differentially expressed genes after hyper- and hypo-salt stress in the halophilic archaeon ...

Differentially expressed genes after hyper- andhypo-salt stress in the halophilic archaeonMethanohalophilus portucalensis

Chao-Jen Shih and Mei-Chin Lai

Abstract: Methanohalophilus portucalensis FDF1 can grow over a range of external NaCl concentrations, from 1.2 to2.9 mol/L. Differential gene expression in response to long-term hyper-salt stress (3.1 mol/L of NaCl) and hypo-salt stress(0.9 mol/L of NaCl) were compared by differential display RT-PCR. Fourteen differentially expressed genes responding tolong-term hyper- or hypo-salt stress were detected, cloned, and sequenced. Several of the differentially expressed geneswere related to the unique energy-acquiring methanogenesis pathway in this organism, including the transmembrane proteinMttP, cobalamin biosynthesis protein, methenyl-H4MPT cyclohydrolase and monomethylamine methyltransferase. Onesignal transduction histidine kinase was identified from the hyper-salt stress cultures. Moreover, 3 known stress-responsegene homologues — the DNA mismatch repair protein, MutS, the universal stress protein, UspA, and a member of theprotein-disaggregating multichaperone system, ClpB — were also detected. The transcriptional analysis of these long-termsalt stress response and adaptation-related genes for cells immediately after salt stress indicated that the expression of theenergy metabolism genes was arrested during hyper-salt shock, while the chaperone clpB gene was stimulated by bothhypo- and hyper-salt shock.

Key words: salt stress response, salt stress adaptation, differential display RT-PCR, halophilic methanogen, molecularchaperone ClpB, universal stress protein USPA, methanogenesis, stringent response.

Resume : Methanohalophilus portucalensis FDF1 peut croıtre sur une gamme de concentrations externes de NaCl de 1,2 a2,9 mol/L. L’expression genique differentielle en reponse a un stress hyper-salin (3,1 mol/L de NaCl) ou hypo-salin(0,9 mol/L de NaCl) prolonge a ete comparee par tri d’ARNm par criblage differentiel. Quatorze genes exprimes de facondifferentielle repondant a un stress hyper-salin ou hypo-salin prolonge ont ete detectes, clones et sequences. Plusieursgenes exprimes de facon differentielle etaient relies au sentier unique de methanogenese permettant un gain d’energie chezcet organisme, notamment la proteine transmembranaire MttP, la proteine de synthese de la cobalamine, la methenyl-H4MPT cyclohydrolase et la monomethylamine methyltransferase. Une kinase d’histidine impliquee dans la transductionde signal a ete identifiee chez les cultures placees en condition de stress hyper-salin. De plus, 3 homologues de genes destress ont ete identifies: la proteine de reparation des appariements d’ADN MutS, la proteine de stress UspA et un membredu systeme multi chaperon de desagregation des proteines, ClpB. L’analyse des transcrits de ces genes relies a la reponseet a l’adaptation a long terme au stress salin, immediatement apres le stress salin, a indique que l’expression des genes liesau metabolisme energetique etait cessee lors du stress hyper-salin, alors que celle du gene chaperon clpB etait stimuleelors d’un choc hypo-salin ou hyper-salin.

Mots-cles : reponse au stress salin, adaptation au stress salin, tri d’ARNm par criblage differentiel (DD RT-PCR), metha-nogene halophile, chaperon moleculaire ClpB, proteine de stress USPA, methanogenese, reponse stringente.

[Traduit par la Redaction]

Introduction

The dominant inhabitants in hypersaline environmentssuch as solar salterns, saline lakes, soda lakes, saline soils,and salt mines are usually defined as extremely halophilicprokaryotes. The prokaryotic organisms that grow best inmedia containing 2.5–5.2 mol/L of NaCl include the aero-bic, extremely halophilic Archaea, the anaerobic, extremelyhalophilic methanogenic Archaea, and the halophilic Bacte-ria (Kamekura 1998). The extremely halophilic, aerobic

Archaea are chemoorganotrophic organisms that require atleast 1.5 mol/L of NaCl for growth and are classified withinthe family Halobacteriaceae and order Halobacteriales. Theanaerobic, extremely halophilic methanogenic Archaea arefound in Methanohalophilus, Methanohalobium, and Metha-nosalsum (Grant 2004).

Living cells have developed 2 principal strategies to minimizewater loss and maintain cell turgor pressure when exposed to in-creasing osmolarity. One is the ‘‘salt-in-cytoplasm’’ strategy thatis restricted mainly to members of the Halobacteriaceae and

Received 26 August 2009. Revision received 18 December 2009. Accepted 26 January 2010. Published on the NRC Research Press Website at cjm.nrc.ca on 15 April 2010.

C.-J. Shih and M.-C. Lai.1 Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, ROC.

1Corresponding author (e-mail: [email protected]).

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Can. J. Microbiol. 56: 295–307 (2010) doi:10.1139/W10-008 Published by NRC Research Press

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anaerobic halophilic bacteria such as Halobacterodies ace-toethylicus. They accumulate high concentrations of KCl (upto 7 mol/L) inside the cytoplasm to achieve osmotic equili-brium with the external high-salt environment (2–5 mol/L ofNaCl). In response to the high internal salt (KCl) concentra-tion, complicated adaptations of macromolecules and intra-cellular machineries are required (Sleator and Hill 2002).The other strategy is to transport or synthesize compatiblesolutes to cope with increasing osmolarity. This strategy iswidespread and evolutionarily well conserved in all 3 do-mains of life (Csonka 1989; Lai et al. 1991; Martin et al.1999; Roberts 2000; Sleator and Hill 2002; Muller et al.2005).

The strict anaerobe Methanohalophilus portucalensisFDF1 can grow over a range of external NaCl concentra-tions, from 1.2 to 2.9 mol/L (Lai et al. 1991; Boone et al.1993). To counter the changing osmotic stress, these cellscan transport betaine from the medium or synthesize be-taine, b-glutamine, and Ne-acetyl-b-lysine as compatible sol-utes in response to the salt concentration of the externalenvironment (Lai et al. 1991, 1999, 2000, 2006). In additionto accumulating organic solutes, the internal K+ concentra-tion also increases from 0.6 to 1.1 mol/L (Lai et al. 1991).For example, compared to growth in 1.7 mol/L of NaCl,when the extremely halophilic methanogen Methanohalophi-lus portucalensis strain Z7302 was grown in 4.1 mol/L ofNaCl, the accumulated internal K+ concentration increasedfrom 1.22 to 3.09 mol/L (Lai and Gunsalus 1992). These ob-servations suggest the use of both ‘‘salt in’’ and ‘‘compatiblesolutes’’ strategies in this halophilic methanogen. Althoughthe detailed molecular mechanisms governing the salt stressresponse and adaptation-related system are not yet fully elu-cidated, one would expect, like the heat shock system, that aglobal gene regulation system should be involved.

The differential display RT-PCR (DDRT-PCR) has beensuccessful in identifying the differentially expressed genesof eukaryotic cells in response to various stress environ-ments (Ammendola et al. 1995; Crawford et al. 1997; Miuraet al. 2000; Tyagi and Chandra 2006). However, because ofthe lack of long stretches of poly(A) tails in bacterialmRNA, few DDRT-PCR analyses have been performed onbacteria (Rindi et al. 1998, 1999; Rivera-Marrero et al.1998; Chia et al. 2001). RNA polyadenylation occurs in thehyperthermophilic archaeon Sulfolobus solfataricus as wellas the halophilic archaeon Halobacterium halobium (Brownand Reeve 1986; Sarkar 1997; Portnoy et al. 2005). In 2methanogens, Methanococcus vannielii and Methanopyruskandleri, the occurrence of RNA polyadenylation has alsobeen demonstrated (Brown and Reeve 1985; Sarkar 1997;Portnoy and Schuster 2006). The average poly(A) length is12 bp in Methanococcus vannielii and 20 bp in Halobacte-rium halobium (Brown and Reeve 1986). Lately, theDDRT-PCR method has been used to identify differentialexpression of genes influenced by changing salinity in theaerobic, halophilic archaeon Haloferax volcanii (Bidle2003). This work led to the identification of a salt stresssensor protein with homology to the eukaryotic Ser/Thr pro-tein kinase.

Recently, genome-wide expression profiles in the saltadaptation of Methanosarcina mazei Go1 suggest that thisnonhalophilic methanogen can up-regulate many different

cellular functions such as solute transport and biosynthesis,import of phosphate, export of Na+, and modification ofDNA and cell surface architecture to adapt to high salinity(Pfluger et al. 2007). Since there is no whole-genome se-quence database available for halophilic methanogens, toobtain a snapshot of gene expression of halophilic methano-gens in response to salt stress, we used the DDRT-PCRmethod to study the differential gene expression of the anae-robic, halophilic Methanohalophilus portucalensis in re-sponse to long-term hyper-salt stress (3.1 mol/L of NaCl)and hypo-salt stress (0.9 mol/L of NaCl). The differentiallyexpressed genes that responded to long-term salt stress inMethanohalophilus portucalensis included several methano-genesis genes. Also detected were several stress responsegene homologues, including the DNA mismatch repair pro-tein, MutS, the universal stress protein, UspA, and a mem-ber of the protein-disaggregating multichaperone system,ClpB.

Materials and methods

Organisms and culture conditionsMethanohalophilus portucalensis strain FDF1 (=DSM7471)

was isolated from the solar saltern of Figueria da Foz,Portugal, by Mathrani and Boone (1985) and provided byRobert A. Mah (Lai et al. 1991). The cells were routinely in-cubated at 37 8C in defined medium that contained 2.1 mol/Lof NaCl and 40 mmol/L of trimethylamine as the sole carbonand energy source (Lai et al. 1991). Sterile medium was pre-pared under a N2–CO2 atmosphere (4:1) by a modification ofthe Hungate technique. The medium (50 mL) was anaerobi-cally dispensed into serum bottles (110 mL) that were thensealed with butyl rubber stoppers and aluminum crimp clo-sures and autoclaved at 121 8C for 20 min. The methanogenictrimethylamine substrate and Na2S�9H2O were prepared underN2 atmosphere and added to the sterile medium just prior tocell inoculation with 40 and 1 mmol/L, respectively. Sealedserum bottles were inoculated with a 5% volume of late-exponential phase culture by using a N2-flushed syringe. Cellgrowth rates were monitored by removing 1 mL of the cul-ture with a N2-flushed syringe, transferring the culture into acuvette (1 cm path length) containing a few crystals ofNa2S2O3, mixing the solution by gentle inversion (Sowers1995), and measuring the optical density of the culture at540 nm.

To analyze the differentially expressed genes of Methano-halophilus portucalensis under long-term hypo- and hyper-saltgrowth conditions, late exponential phase cells of Meth-anohalophilus portucalensis grown at 2.1 mol/L of NaCl(optimal NaCl growth concentration; Lai et al. 1991) con-taining defined medium were transferred into defined me-dium containing 0.9, 2.1, or 3.1 mol/L NaCl and incubatedat 37 8C until these 3 cultures reached mid-exponentialphase. Escherichia coli strain DH5a was used as the cloninghost and was grown in Luria–Bertani medium or on solidmedium at 37 8C (Sambrook and Russell 2001). Ampicillinwas added to the medium at a concentration of 100 mg/mL.

Primers and vectorsThe arbitrary primers used for DDRT-PCR in this study were

designed according to Rindi et al. (1999). The design was based

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on the following criteria: (i) no stop codon was present in anypossible reading frame in both directions and (ii) the sequenceshad minimal tendency to form internal loops. Ten arbitraryprimers, designated P1 (5’-GCCAAGCTCCAG-3’), P2 (5’-GGCGTCATCGAC-3’), P3 (5’-GCCATCCTCGAC-3’), P4(5’-GGCAAGGCACAG-3’), P5 (5’-GCGGTCATCCTG-3’),P6 (5’-GCGCTCAAGCTG-3’), P7 (5’-GGTCTCGCAGTG-3’),P8(5’-GGTGTCACCGAC-3’),P9(5’-GCGACCAAGGTG-3’),and P10 (5’-GAGGCAGTCGAG-3’), as well as the Oligo(dT)11primer, were synthesized by Genset Singapore BiotechnologyPte. Ltd. From these 10 arbitrary primers, 55 combinations ofprimer sets were obtained for DDRT-PCR. The pGEM-T easyvector (Promega, Madison, Wis.) was used to clone PCR prod-ucts according to the manufacturer’s instructions.

Total cell RNA preparations from Methanohalophilusportucalensis

To prepare RNA, the mid-exponential phase cultures ofMethanohalophilus portucalensis strain FDF1 grown at vari-ous salt concentrations (50 mL, OD540 = ~0.6–0.8, about 109

cells) were harvested and lysed with 1 mL of RareRNA(Genepure Technology Co., Ltd, Taichung, Taiwan), a gua-nidinium salts – phenol-based reagent, according to themanufacturer’s instructions and incubated for 5 min at roomtemperature. After the addition of 0.3 mL of chloroform, themixture was shaken vigorously for 15 s, incubated on ice for5 min, and further centrifuged at 12 000g and 4 8C for10 min. The aqueous phase, containing the RNA, was trans-ferred into a new tube, and the nucleic acids were precipi-tated with isopropanol. The RNA pellet was dried by SpeedVac (SC100, Savant Instruments, Holbrook, N.Y.) and thendissolved in 20 mL of DEPC-treated water. The RNA con-centration was determined by absorbance at 260 nm using aUV spectrophotometer (Ultrospec 1000, Pharmacia Biotech,Cambridge, UK). Its quality was assessed in an agarose gelprior to the experiments (Sambrook and Russell 2001).

DDRT-PCR, cloning, and sequencingImmediately after RNA isolation, 5 mg of total RNA was

subjected to reverse transcription in a GeneAmp PCR Sys-tem 2400 (Perkin Elmer Applied Biosystems, Boston,Mass.). The cDNA synthesis reactions were carried out for1 h at 42 8C in 20 mL volumes containing 1� ImProm-IIreaction buffer, 3 mmol/L of MgCl2, 0.5 mmol/L ofeach dNTP, 1 mmol/L of Oligo(dT)11 primer, and 1 mL ofImProm-II reverse transcriptase (Promega). A negative con-trol was performed with the same components as previouslylisted except for the reverse transcriptase. The reaction wasstopped by incubation at 95 8C for 5 min.

Following cDNA synthesis, 20 mL aliquots, including thenegative control, were transferred to 200 mL PCR tubes in afinal volume of 100 mL containing 1� PCR buffer(10 mmol/L of Tris–HCl (pH 8.3), 50 mmol/L of KCl, and1.5 mmol/L of MgCl2), 5 mmol/L of each of the primer sets(1 arbitrary primer or the combinations of 2 arbitrary pri-mers), and 2.5 U of Takara Taq DNA polymerase (TakaraShuzo, Otsu, Japan). The cDNA was amplified using an ini-tial denaturation step of 94 8C for 3 min followed by 30cycles of denaturation at 94 8C for 1 min, 50 8C for 1 min,and 72 8C for 1 min. The resulting products were run in a1.5% TAE–agarose gel. Only the DNA bands detected in

experimental samples (cells grown in medium containing0.9 or 3.1 mol/L of NaCl) or expressed at a level higherthan that in the control samples (cells grown in mediumcontaining 2.1 mol/L of NaCl) were considered differentiallyexpressed fragments. Then, these bands were excised fromthe gel and purified using the GENECLEAN III (BIO 101Systems, Q-BIOgene, Carlsbad, Calif.). Purified cDNA wasfurther ligated into the pGEM-T easy vector (Promega) ac-cording to the manufacturer’s instructions, and positiveclones were sequenced using the T7 and SP6 promoter pri-mers in an automated sequencer (model 373A, Applied Bio-systems, Foster City, Calif.).

Northern analysisFor Northern hybridization, RNA was prepared as de-

scribed previously. The transcriptional level of the 16SrRNA of Methanohalophilus portucalensis FDF1 was usedas an internal control for Northern analysis. The 16S rDNAfragment was generated from Methanohalophilus portuca-lensis FDF1 chromosomal DNA using specific 16S rDNAprimers (coccus1 primer: 5’-CGACTAAGCCATGCG-AGTC-3’; reverse3 primer: 5’-GTGACGGGCGGTGTGTG-CAAG-3’) for the methanogen (Lai et al. 2004), subclonedinto the pGEM-T easy vector, and sequenced. The probesfor 14 differentially expressed genes and 16S rDNA wereprepared with the Renaissance Random Primer Fluoresceinlabeling kit (Perkin Elmer Life Sciences, Inc., Boston,Mass.) according to the manufacturer’s instructions.

The RNA samples (36 mg) were loaded into the formalde-hyde agarose gel prepared as described by Sambrook andRussell (2001). RNA gel electrophoresis was performed at20 V for 16 h, and then the RNA was transferred toHybond-N+ nylon membrane (Amersham, Co., Piscataway,N.J.) with a vacuum transfer system (HYBAID). Further fix-ation, hybridization, washing, and detection were performedas described previously (Lai et al. 2002). TINA software(version 2.09e) was used to analyze the hybridization sig-nals. The relative transcript levels were calculated as thespecific values of the target gene signal divided by the spe-cific value of the 16S rRNA signal. The tests were run induplicate with the same batch of cultures.

Immediate response of the long-term salt stressadaptation genes during salt shock

To examine the immediate salt shock stress response ofthese differentially displayed long-term salt stress adaptationgenes, total RNA was obtained from the culture after the im-mediate hyper-osmotic and hypo-osmotic shocks, and North-ern hybridization was performed with probes of differentiallydisplayed salt stress adaptation genes. Mid-exponential phasecultures of Methanohalophilus portucalensis grown indefined medium with 2.1 mol/L of NaCl at 37 8C wereused. For salt up-shock, the anaerobically prepared definedmedium with 3.85 mol/L of NaCl (66 mL) was added intothe 50 mL Methanohalophilus portucalensis culture grownat 2.1 mol/L of NaCl in defined medium to reach a finalNaCl concentration of 3.1 mol/L; for salt down-shock, thedefined medium without NaCl (66 mL) was added into the50 mL Methanohalophilus portucalensis culture containing2.1 mol/L of NaCl to reach a final NaCl concentration of0.9 mol/L. Control tests were performed by adding the same

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amount of defined medium containing 2.1 mol/L of NaCl.All tested cultures (0.9, 2.1, and 3.1 mol/L of NaCl) werefurther incubated at 37 8C for different time periods. Cellswere collected, and the total RNA was extracted and exam-ined by Northern hybridization, using SU12312, SU34312,and SU26311 as probes.

Computer methods and GenBank accession No. ofnucleotide and amino acid sequences

Sequence comparisons of the differentially expressedgenes obtained with the previous methods were performedby using BLASTX 2.2.15 (Altschul et al. 1997) from theNCBI Web site (http://www.ncbi. nlm.nih.gov/ BLAST/). Aconserved domain search was also performed on the NCBIWeb site (Marchler-Bauer and Bryant 2004). The TMAPprogram (Persson and Argos 1994) from the Biology Work-bench Web site (http://seqtool.sdsc.edu) was used for theprediction of transmembrane segments. The sequences ofthe partial gene fragments identified in this work were de-posited in GenBank under the GenBank accession No. listedin Table 1.

Results and discussion

Identification of long-term salt stress response andadaptation-related genes

Methanohalophilus portucalensis can grow over a saltconcentration range of 1.2–2.9 mol/L of NaCl. To investi-gate salt stress response and adaptation-related genes, a lateexponential phase culture of Methanohalophilus portucalen-sis grown in optimal NaCl (2.1 mol/L) was transferred into adefined medium containing 0.9, 2.1 (control), or 3.1 mol/Lof NaCl and incubated at 37 8C. The growth curves of Meth-anohalophilus portucalensis FDF1 grown in optimal(2.1 mol/L), low (0.9 mol/L), and high (3.1 mol/L) NaClconcentrations were measured (Fig. 1A), yielding specificgrowth rates of 0.072, 0.065, and 0.059 h–1, respectively.Accordingly, the doubling times were 9.6, 10.7, and 11.8 h,respectively. RNA from cultures grown at different salt con-centrations were isolated and examined using 55 differentrandom primer combinations in DDRT-PCR analysis. As in-dicated in Fig. 2, DNA bands with different densities weredetected with the same primer set at the same migration po-sitions shown in agarose gels. These bands indicated thepossible differential mRNA expression for the culture grownin an environment containing 0.9, 2.1 (control), or 3.1 mol/Lof NaCl. Taking the clone SD17092 as an example, with theRT-PCR primer set P1 and P7, a high-density DNA bandwas detected only with RNA obtained from culture grownat a low salt concentration (0.9 mol/L of NaCl), but a bandwas not detected in samples from culture grown at 2.1 mol/Lof NaCl (control) or in a high-salt environment (3.1 mol/Lof NaCl) (Fig. 2). This differentially expressed DNA frag-ment was excised from the gel and further purified, cloned,and sequenced. A total of 20 differentially expressed bands(Fig. 2) out of 184 detected bands, with sizes ranging from1600 to 100 bp, were purified, cloned, and sequenced. Ofthe 20 clones originally isolated, 5 of them were furtherdemonstrated to encode 23S rDNA, and one was the overlapclone of SU37311, which consists of 3 open reading frames(ORFs) related to the ribosomal protein S3 and ribosomal

protein L29 of Methanococcoides burtonii (identity 57%and 73%, respectively) and the ribonuclease P protein com-ponent 1 of Methanocaldococcus jannaschii (identity 65%)(Table 1). There were 2 differential display gene clones(SU78315 and SD56092) whose functions could not be pre-dicted. Clone SU78315 was up-regulated in high salinity andpresented 91% similarity with a hypothetical protein ofPseudomonas aeruginosa (Table 1). The clone SD56092could not be matched with any hypothetical, predicted, orfunctional proteins.

The sequence analysis of these 14 differentially expressedgenes is presented in Table 1. These genes could be clus-tered, based on their predicted functions, into 4 groups.Group I genes are related to the known stress response;group II are related to genes with known functions not di-rectly linked to stress response; group III are related to en-ergy metabolism; and group IV are genes with unknownfunctions. Detailed descriptions and discussion follow.

Group I: stress response genesOf the 14 partial differentially expressed genes (ORFs)

we identified here, 3 (SU12312, SD38092, and SU25311)were related to known stress response genes. CloneSU12312, differentially expressed in culture with a high saltconcentration (3.1 mol/L of NaCl), encodes a protein with anucleotide-binding domain similar to that of the ATPaseAAA-2 of Methanococcoides burtonii (75% identical). TheATPase AAA-2 of Methanococcoides burtonii is a homo-logue of the molecular chaperone ClpB protein. BacterialClpB and its eukaryotic orthologues, plant Hsp 101 andyeast Hsp 104, are heat shock and salt stress induced andare essential proteins for the stress response (Lee et al.2004; Nag et al. 2005). ClpB contains 2 nucleotide-bindingdomains, NBD1 and NBD2. NBDs possess the classicalWalker-type consensus sequences and are highly conservedthroughout all members of ClpB proteins (Schlee and Rein-stein 2002). Clone SU12312 covered the NBD2 region, andthis is the first chaperone gene to be identified in a halo-philic methanogen that might be involved in the salt stressresponse.

The complete putative chaperone clpB gene (2610 bp) wasobtained and used as a probe to analyze the fluctuation oftranscriptional level among different stresses. The transcriptlevel of putative clpB gradually increased with time afterheat shock from 37 to 45 8C, reached a maximal 7-foldincrease within 60 min, and then gradually decreased to4.2-fold above the initial transcript level after 2 h. The puta-tive clpB transcript levels were increased 10-fold with a tem-perature shift from 37 to 55 8C for 15 min. Under heat andhyper-salt stress, the putative clpB transcript was repressedwith the addition of the osmolyte betaine (1 mmol/L) (Shihand Lai 2007).

Clone SU25311, differentially expressed in 3.1 mol/L ofNaCl, encodes a protein that is 49% identical to the DNAmismatch repair protein MutS of Methanosarcina mazei(Table 1). The MutS protein of Thermus aquaticus contains5 structural domains: I, II, III, IV, and V (Obmolova et al.2000). The putative partial MutS gene of Methanohalophilusportucalensis encodes 192 amino acid residues, which corre-sponded with domain V of T. aquaticus MutS. The consensusATP-binding motif Walker A (GxxxxGKT/S), corresponding to



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Table 1. Summary of the sequence comparison of the differentially expressed genes obtained through differential display RT-PCR from Methanohalophilus portucalensis grown atvarious salt concentrations.

Clone Size (bp) GenBank accession No.Predicted gene products and related organism(GenBank accession No.)

AA residues(% identity)* Gene organization

Group I: stress response genesSU12312 1326 DQ387367 ATPase AAA-2; Methanococcoides burtonii

(YP_566814)334/441 (75%)

SU25311 578 EF470288 DNA mismatch repair protein Mut; Methanosarcinamazei (NP_633398)

85/173 (49%)

SD38092 210 EF473724 Universal stress protein; Methanosarcina acetivorans 28/69 (40%)SU12317 531 EF473725 Periplasmic sensor signal transduction histidine ki-

nase; Ralstonia metallidurans (YP_586389)57/169 (33%)

Group II: genes not linked to stress responseSU37311 808 EF469545 (3 ORFs) Ribosomal protein S3; Methanococcoides burtonii

(YP_564781)84/148 (57%)

Partial rps3p

(457 bp)

rpmC

(201 bp)

457

rnp1

(144 bp)

665

Ribosomal protein L29; Methanococcoides burtonii(YP_564782)

49/67 (73%)

Ribonuclease P protein component 1; Methanocal-dococcus jannaschii (Q57903)

31/47 (65%)

SD18092 432 EF472917 Nitrite and sulphite reductase, 4Fe-4S subunit;Methanococcoides burtonii (YP_566139)

112/144 (77%)

SD17092 1063 EF473721 Protein translation elongation factor EF-1, subunit a;Methanococcoides burtonii (YP_565843)

263/317 (82%)

SD44093 467 EF473722 (2 ORFs) Lysyl-tRNA synthetase; Methanosarcina barkeri(Q9C4B7)

36/56 (64%)

Partial lysS

(178 bp)Hypothetical protein

gene (288 bp)

180

Hypothetical protein; Methanococcoides burtonii(YP_566497)

41/97 (42%)

Group III: energy metabolism genesSU34312 930 EF469544 (2 ORFs) Cobalamin biosynthesis protein CbiD; Methanococ-

coides burtonii (YP_566345)143/182 (78%)

Partial cbiE

(294 bp)

637

Partial cbiD (637 bp)

Precorrin-6B methylase CbiE; Methanosarcina ma-zei (NP_633704)

57/85 (67%)

SU11312 566 EF470287 Transmembrane protein MttP; Methanosarcina acet-ivorans (NP_615494)

112/160 (70%)

SD44094 242 EF473723 Methenyl-H4MPT cyclohydrolase; Methanococ-coides burtonii (YP_565374)

67/80 (83%)

SU26311 691 EF470286 (2 ORFs) Corrinoid methyltransferase MtmC; Methanococ-coides burtonii (YP_565548)

86/114 (75%)

Partial mtmC

(356 bp)

Partial mtmB

(201 bp)

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G119 through S127, was also found in this partial MutS. DNAmismatch repair corrects the mispaired and unpaired basesarising from replication errors, physical damage to bases, andhomologous recombination and increases the overall fidelityof DNA synthesis (Stanisławska-Sachadyn and Sachadyn2005; Jun et al. 2006). Eukaryotic MutS homologues havebeen demonstrated to be involved in the repair of oxidativeDNA damage (Earley and Crouse 1998; Ni et al. 1999;Mazurek et al. 2002; Dzierzbicki et al. 2004). In bacteria,Wang et al. (2005) indicated that Helicobacter pylori MutSplays a significant role in repairing oxidative DNA damage.It has been demonstrated that hyperosmolarity in the form ofelevated NaCl causes DNA damage in the form of double-strand breaks in murine kidney cells (Kultz and Chakravarty2001). By screening a library of Tn917–lacZ insertionalmutants to identify Listeria monocytogenes genes involvedin salt tolerance, Gardan et al. (2003) demonstrated thatthe mutS gene was associated with salt stress. Accordingto the DNA microarray analysis, the mutS gene of cyano-bacterium Synechocystis sp. was inducible by hyperosmoticstress (Murata and Los 2006).

Clone SD38092, expressed only in culture containing0.9 mol/L of NaCl, encodes a putative universal stress proteinT

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Fig. 1. Growth curve of Methanohalophilus portucalensis FDF1.(A) Cells were incubated in defined medium with different NaClconcentrations. (&) 0.9 mol/L NaCl; (^) 2.1 mol/L of NaCl; (~)3.1 mol/L of NaCl. (B) Cells were incubated in defined mediumwith 2.1 mol/L of NaCl until the mid-log phase and up-shocked byincreasing the NaCl concentration of the culture from 2.1 to3.1 mol/L with fresh medium containing 3.85 mol/L of NaCl (~)or down-shocked by decreasing the NaCl concentration of the cul-ture from 2.1 to 0.9 mol/L with fresh medium containing 0 mol/Lof NaCl (&). The control culture was incubated in defined mediumcontaining 2.1 mol/L of NaCl (^). The arrow indicates the timewhen the new medium was added. Error bars indicate standard de-viation of 3 independent experiments.



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(UspA) that shares 40% identity with Methanosarcina aceti-vorans UspA (Table 1). By using the Conserved DomainSearch service at the NCBI Web site (Marchler-Bauer andBryant 2004), the deduced amino acid sequences of cloneSD38092 was found to contain a USP-like domain belong-ing to the universal stress protein family, which has an es-sential ATP-binding site. The protein encoded by theMJ0577 gene of the thermophilic archaeon Methanocaldo-coccus jannaschii belonged to the USP family and 4 se-quence motifs surrounded the nucleotide-binding pocket

were identified in the crystal structure of MJ0577 (Zarem-binski et al. 1998). The conserved residues Pro11, Thr12,Asp13, and Val41 in motif 1 of MJ0577 were in contact withthe adenine of ATP, and the conserved Val41 was also foundin the predicted UspA of Methanohalophilus portucalensis(corresponding to Val28). Escherichia coli UspA mutantsare impaired with regard to carbon dissimilation and theirability to survive complete and prolonged growth inhibitioncaused by carbon starvation and a variety of stress condi-tions, including CdCl2, H2O2, 2,4-dinitrophenol, and car-bonyl cyanide m-chlorophenyl hydrazone exposure andosmotic shock (Nystrom and Neidhardt 1996). Using pulselabeling and two-dimensional gel electrophoresis, Diez etal. (2000) demonstrated that DNA damage caused by theagent mitomycin C increased UspA protein production rates,further substantiating that uspA responds to DNA damage.

Clone SU12317, expressed in culture containing both 2.1and 3.1 mol/L of NaCl, has a deduced amino acid sequencethat shares 33% identity with the periplasmic sensor signaltransduction histidine kinase of Ralstonia metallidurans(Table 1). A highly conserved region found in many signalsensor protein sequences also exists in the deduced aminosequences of SU12317, corresponding to Ala30–Thr48. Sev-eral other conserved residues were also found in SU12317(data not shown). The signal transduction histidine kinasebelongs to the two-component system which modulates spe-cific phosphoryl relay systems that couple environmentalsignals to appropriate cellular responses (Mascher et al.2006). The histidine kinase gene we identified in this studymight function to transmit the external salt stress signal tothe intracellular salt stress response genes.

The halophilic methanogen Methanohalophilus portuca-lensis FDF1 has been reported to respond to the changingosmotic stress by transporting betaine or synthesizing be-taine, b-glutamine, and Ne-acetyl-b-lysine de novo as com-patible solutes (Lai et al. 1991, 1999, 2000, 2006).However, there was no synthesis of compatible solutes or re-covery of transport-related transcripts in this survey. Wehave cloned and characterized a compatible solute betaineABC transporter (Bta) in Methanohalophilus portucalensis.Our Northern analysis showed that upon salt up-shock, btaexpression was immediately and specifically stimulated bybetaine (unpublished result). In this report, betaine was notadded, so that the mRNA of the betaine transporter was notdifferentially expressed enough to be detected. We also haveidentified and examined the osmolyte betaine synthesisgenes (gsmt and sdmt) and N-acetyl-b-lysine synthesis genes(ablA and ablB) from Methanohalophilus portucalensis.Northern analysis showed that the expression of thesemRNAs increased slightly with increasing salt concentra-tions (unpublished data). It is likely that under the long-term salt stress condition, the mRNA of compatible solutesynthesis genes might remain quite constant and is not dif-ferentially expressed enough to be detected under the stresssituation in this study.

Group II: genes not linked to stress responseClone SU37311 consisted of 3 ORFs, which were signifi-

cantly down-regulated in culture containing 0.9 mol/L of NaCl(Table 1). Gene organization of the 3 ORFs is the same as thegene cluster of ribosomal proteins from Methanococcoides

Fig. 2. Differentially expressed gene fragments detected by differ-ential display RT-PCR (DDRT-PCR). The sequences of primers arelisted in Materials and methods. DDRT-PCR reactions were per-formed as described using RNA from Methanohalophilus portuca-lensis grown in cultures containing 0.9, 2.1, and 3.1 mol/L of NaCl.The reaction products were then separated in a 1.5% agarose geland stained with ethidium bromide.

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burtonii. The deduced amino acid sequences of the first andsecond ORFs showed 57% and 73% identity with the 30Sribosomal protein S3 and the 50S ribosomal protein L29from Methanococcoides burtonii, respectively (Table 1).Aside from the amino acid identity, the second ORF con-tained a conserved domain, pfam00831.13, which corre-sponded to the ribosomal L29 protein based on the result ofthe Conserved Domain Search (Marchler-Bauer and Bryant2004). L29 can interact with the signal recognition particle,which interacts with the nascent polypeptide chain(Brodersen and Nissen 2005). In E. coli, ribosomal proteinS3 is known to be involved in the binding of initiator Met-tRNA. The deduced amino acid sequences of the third ORFhad 65% identity with the ribonuclease P protein component1 of Methanocaldococcus jannaschii (Table 1). RNase P isan endoribonuclease responsible for generating the 5’ end ofmature tRNA molecules (Brannvall et al. 2002). Under ex-treme stress, if bacteria do not have sufficient amino acidsfor biosynthesis, cells will initiate a ‘‘stringent response’’ todown-regulate many genes that encode the transcription andtranslation apparatus (Chang et al. 2002). The down-regulatedgene expression of ribosomal proteins could be related to thestringent response of Methanohalophilus portucalensis in thediluted salt environment.

Clone SD17092 appears to encode a protein that had 82%identity with the protein translation elongation factor EF-1,subunit a from Methanococcoides burtonii (Table 1). EF-1a has 3 structural domains, I, II, and III. The analysis fromthe Conserved Domain Search indicated that all 3 domainswere contained in the deduced amino acid sequences ofSD17092. EF-1 a in Archaea and Eukarya (and the bacteriaortholog EF-Tu) is a highly conserved and indispensableGTPase involved in the translation process (Andersen et al.2003; Inagaki et al. 2006). Clone SD44093 consisted of 2ORFs, one of which encoded a protein containing a LysSconserved domain that shared 64% identity with a lysyl-tRNA synthetase of Methanosarcina barkeri, while the otherencoded protein having the highest identity (42%) with a hy-pothetical protein from Methanococcoides burtonii (Table 1).The conserved residues Phe7 and Tyr11, corresponding to thePhe487 and Tyr491, which are the tRNA recognition sites ofPyrococcus horikoshii LysRS-I, were also found in the puta-tive lysyl-tRNA synthetase of Methanohalophilus portuca-lensis (Terada et al. 2002). Lysyl-tRNA synthetase catalyzesthe formation of lysyl-transfer RNA, which then is ready toinsert lysine into proteins (Freist and Gauss 1995). Based onthe Conserved Domain Search, the deduced amino acid se-quences of Clone SD18092 contained 2 conserved domainsthat corresponded to pfam03460.12, a nitrite and sulfite re-ductase ferredoxin-like half-domain, and pfam01077.13, anitrite and sulfite reductase 4Fe–4S domain. From the Blastresult, clone SD18092 had 77% amino acid sequence iden-tity with the 4Fe–4S subunit of nitrite and sulfite reductasefrom Methanococcoides burtonii (Table 1). This reductase,identified from hypo-salt culture, is a member of the sulfiteassimilation pathway.

Differentially expressed genes of 30S ribosomal proteinS3, 50S ribosomal protein L29, ribonuclease P protein,translational elongation factor EF-1, lysyl-tRNA synthetase,as well as nitrite and sulphite reductase have never beenmentioned in relation to a salt stress response before. These

results indicated that the protein synthesis machine and inor-ganic nitrogen and sulfur assimilation might also be in-volved in the long-term salt adaptation process.

Group III: energy metabolism genesThe differentially expressed gene clone SU11312 is re-

lated to transmembrane protein MttP of Methanosarcinaacetivorans (70% identity) (Table 1). According to theTMAP program from Biology Workbench (Persson and Ar-gos 1994), 4 transmembrane segments, namely TM1 (Val4–Ala26), TM2 (Arg42–Gly62), TM3 (Thr77–Lys97), and TM4(Ala118–Phe146), were predicted in the deduced amino acidsequences of clone SU11312. This transmembrane proteinis a putative trimethylamine permease that might transporttrimethylamine into the cytoplasm for use as a methanogen-esis substrate (Paul et al. 2000). Clone SU26311 consists of2 ORFs. One is a sequence closely related to the corrinoidmethyltransferase of Methanococcoides burtonii (75% iden-tity), a homologue of Methanosarcina barkeri monomethyl-amine corrinoid protein (MtmC), and the other is predictedas a monomethylamine methyltransferase MtmB from Meth-anosarcina barkeri (80% identity). The gene organizationwas the same as that of the mtm operon from Methanosar-cina barkeri (Burke et al. 1998) (Table 1). A B12-bindingdomain with the invariant cobalt ligand DxHxxG (Drennanet al. 1994) was found in the putative MtmC of Methanoha-lophilus portucalensis, while the conserved aspartic acid wasreplaced by valine. These 2 predicted protein genes fromSU26311 are involved in the methyl transfer in methanogen-esis in this organism. The MtmB uses monomethylamine asa methyl donor to methylate its cognate corrinoid protein,MtmC. Subsequently, the methylated MtmC is then deme-thylated by the CoM-methylating protein MtbA to methylateCoM. Finally, the methyl-CoM is reduced by methyl-CoMreductase MCR and ATP is generated (Burke and Krzycki1997; Paul et al. 2000) (Fig. 3). Similarly, the global tran-scriptional analysis of nonhalophilic Methanosarcina mazeistrain Go1 also showed the energy metabolism genes, mtmBand mtmC, induced by nitrogen limitation (Veit et al. 2006).

Clone SU34312 contains 2 partial genes: one is related tothe cobalamin biosynthesis protein CbiD from Methanococ-coides burtonii (78% identity), and the other is related to theprecorrin-6B methylase CbiE from Methanosarcina mazei(67% identity) (Table 1); both are needed for methylaminemetabolism. According to the Conserved Domain Search, the2 partial genes contain the conserved domains pfam01888.13(CbiD domain) and COG2241.2 (CobL domain), respectively.These 2 predicted proteins are involved in C-1 methylation inthe anaerobic pathway to cobalamin (Roessner et al. 2005),which is also related to the cofactor of methyltransferase formethanogenesis (Deppenmeier et al. 1999). Clone SD44094encodes a partial protein that shares 83% identity with theMethanococcoides burtonii methenyl-H4MPT cyclohydrolase(Table 1). A putative conserved domain similar to the MCHdomain was detected in the deduced amino acid sequences ofSD44094 by the Conserved Domain Search. In the predictedMethanohalophilus portucalensis methenyl-H4MPT cyclo-hydrolase, residue Lys77 corresponds to the Lys118 of theMethanopyrus kandleri methenyl-H4MPT cyclohydrolasethat binds to the phosphate moiety of H4MPT (Grabarseet al. 1999). This enzyme catalyzes the hydrolysis of



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methenyl-H4MPT to formyltetrahydromethanopterin (formyl-H4MPT) (Donnelly et al. 1985), which participates in thepathway of methyl-group oxidation for transferring metha-nogenesis substrates into biosynthesis substrates (Fig. 3).

The differential expression of the methanogenesis-relatedgenes identified from long-term hyper-salt stress adaptation ofMethanohalophilus portucalensis indicated that energy conser-vation is important for a cell facing long-term salt stress. Toverify the differential expression of methanogenesis-relatedgenes, total RNA from cultures containing 0.9, 2.1, and3.1 mol/L of NaCl were prepared and Northern hybridizationwas performed using SU26311, SU34312, and SD44094 asprobes, as described in the Materials and methods. Accordingto the results of the Northern hybridization, the transcriptlevels of SU26311 and SU34312 were both up-regulated inlong-term hyper- and hypo-salt cultures (Fig. 4), which wasdifferent from the DDRT-PCR results showing that expressionwas up-regulated only with an increase in salt concentration.However, the transcript levels of SD44094 and SU11312were up-regulated only in the hypo-and hyper-salt cultures(Fig. 4), respectively, and were consistent with the results ofDDRT-PCR (Fig. 2). Based on the results of Northern hybrid-ization, the transcription of genes of putative trimethylaminepermease, monomethylamine methyltransferase, monomethyl-amine corrinoid protein, cobalamin biosynthesis protein, andprecorrin-6B methylase were stimulated in long-term adapta-tion to both hyper- and hypo-salt stress, whereas the transcrip-tion of methenyl-H4MPT cyclohydrolase was repressed underhyper-salt stress (Fig. 3). These results suggest more substrate

demand for salt stress adaptation (both hypo- and hyper-salinestress). Under hyper-salt stress conditions, more energy frommethanogenesis might be needed, leading to the down-regulationof cellular biosynthesis pathways.

Immediate expression of the long-term salt response andadaptation genes under salt shock

To differentiate gene expression between long-term saltadaptation and immediate salt shock response, the 14 differ-entially expressed long-term salt response and adaptationgenes were used as probes to survey their expression immedi-ately after osmotic shock of cultures of Methanohalophilusportucalensis. Growth of Methanohalophilus portucalensisunder increased salt shock (from 2.1 to 3.1 mol/L of NaCl)and decreased salt shock (from 2.1 to 0.9 mol/L of NaCl) isshown in Fig. 1B. Immediately after a hyperosmotic shock, alag in growth was observed; this was not observed in the con-trol (from 2.1 to 2.1 mol/L of NaCl) and in hypo-osmoticallyshocked cultures (Fig. 1B). Among all 14 of the differentiallyexpressed genes, only SU12312 (putative clpB), SU34312(putative cbiD, cbiE), and SU26311 (putative mtmBC) couldbe detected by Northern hybridization. The other differen-tially expressed long-term salt response and adaptationgenes did not respond immediately after salt shock. Theymight only be expressed for salt adaptation or need morethan 2 h for expression, and (or) the transcripts might betoo rare to be detected.

In the SU12312 (putative clpB) response to immediatesalt shock, gene expression increased 2.01-fold within

Fig. 3. Placement of the differentially expressed genes related to methanogenesis, the unique energy-acquiring process of Methanoarchaea.

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Fig. 4. Transcriptional levels of SU26311 (mtmC, mtmB), SU11312(mttP), SU34312 (cbiD), and SD44094 (MCH gene) from Metha-nohalophilus portucalensis during long-term salt adaptation wereassessed by Northern blotting. Total RNA (36 mg) was subjected todenaturing agarose gel electrophoresis. After transfer to nylonmembranes, RNA was hybridized with the SU26311 probe (A) andthe 16S rRNA probe (B). The relative transcript levels of (C)SU26311, (D) SU11312, (E) SU34312, and (F) SD44094 fromMethanohalophilus portucalensis during salt shock were calculatedwith TINA software.

Fig. 5. Transcriptional levels of SU12312 (putative clpB) fromMethanohalophilus portucalensis during salt shock were analyzedby Northern blotting. Total RNA (36 mg) was subjected to denatur-ing agarose gel electrophoresis. After transfer to nylon membranes,RNA was hybridized with the clpB-2.6 probe (A) and the 16SrDNA probe (B). The relative transcript levels of (C) SU12312(clpB), (D) SU34312 (putative cbiDE), and (E) SU26311 (mtmCB)from Methanohalophilus portucalensis during salt shock were cal-culated with TINA software. White bars, relative transcript levelsafter 60 min incubation; gray bars, relative transcript levels after120 min incubation.



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60 min after increasing the external salt concentration andincreased 2.35-fold after decreasing the external salt concen-tration (Fig. 5A–5C; Shih and Lai 2007). After 120 min, theexpression of SU12312 (putative clpB) decreased to 1.36-fold of the original value following hyperosmotic stress andto 1.53-fold of the original value following hypo-osmoticstress (Fig. 5A–5C; Shih and Lai 2007). Clearly, the tran-scription of the chaperone ClpB gene was immediately sti-mulated and up-regulated to respond to the sudden saltstress. The relative transcript levels of the methanogenesis-related genes, including cobalamin biosynthesis protein(SU34312) and the monomethylamine corrinoid protein(SU26311), were largely reduced under salt up-shock(Fig. 5D, 5E). Except for the putative chaperone ClpB gene,the other immediately expressed genes (the unique energymetabolism related genes for methanogenesis) were alldown-regulated during hyper-salt shock. These results likelyrelate to the slower growth rate during hyperosmotic saltshock (Fig. 1B).

In conclusion, 14 clones with 19 genes from the func-tional groupings possibly involved in protein synthesis(SU37311), signal transduction (SU12317), protein refoldingand DNA damage repair (SU12312, SU25311, andSD18092), methanogenesis (SU34312, SU11312, SU26311and SD44094), sulfite reduction (SD38092), translation(SD17092), and aminoacyl-tRNA synthesis (SD44093) wereexpressed to facilitate long-term survival and adaptation ofMethanohalophilus portucalensis FDF1 under conditions ofchanging salinity. When cells meet the sudden salt shockstress, the signal transduction system would transmit thestress signal, then stress response genes such as clpB, mutS,and uspA would be expressed to repair the proteins andDNA damaged by salt stress. During the adaptation processfor long-term salt stress, the methanogenesis-related geneswould be up-regulated to supply sufficient energy to over-come the stress condition.

Bidle (2003) used RNA arbitrarily primed PCR to detect thedifferential expression of genes influenced by changing salinityin the extreme halophile Haloferax volcanii. Only 7 cloneswere analyzed, and 2 appeared to have some involvement withenvironmental stress signaling. We used a similar DDRT-PCRstrategy with 55 primer sets to study the differential gene ex-pression of long-term adaptation and immediate salt shockunder hyperosmotic stress (3.1 mol/L of NaCl), optimal externalsalt conditions (2.1 mol/L of NaCl), and under hypo-osmoticsalt stress (0.9 mol/L of NaCl) in the halophilic methanogenMethanohalophilus portucalensis and obtained 14 clones with19 genes. Our investigation not only explored the differencesin long-term salt adaptation and immediate salt shock responses,but also revealed that many facets of cellular metabolism, suchas DNA damage repair, protein chaperone systems, energy con-servation, protein synthesis, stringent response, and signal trans-duction, are all involved in osmoregulation. Compared with theresults of Coker et al. (2007), who performed a whole-genomeDNA microarray assay for changes in gene expression underdifferential growth conditions of Halobacterium sp. NRC-1 inresponse to salinity and temperature stresses we did not detectany differentially expressed ion or peptide transporters underlow- or high-salinity conditions. Interestingly, the transcripts of50S ribosomal protein L29 were down-regulated by low salinity

conditions in both Halobacterium sp. NRC-1 (Coker et al.2007) and Methanohalophilus portucalensis FDF1 (this study).

AcknowledgementsThis work was partially supported by grant NSC 93-2311-

B-005-016 from the National Council of Science, Taiwan,Republic of China.

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differentially expressed genes after hyper- and hypo-salt stress in the halophilic archaeon ...

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