Characterization of an alcohol dehydrogenase from the cyanobacterium Synechocystis sp. strain PCC 6803 that responds to environmental stress conditions via the Hik34-Rre1 two-component system

JOURNAL OF BACTERIOLOGY, July 2009, p. 4383–4391 Vol. 191, No. 13 0021-9193/09/$08.00⫹0 doi:10.1128/JB.00183-09 Copy igh © 2009, Ame ican Socie y o Mic obiology. All Righ s Rese ed. Cha ac e iza ion o an Alcohol Dehyd ogenase om he Cyanobac e ium Synechocys is sp. S ain PCC 6803 Tha Responds o En i onmen al S ess Condi ions ia he Hik34-R e1 Two-Componen Sys em 䌤 † Rebeca Vidal, Luis Lo´pez-Mau y,‡ Miguel G. Gue e o, and F ancisco J. Flo encio* Ins i u o de Bioquímica Vege al y Fo osín esis, CSIC—Uni e sidad de Se illa, A . Ame´ ico Vespucio 49, E-41092 Se illa, Spain Recei ed 11 Feb ua y 2009/Accep ed 22 Ap il 2009 The sl 1192 (adhA) gene om Synechocys is sp. s ain PCC 6803 encodes a membe o he medium-chain alcohol dehyd ogenase/ educ ase amily. The gene p oduc AdhA exhibi s NADP-dependen alcohol dehyd o- genase ac i i y, ac ing on a b oad a ie y o a oma ic and alipha ic p ima y alcohols and aldehydes bu no on seconda y alcohols o ke ones. I exhibi s supe io ca aly ic e iciency o aldehyde educ ion compa ed o ha o alcohol oxida ion. The enzyme is a cy osolic p o ein p esen in pho oau o ophically g own Synechocys is cells. The exp ession o AdhA is enhanced upon he exposu e o cells o di e en en i onmen al s esses, al hough i is no essen ial o su i al e en unde such s ess condi ions. The induc ion o he exp ession o he adhA gene is dependen on he Hik34-R e1 wo-componen sys em, as i is se e ely impai ed in mu an s ains lacking ei he he his idine kinase Hik34 o he esponse egula o R e1. In i o DNA-p o ein in e ac ion analysis e eals ha he esponse egula o R e1 binds speci ically o he p omo e egion o he adhA gene. Medium-chain dehyd ogenases/ educ ases (MDR) cons i- u e a supe amily o alcohol dehyd ogenases ha ca alyze he e e sible NAD(P)-dependen oxida ion o alcohols o alde- hydes o ke ones. I includes a la ge numbe o s uc u ally ela ed p o eins, which ca alyze se e al ypes o enzyma ic ac i i y (23, 41, 44). Sc eening o comple e genome sequences has e ealed ha his amily is widesp ead, complex, and o ancien o igin (22, 44). MDR alcohol dehyd ogenases a e ound in mammals, plan s, ungi, and bac e ia (52). The alco- hol dehyd ogenases ul ill an as onishing a ie y o unc ions in cell me abolism (21), also being a key enzyme in e hanol gen- e a ion by Saccha omyces ce e isiae (6) and bac e ia (10). Fu - he mo e, he gene a ion o bio uels by pho oau o ophic mi- c oo ganisms is o g ea bio echnological in e es (43). Complemen a ion o a cyanobac e ium’s enzyme machine y wi h a speci ic exogenous gene(s) can esul in he abili y o gene a e bioe hanol om pho osyn he ically ixed CO 2 (11). No wi hs anding, cu en knowledge o cyanobac e ial alcohol dehyd ogenases is a he limi ed. In he cyanobac e ium Syn- echocys is sp. s ain PCC 6803 ( e e ed he e as Synechocys is), he sl 1192 gene encodes a pu a i e MDR alcohol dehyd oge- nase. Acco ding o in silico analyses (38, 44), he sl 1192 p o- ein has simila i y wi h wo sub amilies o MDRs: he yeas ADH amily (Y-ADH) and he cinnamyl ADH amily (CADH). Y-ADH- ela ed enzymes ha e ca abolic unc ions and a e in ol ed mainly in he me abolism o e hanol o sho - chain alcohols o which hey exhibi b oad subs a e speci ic- i y. CADH and ela ed enzymes, on he o he hand, pe o m anabolic unc ions and pa icipa e in biosyn he ic pa hways in plan s and bac e ia (5, 25, 44). In Synechocys is, he exp ession o sl 1192 is induced by osmo ic (35) o sal (48) s ess. In highe plan s, alcohol dehy- d ogenase ac i i y appea s o be in ol ed in ae obic me abo- lism unde ce ain s ess condi ions (26, 56) such as low em- pe a u e, wa e de ici , o ozone exposu e, bu i s unc ion emains unknown. A empe a u e dec ease seems o induce he accumula ion o alcohol dehyd ogenase mRNA in A abi- dopsis haliana (20), co n, and ice (9). In gene al, cyanobac e ia pe cei e and espond o en i on- men al changes by means o wo-componen egula o y sys- ems, a ubiqui ous signal ansduc ion pa hway ha ep esen s a p e alen signaling mechanism in bac e ia (8, 61). Two-com- ponen sys ems consis o a his idine kinase (Hik) and a e- sponse egula o (R e) and gene ally induce o ep ess he exp ession o speci ic genes in esponse o en i onmen al s im- uli. The his idine kinase au ophospho yla es a conse ed his- idine esidue in esponse o he en i onmen al signal and hen ans e s he phospha e g oup o a conse ed aspa a e esidue o he esponse egula o , which media es he ans e o he signal. In Synechocys is, di e en Hik-R e sys ems ha e been iden i ied as being egula o s o he esponse o di e en en- i onmen al s esses (37). A memb ane-bound his idine ki- nase, Hik33, is in ol ed in he pe cep ion o cold, sal , and osmo ic s ess (33, 35, 39, 48, 54). A cy osolic his idine kinase, Hik34, has been shown o be in ol ed in he pe cep ion o sal and hype osmo ic s ess (33, 39, 48) as well as hea shock (53). Speci ically, he couples Hik33-R e31, Hik10-R e13, Hik16 Hik41-R e17, Hik34-R e1, and a pu a i e Hik2-R e1 ha e been iden i ied as being elemen s in ol ed in he pe cep ion * Co esponding au ho . Mailing add ess: Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa—CSIC, A . Ame ico Vespucio 49, E-41092 Se ille, Spain. Phone: 34 954489509. Fax: 34 954460065. E-mail: [email p o ec ed]. † Supplemen al ma e ial o his a icle may be ound a h p://jb .asm.o g/. ‡ P esen add ess: Fission Yeas Func ional Genomics, Cance Re- sea ch UK, Wellcome T us Sange Ins i u e, Mo gan Building, Hinx- on, Camb idge CB10 1HH, Uni ed Kingdom. 䌤 Published ahead o p in on 1 May 2009. 4383 on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om and ansduc ion o signals p omo ed by hype osmo ic and sal s ess (39, 48). In he p esen wo k, a biochemical cha ac e iza ion o he sl 1192 p o ein (designa ed AdhA he e) om Synechocys is has been pe o med, e ealing ha he e ame ic 140-kDa en- zyme is ac i e owa d linea and a oma ic p ima y alcohols and ha i p e e en ially educes aldehydes a he han oxidizing alcohols. In addi ion, an ex ensi e analysis o he exp ession o he adhA gene has e i ied i s induc ion in esponse o hea shock, hype osmo ic s ess, sal s ess, and he addi ion o benzyl alcohol (BA). The Hik34-R e1 wo-componen sys em has been shown o play a ele an ole in he egula ion o he exp ession o he adhA gene unde hese s ess condi ions. A speci ic in e ac ion o R e1 wi h he p omo e egion o he adhA gene has also been demons a ed. In ligh o his inding and addi ional in o ma ion p esen ed he e, he physiological ole o AdhA in Synechocys is is discussed. MATERIALS AND METHODS S ains and cul u e condi ions. Synechocys is sp. s ain PCC 6803 cells we e g own pho oau o ophically on BG-11c medium (42) a 30°C unde condi ions o con inuous illumina ion (50 ␮Em ⫺2 s ⫺1 ) and bubbled wi h 1% CO 2 in ai . He e o ophic g ow h was pe o med as desc ibed p e iously (4). The ⌬hik34 and ⌬ e1 mu an s ains we e kindly p o ided by Iwane Suzuki (G adua e School o Li e and En i onmen al Sciences, Uni e si y o Tsukuba, Japan) (55). Fo p ecul u es o he ⌬adhA o ⌬ e1 s ain, kanamycin was added o a inal concen a ion o 100 ␮gml ⫺1 , and o p ecul u es o he ⌬hik34 s ain, spec i- nomycin was added o a inal concen a ion o 5 ␮gml ⫺1 . Expe imen s we e pe o med using cul u es om he mid-loga i hmic phase (op ical densi y a 730 nm o 0.6 o 0.7 and 3 o 5 ␮g chlo ophyll ml ⫺1 ). Fo high-ligh ea men , cell suspensions we e dilu ed wi h esh medium o an op ical densi y a 730 nm o 0.35, and he cul u es we e placed in a empe a u e-con olled chambe a 30°C and exposed o an i adiance o 500 ␮Em ⫺2 s ⫺1 . Unde hea shock condi ions, cul u es we e incuba ed in a wa e ba h a 45°C. Fo sal o hype osmo ic shock, NaCl o so bi ol, espec i ely, was added o he cell suspension o a inal con- cen a ion o 0.5 M. BA was added o a inal concen a ion o 30 mM. Fo oxida i e s ess, H 2 O 2 was added o a inal concen a ion o 1 mM. Glucose and 3-(3P,4P-dichlo ophenyl)-1,1P-dime hylu ea we e added o inal concen a ions o 5 mM and 10 ␮M, espec i ely. Esche ichia coli DH5␣and E. coli BL21(DE3) cells we e g own in LB b o h medium as desc ibed p e iously (46) and supplemen ed wi h 100 ␮gml ⫺1 o ampicillin o 50 ␮gml ⫺1 o kanamycin when equi ed. M9 minimal medium (46) was used o he me al dependence analysis o His-AdhA exp ession. Cons uc ion o he adhA dis up ion mu an . To inac i a e he adhA gene, he en i e coding egion was ampli ied by PCR o yield a agmen o 1,011 bp using p ime pai s ADH1 and ADH2. All he p ime s used in his wo k a e lis ed in Table 1. The PCR p oduc was cloned in o he pGEM-T ec o o gene a e plasmid pADH1. A DNA agmen con aining he np gene (neomycin phospho ans e ase) unde he con ol o he Synechocys is sp. s ain PCC 6803 psbA p omo e (13) was in oduced in o he HindIII si e o he coding egion o adhA (712 bp downs eam o he s a codon), gene a ing a cons uc ha was used o ans o m Synechocys is sp. s ain PCC 6803 (14). T ans o man s we e selec ed by sc eening o esis ance o 50 ␮gml ⫺1 o kanamycin in BG-11 medium pla es. Seg ega ion o he inac i a ed adhA gene was moni o ed by Sou he n blo ing using s anda d p ocedu es (46). To al DNA om cyanobac e ia was isola ed as p e iously desc ibed (7). Exp ession and pu i ica ion o he AdhA p o ein. To gene a e a plasmid o he exp ession o ecombinan N- e minal His- agged AdhA, he PCR agmen ob ained using p ime s ADH1 and ADH2 was diges ed wi h NdeI and SalI and cloned in o he same es ic ion si es o he pET-28a(⫹) ec o (No agen) o gene a e plasmid pETAdhA. The DNA agmen s cloned we e o ally sequenced o ensu e ha no modi ica ions in he nucleo ide sequence occu ed du ing cloning. Fo he exp ession o he AdhA p o ein, E. coli BL21(DE3) was ans- o med wi h plasmid pETAdhA. Fo la ge-scale p o ein p oduc ion, he ans- o man cells we e g own a 37°C in LB b o h o an op ical densi y a 580 nm o 0.5, 1 mM isop opyl-␤-D- hiogalac opy anoside (IPTG) was added, and he cul- u e was incuba ed o 2.5 h. The ea e , cells we e ha es ed by cen i uga ion, esuspended in 20 mM T is-HCl (pH 7.9) supplemen ed wi h 50 mM NaCl and 1 mM phenylme hylsul onyl luo ide, and b oken by sonica ion. The insoluble deb is was pelle ed by cen i uga ion a 18,000 ⫻g o 30 min, and he supe - na an was used immedia ely o p o ein pu i ica ion. AdhA was pu i ied by anion-exchange ch oma og aphy using DEAE-cellulose esin equilib a ed wi h 20 mM T is-HCl (pH 7.5) and applying a 0 o 0.5 M NaCl g adien . The elu ed ac ions con aining he p o ein we e loaded on o Ni-ni ilo iace ic acid (NTA) esin (No agen) and elu ed wi h 0.4 M imidazole. P o ein samples we e examined by 12% sodium dodecyl sul a e-polyac ylamide gel elec opho esis (SDS-PAGE) (46). Gel il a ion. A 200-␮l aliquo o na i e AdhA (30 ␮M) was applied o a Supe ose 6 10/300 GL (GE Heal hca e) column connec ed o a as -pe o mance liquid ch oma og aph (A ¨k a; Ame sham Biosciences) a 0.8 ml min ⫺1 . The column was p e iously equilib a ed wi h 30 mM T is-HCl (pH 8.0) supplemen ed wi h 0.5 M NaCl. The molecula mass s anda ds used we e bo ine hy oglobulin (670,000 Da), bo ine ␥-globulin (158,000 Da), chicken o albumin (44,000 Da), ho se myoglobin (17,000 Da), and i amin B 12 (1,350 Da). Exp ession and pu i ica ion o he R e1 p o ein. A DNA agmen co e ing om ⫹1 o⫹846 bp o sl 1783 (Cyanobase) was ampli ied by PCR using p ime s RR1 and RR2, diges ed wi h BamHI and Ps I, and cloned in o he same es ic- ion si es o ec o pQE-80L o gene a e plasmid pQR1, which was used o p oduce a ecombinan N- e minal His- agged R e1 p o ein. The DNA agmen cloned was o ally sequenced o ensu e ha no modi ica ions in he nucleo ide sequence occu ed du ing cloning. Fo he exp ession o he R e1 p o ein, IPTG-inducible E. coli s ain DH5␣ ans o med wi h plasmid pQR1 was g own a 37°C in LB b o h o an op ical densi y a 580 nm o 0.5, 1 mM IPTG was hen added, and he cul u e was incuba ed o 2.5 h. The ea e , cells we e ha es ed by cen i uga ion, esuspended in 20 mM T is-HCl (pH 7.9) supplemen ed wi h 50 mM NaCl and 1 mM phenylme hylsul onyl luo ide, and b oken by sonica ion. The insoluble deb is was pelle ed by cen i uga ion a 18,000 ⫻g o 30 min, and he supe na an was used immedia ely o p o ein pu i ica ion. R e1 was pu i ied by a ini y ch oma og aphy using Ni-NTA esin (No agen). Alcohol dehyd ogenase assay. Enzyme ac i i y was assayed a 25°C by moni- o ing changes in he abso bance o NAD(P)H a 340 nm in a Pha macia LKB Ul ospec Plus spec opho ome e wi h a 1-cm-ligh -pa h cu e e. Reac ions we e pe o med using 30 mM T is-HCl bu e (pH 8.0) con aining 0.2 mM NAD(P)H o he educ ion o aldehydes o 0.5 mM NAD(P) ⫹ o he oxida ion o alcohols. The kine ic cons an s we e de e mined by i ing he ini ial a es, calcula ed in he linea ange o p o ein concen a ion and eac ion ime, o he Michaelis-Men en equa ion, and he co esponding alues a e exp essed as he means ⫾s anda d de ia ions o h ee independen de e mina ions. RNA isola ion and No he n blo analysis. To al RNA was isola ed om 30-ml samples o Synechocys is sp. s ain PCC 6803 cul u es in he mid-exponen ial g ow h phase (3 o 5 ␮g chlo ophyll ml ⫺1 ) as p e iously desc ibed (17). Fo No he n blo ing, 15 ␮g o o al RNA was loaded pe lane in 1% (w / ol) aga ose dena u ing o maldehyde gels and ans e ed on o nylon memb anes (Hybond N ⫹ ; Ame sham Biosciences). Hyb idiza ion was pe o med a 65°C. The adhA, sll1106, and sll1107 p obes we e syn hesized by PCR using p ime pai s ADH1/ADH2, S061/S062, and S071/S072, espec i ely, and 32 P labeled wi h a andom-p ime ki (Ame sham Biosciences) wi h [␣- 32 P]dCTP (3,000 Ci mmol ⫺1 ). All il e s we e s ipped and ehyb idized wi h he cons i u i ely ex- p essed npB gene om Synechocys is sp. s ain PCC 6803 (59). P epa a ion o AdhA polyclonal an ibodies. A homogeneous sample o he p o ein was used o he p oduc ion o an ibodies a he Animal P oduc ion and Expe imen a ion Se ice o Se ille Uni e si y (Se ille, Spain). P epa a ion o cy osolic ex ac s om Synechocys is and Wes e n blo analysis. Cy osolic p o ein ex ac s om Synechocys is we e ob ained as desc ibed p e i- ously (40). Cy osolic p o eins (5 ␮g) om Synechocys is cul u es exposed o TABLE 1. P ime s used in his wo k Oligonucleo ide Sequence (5⬘33⬘) ADH1.....................ACTCTATTACATATGATTAAAGCC ADH2.....................TGACCATTCCACGTCGACAGAAGC ADP1......................TATTCATCGGATCCGATAACA ADP2......................GGCAGCGTAGGCTTGGATCCTGG PEX1 ......................TTCCGTTGGCTTCCAGGGCAGCG PEX2 ......................CCTCTGCCTTGTCTTCATCATCGT RR1 ........................CAAGGATCCGTGGGGTTGAGTTTGCTG RR2 ........................TAATTGCTGCAGACTTGTCATAGTTAT S061 ........................CATGGAGCCTAGTAATCACC S062 ........................GCGCTGGCATAAACCAGACC S071 ........................GATCGTTAGGATCATTGCG S072 ........................CGTCTAGAACTGTCTGCGGGC 4384 VIDAL ET AL. J. BACTERIOL. on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om di e en condi ions we e sepa a ed by SDS-PAGE and ans e ed on o ni o- cellulose memb anes. Memb anes we e incuba ed wi h dilu ed an i-AdhA (1: 1,000) o e nigh a 4°C. De ec ion was pe o med using ECL Plus Wes e n blo ing de ec ion eagen s (Ame sham Biosciences) acco ding o ins uc ions p o ided by he manu ac u e . Gel mobili y shi assay. A p obe was syn hesized by PCR using oligonucleo- ide pai ADP1 and ADP2, bo h con aining a BamHI es ic ion si e. The PCR p oduc was diges ed wi h BamHI, gene a ing a agmen om posi ions ⫺215 o ⫹22 wi h espec o he ansc ip ion s a poin o adhA, which was end labeled wi h [␣- 32 P]dCTP (3,000 Ci mmol ⫺1 ) by using he Klenow agmen o DNA polyme ase. The binding eac ion was ca ied ou using a inal olume o 30 ␮l con aining 0.1 ng o labeled DNA (2 pM) and 3 ␮g o poly(dI-dC) in 50 mM T is-HCl (pH 8.0), 100 mM KCl, 15 mM di hio h ei ol, 10% glyce ol, and di e en amoun s o p o ein. Reac ion mix u es we e incuba ed o 30 min a oom empe a u e, and samples we e loaded on o a nondena u ing 6% poly- ac ylamide gel. Elec opho esis was ca ied ou a 4°C and 180 V in 22 mM T is-bo a e–0.5 mM EDTA. The gel was ans e ed on o a Wha man 3-mm il e and d ied p io o au o adiog aphy. Fo compe i ion assays, a 222-bp DNA agmen om pGEM-T diges ed wi h SspI was used as nonspeci ic compe - i o DNA. P ime ex ension analysis. Oligonucleo ides PEX1 and PEX2 we e end la- beled wi h T4 polynucleo ide kinase and [␥- 32 P]ATP (3,000 Ci mmol) and used o p ime ex ension analysis o he adhA and sll1106 genes, espec i ely. P ime ex ension eac ions we e ca ied ou as p e iously desc ibed (31). One-hal o he eac ion mix u e was subjec ed o elec opho esis on a 6% polyac ylamide se- quencing gel, and he adhA and sll1106 p omo e egions we e subjec ed o a sequencing eac ion using he oligonucleo ides desc ibed abo e and plasmid pEXAdh as a empla e. RESULTS AdhA is a e ame ic medium-chain Zn-con aining alcohol dehyd ogenase. In o de o gain knowledge on he ac i i y and p ope ies o he AdhA p o ein, he adhA gene was cloned, and a His 6 - agged e sion o he p o ein was exp essed in E. coli cells. Homogeneous p epa a ions o AdhA we e ob ained by DEAE-cellulose ion-exchange ch oma og aphy ollowed by a ini y ch oma og aphy on Ni-NTA (Fig. 1A). Pu i ica ion a - emp s using ex ac s o E. coli cells g own on M9 chemically de ined medium e ealed ha he p esence o di alen me als in he medium was a equisi e o he a ailabili y o he enzyme in a soluble o m (Fig. 1B). Thus, when me als we e absen om he g ow h medium, exp essed AdhA did no appea in he soluble ac ion bu appea ed he e, howe e , i he me- dium was supplemen ed wi h zinc, cobal , o e ous ions (Fig. 1B). Di e ences in he speci ic ac i i ies o he enzyme pu i ied om cul u es g own on media wi h di e en me al con en s showed ha AdhA can use ei he zinc o cobal wi h analogous e ec i enesses (2 ⫾0.2 o 1.9 ⫾0.12 mU 䡠mg o al p o ein ⫺1 , espec i ely), while e ous ion was less e icien (0.3 ⫾0.05 mU 䡠mg o al p o ein ⫺1 ). Analysis o he amino acid se- quences (see Fig. S1 in he supplemen al ma e ial) e ealed he p esence o conse ed esidues in ol ed in he binding o bo h ca aly ic (Cys 55 , His 78 , Cys 176 , Asp 58 , and Glu 79 ) and s uc u al (Cys 111 , Cys 114 , Cys 117 , and Cys 125 ) zinc ions (23, 30, 52). To es ablish he qua e na y s uc u e o he p o ein, gel il a ion on a Supe ose 6 10/300 GL column we e pe o med using pu i ied p epa a ions o ecombinan AdhA, and an ap- pa en molecula mass o 140 kDa was de e mined. Taking in o accoun he polypep ide mass (36.7 kDa) o he ecombi- nan p o ein calcula ed om mig a ion on SDS-PAGE gels (Fig. 1A), a e ame ic s uc u e is sugges ed o he AdhA ac i e enzyme. A ho ough subs a e speci ici y analysis as well as a kine ic cha ac e iza ion o AdhA ha e been pe o med. As shown in Table 2, he enzyme was ac i e owa d a wide a ie y o p i- ma y alcohols and hei co esponding aldehydes, bu nei he ke ones no seconda y alcohols we e e ec i e subs a es. Ali- pha ic compounds we e e icien ly p ocessed by he enzyme, wi h he ac i i y inc easing wi h he leng h o he chain up o FIG. 1. Pu i ica ion and me al dependence o AdhA. (A) Coomas- sie-s ained SDS-PAGE gel o di e en ac ions co esponding o s eps o AdhA pu i ica ion (0.5 ␮g o al p o ein pe lane). M, molecula mass ma ke s. Lane 1, c ude ex ac om E. coli cells ha o e exp ess His- agged AdhA; lane 2, pooled DEAE-cellulose ch oma og aphy ac ions con aining His- agged AdhA; lane 3, pooled a ini y ch oma- og aphy (Ni-NTA) ac ions con aining His- agged AdhA. (B) Wes - e n blo ing o soluble ex ac s o AdhA-o e exp essing E. coli cells g own on M9 medium. ⫺, no me al added; Fe, 5 ␮M (NH 4 ) 2 Fe(SO 4 ) 2 ; Zn, 5 ␮M ZnSO 4 ; Co, 5 ␮M CoCl 2 . TABLE 2. Subs a e speci ici y o AdhA om Synechocys is c Reduc ion a Rela i e ac i i y (%) Oxida ion b Rela i e ac i i y (%) 0.1 mM 1 mM 1 mM 10 mM Ace aldehyde 1 23 E hanol 19 91 P opanal 26 87 P opanol 21 84 Bu anal 73 120 Bu anol 23 70 Pen anal 100 107 Pen anol 31 52 Hexanal 53 100 Hexanol 27 40 Oc anal 42 80 Oc anol 8 17 Decanal 21 22 Decanol 3 6 Benzaldehyde 30 14 Benzyl alcohol 42 79 Cinnamaldehyde 61 7 Cinnamyl alcohol 100 116 Ace one ND ND Phenyle hanol 15 24 3-Pen anone ND ND 2-Me hylbu anol 24 22 d-Glucose ND ND 3-Pen anol ND ND a The educ ion ac i i ies we e measu ed using 30 mM T is-HCl (pH 8.0) con aining 0.1 mM o 1 mM subs a e in he p esence o 0.2 mM NADPH. The ac i i y owa d 0.1 mM pen anal (67.7 U mg ⫺1 p o ein) was conside ed o be 100%. b The oxida ion ac i i ies we e measu ed using 30 mM T is-HCl (pH 8.0) con aining 1 mM o 10 mM subs a e in he p esence o 0.5 mM NADP ⫹ . The ac i i y owa d 1 mM cinnamyl alcohol (26.1 U mg ⫺1 p o ein) was conside ed o be 100%. c ND, no de ec ed. VOL. 191, 2009 ALCOHOL DEHYDROGENASE IN CYANOBACTERIA 4385 on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om i e ca bon a oms and dec easing o longe chains. A oma ic alcohols and aldehydes we e also subs a es o he enzyme, as was he case o he BA/benzaldehyde o cinnamyl alcohol/ cinnamaldehyde. B anched-chain p ima y alcohols, such as 2-me hyl-bu anol, we e p ocessed less e icien ly han he lin- ea subs a e (Table 2). The K m and k ca alues we e calcula ed o se e al subs a e pai s o alcohols and hei co esponding aldehydes, and hey a e shown in Table 3. The alues o k ca we e signi ican ly highe o aldehydes han o alcohols. Cin- namaldehyde and pen anal beha ed as he subs a es mos e icien ly p ocessed by he AdhA p o ein, wi h he highes k ca /K m a ios. The enzyme could use bo h NADPH and NADH as elec on dono s, al hough NADPH (K m ⫽0.025 mM) was p e e ed o NADH (K m ⫽1.3 mM) as he educ an o aldehydes. Equally, ha a ini y o NADP ⫹ was highe han ha o NAD ⫹ as an alcohol oxidan . Thus, i is likely ha AdhA wo ks o educe aldehydes o alcohols. Exp ession o adhA is induced unde a ious s ess condi- ions. The adhA gene was p e iously iden i ied by DNA mi- c oa ay analysis o be a hype osmo ic-s ess- and sal s ess- inducible gene (35, 48). Using No he n blo hyb idiza ion (Fig. 2), we ha e con i med ha he le el o ansc ip ion o adhA inc eases in esponse o ei he hype osmo ic s ess (0.5 M so bi ol) o sal s ess (0.5 M NaCl). In o de o de e mine whe he adhA exp ession is speci ically enhanced in esponse o he osmo ic s ess induced by he addi ion o so bi ol o NaCl o as a pa o a gene al s ess esponse, Synechocys is cells we e exposed o di e en s ess condi ions such as hea shock, high-ligh condi ions, and oxida i e s ess, and changes in le els o adhA ansc ip s we e moni o ed. The exposu e o cells o a sudden inc ease in he ambien empe a u e (45°C) also esul ed in enhanced adhA ansc ip le els (Fig. 2). How- e e , nei he high-ligh ea men no oxida i e s ess (1 mM H 2 O 2 ) had an in luence on he ansc ip ion o he adhA gene (da a no shown). I was p e iously epo ed (19) ha many o he genes ha espond o hea shock a e also induced when BA, a memb ane- luidizing agen (18), is added o he cul u e medium. As shown in Fig. 2, we ha e con i med ha BA also induces adhA gene exp ession, al hough he kine ics o adhA ansc ip induc ion we e di e en . Thus, he induc ion o adhA exp ession was ansien unde condi ions o hea shock exposu e, wi h he maximum ansc ip le el being eached in less han 1 h and e u ning o he s eady-s a e le el in 2 h (Fig. 2). Howe e , o he esponse o BA, he maximum ansc ip le el was eached a 5 h. On he o he hand, he maximum le el o adhA ansc ip was eached in less han 1 h and hen dec eased a 2 h unde condi ions o sal s ess. In esponse o hype osmo ic shock, he kine ics we e e y simila o hose o sal s ess, bu he dec ease in he ansc ip le el a 2hwas ollowed by ano he inc ease o each he maximum le el a 5 h. In bo h cases, he ansc ip ion o he adhA gene emained induced a leas 8 h a e ea men and e u ned o basal le els a e 24 h (da a no shown). In addi ion o he in o ma ion on he induc ion o he exp ession o adhA, Fig. 2 includes also da a o he exp ession o he nea by sll1106 and sll1107 genes, which exhibi ed a pa e n analogous o ha o adhA. This ci cums ance is conside ed below. In o de o analyze he ela ionship be ween adhA ansc ip and AdhA p o ein le els, Wes e n blo expe imen s we e pe - o med. A basal amoun o AdhA was de ec ed in Synechocys is cells g own unde pho oau o ophic condi ions. As shown in FIG. 2. No he n blo ing analysis o he s ess-inducible exp ession o he adhA, sll1106, and sll1107 genes. Synechocys is sp. s ain PCC 6803 cells a he exponen ial phase o g ow h we e subjec ed o a ious condi ions, and o al RNA was ex ac ed a di e en ime poin s. Fi een mic og ams o o al RNA was dena u ed, sepa a ed by elec opho esis in a 1.2% aga ose gel, blo ed, and hyb idized wi h p obes o he adhA, sll1106, and sll1107 genes. The il e s we e s ipped and hyb idized wi h an npB gene p obe as a loading con ol. All p obes we e syn hesized as desc ibed in Ma e ials and Me hods. Th ee independen expe imen s we e pe o med, and da a plo ed a e he a e ages ⫾s anda d e o s. TABLE 3. Kine ic pa ame e s o AdhA om Synechocys is a Subs a e Mean K m (mM) ⫾SD a Mean k ca (min ⫺1 )⫾SD k ca /K m a io (min ⫺1 mM ⫺1 ) Ace aldehyde 0.510 ⫾0.050 9,458 ⫾138 18,545 Bu anal 0.110 ⫾0.008 9,398 ⫾181 84,664 Pen anal 0.075 ⫾0.002 9,547 ⫾185 123,295 Cinnamaldehyde 0.017 ⫾0.001 6,475 ⫾131 380,882 E hanol 18.800 ⫾3.300 1,641 ⫾33 87 Bu anol 3.280 ⫾0.100 1,023 ⫾21 312 Pen anol 1.540 ⫾0.060 789 ⫾15 512 Cinnamyl alcohol 0.270 ⫾0.010 3,647 ⫾73 13,507 NADPH 0.025 ⫾0.007 NADP 0.050 ⫾0.007 NADH 1.310 ⫾0.060 NAD 9.700 ⫾0.800 a Reac ions we e pe o med using 30 mM T is-HCl (pH 8.0). Fo he educ- ion o aldehydes and oxida ion o alcohols, 0.15 mM NAD(P)H and 0.4 mM NAD(P) ⫹ we e used, espec i ely. Kine ic cons an s o NAD(P) ⫹ and NAD(P)H we e de e mined wi h 100 mM e hanol and 25 mM ace aldehyde, espec i ely. 4386 VIDAL ET AL. J. BACTERIOL. on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om Fig. 3, he AdhA p o ein le el inc eased unde condi ions o sal and hype osmo ic s esses in acco dance wi h he obse ed induc ion o ansc ip le els. The kine ics o AdhA accumu- la ion di e ed depending on he s ess condi ion imposed (Fig. 3). While unde hype osmo ic condi ions, an inc ease in AdhA le els was eadily obse ed a e 2ho ea men , unde sal s ess condi ions, he p o ein s a ed o accumula e a e 8 h (Fig. 3). This di e ence could be explained by he ac ha p o ein syn hesis was p e iously epo ed o be nea ly blocked wi hin 4 h a e he onse o sal s ess (15). In con as , he amoun o he AdhA p o ein emained i ually unal e ed un- de condi ions o hea shock and upon he addi ion o BA. Induc ion expe imen s pe o med in he p esence o chlo am- phenicol con i med ha no deg ada ion o AdhA ook place unde any condi ion es ed (da a no shown). The sll1106 and sll1107 genes sha e he same exp ession pa e n as adhA.Immedia ely ups eam o he adhA gene in he opposi e DNA s and a e wo open eading ames, sll1106 and sll1107 (Fig. 4A). One o hem, sll1107, was also iden i ied, oge he wi h he adhA gene, as being a sal -inducible gene in a genome-wide DNA mic oa ay assay (48); bo h genes we e shown o be unde he con ol o he Hik34-R e1 wo-compo- FIG. 3. Wes e n blo ing analysis o he s ess-inducible exp ession o he AdhA p o ein. Synechocys is sp. s ain PCC 6803 cells a he expo- nen ial phase o g ow h we e subjec ed o a ious condi ions as indica ed. Cells we e ha es ed a he imes indica ed, and 5 ␮g o o al p o ein om soluble ex ac s was sepa a ed by 12% SDS-PAGE and subjec ed o Wes e n blo ing. FIG. 4. P ime ex ension analysis o adhA and sll1106 ansc ip s. (A) Schema ic ep esen a ion o he genomic egion co esponding o he adhA, sll1106, and sll1107 genes. (B) P ime ex ension analysis o adhA ansc ip s om Synechocys is cells exposed o 1h o30mMBA(lane 1), hea shock (45°C) (lane 2), 0.5 M NaCl (lane 3), and 0.5 M so bi ol (lane 4) and om nonexposed cells (lane 5). (C) P ime ex ension analysis o sll1106 ansc ip s om Synechocys is cells exposed o hea shock (45°C) (lane 1) and 0.5 M so bi ol (lane 2) and om nonexposed cells (lane 3). (D) Sequence o he adhA-sll1106 in e genic egion, wi h ansla ion s a codons in bold ace ype. The ansc ip s s a poin s a e ma ked wi h a ows. Pu a i e ⫺10 and ⫺35 boxes a e indica ed wi h ec angles. VOL. 191, 2009 ALCOHOL DEHYDROGENASE IN CYANOBACTERIA 4387 on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om nen sys em. The p oximi y be ween sll1106 and adhA and he co egula ion o sll1107 p omp ed us o es o a possible co- o dina ed egula ion o he sll1106, sll1107, and adhA genes. In o de o es his hypo hesis, il e s used o No he n hyb id- iza ions wi h adhA we e ehyb idized wi h p obes co espond- ing o sll1106 and sll1107. The esul s (Fig. 2) showing he sll1106 and sll1107 genes o be induced unde he same con- di ions ha induced he exp ession o adhA sugges a possible co egula ion in which he sll1106-adhA in e genic egion could con ain he p omo e and he egula o y sequences o he exp ession o he h ee genes. Howe e , ansc ip s o di e en sizes (516 nucleo ides o sll1106 and 777 nucleo ides o sll1107) we e de ec ed by No he n blo ing, indica ing ha bo h genes a e p obably ansc ibed unde he con ol o in- dependen p omo e s. On he o he hand, he sll1106 and adhA genes a e sepa a ed by a 200-bp egion. In o de o es ablish he loca ion o he adhA and sll1106 p omo e (s), s a ing poin s o adhA and sll1106 ansc ip s we e mapped by p ime ex ension expe imen s. One single ex ension p oduc , which appea ed as a double band, was ob ained o bo h genes when RNA ex ac ed om cells subjec ed o s ess condi ions was used (Fig. 4B and C). The adhA ansc ip ion s a si e was loca ed 69 bp ups eam o he ATG s a codon, while he sll1106 s a si e was loca ed 31 bp ups eam o he co espond- ing ATG s a codon (Fig. 4D). Bo h si es we e sepa a ed by a egion o 100 bp, which migh con ain he p omo e s o bo h genes. The wo-componen sys em Hik34-R e1 egula es he induc- ion o adhA exp ession in esponse o s ess condi ions. The adhA gene was p e iously iden i ied as being a sal -s ess- inducible gene unde he con ol o he Hik34-R e1 wo-com- ponen sys em (48). In o de o cla i y i he his idine kinase Hik34 and he esponse egula o R e1 egula e he exp ession o he adhA gene in esponse o he o he condi ions es ed (Fig. 2), he pa e n o exp ession o he adhA gene in wild- ype cells was compa ed o ha o ⌬hik34 and ⌬ e1 mu an s ains. As shown in Fig. 5, he exp ession le els o he adhA gene in esponse o hea shock o BA we e simila o bo h he ⌬hik34 and ⌬ e1 mu an s and di e ed om ha o he wild- ype s ain. In he case o hea shock, he induc ion o he exp es- FIG. 5. Exp ession o he adhA gene unde s ess condi ions in wild- ype (WT), ⌬hik34, and ⌬ e1 s ains. (A) No he n blo analysis. Cul u es a he exponen ial phase o g ow h we e subjec ed o a ious s ess condi ions, and o al RNA was ex ac ed a di e en ime poin s. Fi een mic og ams o o al RNA was dena u ed, sepa a ed by elec opho esis in a 1.2% aga ose gel, blo ed, and hyb idized wi h adhA. As a loading con ol, all he il e s we e ehyb idized wi h npB. (B) Kine ics o exp ession o he adhA ansc ip s. The alues in he g aphs indica e he a io o adhA ansc ip s in s essed cells o hose in con ol cells. Th ee independen expe imen s we e pe o med, and da a plo ed a e he a e ages ⫾ s anda d e o s. F, wild- ype s ain; ,⌬hik34 mu an s ain; Œ,⌬ e1 mu an s ain. 4388 VIDAL ET AL. J. BACTERIOL. on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om sion o he adhA gene was comple ely abolished in bo h mu- an s. In he case o BA, he e ec o ei he mu a ion was no iceable only a 5 h a e ea men (Fig. 5). When mu an cells we e exposed o ei he sal o hype osmo ic shock, he ea ly induc ion o he adhA gene (30 min a e exposu e o s ess) was abolished. In he case o he ⌬ e1 mu an , his si ua ion was main ained wi h ime, al hough his was no he case o he ⌬hik34 mu an . R e1 binds o he p omo e egion o he adhA gene. In o de o es i he esponse egula o R e1 a ec s he exp ession o he adhA gene di ec ly by in e ac ions wi h i s p omo e egion, a DNA binding shi assay was pe o med. A adiolabeled DNA agmen co esponding o he sll1106-adhA in e genic egion was incuba ed wi h inc easing amoun s o he pu i ied R e1 p o ein (see Ma e ials and Me hods). A single shi ed band ep esen ing a DNA-p o ein complex was obse ed upon he addi ion o R e1 (Fig. 6A). Sequence-speci ic binding was e iden om compe i ion assays in he p esence o di e en concen a ions o ei he speci ic o nonspeci ic compe i o DNA. The R e1-dependen band shi was diminished in he p esence o a 200- old excess o he same unlabeled agmen bu was una ec ed by he p esence o an excess o he un e- la ed DNA agmen (Fig. 6B). DISCUSSION To he bes o ou knowledge, his is he i s epo o he molecula cha ac e iza ion o an alcohol dehyd ogenase in cya- nobac e ia. We show ha he p o ein encoded by he Synecho- cys is adhA gene is a zinc-dependen homo e ame ic enzyme wi h NADP-dependen alcohol dehyd ogenase ac i i y ac ing on a b oad a ie y o subs a es. The highe ca aly ic e iciency o Synechocys is AdhA o aldehyde educ ion, wi h pen anal and cinnamaldehyde as he p e e ed subs a es, poin s o he educ ion o a oma ic and medium-chain alipha ic aldehydes as a mos plausible in i o ac i i y o he enzyme. Synechocys is AdhA exhibi s a p e e en ial speci ici y o NADP(H) bu is also able o use NAD(H). This may be explained by he p es- ence o a h eonine a posi ion 226 (see Fig. S1 in he supple- men al ma e ial), which is mo e simila o he se ine esidue o he known NADPH-dependen ADH (52) han o he aspa - a e o glu ama e esidue o NADH-dependen ADH (29, 34). Thus, he Synechocys is AdhA enzyme sha es biochemical ea- u es wi h bo h Y-ADH enzymes, which a e e ame ic NAD- dependen enzymes, and CADH enzymes, which a e dime ic and NADP dependen . A sea ch pe o med wi h he AdhA sequence (sl 1192 p o- ein) as a que y using a non edundan p o ein sequence da a- base o he NBCI e ealed mo e han 60% iden i y wi h pu a- i e MDRs om gammap o eobac e ia. Among cyanobac e ia, homologous sequences showing abou 50 o 60% iden i y we e ound in some s ains o Mic ocys is and Synechococcus. Se- quence alignmen o AdhA wi h ela ed membe s o he MDR amily om bac e ia, plan s, and yeas s yielded di e en clus- e s, wi h bac e ial sequences o ming a closed g oup indepen- den om CADH and Y-ADH g oups (see Fig. S2 in he supplemen al ma e ial). In plan s, he unc ion o he CADH amily is ela ed o lignin biosyn hesis (5, 25). In bac e ia, om which lignin is absen , CADH-like genes a e ela ed o he biosyn hesis o lipids o he cell en elope (16, 62), bu i is possible ha hey ul ill o he speci ic unc ions. An alcohol dehyd ogenase om Acine obac e ha sha es 46% iden i y wi h he adhA gene om Synechocys is has been p oposed o be in ol ed in he biosyn hesis o wax es e s as cell ese es when cells a e g own on n-alkanes (57). On he o he hand, some Y-ADH- ela ed enzymes, which show b oad subs a e speci ici y, ul ill a unc- ion ela ed o p imo dial me abolic pa hways such as alcohol e men a i e ac i i y (49). A a ie y o di e en e men a ion pa hways yielding p oduc s such as CO 2 ,H 2 , o ma e, ace a e, lac a e, and e hanol ha e been epo ed o exis in cyanobac- e ia (50). A numbe o cyanobac e ia a e capable o he e o- ophic g ow h unde da k condi ions a he expense o ei he endogenous glycogen o exogenous subs a es. In mos cases, he e o ophic g ow h akes place unde ae obic condi ions, bu some o he acul a i e he e o ophic cyanobac e ia a e able o su i e unde da k anoxic condi ions ia e men a ion o he glucose de i ed om endogenous subs a es such as glycogen o some osmop o ec an compounds (50). Thus, AdhA migh pa icipa e in any o such e men a i e pa hways in Synecho- cys is. He e we show ha a soluble AdhA p o ein is p esen in Synechocys is cells and ha i s exp ession is enhanced upon he exposu e o cells o s ess condi ions. Al hough he le el o he adhA ansc ip inc eases, he AdhA p o ein le el emains unchanged unde condi ions o hea shock, making i unlikely ha AdhA plays a ole in he esponse o his condi ion. On he o he hand, he induc ion o adhA ansc ip ion unde condi- ions o sal s ess o hype osmo ic s ess is accompanied by an inc ease in he amoun o AdhA. Bo h sal and hype osmo ic s ess cause al e a ions in he physical in eg i y o he plasma memb ane (24) and a ec he ac i i y o pho osys em I (PSI) FIG. 6. Binding o R e1 o he adhA-sll1106 p omo e egion. (A) Mobili y shi assay o he adhA-sll1106 p omo e -ope a o egion wi h inc easing amoun s o R e1. (B) Compe i i e assays we e pe - o med wi h R e1 (1.6 ␮M) in he p esence o ei he a 50- old (lanes 3 and 5) o a 200- old (lanes 4 and 6) excess o unlabeled sel -com- pe i o agmen (lanes 3 and 4) o unlabeled nonspeci ic compe i o agmen (lanes 5 and 6). VOL. 191, 2009 ALCOHOL DEHYDROGENASE IN CYANOBACTERIA 4389 on July 21, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om and pho osys em II in cyanobac e ia (1–3, 32), hus al e ing ATP p oduc ion and he NADP/NADPH balance. In Synecho- cys is, sal s ess has been epo ed o enhance PSI-dependen cyclic elec on low (58) ha gene a es ATP bu no NADPH (36), hus pa icipa ing in de e mining he ATP/NADPH a io when pho osyn hesis ope a es unde changing en i onmen al condi ions (45). The ac ha AdhA om Synechocys is oxi- dizes NADPH much mo e e icien ly han i educes NADP ⫹ suppo s a possible ole o his enzyme in oxidizing NADPH unde such condi ions and hus con ibu ing o he main e- nance o he ATP/NADPH balance, as was also p e iously p oposed o wo membe s o he CADH amily in Saccha o- myces (27, 28). We ha e compa ed he g ow h o an ⌬adhA mu an wi h ha o he wild- ype s ain unde a a ie y o condi ions. No di e ences in esponses o hea shock, sal , o hype osmo ic s ess ha e been obse ed. Bo h s ains exhibi analogous g ow h a es when es ed in he ligh unde au o ophic, mix- o ophic (added glucose), o pho ohe e o ophic [added glu- cose and 3-(3P,4P-dichlo ophenyl)-1,1P-dime hylu ea] condi- ions. Ne e heless, he adhA mu an s ain was unable o g ow he e o ophically (added glucose) in da kness, con a y o he wild- ype s ain (ou unpublished da a). These esul s s ongly sugges an in ol emen o AdhA in he e o ophic me abolism in Synechocys is, which calls o u he in es iga ion. The his idine kinase Hik34 is in ol ed in he egula ion o he exp ession o hea shock genes in Synechocys is (53). Fou wo-componen sys ems a e in ol ed in he pe cep ion and ansduc ion o sal and hype osmo ic s ess signals in Synecho- cys is, wi h he Hik34-R e1 sys em being he one esponsible o con olling he exp ession o he adhA and sll1107 genes unde condi ions o sal s ess (39, 48). The esul s ob ained in he p esen wo k conce ning he e ec o a mu a ion o ei he hik34 o e1 on he exp ession o he adhA gene show ha he Hik34-R e1 sys em is in ol ed in he ea ly induc ion o adhA exp ession unde condi ions o hea shock and hype osmo ic s ess, a ac no ye epo ed. Al hough his sys em is essen ial o he esponse o he adhA gene o hea shock, he enhance- men o ansc ip ion o adhA in esponse o sal s ess, hype - osmo ic s ess, o he p esence o BA is only in pa dependen on he ope a ion o he Hik34-R e1 sys em. Mo eo e , he di e ences obse ed be ween hik34 and e1 mu an s wi h e- ga d o adhA gene ansc ip ion in esponse o hype osmo ic and sal s ess sugges he in ol emen o an addi ional ac o in he ac i a ion o R e1. We ha e demons a ed ha he R e1 p o ein isola ed om E. coli binds speci ically o he p omo e egion o he adhA gene in i o. Al hough in gene al, i is accep ed ha esponse egula o s exis in wo con o ma ions, an inac i e s a e and an ac i e s a e, wi h phospho yla ion se ing o shi he equilib- ium owa d he ac i e s a e (60), ecen indings (12) suppo he con en ion o an in e media e con o ma ion be ween inac- i e and ac i e s a es (51). This is he case o he CheY esponse egula o , which binds o i s a ge in he unphospho - yla ed o m and displays a basal le el o kinase-independen ac i i y in i o (47). The phosphoaccep o domain o R e1 is a homolog o ha o CheY. The a ini y o he speci ic DNA sequence in he adhA p omo e o unphospho yla ed R e1 would be su icien o he obse ed binding. Ne e heless, he speci ic sequence o binding emains o be elucida ed. The ups eam di e gen ly ansc ibed sll1106 gene, which shows he same exp ession pa e n, could sha e he egula o y sequences wi h he adhA gene, as sugges ed by he p oximi y o hei co esponding ⫺35 boxes. Two epea ed palind omic GTTG sequences loca ed be ween he pu a i e ⫺35 boxes o bo h he adhA and sll1106 p omo e s (Fig. 4D) migh be he ac ual binding si es o R e1, allowing he ac i a ion o bo h p omo - e s. In silico analysis p edic s ha he sll1106 gene encodes a memb ane p o ein ha highly conse ed among cyanobac e ia. Un o una ely, he lack o in o ma ion abou i s unc ion ham- pe s he es ablishmen o any unc ional ela ionship be ween he sll1106 and AdhA p o eins. On he o he hand, he as- signed unc ion o he sll1107 gene p oduc , a ype IV pilus biogenesis p o ein, excludes a di ec unc ional ela ionship wi h he AdhA p o ein. In conclusion, Synechocys is posses a zinc-dependen alcohol dehyd ogenase (AdhA), which has a p e e ence o NADPH as a educ an and a oma ic and medium-chain alipha ic alde- hydes as oxidan s. The co esponding gene is up egula ed in esponse o ce ain s ess condi ions ia he Hik34-R e1 wo- componen sys em. I s speci ic ole in he he e o ophic g ow h o Synechocys is emains o be es ablished. ACKNOWLEDGMENTS This wo k was suppo ed by a g an om he Ramo´n A eces Foun- da ion ( o M.G.G.), g an s BFU2004-0635 and BFU2007-60300 om he Minis y o Science and Educa ion o Spain ( o F.J.F.), and unds o Jun a de Andalucia (BIO131 and BIO284). We acknowledge Iwane Suzuky o kindly p o iding he ⌬hik34 and ⌬ e1 mu an s ains. We acknowledge Anna Lindahl o he c i ical eading o he manusc ip . REFERENCES 1. Allakh e die , S. I., Y. Nishiyama, I. Suzuki, Y. Tasaka, and N. Mu a a. 1999. Gene ic enginee ing o he unsa u a ion o a y acids in memb ane lipids al e s he ole ance o Synechocys is o sal s ess. P oc. Na l. Acad. Sci. USA 96:5862–5867. 2. Allakh e die , S. I., A. Sakamo o, Y. Nishiyama, M. Inaba, and N. Mu a a. 2000. Ionic and osmo ic e ec s o NaCl-induced inac i a ion o pho osys ems I and II in Synechococcus sp. Plan Physiol. 123:1047–1056. 3. Allakh e die , S. I., A. Sakamo o, Y. Nishiyama, and N. 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