a two-component signal transduction system involved in nickel sensing in the cyanobacterium...

© 2002 Blackwell Science Ltd

A two-component signal transduction system involved in nickel sensing in the cyanobacteriumSynechocystis sp. PCC 6803

years on the biological roles of nickel and the structure ofnickel metalloenzymes (Maroney, 1999). Ni atoms formpart of the active site of a number of enzymes includingglyoxalases I, peptide deformylases, methyl-CoM reduc-tase and ureases, as well as some superoxide dismu-tases and hydrogenases (Ermler et al., 1998). However,at high concentration, Ni2+ ions induce different types ofharmful effects including generation of free radicals, inhi-bition of enzyme activity and DNA damage, leading togenetic instability, developmental defects and cancer (VonBurg, 1997; Costa, 1991, 1998). Because of these oppo-site effects, organisms have to carefully regulate the intra-cellular concentration of Ni2+.

Microbial Ni2+ uptake is mediated by non-specific transport systems for divalent cations and by high affin-ity specific systems (for two recent reviews, see Nies,1999; Eitinger and Mandrand-Berthelot, 2000). Two typesof high affinity transporters have been identified: (i) multicomponent ABC (ATP-binding cassette) transportsystems (such as the NikABCDE system of Escherichiacoli) (Navarro et al., 1993); and (ii) one-component per-meases (such as NixA, UreH, HupN and HoxN) (Eitingerand Friedrich, 1991; Fu et al., 1994; Mobley et al., 1995)which are integral membrane proteins with eight trans-membrane spanning helices. Most of the one-componentpermeases have been shown to share a conserved His-X4-Asp-His sequence in the second transmembrane helixinvolved in Ni2+ ligation. In the case of the Nik system, theperiplasmic NikA protein is also involved in Ni2+ binding butthe amino acid determinant of the interaction has not yetbeen identified. Beside these uptake systems, various Ni2+

export systems have also been described. The best knownnickel resistance systems belong to bacteria of the genusRalstonia. The Cnr (cobalt, nickel) and the Ncc (nickel,cobalt, cadmium) systems are membrane protein com-plexes that carry out Ni2+ efflux driven by proton/cationantiport (Collard et al., 1993; Liesegang et al., 1993;Schmidt and Schlegel, 1994; Grass et al., 2000). Bothsystems are closely related to the cobalt/zinc/cadmiumresistance system Czc from Ralstonia sp. CH34 strain. Inthis system, CzcA is the transmembrane protein thatcarries out the antiport. CzcB is a membrane fusion proteinthat may span the peri-plasmic space and CzcC seems tobe attached to the outer membrane (Nies, 1992; 1995;Rensing et al., 1997).

Molecular Microbiology (2002) 43(1), 247–256

Luis López-Maury, Mario García-Domínguez,Francisco J. Florencio and José C. Reyes*Instituto de Bioquímica Vegetal y Fotosíntesis,Universidad de Sevilla-CSIC, E-41092 Sevilla, Spain.

Summary

In the cyanobacterium Synechocystis sp. PCC 6803,genes for Ni2+, Co2+, and Zn2+ resistance are groupedin a 12 kb gene cluster. The nrsBACD operon is com-posed of four genes, which encode proteins involvedin Ni2+ resistance. Upstream from nrsBACD, and inopposite orientation, a transcription unit formed bythe two genes rppA and rppB has been reported previously to encode a two-component signal trans-duction system involved in redox sensing. In thisreport, we demonstrate that rppA and rppB (hereredesigned nrsR and nrsS respectively) control theNi2+-dependent induction of the nrsBACD operon and are involved in Ni2+ sensing. Thus, expression ofthe nrsBACD operon was not induced by Ni2+ in anrsRS mutant strain. Furthermore, nrsRS mutant cells showed reduced tolerance to Ni2+. Whereas thenrsBACD operon is transcribed from two differentpromoters, one constitutive and the other dependenton the presence of Ni2+ in the medium, the nrsRSoperon is transcribed from a single Ni2+-inducible promoter. The nrsRS promoter is silent in a nrsRSmutant background suggesting that the system isautoregulated. Purified full length NrsR protein isunable to bind to the nrsBACD-nrsRS intergenicregion; however, an amino-terminal truncated proteinthat contains the DNA binding domain of NrsR bindsspecifically to this region. Our nrsRS mutant, whichcarries a deletion of most of the nrsR gene and partof the nrsS gene, does not show redox imbalance or photosynthetic gene mis-expression, contrastingwith the previously reported nrsR mutant.

Introduction

Increasing interest has been developed during the last

Accepted 5 October, 2001. *For correspondence. E-mailjcreyes@ibvf.csic.es; Tel. (+34) 954489573; Fax (+34) 954460065.

In the unicellular cyanobacterium Synechocystis sp.PCC 6803 (hereafter referred to as Synechocystis), anickel resistance operon (nrsBACD) formed by four openreading frames (ORFs) has been described previously byus (García-Domínguez et al., 2000). NrsB and NrsA pro-teins are homologues to CzcB and CzcA, respectively,and they very probably form a membrane-bound proteincomplex catalysing Ni2+ efflux by a proton/cation antiport.NrsC is not homologous to proteins encoded by the czcor related operons, and its role in Ni2+ export is unknown.Finally, NrsD is a membrane protein belonging to themajor facilitator superfamily of transport proteins. NrsD ishighly homologous to NreB from Achromobacter xylosox-idans (Grass et al., 2001). Expression of NreB in E. coliconfers resistance to Ni2+, suggesting that this permeaseis able to carry out Ni2+ export. Interestingly the carboxyterminal part of NrsD contains 14 histidine residuesinvolved in Ni2+ binding (García-Domínguez et al., 2000).The nrsBACD operon is integrated into the Syne-chocystis metal resistance cluster which includes ninegenes involved in Zn2+, Co2+ and Ni2+ resistance (Fig. 1)(Thelwell et al., 1998; Rutherford et al., 1999; García-Domínguez et al., 2000). Two ORFs, rppA (sll0797) andrppB (sll0798), are found 118 bp upstream from thenrsBACD operon and transcribed in the opposite direction(Fig. 1). Whereas rppA encodes a response regulator,rppB encodes a sensor histidine kinase, suggesting thatboth proteins form a classical two-component signaltransduction system (Stock et al., 2000). A role of thesetwo proteins on redox sensing has been postulated pre-viously, based on the analysis of an insertional mutant ofthe response regulator rppA. However, the same pheno-type was not obtained in a mutant of the sensor histidinekinase rppB (Li and Sherman, 2000).

In this work, we demonstrate that the signal transduc-tion system formed by the rppA and rppB products is con-trolling the expression of the nrsBACD operon, and isinvolved in Ni2+ sensing. A rppAB mutant with a deletionof most of the rppA gene and part of the rppB gene didnot show any kind of redox imbalance, suggesting that the previously reported phenotype is the consequence of unspecific cross-talk. Therefore, we propose the re-designation of rppAB as nrsRS.

Results

Ni2+-dependent expression of nrsR and nrsS genes

The stop codon of the nrsR (sll0797) gene overlaps withthe start codon of the nrsS (sll0798) gene, strongly sug-gesting that they form a single transcription unit. This wasfurther demonstrated by the size of the mRNA transcriptdetected in Northern blotting experiments (about 2000nucleotides). Metal-dependent expression of the nrsRSoperon was analysed by Northern blotting. For this, aprobe of the nrsRS operon was used to hybridize totalRNA obtained from mid-log Synechocystis cells grown in BG11C medium and exposed for 1 h to 17 mM of either ZnSO4, CdCl2, CoCl2, CuSO4, NiSO4 or MgCl2.Control cells were not exposed to added metals. Asshown in Fig. 2A, the nrsRS transcript was stronglyinduced in the presence of Ni2+ and weakly induced in thepresence of Co2+. Therefore, the nrsRS operon follows thesame pattern of expression as the nrsBACD operon(Fig. 2A). A time-course experiment demonstrated thatnrsRS and nrsBACD transcripts showed differentinducibility. Thus, the amount of the nrsBACD transcriptincreased about 10 fold, 4 h after Ni2+ addition, whereasthe amount of the nrsRS mRNA increased only threefoldafter Ni2+ addition (Fig. 2B).

The nrsRS system controls the Ni2+-dependentexpression of the nrsBACD operon

To identify the signal transduced by the nrsRS two-component system, a nrsRS mutant was generated byreplacing most of the nrsR sequence and part of the nrsSsequence by a kanamycin resistance cassette (C.K1) inboth orientations (Fig. 3A and B). Similar results wereobtained for both mutants and, therefore, only data about the mutant with the kanamycin resistance gene (npt) in the opposite orientation with respect to the nrsRS genes are shown, except when indicated. ThenrsRS::C.K1 Synechocystis strain was viable and itsgrowth rate in BG11C medium was similar to that of thewild-type strain (data not shown). As expression of thenrsRS operon is induced in the presence of Ni2+ and Co2+,growth of nrsRS::C.K1 mutant was examined in Ni2+ and

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Fig. 1. ORF organization of the metal-resistance gene cluster from Synechocystis. The nrsBACD operon was described in García-Domínguezet al. (2000), the corR (coaR) and corT (also called coaT) genes were described in Rutherford et al., 1999 and García-Domínguez et al.(2000), and the zia genes were described in Thelwell et al. (1998). Cyanobase database ORFs numbers are also included (Kaneko et al.,1996).

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Co2+ supplemented BG11C medium. Normal growth wasobserved in Co2+-containing medium (data not shown);however, nrsRS::C.K1 cells were clearly less tolerant tothe presence of Ni2+ in the medium (Fig. 3C). The effectof a number of other metals on the growth of wild-typeSynechocystis and nrsRS::C.K1 cells was also tested, butnone of them had a differential effect on the growth ofnrsRS::C.K1 cells with respect to the effect observed forwild-type cells (data not shown).

How can the nrsRS system affect Ni2+ tolerance? Oneobvious possibility is that NrsR and NrsS are control-ling the expression of the nickel resistance operon

(nrsBACD). Thus, Northern blotting experiments demon-strated that the Ni2+-dependent inducibility of thenrsBACD operon was completely abolished in thenrsRS::C.K1 mutant (Fig. 3D).

Analysis of the nrsRS-nrsBACD intergenic region

The 118 bp-nrsRS-nrsBACD intergenic region shouldcontain the promoter and the regulatory sequencesresponsible for the response to Ni2+. To identify the promoters of both nrsRS and the nrsBACD operons, transcription start-points were determined by primerextension (Fig. 4A). In the absence of Ni2+, one only tran-scription start site was found 49 bp upstream of the nrsBATG start codon. This transcription start-point was unaltered in the nrsRS::C.K1 mutants. However, in thepresence of Ni2+, two different mRNAs were found. Onestarts in the same position as the transcript found undernon-induced conditions. The second, much more abun-dant than the previous one, starts 33 bp upstream of thenrsB ATG start codon. The inducible transcription start sitewas absent in the nrsRS::C.K1 mutant. These data indi-cate that the nrsBACD operon has two promoters; oneconstitutive and a second one inducible by the nrsRSsystem in the presence of Ni2+. Putative –10 boxes in theform TTTCAT and CAGACT were found 7 and 6 bpupstream of the Ni2+-independent and the Ni2+-dependenttranscription start-points respectively. No obvious –35boxes were detected at the appropriated positions(Fig. 4C).

Primer extension analysis demonstrated the existenceof one only transcription start point for the nrsRS tran-scription unit 10 bp upstream of the nrsR translation startcodon (Fig. 4B). As shown in RNA blotting experiments,primer extension products from the nrsRS promoter weremore abundant in samples from cells exposed to Ni2+,than in non-exposed cells. Ni2+-dependent tran-scription from the nrsRS promoter was abolished in thenrsRS::C.K1 mutants indicating that expression of nrsRSgenes is autoregulated. A putative –10 box in the formCGTTAT was found 5 bp upstream of the transcriptionstart point (Fig. 4C).

NrsR binds to the nrsRS-nrsBACDintergenic region

NrsR belongs to the PhoB/OmpR subfamily of responseregulators (Martinez-Hackert and Stock, 1997). Mostresponse regulators consist of multiple domains: an N-terminal receiver domain that contains the asparticresidue that is phosphorylated by the histidine kinase anda C-terminal output domain that often binds DNA and acti-vates transcription. To verify whether NrsR is able to inter-act with the nrsRS-nrsBACD intergenic region, we purified

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Fig. 2. Metal-dependent regulation of nrsBACD and nrsRS.A. Northern blot analysis of nrsBACD and nrsRS expression frommid-log phase Synechocystis sp. PCC 6803 cells were exposed to17 mM of different metals for 1 h. Control cells were not exposed toadded metals.B. Time-course expression of nrsBACD and nrsRS after 17 mMNiSO4 addition. Radioactive signals from a time-course northern blotanalysis of nrsBACD and nrsRS transcripts were quantified using anInstant Imager Electronic Autoradiography apparatus (PackardInstrument Company). Levels of nrsBACD (�) and nrsRS signals(�) were normalized to the rnpB signal as indicated in Experimentalprocedures. It should be noted that 100% corresponds to themaximal signal of hybridization for each probe, and that signals fromdifferent probes can not be compared. Average of two independentdeterminations is shown.

a recombinant amino-terminal His-tagged version of NrsRexpressed in E. coli (Fig. 5A and B). DNA binding wastested by gel retardation assays using a fragment encom-passing the 118 bp nrsRS-nrsBACD intergenic region.Full-length NrsR was not able to bind to this region(Fig. 5C), even in the presence 100 mM of acetyl phos-phate. Similar results were obtained using a carboxy terminal His-tagged NrsR protein (data not shown). In a number of cases, it has been proposed that the unphosphorylated receiver domain functions as anintramolecular repressor of the DNA binding and trans-activation activities of the N-terminal domain (Baikalov et al., 1996; Ames et al., 1999). This repressive effect is naturally released upon phosphorylation. A repres-sive role has been further supported by the fact that deletion of the N-terminal receiver domain leads to a constitutive DNA binding activity (Perez-Martin and deLorenzo, 1996; Ellison and McCleary, 2000). Therefore,we generated a truncated form of NrsR deleted from thefirst 117 amino acids of the N-terminal part (Fig. 5A and

B). This truncated version, named NrsR∆N, bound specifi-cally to the nrsRS-nrsBACD intergenic region (Fig. 5C).The NrsR∆N-dependent band shift was severely dimini-shed in the presence of a 10-fold excess of the sameunlabelled fragment and it was unaffected by the pres-ence of an excess of an unrelated DNA fragment (datanot shown).

The nrsRS::C.K1 Synechocystis strain showed normal expression of photosynthetic genes

Li and Sherman have previously reported the inactiva-tion of the rppA (nrsR) and rppB (nrsS) genes by inser-tional mutagenesis (Li and Sherman, 2000). Surprisingly,whereas ∆rppA cells showed a pleiotropic phenotypecharacterized by reduced chlorophyll and phycobiliproteincontent and mis-expression of photosynthetic genes,∆rppB cells behaved as wild-type cells. Thus, expressionof the psbA (gene encoding the D1 protein of photosys-tem II) and nblA (gene encoding a polypeptide involved

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Fig. 3. Loss of Ni2+ induction of the nrsBACD operon and nickel-reduced tolerance of nrsRS deletion mutant.A. Schematic representation of the nrs genomic regions in the wild type and in the nrsRS::C.K1 mutant strains.B. Southern blot analysis of Synechocystis wild type and nrsRS::C.K1 mutant. Genomic DNA was digested with HincII and hybridized usingthe DNA fragment indicated in panel A as a probe. The size of the hybridizing bands is indicated.C. Ni2+ tolerance of wild-type and nrsRS::C.K1 strains. Ten-fold culture dilutions were spotted on BG11C plates supplemented with theindicated concentration of NiSO4 and photographed after 10 days of growth.D. Northern blot analysis of nrsBACD expression in wild type or in nrsRS::C.K1 mutant cells exposed to 17 mM Ni2+, 17 mM Co2+ for 2 h, or inthe absence of metal added.

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in degradation of phycobiliproteins) was strongly upregu-lated (around 5- and 100-fold respectively) in ∆rppA cellscompared with wild-type cells. Li and Sherman sug-gest that RppA (NrsR) is controlling the stoichiometrybetween photosystem I and photosystem II. OurnrsRS::C.K1 Synechocystis strain is a double mutantlacking both NrsR and NrsS products; however, we didnot observe any effect on growth or reduction in thechlorophyll (31 ± 2.10–3 mg Chl mg–1 total protein for the

nrsRS::C.K1 cell versus 33 ± 3.10–3 mg Chl mg –1 totalprotein for the wild-type cells) and phycobiliprotein (PC)content (306 ± 35.10–3 mg PC mg–1 total protein for thenrsRS::C.K1 cell versus 352 ± 11.10–3 mg PC mg–1 totalprotein for the wild-type cells). Furthermore, Northern blotexperiments using psbA and nblA probes demonstratedthat both genes were equally expressed in nrsRS::C.K1and wild-type cells (Fig. 6). Therefore, our results contrastwith the data published for the ∆rppA mutant strain. Takinginto account our results, one simple explanation could bethat the pleiotropic phenotype reported for the ∆rppAmutant was the consequence of a high intracellular accu-mulation of Ni2+ and that this phenotype was not observedin the nrsRS::C.K1 mutant because of differences in theNi2+ concentration of our BG11C medium. To test thishypothesis, expression of nblA and psbA genes wasanalysed in the presence of a growth-inhibitory Ni2+ con-centration. Three different mutants presenting low toler-ance to Ni2+ were analysed: nrsRS::C.K1, nrsA:C.K1 andnrsD::C.K1 (García-Domínguez et al., 2000). As shown inFig. 6, neither nblA nor psbA gene expression wasaffected by the Ni2+ concentration in any of the analysedstrains.

Discussion

We have reported previously that Synechocystis containsa metal resistance gene cluster formed by nine genesinvolved in Ni2+, Co2+ and Zn2+ resistance (García-Domínguez et al., 2000). The Co- and Zn-dependent P-type ATPases, encoded by the genes corT (also calledcoaT ) and ziaA, are regulated in a metal-dependent wayby transcription factors also encoded by genes of thecluster (corR/coaR and ziaR) (Thelwell et al., 1998;Rutherford et al., 1999; García-Domínguez et al., 2000)(see Fig. 1). However, genes controlling the Ni2+-dependent induction of the nickel resistance system(nrsBACD) were unknown. In this study, we demonstratethat ORFs sll0797 (nrsR) and sll0798 (nrsS) also formpart of the same gene cluster and constitute a two-component signal transduction system involved in Ni2+

sensing and regulation of the nrsBACD operon. Thus, thenrsRS::C.K1 mutant displays a reduced tolerance to Ni2+

and it is unable to induce the nrsBACD operon in the pres-ence of Ni2+. Whereas the carboxy terminal part of theNrsS protein shares homology with the histidine kinasesof the PhoR subfamily, the amino terminal half appears tobe a periplasmic domain, as predicted by two possibletransmembrane helices (amino acids 14–34 and 187–214). This putative periplasmic domain shows significantsequence similarity with the alpha subunit of the methylCoM reductase (MCR) from several Methanobacteria(Ermler et al., 1997) (Fig. 7). MCR is the final step enzymeof the methanogenesis pathway and it catalyses the

© 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 247–256

Fig. 4. Primer extension analysis of the nrsBACD and nrsRStranscripts. Primer extension analysis of the nrsBACD (A) andnrsRS (B) transcripts from Synechocystis wild type, nrsRS::C.K1(+)and nrsRS::C.K1(–) mutant cells exposed to 17 mM Ni2+ for 2 h (+)or control cells (–).To avoid polar effects, insertional mutants withthe npt gene of the C.K1 cassette in the same orientation[nrsRS::C.K1(+)] or in opposite orientation [nrsRS::C.K1(–)] thanthe nrsRS genes were used. Sequencing ladders generated withthe same oligonucleotides used for primer extension are alsoshown.C. Schematic representation and sequence of the nrs intergenicregion. Transcripts stars points are marked with an arrow. Putative–10 boxes based on the transcription start site are boxed. Directrepeats are shaded. The translation start codons are indicated inboldface type.

reduction of methyl-CoM to methane. Interestingly, one ofthe prosthetic groups of the enzyme is a tetrapyrrole ringof coenzyme F430 which co-ordinates a nickel atom. Inaddition to the four tetrapyrrole nitrogens, nickel is alsoco-ordinated by a Gln residue of the MCR alpha subunit(marked in Fig. 7) (Ermler et al., 1997) which is locatedin the region that aligns with the NrsS periplasmicdomains. These data raise the interesting possibility thatthe Ni2+-sensing domain of NrsS is phylogeneticallyrelated to the MCR alpha subunit. Whether the periplas-mic domain of NrsS contains a tetrapyrrole ring involvedin Ni2+ binding is currently unknown. NrsR is a responseregulator of the OmpR/PhoB subfamily (Martinez-Hackertand Stock, 1997). PhoB-like factors contain two functionaldomains, an N-terminal phosphorylation domain (receiverdomain) and a C-terminal DNA-binding/transactivationdomain. In several cases, it has been demonstrated thatphosphorylation of the receiver domain induces dimeri-zation and binding to the DNA, whereas unphosphory-lated proteins are unable to bind DNA (Fiedler and Weiss,1995; Baikalov et al., 1996; Da Re et al., 1999). It hasalso been shown that deletion of the receiver domainyields proteins that can bind to the DNA constitutively,suggesting that the unphosphorylated receiver domainfunctions as an intramolecular repressor of the DNAbinding; (Perez-Martin and de Lorenzo, 1996; Ellison andMcCleary, 2000). In agreement with this view, recombi-nant NrsR purified from E. coli was unable to bind to thenrsRS-nrsBACD intergenic region; however, a mutantprotein deleted of the first 117 amino acids bound specifi-cally to this region. PhoB binds DNA tandemly to twodirect repeated sequences placed around 10 bp upstream

of the –10 box (Okamura et al., 2000). Upon sequenceexamination, two direct repeats in the formGA(T/A)TTTCA, separated by 3 bp, were found 12 bpupstream of the –10 box of the inducible nrsBACD pro-moter (Fig. 4B). In the same region, but in the oppositestrand, two direct repeats also separated by 3 bp, andrelated to the previous one (GAAA(T/A)TC(A/T), werefound 22 bp upstream of the –10 box of the nrsRS pro-moter (Fig. 4C). The long distance between the activatorsite and the nrsRS –10 box might account for the lowerinducibility of the nrsRS promoter with respect to the

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Fig. 5. NrsRDN binds to the nrsRS-nrsBACDintergenic region.A. Schematic representation of NrsR andNrsRDN recombinant proteins domainorganization.B. SDS–PAGE of purified NrsR and NrsRDN.Lane M, molecular mass markers.C. Band-shift assay of the nrsRS-nrsBACDintergenic region with increasing quantities(from 0.07 to 5.6 mM) of purified NrsR (left)and NrsRDN (right).

Fig. 6. nblA and psbA expression in nrs mutants. Northern blotanalysis of nblA and psbA expression in Synechocystis wild type,nrsRS::C.K1, nrsA::C.K1 and nrsD::C.K1 (García-Domínguez et al.,2000) cells under normal growth conditions or after treatment with17 mM Ni2+ for 6 h. The filters were stripped and hybridized with arnpB gene probe as control.

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nrsBACD promoter (see Fig. 2B). Thus, this sequenceanalysis suggests that four binding sites for NrsRmonomers may exist in the nrsRS-nrsBACD intergenicregion. Therefore, our current model suggests that thepresence of Ni2+ in the medium stimulates the kinaseactivity of NrsS which transfers a phosphate group toNrsR. Phosphorylated NrsR binds (probably as a dimer)to the nrsRS-nrsBACD intergenic region activating thetranscription of nrsBACD genes and positively autoregu-lating its own synthesis.

Our results concerning the role of the NrsR and NrsSproteins contrast with those of Li and Sherman (2000).Their nrsR insertional mutant (∆rppA) presents a redoximbalance characterized by altered levels of phyco-biliproteins and chlorophylls, upregulation of genesencoding photosystem II proteins, downregulation ofgenes encoding photosystem I proteins and a 100-foldinduction of the nblA gene (NblA is a small polypeptideinvolved in degradation of phycobiliproteins). Strikingly,the nrsS mutant (∆rppB) showed wild-type phenotype. To explain this result, the authors suggest that RppAmay be phosphorylated by another histidine kinase. OurnrsRS::C.K1 mutant harbours a replacement of most ofthe nrsR sequence and part of the nrsS sequence by akanamycin resistance cassette and, therefore, is a doublemutant. However, this strain showed normal growth rate,normal levels of phycobiliproteins and chlorophylls andnormal expression of psbA and nblA genes. Therefore,the phenotype observed by Li and Sherman (2000) in the∆rppA mutant cannot be attributed to the lack of NrsR, butto the expression of NrsS in the absence of its naturalsubstrate (NrsR). Thus, a cross-talk between NrsS and a response regulator involved in redox control may beresponsible for the phenotype observed in the ∆rppAmutant.

The results shown here demonstrate conclusively that the NrsRS two-component system is controlling thenickel-dependent expression of the nrsBACD operon.How nickel sensing is carried out and how the signal istransduced is currently being investigated.

Experimental procedures

Bacterial strains and growth conditions

Synechocystis sp. strain PCC 6803 was grown photo-autotrophically at 30∞C in BG11 (Rippka et al., 1979) mediumsupplemented with 1 g per litre of HCO3Na (BG11C) andbubbled with a continuous stream of 1% (v/v) CO2 in air undercontinuous fluorescent illumination (50 mmol of photons perm2 per second, white light). For plate cultures, BG11C liquidmedium was supplemented with 1% (wt/vol) agar. Kanamycinwas added to a final concentration of 50–200 mg ml–1 whenrequired. BG11C medium was supplemented with differentconcentrations of ZnSO4, CdCl2, CoCl2, CuSO4, NiSO4 andMgCl2 when indicated.

Escherichia coli DH5a (Bethesda Research Laboratories),grown in Luria-Bertani broth (LB broth) medium as describedin Sambrook et al. (1989) was used for plasmid constructionand replication. Escherichia coli BL21(DE3) grown in LBbroth medium was used for expression of NrsR and NrsRDN His-tagged proteins. Escherichia coli was supplemented with100 mg ml–1 ampicillin or 50 mg ml–1 kanamycin when required.

Insertional mutagenesis of Synechocystis genes

Loci sll0797 and sll0798 were inactivated by replacing a535 bp HpaI–EcoRI fragment by a kanamycin resistance cassette (C.K1) (Elhai and Wolk, 1988). For this, a genomicDNA fragment was polymerase chain reaction (PCR)-ampli-fied with oligonucleotides NIW1-NIW2 from the cosmidcs1377 (provided by Kazusa DNA Research Institute) andcloned into pGEM-T (Promega) to generate pNIQ6. The tar-geting vector was generated by inserting the C.K1 cassetteinto HpaI–EcoRI-digested pNIQ6, in the same orientation asthe sll0797 gene (pNIQ9+) or in the inverse orientation(pNIQ9–). Synechocystis was transformed with these plas-mids to generate the nrsRS::C.K1(+) and nrsRS::C.K1(–)mutant strains. Correct integration and complete segregationof the mutant strain was tested by Southern blotting. For this,total DNA from the cyanobacteria was isolated as describedpreviously (Cai and Wolk, 1990). DNA was digested withHincII and electrophoresed in 0.7% agarose gels in a Tris-borate-EDTA buffer system (Sambrook et al., 1989), thenDNA was transferred to nylon Z-probe membranes (Bio-Rad).DNA probes were 32P-labelled with a random-primer kit(Pharmacia) using [a-32P]-dCTP (3000 Ci mmol–1).

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Fig. 7. Sequence alignment of the NrsS N-ter-minal periplasmic domain with the alphasubunit of the methyl CoM reductases from dif-ferent methanobacteria. metvo, Methanococ-cus voltae; barker, Methanosarcina barkeri;metka, Methanopyrus kandleri. Glutamineresidue involved in nickel co-ordination ismarked with a vertical arrow. Identical aminoacids are marked with (*), conservativechanges are marked with ‘:’ or ‘.’ as defined byCLUSTAL X (Thompson et al., 1997).

RNA isolation and Northern blot analysis

Total RNA was isolated from 25 ml samples of Synechocys-tis cultures at the mid-exponential phase (3–5 mg chlorophyllml–1). Extractions were performed by vortexing cells in thepresence of phenol–chloroform and acid-washed bakedglass beads (0.25–0.3 mm diameter; Braun) as describedpreviously (García-Domínguez and Florencio, 1997).

For Northern blotting, 15 mg of total RNA was loaded per lane and electrophoresed in 1.0% agarose denaturingformaldehyde gels, and transferred to nylon membranes(Hybond N-plus). Prehybridization, hybridization and washeswere performed as described in the Amersham instructionmanual.

Probes for Northern blot hybridization were PCR-synthesized using the following oligonucleotides pairs: NIA3-NIA4 (García-Domínguez et al., 2000) for the nrsBprobe; NIW1-NIW2 for the nrsRS probe; NBLA1-NBLA2 fornblA probe; and PSBA1-PSBA2 for the psbA probe (Table 1).As a control, in all the cases the filters were stripped andreprobed with a HindIII–BamHI 580 bp probe from plasmidpAV1100 that contains the constitutively expressed RNase PRNA gene (rnpB) from Synechocystis (Vioque, 1992). DNAprobes were 32P-labelled with a random-primer kit (Pharma-cia, Sweden) using [a-32P]-dCTP (3000 Ci mmol–1). To deter-mine cpm of radioactive areas in Northern blot hybridizations,whole band radioactivity was quantified using an InstantIm-ager Electronic Autoradiography apparatus (Packard In-strument Company). The signal was then normalized bycalculating the ratios between the mRNA signal and rnpBsignal for each lane, to avoid loading effects. Then, maximalratio was considered as 100% induction for each mRNA.

Primer extension analysis of nrsRS and nrsBACDtranscripts

Oligonucleotides NIW3 and NIA1, end-labelled with T4polynucleotide kinase and [g-32P]-dATP (3000 Ci mmol–1) following standard procedures (Sambrook et al., 1989), wereused for primer extension analysis of nrsRS or nrsBACD pro-moters respectively. For annealing a 10 ml mixture containing0.15 M HCl, 10 mM Tris HCl pH 8.0, 1 mM EDTA, 20 mg oftotal RNA and about 2 pmoles of oligonucleotide (106 cpm)were prepared. The annealing mixture was heated for 2 min

at 90∞C in a water bath and cooled slowly to 50∞C. For exten-sion, a 10 ml mixture was prepared with half of the annealingmixture: 10 mM DTT, 0.5 mM each dNTP, 2 mg of ActinomycinD, 50 mM Tris HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 and 100 Uof Superscript™ II RNase H-Reverse Transcriptase (GibcoBRL). The mixture was incubated for 45 min at 45∞C, and thereaction was stopped by adding 4 ml of formamide-loadingbuffer. Half of the reaction was electrophoresed on a 6% poly-acrylamide sequencing gel together with a sequencing reac-tion of the nrsRS or nrsBACD promoter region, using theNIW3 or NIA1 oligonucleotide respectively.

Cloning and purification of NrsR and NrsRDN His-tagged proteins

The complete nrsR ORF was PCR-amplified with oligo-nucleotide TCP1 (which introduces a NdeI restriction site)and oligonucleotide TCP5 (which introduces a BamHI restric-tion site) (Table 1), digested with NdeI–BamHI and clonedinto the NdeI–BamHI-digested pET28a (Novagen) to gener-ate pTCP3. The NrsR protein expressed from this plasmidcontains a six-histidine tag in the amino terminus. nrsRDNwas cloned by amplifying a 386 bp (from amino acid 118 tothe end of the protein) using oligonucleotide TCP4 (whichintroduces a NdeI restriction site) and oligonucleotide TCP5(which introduces a BamHI restriction site), digested withNdeI–BamHI and cloned into the NdeI–BamHI-digestedpET28a (Novagen) generating pTCP2. The NrsRDN proteinexpressed from this plasmid contains a six-histidine tag in theamino terminus. NrsR and NrsRDN were expressed in E. coliBL21 from the plasmids pTCP3 and pTCP2 respectively.Then, 200 ml of culture was grown in L broth to an opticaldensity at 600 nm of 0.6, induced with 1 mM isopropyl-b-D-thiogalactopyranoside for 2.5 h, harvested by centrifugationand resuspended in 8 ml of Tris-HCl pH 8.0 50 mM, KCl 50mM, 10% glycerol, 0.1% Triton X-100, 5 mM imidazol (bufferA) supplemented with 1 mM phenylmethylsulphonyl fluoride.Cells were broken by sonication, and insoluble debris waspelleted by centrifugation at 18 000 g for 15 min. The super-natant was then applied to a Ni2+-charged, His·Bind, beadscolumn (Novagen) (1 ml bead volume), washed with buffer Aand eluted with the same buffer containing 200 mM imidazol.Imidazol was removed dialysing the samples against bufferA without imidazol. The purity of the samples was about 90%,as determined by scanning densitometry from the Coomassieblue-stained gel.

Gel retardation assays

The probe was PCR-synthesized using oligonucleotidesNIP1-NIP2 (Table 1), which introduce StyI and NcoI restrictionsites, respectively, from cosmid cs1377 (provided by KazusaDNA Research Institute), and the resulting DNA was digestedwith either of the enzymes and end-labelled with [a-32P]-dCTP(3000 Ci mmol–1) using Klenow fragment. The binding reac-tion was carried out in a final volume of 25 ml containing 4 ngof labelled DNA and 4 mg salmon sperm DNA in 20 mM Tris-HCl pH 8.0, 150 mM KCl, 10 mM spermidine, 10 mM DTT, 1 mM EDTA, 10% glycerol and different amounts of partially purified NrsR and NrsRDN. The mixtures were incu-

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254 L. López-Maury et al.

Table 1. Oligonucleotides used in this work.

Oligonucleotide Sequence

NBLA1 5¢-GAAAGGTAGTCGCCTTGGAGGGC-3¢NBLA2 5¢-CCTGTTGCAAACACTGCAGTTG-3¢NIA1 5¢-GATTGGGAGTTCCTAGGGTC-3¢NIP1 5¢-GTTTCCATGGCACCACCTC-3¢NIP2 5¢-TCTTCCATGGGCAAAATTCGC-3¢NIW1 5¢-TTTCCAGTCTGCCAAAGGGG-3¢NIW2 5¢-TCTGTATTACCGGCGCTATGC-3¢NIW3 5¢-ATCCGTCACCCAATCCACCAC-3¢PSBA1 5¢-ATGACAACGACTCTCCA-3¢PSBA2 5¢-GCAGGAGCGGTCAAAGCC-3¢TCP1 5¢-GCAGAGCATATGCGACGATTTTG-3¢TCP4 5¢-GGGCACTACATATGCCGATCGCC-3¢TCP5 5¢-CTACGGGCAAGGATCCGACGGG-3¢

Nickel sensing in cyanobacteria 255

bated for 25 min at 4∞C and loaded on a non-denaturing 6% polyacrylamide gel. Electrophoresis was carried out at 4∞C and 280 V in 10 mM Na2HPO4 pH 6.0. Gels were transferred to a Whatman 3 MM paper, dried and autoradiographed.

Acknowledgements

We thank Kazusa DNA Research Institute and Dr S. Tabatafor providing cs1377 cosmid DNA. We thank Marika Lindahlfor critical reading of the manuscript. Luis López-Maury is therecipient of a fellowship from the Spanish Ministerio de Edu-cación Cultura y Deporte. This work was supported by grantPB97–0732 from DGESIC and by Junta de Andalucía (groupCV1–0112).

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