 ´ ros 1 , Gilles Peltier 2 and Korne ´ l L. Kova ´ cs 1 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, H 2 , 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 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 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 H 2 -driven signal transduction system and to the regulatory hydrogenases of Ralstonia eutropha and Rhodobacter capsulatus, respect- ively. Although the genes of the entire H 2 sensing and regulation system were present, the expression of the hupSL genes was not affected by the presence or absence of H 2 . 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 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 H 2 -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 H 2 . 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 H 2 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 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- ence of hydrogen. In order to study directly the H 2 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 H 2 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 H 2 (G. Ra ´ khely and K. L. Kova ´ cs, 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. 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 ´ 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. Strain or plasmid Relevant genotype or phenotype Reference or source Thiocapsa roseopersicina GB11 hynSLD::Sm r [11] GB1121 hynSLD::Sm r , hupSLD::Gm [11] HRMG hupR::Em r , GB11 This work RPON rpoN::Gm r , GB11 This work HUVMG hupUVD, GB11 This work HTMG hupTD, GB11 This work 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 (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 + , sacB + [29] p34S-Gm Cloning vector carrying Gm r [36] pRL271 Cloning vector carrying sacB,Em r (ermC), Cm r GenBank no. L05081 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 pBBRMCS2 Km r , 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 + , 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 Km r , 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). Strain Inactivated genes HupSL hydrogenase activity a LacZ activity b –H 2 +H 2 –H 2 +H 2 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::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 ). Fur- ther upstream from the r 54 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 N 2 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 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 H 2 -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. 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 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 (pTrTUV C 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 (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. A ´ . T. Kova ´ cs et al. Transcription regulation of HupSL hydrogenase FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS 4811 HupT. Production of HupTUV from a similar plasmid construction, however, did not alter the HupSL hydrogenase activity, i.e. HupSL was not regulated by H 2 (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 pTrTUV C 1, pTrTUV C 2 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 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- lism is strictly linked to H 2 , 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 H 2 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 r 54 promoter element. The r 54 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 expression is a sign for the biosynthesis of the enzyme being under the H 2 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. H 2 uptake 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 –H 2 +H 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 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 H 2 . 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 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 of HupSL does not apparently depend on the pres- ence or absence of H 2 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 H 2 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% NH 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 ´ cs et al. Transcription regulation of HupSL hydrogenase FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS 4813 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 Eco RV– 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 pTrTUV C 1. To express the hupT gene only the hupUV genes were deleted from pTrTUV C 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 2 O. 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 H 2 and HD formation, resulting from exchange between D 2 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 (D 600 ¼ 0.464 ± 0.034 of 10-times diluted cultures) culture was used. Hydrogenase activity based on the rates of H 2 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 Ro ´ zsa Verebe ´ ly for excel- lent technical assistance. References 1 Dischert W, Vignais PM & Colbeau A (1999) The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two-component HupT ⁄ HupR sys- tem. Mol Microbiol 34, 995–1006. 2 Lenz O, Bernhard M, Buhrke T, Schwartz E & Frie- drich B (2002) The hydrogen-sensing apparatus in Ralstonia eutropha. J Mol Microbiol Biotechnol 4, 255–262. 3 Kleihues L, Lenz O, Bernhard M, Buhrke T & Friedrich B (2000) The H 2 sensor of Ralstonia eutropha is a mem- ber of the subclass of regulatory [NiFe] hydrogenases. J Bacteriol 182, 2716–2724. 4 Bernhard M, Buhrke T, Bleijlevens B, De Lacey AL, Fernandez VM, Albracht SP & Friedrich B (2001) The H 2 sensor of Ralstonia eutropha. Biochemical character- istics, spectroscopic properties, and its interaction with a histidine protein kinase. J Biol Chem 276, 15592– 15597. 5 Buhrke T, Bleijlevens B, Albracht SPJ & Friedrich B (2001) Involvement of hyp gene products in maturation of the H 2 -sensing [NiFe] hydrogenase of Ralstonia eutro- pha. J Bacteriol 183, 7087–7093. 6 Elsen S, Duche O & Colbeau A (2003) Interaction between the H 2 sensor HupUV and the histidine kinase HupT controls HupSL hydrogenase synthesis in Rhodo- bacter capsulatus. J Bacteriol 185, 7111–7119. 7 Buhrke T, Lenz O, Porthun A & Friedrich B (2004) The H 2 -sensing complex of Ralstonia eutropha: interac- tion between a regulatory [NiFe] hydrogenase and a histidine protein kinase. Mol Microbiol 51, 1677–1689. 8 Elsen S, Colbeau A, Chabert J & Vignais PM (1997) The hupTUV operon is involved in negative control of hydrogenase synthesis in Rhodobacter capsulatus. J Bacteriol 178, 5174–5181. 9Ra ´ khely G, Colbeau A, Garin J, Vignais PM & Kova ´ cs KL (1998) Unusual organization of the genes coding for HydSL, the stable [NiFe] hydrogenase in the photosyn- thetic bacterium Thiocapsa roseopersicina BBS. J Bacte- riol 180, 1460–1465. 10 Colbeau A, Kova ´ cs KL, Chabert J & Vignais PM (1994) Cloning and sequencing of the structural (hupSLC) and accessory (hupDHI) genes for hydroge- nase biosynthesis in Thiocapsa roseopersicina. Gene 140, 25–31. 11 Ra ´ khely G, Kova ´ cs A ´ T, Maro ´ ti G, Latinovics D, Fodor BD, Csana ´ di G & Kova ´ cs KL (2004) A hetero- pentameric NAD + reducing [NiFe] hydrogenase in the purple sulfur photosynthetic bacterium, Thiocapsa rose- opersicina. Appl Environ Microbiol 70, 722–728. 12 Fodor B, Ra ´ khely G, Kova ´ cs A ´ T & Kova ´ cs KL (2001) Transposon mutagenesis in purple sulfur photosynthetic bacteria: identification of hypF, encoding a protein cap- able of processing [NiFe] hydrogenases in a, b and c A ´ . T. Kova ´ cs et al. Transcription regulation of HupSL hydrogenase FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS 4815 subdivision of the proteobacteria. Appl Environ Micro- biol 67, 2476–2483. 13 Maro ´ ti G, Fodor BD, Ra ´ khely G, Kova ´ cs A ´ T, Arvani S & Kova ´ cs KL (2003) Selectivity and cooperativity of accessory proteins in the biosynthesis of [NiFe] hydro- genases. Eur J Biochem 270, 2218–2227. 14 Kova ´ cs KL, Fodor B, Kova ´ cs A ´ T, Csana ´ di G, Maro ´ ti G, Balogh J, Arvani S & Ra ´ khely G (2002) Hydro- genases, accessory genes and the regulation of [NiFe] hydrogenase biosynthesis in Thiocapsa roseopersicina. Int J Hydrogen Energy 27, 1463–1469. 15 Kova ´ cs A ´ T, Ra ´ khely G, Browning DF, Fu ¨ lo ¨ p A, Mar- o ´ ti G, Busby SJW & Kova ´ cs KL (2005) An FNR-type regulator controls the anaerobic expression of Hyn hydrogenase in Thiocapsa roseopersicina. J Bacteriol 187, 2618–2627. 16 Vignais PM & Colbeau A (2004) Molecular biology of microbial hydrogenases. Curr Issues Mol Biol 6, 159–188. 17 Baginsky C, Palacios JM, Imperial J, Ruiz-Argueso T & Brito B (2004) Molecular and functional characteriza- tion of the Azorhizobium caulinodans ORS571 hydroge- nase gene cluster. FEMS Microbiol Lett 237, 399–405. 18 Vignais PM, Dimon B, Zorin NA, Colbeau A & Elsen S (1997) HupUV proteins of Rhodobacter capsulatus can bind H 2 : evidence from the H-D exchange reaction. J Bacteriol 179, 290–292. 19 Cammack R, Frey M & Robson R, eds. (2001) Hydro- gen as a fuel. Learning from Nature. London: Taylor & Francis. 20 Kova ´ cs A ´ T, Ra ´ khely G, Balogh J, Maro ´ ti G, Fu ¨ lo ¨ pA & Kova ´ cs KL (2005) Anaerobic regulation of hydro- genase transcription in different bacteria. Biochem Soc Transact 33, 36–38. 21 Black LK & Maier RJ (1994) Sequences and characteri- zation of hupU and hupV genes of Bradyrhizobium japo- nicum encoding a possible nickel-sensing complex involved in hydrogenase expression. J Bacteriol 176, 7102–7106. 22 Van Soom C, de Wilde P & Vanderleyden J (1997) HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum. Mol Microbiol 23, 967–977. 23 Ro ¨ mmermann D, Warrelmann J, Bender RA & Frie- drich B (1989) An rpoN-like gene of Alcaligenes eutro- phus and Pseudomonas facilis controls expression of diverse metabolic pathways, including hydrogen oxida- tion. J Bacteriol 171, 1093–1099. 24 Black LK & Maier RJ (1995) IHF- and RpoN-depen- dent regulation of hydrogenase expression in Bradyrhi- zobium japonicum. Mol Microbiol 16, 405–413. 25 Lenz O & Friedrich B (1998) A novel multicomponent regulatory system mediates H 2 sensing in Alcaligenes eutrophus. Proc Natl Acad Sci USA 95, 12474–12479. 26 Giraud E, Zappa S, Jaubert M, Hannibal L, Fardoux J, Adriano JM, Bouyer P, Genty B, Pignol D & Verme ´ glo A (2004) Bacteriophytochrome and regulation of the synthesis of the photosynthetic apparatus in Rhodopseu- domonas palustris: pitfalls of using laboratory strains. Photochem Photobiol Sci 3, 587–591. 27 Kova ´ cs A ´ T, Ra ´ khely G & Kova ´ cs KL (2003) Genes involved in the biosynthesis of photosynthetic pigments in the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina. Appl Environ Microbiol 69, 3093–3102. 28 Scha ¨ fer A, Tauch A, Jager W, Kalinowski J, Thierbach G & Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plas- mids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145, 69–73. 29 Lenz O, Schwartz E, Dernedde J, Eitinger M & Frie- drich B (1994) The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation. J Bacteriol 176, 4385–4393. 30 Fodor B, Kova ´ cs A ´ T, Csa ´ ki R, Hunyadi-Gulya ´ sE ´ , Klement E ´ , Maro ´ ti G, Me ´ sza ´ ros LS, Medzihradszky KF, Ra ´ khely G & Kova ´ cs KL (2004) Modular broad- host-range expression vectors for single-protein and pro- tein complex purification. Appl Environ Microbiol 70, 712–721. 31 Jouanneau Y, Kelley BC, Berlier Y, Lespinat PA & Vignais PM (1980) Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rho- dopseudomonas capsulata. J Bacteriol 143, 628–636. 32 Vignais PM, Cournac L, Hatchikian EC, Elsen S, Sere- bryakova L, Zorin N & Dimon B (2002) Continuous monitoring of the activation and activity of [NiFe]- hydrogenases by membrane-inlet mass spectrometry. Int J Hydrogen Energy 27, 1441–1448. 33 Cournac L, Guedeney G, Peltier G & Vignais PM (2004) Sustained photoevolution of molecular hydrogen in a mutant of Synechocystis sp. strain PCC 6803 defi- cient in the type I NADPH-dehydrogenase complex. J Bacteriol 186, 1737–1746. 34 Miller J (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 35 Herrero M, Lorenzo V & Timmis KN (1990) Transpo- son vectors containing non antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol 172, 6557–6567. 36 Dennis JJ & Zylstra GJ (1998) Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 64, 2710–2715. 37 Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop IIRM & Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176. Transcription regulation of HupSL hydrogenase A ´ . T. Kova ´ cs et al. 4816 FEBS Journal 272 (2005) 4807–4816 ª 2005 FEBS . and hupUV genes are apparently intact, yet the hydrogen sensing system is not functional in T. roseopersicina BBS. Results Hydrogen independent hupSL expression The. Hydrogen independent expression of hupSL genes in Thiocapsa roseopersicina BBS A ´ kos T. Kova ´ cs 1 ,Ga ´ bor Ra ´ khely 1 ,