The narA locus of Synechococcus sp. strain PCC 7942 consists of a cluster of molybdopterin biosynthesis genes

JOURNAL OF BACTERIOLOGY, 0021-9193/98/$04.0010Ma . 1998, p. 1200–1206 Vol. 180, No. 5 Copy igh © 1998, Ame ican Socie y o Mic obiology The na A Locus o Synechococcus sp. S ain PCC 7942 Consis s o a Clus e o Molybdop e in Biosyn hesis Genes LUIS M. RUBIO, ENRIQUE FLORES, AND ANTONIA HERRERO* Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Consejo Supe io de In es igaciones Cien ı´ icas—Uni e sidad de Se illa, Se ille, Spain Recei ed 15 Augus 1997/Accep ed 18 Decembe 1997 The na A locus equi ed o ni a e educ ion in Synechococcus sp. s ain PCC 7942 is shown o consis o a clus e o genes, namely, moeA,moaC,moaD,moaE, and moaA, in ol ed in molybdenum co ac o biosyn hesis. The p oduc o he moaC gene o s ain PCC 7942 shows homology in i s N- e minal hal o MoaC om Esche ichia coli and in i s C- e minal hal o MoaB o Mog. O e exp ession o he Synechococcus moaC gene in E. coli esul ed in he syn hesis o a polypep ide o 36 kDa, a size ha would con o m o a p o ein esembling a usion o he MoaC and MoaB o Mog polypep ides o E. coli. Inse ional inac i a ion o he moeA,moaC, moaE, and moaA genes showed ha he moeA-moa gene clus e is equi ed o g ow h on ni a e and exp ession o ni a e educ ase ac i i y in s ain PCC 7942. The moaCDEA genes cons i u e an ope on which is ansc ibed di e gen ly om he moeA gene. Exp ession o he moeA gene and he moa ope on was li le a ec ed by he ni ogen sou ce p esen in he cul u e medium. Ni a e is p obably he mos abundan sou ce o combined ni ogen o cyanobac e ial nu i ion, i s assimila ion being a p ocess closely linked o pho osyn hesis (9). Ni a e is ans- po ed in o he cyanobac e ial cell by a mul icomponen ans- po sys em o he ABC (ATP-binding casse e) ype (34). Once inside he cell, ni a e is educed o ammonium by wo sequen ial eac ions ca alyzed by ni a e educ ase and ni i e educ ase, espec i ely. Ammonium is inco po a ed in o ca - bon skele ons mainly ia he glu amine syn he ase/glu ama e syn hase cycle (9). In Synechococcus sp. s ain PCC 7942, he ni gene encoding ni i e educ ase (22), he n ABCD genes encoding he com- ponen s o he ni a e anspo sys em (34), and he na B gene encoding ni a e educ ase (1, 45) a e clus e ed oge he and cons i u e an ope on (24, 51). Two o he loci, na A and na C, in ol ed in ni a e educ ion in Synechococcus sp. s ain PCC 7942 ha e been iden i ied and cloned by means o complemen- a ion o ni a e educ ase-de icien , Tn901-induced mu an s wi h a gene lib a y om s ain PCC 7942 (20, 21). These loci a e no clus e ed oge he in he Synechococcus genome. Wi h ega d o egula ion, ammonium ac s as a nu i ional ep esso o he ni a e assimila ion sys em (9). The N cA p o ein ound in cyanobac e ia (9, 10, 52) ac s as a ansc ip ional ac i a o ha con ols he exp ession o cyanobac e ial genes subjec ed o ep ession by ammonium such as he ni ope on (23). Ni a e educ ases om cyanobac e ia a e monome ic mo- lybdoenzymes o abou 75 kDa ha use educed e edoxin as a physiological elec on dono (9). Molybdoenzymes o he han ni ogenase ca alyze ei he oxida i e hyd oxyla ions o educ i e dehyd oxyla ions, and i s molybdenum cen e is cons i u ed by a molybdenum-p e in co ac o in he o m o molybdop e in (MPT), molybdop e in guanine dinucleo ide (MGD), molybdop e in cy osine dinucleo ide, o o he s (40). In Esche ichia coli, he pa hway o molybdenum co ac o bio- syn hesis is he subjec o in ense esea ch. The genes espon- sible o he anspo o molybda e (modABC) (29), o MPT biosyn hesis (moaABCDE and moeAB) (33, 36, 44), and o assembly o molybdenum in o MPT (mog) (18) ha e been iden i ied and sequenced, as is also he case o mobA, which is in ol ed in he addi ion o GMP o MPT du ing he syn hesis o he MGD o m o he molybdenum co ac o (16, 17, 35). In cyanobac e ia, in o ma ion abou molybdenum co ac o s is sca ce. Some molybdenum co ac o , pa ially bound o a ca ie p o ein ha would s abilize he co ac o , has been e- po ed o be p esen in he soluble ac ion o Nos oc musco- um (3). In Anabaena a iabilis, he exis ence o common and speci ic genes o he syn hesis o he i on-molybdenum (o ni ogenase) and molybdenum co ac o s has been in e ed (28); in Anabaena sp. s ain PCC 7120, inac i a ion o a moeA gene leads o loss o ni a e educ ase ac i i y (41), while he moeB-like hesA gene ound downs eam o he ni HDK ope on seems o be necessa y o a aining ull ni ogenase ac i i y (5). Recen ly, he en i e genome o Synechocys is sp. s ain PCC 6803 has been sequenced (19), and a clus e o open eading ames (ORFs) showing simila i y o he moeA,moaA,moaC, and moaE genes o E. coli has been ound close o he ni gene. Al hough no iden i ied by he au ho s, ORF ss 1527, also ound in his gene clus e , would encode a pu a i e MoaD homolog (see below). In his epo , we desc ibe a gene ic analysis o he Synecho- coccus sp. s ain PCC 7942 na A locus and show ha i consis s o i e genes whose p oduc s a e essen ial o ni a e educ ion and would be in ol ed in he biosyn hesis o molybdop e in. MATERIALS AND METHODS O ganisms, g ow h condi ions, and plasmids. Synechococcus sp. s ain PCC 7942 was ou inely g own pho oau o ophically unde whi e ligh wi h shaking (90 pm) a 30°C in he BG11 medium (17.6 mM NaNO 3 as he ni ogen sou ce) desc ibed p e iously (43). When ammonium was used as he ni ogen sou ce, ni a e was omi ed and 4 mM NH 4 Cl and 8 mM N- is(hyd oxyme hyl)me hyl- 2-aminoe hane sul onic acid (TES)-NaOH bu e (pH 7.5) we e supplied, en- de ing medium BG11 0 NH 41 . Fo g ow h on pla es, he medium was solidi ied wi h sepa a ely au ocla ed 1% aga (Di co Labo a o ies). The pla es we e incu- ba ed a 30°C in he ligh . Synechococcus s ains as well as plasmids used in his wo k a e lis ed in Table 1. Mu an s ain FM6 was g own in BG11 0 NH 41 medium, and mu an s ains CSLM26, CSLM27, CSLM35, and CSLM40 we e g own in BG11 0 NH 41 medium supplemen ed wi h 10 mg o kanamycin/ml. Mu an s ains CSLM32 and CSLM34 we e g own in BG11 0 NH 41 medium supplemen ed wi h 2 mg o s ep omycin and 2 mg o spec inomycin/ml. Mu an * Co esponding au ho . Mailing add ess: Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Cen o de In es igaciones Cien ı´ icas Isla de la Ca uja, A da. Ame´ ico Vespucio s/n, E-41092 Se ille, Spain. Phone: 34-5 448 95 22. Fax: 34-5 446 00 65. E-mail: [email p o ec ed]. 1200 on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om s ain CSLM37 was g own in BG11 0 NH 41 medium supplemen ed wi h 2 mgo s ep omycin/ml, 2 mg o spec inomycin/ml and 10 mg o kanamycin/ml. Fo b-galac osidase assays, Synechococcus s ains we e g own in 70-ml glass ubes con aining 35 ml o he medium indica ed in each expe imen , bubbled wi h ai -CO 2 (98:2) a 30°C in he ligh . A he mid-exponen ial g ow h phase (cul u es wi h abou 3 o 5 mg o chlo ophyll/ml), samples con aining an amoun o cells co esponding o abou 2 mg o chlo ophyll we e wi hd awn o de e - mina ion o p o ein con en and b-galac osidase ac i i y. Fo ni a e educ ase assays and o g ow h a e de e mina ions, Synechococ- cus s ains we e g own in 70-ml glass ubes con aining 35 ml o BG11 0 NH 41 medium wi hou an ibio ics; a e ex ensi e washing, he cells we e ans e ed o and incuba ed in he medium in each expe imen bubbled wi h ai -CO 2 (98:2) a 30°C in he ligh . Fo isola ion o DNA and RNA, Synechococcus s ains we e g own in 240-ml glass lasks con aining 150 ml o BG11 medium bubbled wi h ai a 30°C in he ligh . Cul u es wi h a cell densi y co esponding o 3 o 5 mg chlo ophyll/ml we e used. E. coli DH5a, GM48, BL21, and HB101 we e g own in Lu ia-Be ani medium a 37°C wi h shaking (200 pm). Fo g ow h o E. coli on pla es, medium solid- i ied wi h 1.5% aga was used. An ibio ics we e used a s anda d concen a ions (2). G ow h a es we e es ima ed om he inc ease o p o ein concen a ion in he cul u es. The g ow h a e cons an co esponds o ln2/ d , whe e d ep esen s he doubling ime. Gene a ion o mu an s ains. Inse ions o ei he HincII-ended gene casse e C.S3 om plasmid pRL463 (8) o SmaI-ended gene casse e lacZ-C.K3 om plasmid pPE20 (48) a he S uI es ic ion si e, which is in e nal o he Synecho- coccus moeA gene, o plasmid pCSLM6 we e made o ende plasmids pCSLM32 (moeA::C.S3) and pCSLM27 (moeA::lacZ-C.K3), espec i ely. Simila inse ions we e made in o he moaC gene, dis up ing i a he HpaI es ic ion si e o pCSLM6 o ende plasmids pCSLM34 (moaC::C.S3) and pCSLM35 (moaC:: lacZ-C.K3). The moaA gene was also mu a ed by subs i u ion o a 335-bp, NheI DNA agmen a he 39 e minus o he gene, a e diges ion o pCSLM6 wi h NheI and ea men wi h he Klenow enzyme (2), by SmaI-ended gene casse e lacZ-C.K3, ende ing plasmid pCSLM26. The ni gene was mu a ed by subs i- u ion o a 99-bp, NaeI DNA agmen by SmaI-ended gene casse e lacZ- C.K3, ende ing plasmid pCSLM40 (Table 1). Res ic ion analysis o plas- mids pCSLM26, pCSLM27, pCSLM35, and pCSLM40 con i med ha he an ibio ic esis ance genes p esen in he inse ed gene casse es we e, in e e y case, in he same o ien a ion as he Synechococcus genes dis up ed by he gene casse e. Plasmids pCSLM26, pCSLM27, pCSLM32, pCSLM34, pCSLM35, and pCSLM40 we e ans e ed o Synechococcus sp. s ain PCC 7942 by means o ans o ma ion (12) o gene a ion o mu an s ains CSLM26, CSLM27, CSLM32, CSLM34, CSLM35, and CSLM40, espec i ely. Fo gene a ion o mu an s ain CSLM37, pCSLM34 was ans e ed o s ain CSLM26 (Table 1). A e ans o ma ion, cells we e sp ead on o ni ocellulose il e s (Nuclepo e; REC85) se successi ely a op BG11 0 NH 41 solid medium (incuba ed o 48 h) and BG11 0 NH 41 wi h he app op ia e an ibio ics (incuba ed o 3 weeks). Indi- idual colonies we e selec ed and, a e ecloning, main ained in BG11 0 NH 41 solid medium wi h an ibio ics. DNA manipula ions. PCR using EcoTaq DNA polyme ase (EcoGen S.R.L.), plasmid cons uc ions, DNA elec opho esis, isola ion o DNA agmen s om aga ose gels, liga ion, and ans o ma ion o E. coli we e ca ied ou by s anda d me hods (2). Res ic ion endonucleases we e used acco ding o he manu ac u - e ’s ecommenda ions o by s anda d me hods (2). Sequencing was pe o med in double-s anded DNA by he chain e mina ion me hod wi h a T7 Sequencing ki (Pha macia LKB) and [ 35 S]deoxyadenosine 59-(a- hio) iphospha e (1,000 o TABLE 1. Cyanobac e ial s ains and plasmids used in his wo k S ain o plasmid O igin and ele an cha ac e is ics Re e ence o sou ce S ains PCC 7942 Wild- ype Synechococcus s ain 42 CSLM26 Km de i a i e o s ain PCC 7942; moaA::lacZ-C.K3 gene usion This wo k CSLM27 Km de i a i e o s ain PCC 7942; moeA::lacZ-C.K3 gene usion This wo k CSLM32 Sm Sp de i a i e o s ain PCC 7942; gene casse e C.S3 inse ed in o he moeA gene This wo k CSLM34 Sm Sp de i a i e o s ain PCC 7942; gene casse e C.S3 inse ed in o he moaC gene This wo k CSLM35 Km de i a i e o s ain PCC 7942; moaC::lacZ-C.K3 gene usion This wo k CSLM37 Km Sm Sp de i a i e o s ain CSLM26; double mu an (gene casse e C.S3 inse ed in o he moaC gene and moaA::lacZ-C.K3 gene usion) This wo k CSLM40 Km de i a i e o s ain PCC 7942; ni ::lacZ-C.K3 gene usion This wo k FM6 De i a i e o s ain PCC 7942; ansposon Tn901 inse ed in o he na A locus; ans o mable o he wild- ype pheno ype by plasmid pNR12 26 Plasmids pBluesc ip SK(1) Cloning ec o S a agene pCSLM6 4,758-bp XhoI DNA agmen om pNR1211 cloned in o he XhoI si e o pBluesc ip SK(1) This wo k pCSLM8 Synechococcus sp. s ain PCC 7942 ni gene cloned in o he NcoI si e o exp ession ec o pT c99A 46 pCSLM26 De i a i e o pCSLM6; 335-bp NheI DNA agmen in e nal o he moaA gene in pCSLM6 e- placed by he lacZ-C.K3 ansc ip ional epo e casse e om pPE20 (see below); used o gen- e a e mu an s ain CSLM26 This wo k pCSLM27 De i a i e o pCSLM6; lacZ-C.K3 ansc ip ional epo e casse e om pPE20 inse ed in o he S uI si e o he moeA gene; used o gene a e mu an s ain CSLM27 This wo k pCSLM32 De i a i e o pCSLM6; gene casse e C.S3 inse ed in o he S uI si e o he moeA gene; used o gene a e mu an s ain CSLM32 This wo k pCSLM34 De i a i e o pCSLM6; gene casse e C.S3 om pRL463 (see below) inse ed in o he HpaI si e o he moaC gene; used o gene a e mu an s ain CSLM34 This wo k pCSLM35 De i a i e o pCSLM6; ansc ip ional epo e casse e lacZ-C.K3 om pPE20 inse ed in o he HpaI si e o he moaC gene; used o gene a e mu an s ain CSLM35 This wo k pCSLM40 De i a i e o pCSLM8; a 99-bp NaeI DNA agmen in e nal o he ni gene in pCSLM8 subs i- u ed by he lacZ-C.K3 ansc ip ional epo e casse e om pPE20; used o gene a e mu an s ain CSLM40 This wo k pCSLM43 Synechococcus sp. s ain PCC 7942 moaC gene cloned in o he BamHI si e o he exp ession ec o pGEX-4T-2 (see below) This wo k pGEX-4T-2 Exp ession ec o o he p oduc ion o GST- used p o eins Pha macia pNR12 Cosmid con aining 19-kb SalI DNA agmen om he genome o s ain PCC 7942 ha includes he na A locus 20 pNR1211 XhoI DNA agmen o 4,758 bp om pNR12 including he na A locus cloned in o he XhoI si e o pACYC177 20 pPE20 P omo e less lacZ gene ollowed by gene casse e C.K3 ( ansc ip ional epo e casse e) cloned in o he BamHI si e o pUC18/19 48 pRL463 Plasmid pUC18/19 con aining he C.S3 gene casse e cloned in o he BamHI si e o he L.EHE1 polylinke 8 pT c99A Exp ession ec o Pha macia VOL. 180, 1998 moeA-moaCDEA GENE CLUSTER IN A CYANOBACTERIUM 1201 on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om 1,500 Ci/mmol). Bo h s ands o he DNA we e sequenced. Compu e sea ching o homologies was made by using he FASTA and TFASTA algo i hms included in he Gene ics Compu e G oup package (7). Isola ion o DNA om Synecho- coccus s ains was pe o med essen ially as desc ibed by Cai and Wolk (6). Fo Sou he n blo s, es ic ion endonuclease-diges ed DNA o PCR p oduc s we e subjec ed o elec opho esis in aga ose gels and ans e ed o Genesc een Plus memb anes (Dupon ) as ins uc ed by he manu ac u e . P obes we e labeled wi h [a- 32 P]dCTP (3,000 Ci/mmol). P ehyb idiza ion and hyb idiza ion we e pe o med essen ially as desc ibed by F ı´as e al. (10) in a solu ion con aining 53 SSPE (0.8 M NaCl, 10 mM sodium phospha e, 1 mM EDTA [pH 7.4]), 53 Denha d ’s solu ion (47), 0.5% (w / ol) sodium dodecyl sul a e (SDS), and 100 mg o nonhomologous DNA/ml, unde high-s ingency condi ions a 65°C and unde low-s ingency condi ions a 55°C. RNA isola ion and RT-PCR analysis. Isola ion o RNA om Synechococcus sp. s ain PCC 7942 was pe o med as desc ibed by Mohamed and Jansson (32), wi h he modi ica ions desc ibed in Luque e al. (23). Samples we e ea ed wi h RNase- ee DNase I ( om bo ine panc eas; Boeh inge ) o elimina ion o any emaining DNA. Fo e o ansc ip ion-PCR (RT-PCR) expe imen s, 4 mgo s ain PCC 7942 o al RNA was mixed wi h 20 pmol o he oligonucleo ide 59-ATTGACCTTGAGGATCGGTAAGCG-39(complemen a y o nucleo ides 479 o 456 wi h espec o he ansla ion s a o he moaA gene) in he p esence o 50 mM T is-HCl, 8 mM MgCl 2 , 30 mM KCl, and 1 mM di hio h ei ol, pH 8.5 (AMV [a ian myeloblas osis i us] bu e ), hea ed o 2 min a 90°C, and imme- dia ely cooled down o 55°C. Then 1 mM each deoxynucleoside iphospha e, 20 U o RNA Gua d (Pha macia), and 50 U o AMV e e se ansc ip ase (Boeh - inge ) we e added, and he ex ension eac ion was de eloped o 1ha 54°C in a olume o 20 ml. To con ol o he p esence o con amina ing DNA, samples con aining 4 mg o he RNA p epa a ion, 20 pmol o he same oligonucleo ide, and 1 mg o RNase A (DNase ee; Boeh inge ) we e incuba ed, in a 20-ml eac ion olume, a 37°C o 1 h. PCR was ca ied ou wi h 35 ml o e o ans- c ip ion mix u e (dilu ed 10- old wi h 10 mM T is-HCl, 1 mM EDTA [pH 8.0]) o RNase- ea ed sample (see abo e) as he empla e, and oligonucleo ides 59-ATTGACCTTGAGGATCGGTAAGCG-39(complemen a y o nucleo ides 479 o 456 wi h espec o he moaA ansla ion s a ; same as abo e) and 59-GGTCTATCAGCGCGTTACTCAAGG-39(complemen a y o nucleo ides 74 o 97 wi h espec o he moaC ansla ion s a ) as p ime s. Con ol samples con aining he same oligonucleo ides and s ain PCC 7942 genomic DNA as he empla e we e un in pa allel. PCR consis ed o 35 cycles o empla e dena u - a ion a 95°C o 1 min, annealing wi h he oligonucleo ides o 1 min a 69°C, and DNA ex ension a 72°C o 2 min. One hal o each sample was esol ed by elec opho esis in 1% aga ose gels and ans e ed o memb anes o Sou he n blo analysis. A 0.53-kb, P uII DNA agmen om plasmid pNR1211 (Table 1), in e nal o he moa ope on (see Fig. 2), was used as he p obe. Exp ession o he Synechococcus moaC gene in E. coli. A 1,030-bp DNA ag- men , con aining he moaC gene and pa o he moaD gene, om an unme h- yla ed pCSLM6 plasmid es ic ed wi h ClaI and SacI and ea ed wi h Klenow enzyme was isola ed and cloned in o he BamHI si e o plasmid pGEX-4T-2 (Pha macia), made blun ended wi h Klenow enzyme, o ende plasmid pCSLM43. E. coli BL21 ca ying plasmid pCSLM43 was used o o e p oduc ion o he glu a hione S- ans e ase (GST)–MoaC usion p o ein a e induc ion wi h 1 mM isop opyl-b-D- hiogalac oside (IPTG). P epa a ion o cell ex ac s om BL21(pCSLM43), pu i ica ion o GST-MoaC p o ein by using bulk glu a hione- Sepha ose 4B in ba ch, and h ombin clea age o usion p o eins we e ca ied ou as ecommended by he manu ac u e . P o eins we e esol ed by SDS- polyac ylamide gel elec opho esis (PAGE) as desc ibed by Schlei and Wensink (49), using a 12% ac ylamide unning gel wi h an uppe 4% ac ylamide s acking gel. Enzyme assays and analy ical p ocedu es. Ni a e educ ase ac i i y was de- e mined by using di hioni e- educed me hyl iologen as he educ an in alkyl- ime hylammonium b omide-pe meabilized Synechococcus cells (14). b-Galac- osidase ac i i y was de e mined as desc ibed by Schae e and Golden (48) by colo ime ic assay wi h o-ni ophenyl-b-D-galac opy anoside (ONPG) (31). One uni o enzyma ic ac i i y co esponds o he o ma ion o 1 mmol o p oduc (ni i e o o-ni ophenol) pe min. P o ein quan i ica ions we e made by a mod- i ied Low y me hod (27), using bo ine se um albumin as he s anda d. Chlo o- phyll ade e mina ions we e made in me hanolic ex ac s as desc ibed by Mac- Kinney (25). Nucleo ide sequence accession numbe . The nucleo ide sequence o he moeA and moa genes epo ed in his pape will appea in he EMBL/GeneBank/DDBJ nucleo ide sequence da a lib a ies unde accession no. X99625. RESULTS Iden i ica ion o he na A locus. FM6 is a Tn901-induced mu an de i ed om Synechococcus sp. s ain PCC 7942 ha is impai ed in ni a e educ ase ac i i y. This mu an is eadily ans o mable o he wild- ype pheno ype by he 4.7-kb, XhoI DNA agmen om s ain PCC 7942 ha is cloned in plasmid pNR1211. This gene ic locus has been named na A (20). Fo localiza ion and iden i ica ion o he ni a e educ ion- ela ed gene(s) co esponding o he na A locus, genomic DNA om mu an s ain FM6 was es ic ed wi h he endonucleases XhoI, BglII, and P uII and subjec ed o Sou he n blo analysis using as a p obe he 4.7-kb, s ain PCC 7942 DNA agmen o pNR1211. Compa ed o s ain PCC 7942 DNA, mu an FM6 DNA showed a clea change in he hyb idiza ion pa e n in- dica i e o a Tn901 inse ion in o an 0.53-kb, P uII DNA agmen (Fig. 1). Sequencing o his P uII agmen e ealed he exis ence o wo ORFs. The pu a i e p oduc o one o hem showed homology o he la ge subuni o he MPT- con e ing ac o , he MoaE polypep ide o E. coli. Sequencing o he en i e Synechococcus sp. s ain PCC 7942 DNA agmen cloned in pNR1211 was ca ied ou by using se e al syn he ic oligonucleo ides as p ime s and plasmid pNR1211 as he empla e. Sequence analysis e ealed he ex- is ence o i e ORFs in ha agmen (Fig. 2). Pu a i e ibo- some binding si es could be ound only in on o ORF1, ORF4, and ORF5. No o he ORF was ound a e sequencing 300 bp o he Synechococcus DNA adjacen o he XhoI si e closes o ORF5, using as empla e cosmid pNR12 (20), whose inse includes ha o plasmid pNR1211. As summa ized in Table 2, ORF1 would encode a polypep- ide o 403 amino acids ha shows homology o MoeA poly- pep ides om E. coli (33) and Anabaena sp. s ain PCC 7120 (41). ORF2 would encode a 319-amino-acid polypep ide whose N- e minal hal shows homology o he MoaC polypep ide o E. coli (44) and whose C- e minal hal shows homology o MoaB and Mog polypep ides o E. coli (i should be no ed ha MoaB and Mog a e hemsel es homologous o each o he ) (44, 53). ORF3 would encode a polypep ide o 90 amino acids ha in i s C- e minal pa (amino acids 60 h ough 90) shows ho- mology o he MoaD polypep ide o E. coli (44). The pu a i e p oduc o ORF4 (165 amino acids) shows homology o he E. coli MoaE polypep ide (44). ORF5 would encode a poly- pep ide o 327 amino acids homologous o E. coli MoaA (44), FIG. 1. Localiza ion o Tn901 in he na A locus o mu an s ain FM6. Genomic DNA om he indica ed s ain was simul aneously diges ed wi h XhoI, BglII, and P uII and subjec ed o Sou he n blo analysis using he 4.7-kb XhoI inse o plasmid pNR1211 as a p obe. The posi ions and sizes (in kilobases) o some s anda ds a e indica ed o he le . 1202 RUBIO ET AL. J. BACTERIOL. on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om o Bacillus sub ilis Na A (11), and o Cnx2 om A abidopsis haliana (15). All Moa, Moe, and Mog polypep ides o E. coli ha e been shown o be in ol ed in he biosyn hesis o Mo-MPT (39). Because o he homologies desc ibed abo e, we p opose o name ORF1 as moeA, ORF2 as moaC (whose p oduc would bea a domain homologous o MoaC and ano he one homol- ogous o MoaB and Mog om E. coli), ORF3 as moaD, ORF4 as moaE, and ORF5 as moaA. No e idence o he exis ence in s ain PCC 7942 o o he genes homologous o hose p esen in he moeA-moa clus e he e desc ibed could be ob ained by means o Sou he n blo analysis unde low-s ingency condi ions using a 3,458-bp, EcoRV DNA agmen con aining mos o he moa-moe gene clus e (Fig. 2) as a p obe (no shown). O e exp ession o he Synechococcus moaC gene in E. coli. The MoaC polypep ide om s ain PCC 7942 was p oduced in E. coli BL21(pCSLM43) cells as a GST-MoaC usion p o ein o abou 60 kDa. A e pu i ica ion o he GST-MoaC p o ein and clea age wi h h ombin, a 36-kDa MoaC p o ein was e- leased (Fig. 3). This p o ein would di e om he na i e MoaC only in he i s wo amino acids, which we e changed om Me -Ile in he na i e p o ein o Gly-Se in he ecombinan p o ein. The size o MoaC polypep ide de i ed om he nu- cleo ide sequence o he Synechococcus moaC gene would be 33 kDa. Mu a ional analysis o he moeA and moa genes. Th ee o he genes ound in he inse o pNR1211 we e mu a ed by in i o gene casse e inse ion (Fig. 2 and Table 1) o es hei in ol emen in exp ession o ni a e educ ase ac i i y. Syn- echococcus s ains bea ing hose mu a ions in he moeA o moa genes we e ob ained by gene ic ans o ma ion o s ain PCC 7942 wi h plasmids bea ing he inac i a ed genes (see Fig. 2 and Ma e ials and Me hods o de ails). S ain CSLM26 bea s gene casse e lacZ-C.K3, which does no ca y ansc ip ional e mina o s (30), subs i u ing o he 335-bp NheI agmen in e nal o he moaA gene; s ain CSLM27 bea s gene casse e lacZ-C.K3 inse ed in o he S uI si e o he moeA gene; s ain CSLM32 bea s gene casse e C.S3, which ca ies ansc ip- ional e mina o s (38), inse ed in o he S uI si e wi hin he moeA gene; s ain CSLM34 bea s gene casse e C.S3 inse ed in o he HpaI si e wi hin he moaC gene; s ain CSLM35 bea s gene casse e lacZ-C.K3 inse ed a he same HpaI si e wi hin he moaC gene. We also cons uc ed a double mu an , s ain CSLM37, ha bea s he mu a ions p esen in s ains CSLM34 and CSLM26. The gene ic s uc u e o each o he mu an FIG. 2. S uc u e o a genomic egion o Synechococcus sp. s ain PCC 7942 ha con ains moeA and se e al moa genes. The iden i ies and o ien a ions o gene casse es inse ed a some es ic ion si es o he gene a ion o cyanobac e ial mu an s a e indica ed oge he wi h he CSLM denomina ion o he esul ing mu an s ain. The loca ion o Tn901 in mu an s ain FM6 is also indica ed. B, BglII; E, EcoRV; H, HpaI; N, NheI; P, P uII; S, S uI; X, XhoI. FIG. 3. Exp ession in E. coli and pu i ica ion o a GST-MoaC usion p o ein. (A) SDS-PAGE o a cell ex ac om s ain BL21(pGEX-4T-2) con aining GST (lane 1), cell ex ac om BL21(pCSLM43) con aining GST-MoaC (lane 2), pu i ied GST (lane 3), and pu i ied GST-MoaC p o ein (lane 4). (B) SDS-PAGE o pu i ied GST-MoaC p o ein (lane 1) and h ombin- ea ed GST-MoaC p o- ein (lane 2). The a owhead poin s o he ca. 36-kDa Synechococcus MoaC p o ein. Posi ions and sizes (in kilodal ons) o some molecula weigh ma ke s a e indica ed o he le o each panel. TABLE 2. ORFs in he na A locus o Synechococcus sp. s ain PCC 7942 ORF P oposed gene name Size (no. o amino acids) Homologous p o ein (o ganism) % Iden- i y a O e - lapping agmen b ORF1 moeA 403 MoeA (E. coli) 36 1–403 MoeA (Anabaena sp.) 32 1–403 ORF2 moaC 319 MoaC (E. coli) 47 1–145 MoaB (E. coli) 27 163–303 Mog (E. coli) 28 156–300 ORF3 moaD 90 MoaD (E. coli) 34 60–90 ORF4 moaE 165 MoaE (E. coli) 35 10–156 ORF5 moaA 327 MoaA (E. coli) 46 1–327 Na A (B. sub ilis) 31 1–327 Cnx2 (A. haliana) 35 1–327 a As deduced om he aligned agmen . b Posi ion in he Synechococcus polypep ide o he amino acids ha could be aligned wi h he indica ed homologous p o ein (o wi h a agmen o ha p o- ein). VOL. 180, 1998 moeA-moaCDEA GENE CLUSTER IN A CYANOBACTERIUM 1203 on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om s ains in he moeA-moa egion, as well as he absence o wild- ype ch omosomes in hem, was con i med by PCR anal- ysis using p ime s lanking each mu a ion (no shown). In ad- di ion, s ain FM6, which bea s Tn901 inse ed in o he 0.53-kb P uII agmen , was analyzed by PCR using se e al p ime s in e nal o he moaE and moaA genes. Resul s ob ained (no shown) indica ed ha Tn901 in s ain FM6 is inse ed in o he moaE gene (Fig. 2). S ains CSLM26, CSLM27, CSLM32, CSLM34, CSLM35, and CSLM37 we e unable o g ow wi h ni a e as he sole ni ogen sou ce (Table 3). Mo eo e , in con as o s ain PCC 7942, none o he mu an s exhibi ed ni a e educ ase ac i i y upon incuba ion in medium con aining ni a e as he sole ni- ogen sou ce (Table 3). Exp ession o he moeA and moa genes. Because we we e unable o de ec any moa ansc ip in Synechococcus sp. s ain PCC 7942 by means o No he n analysis, we s udied he ex- p ession o moa genes by subjec ing mRNA o RT-PCR (see Ma e ials and Me hods o de ails). Fo e o ansc ip ion, an oligonucleo ide complemen a y o sequences in e nal o he moaA gene was used as he p ime . The esul ing cDNA was hen ampli ied by PCR using he same p ime used o e o- ansc ip ion and ano he one ha should anneal a he begin- ning o he moaC gene. A e elec opho esis o he RT-PCR p oduc s on an aga ose gel, a band o he expec ed size, 2.1 kb, was obse ed. This band was e i ied by Sou he n blo analysis o hyb idize o a p obe o he moaE gene (Fig. 4, lane 3). Since his RT-PCR p oduc was s ic ly dependen on he p esence o RNA (Fig. 4, lane 2), i canno be due o ampli ica ion o con amina ing genomic DNA. These esul s sugges ha he moaC,moaD,moaE, and moaA genes o s ain PCC 7942 (Fig. 2) a e co ansc ibed in o a single mRNA species. I is wo h no ing ha o e lapping o e mina ion and s a codons is ound be ween he moaC and moaD genes, as well as be ween he moaE and moaA genes, o s ain PCC 7942. The e ec o he p esence o ammonium o ni a e in he ex acellula medium on he exp ession o he moeA and moa genes o s ain PCC 7942 was s udied by measu ing b-galac- osidase ac i i y in mu an s ains bea ing gene usions o he lacZ-C.K3 gene casse e. Ammonium-g own cells o mu an s ains CSLM26 (moaA::lacZ-C.K3), CSLM27 (moeA::lacZ- C.K3), CSLM35 (moaC::lacZ-C.K3) (Fig. 2), and, as a con ol, CSLM40 (ni ::lacZ-C.K3) (Table 1) we e incuba ed o 14 h in media con aining ei he ammonium o ni a e as he sole ni- ogen sou ce, and p o ein and b-galac osidase ac i i ies we e de e mined (Table 4). While he ac i i y le el o b-galac osi- dase in CSLM40 (ca ying he ni ::lacZ-C.K3 usion) was abou 4.2- old highe in ni a e- han in ammonium-incuba ed cells, only 1.7- o 1.9- old-highe le els we e ound in ni a e- han in ammonium-incuba ed cells o he s ains ca ying lacZ used o he moeA o moa genes. Da a in Table 4 should be in e p e ed wi h cau ion, howe e , since b-galac osidase basal le els can be ela i ely high in Synechococcus cells ca ying lacZ.Asan example, b-galac osidase ac i i y in ammonium-g own cells o s ain CSLM37, in which lacZ is inse ed wi hin he moa ope on abou 1.8 kb downs eam om he ansc ip ional e - mina o p esen in he C.S3 gene casse e, was abou 60 mU/mg o p o ein. No b-galac osidase ac i i y was de ec ed in wild- ype s ain PCC 7942. DISCUSSION Some yea s ago, h ee gene ic loci, na A,na B, and na C, whose mu a ion leads o impai men o ni a e educ ase ac- i i y in he unicellula cyanobac e ium Synechococcus sp. s ain PCC 7942 we e iden i ied and cloned (20, 21). The na B locus was la e iden i ied as he s uc u al gene o ni a e educ ase (1, 45) ha is pa o an ope on o ni a e assimila- FIG. 4. Sou he n analysis o RT-PCR p oduc s o he moa gene clus e o Synechococcus sp. s ain PCC 7942, using a moaE gene p obe. The p ime s used o he RT-PCR co esponded o DNA sequences o he moaA and moaC genes (see Ma e ials and Me hods o p ime s used). Lane 1, PCR-ampli ied s ain PCC 7942 genomic DNA; lane 2, PCR-ampli ied, RNase- ea ed s ain PCC 7942 o al RNA; lane 3, RT-PCR-ampli ied RNA. Samples o lanes 2 and 3 we e incuba ed wi h DNase be o e he RNase ea men o he e o ansc ip ion eac ion. The size o he ampli ied DNA agmen is indica ed o he le . TABLE 3. G ow h a es and ni a e educ ase ac i i ies o mu an s ains de i ed om Synechococcus sp. s ain PCC 7942 S ain Rele an geno ype G ow h a e (days 21 ) a Ni a e educ ase (mU/mg o p o ein) b Ammonium Ni a e PCC 7942 Wild ype 2.76 3.07 36.5 CSLM26 moaA::lacZ-C.K3 2.10 ,0.01 ,0.1 CSLM27 moeA::lacZ-C.K3 1.61 ,0.01 ,0.1 CSLM32 moeA::C.S3 2.90 ,0.01 ,0.1 CSLM34 moaC::C.S3 2.06 ,0.01 ,0.1 CSLM35 moaC::lacZ-C.K3 3.26 ,0.01 ,0.1 CSLM37 moaC::C.S3, moaA::lacZ-C.K3 1.80 ,0.01 ,0.1 FM6 c moaE::Tn901 1.80 ,0.01 ,0.1 a Ammonium-g own cells o he wild- ype and mu an s ains we e washed wice wi h medium lacking combined ni ogen and used o inocula e, a a inal concen a ion o 0.2 mg o chlo ophyll/ml, cul u es wi h he indica ed ni ogen sou ce. The cul u es we e bubbled wi h ai -CO 2 (98:2) a 30°C in he ligh o 24 h and hen dilu ed eigh old wi h esh cul u e medium wi h he same ni ogen sou ce. To es ima e he g ow h a e, p o ein con en was measu ed in aliquo s sampled pe iodically om he las se o cul u es. b Fo ni a e educ ase ac i i y de e mina ions, ammonium-g own cells o he wild- ype and mu an s ains we e washed wice wi h medium lacking combined ni ogen and incuba ed o 6hinmedium con aining ni a e as he sole ni ogen sou ce, bubbled wi h ai -CO 2 (98:2) a 30°C in he ligh . Ni a e educ ase ac i i y was hen de e mined in aliquo s o each cul u e. c Al hough he pheno ype o his s ain has been p e iously epo ed (26), i is included in his expe imen o he sake o compa ison. TABLE 4. b-Galac osidase ac i i ies o Synechococcus sp. mu an s ains CSLM40, CSLM26, CSLM27, and CSLM35 incuba ed wi h ammonium o ni a e a S ain Rele an geno ype b-Galac osidase (mU/mg o p o ein) Ammonium Ni a e CSLM40 ni ::lacZ-C.K3 113 (18) 475 (90) CSLM26 moaA::lacZ-C.K3 110 (11) 190 (40) CSLM27 moeA::lacZ-C.K3 106 (10) 200 (31) CSLM35 moaC::lacZ-C.K3 116 (22) 216 (19) a Ammonium-g own cells we e collec ed and ans e ed o media con aining he indica ed ni ogen sou ce. A e 14 h o incuba ion unde cul u e condi ions, o al p o ein and b-galac osidase ac i i y we e measu ed. Da a a e he medians o ou independen expe imen s (s anda d de ia ions a e shown in o pa en he- ses). 1204 RUBIO ET AL. J. BACTERIOL. on July 25, 2017 by USE/BTCA.GENERAL UNIVERSITARIA Se illah p://jb.asm.o g/Downloaded om ion genes which includes, in addi ion o na B, he ni and n ABCD genes (22, 24, 34). Up o now, no hing was known abou he ac ual unc ion o he genes in he na A o na C locus. We ha e mapped he si e o inse ion o Tn901 in a p e i- ously epo ed na A mu an o Synechococcus sp. s ain PCC 7942, s ain FM6 (26), and ha e de e mined ha he inac i- a ed genomic egion ca ies a clus e o genes whose pu a i e polypep ide p oduc s show simila i y o genes in ol ed in he biosyn hesis o he molybdenum co ac o o ni a e educ ase and o he molybdoenzymes. The genes iden i ied include ho- mologs o he moaA,moaB and mog,moaC,moaD,moaE, and moeA genes om E. coli and some o he biological sou ces. The syn hesis o all molybdenum co ac o s o molybdoen- zymes, excep he i on-molybdenum co ac o o ni ogenase, comp ises he syn hesis o Mo-MPT, which p esumably is com- mon o all molybdoenzyme-con aining o ganisms, and, in some cases, he pos e io o ma ion o di e en dinucleo ide a i- an s. Syn hesis o MPT akes place h ough he o ma ion o a sul u - ee p e in p ecu so , e med p ecu so Z, ha is hen sul u yla ed, leading o MPT and, a e inco po a ion o mo- lybdenum, o he Mo-MPT complex (39). The moaABC genes o E. coli, which a e pa o he moaABCDE ope on, a e in ol ed in he biosyn hesis o p ecu so Z. The moa ope on o Synechococcus sp. s ain PCC 7942 desc ibed in his wo k con- ains genes homologous o moaABC. While Synechococcus MoaA would be simila o o he MoaA (o Cnx2) p o eins, Synechococcus MoaC is unique in ha i esembles a usion p o ein o MoaC (N- e minal hal ) and MoaB o Mog (C- e minal hal ). Sequence simila i ies do no allow us o con- clude whe he he C- e minal hal o Synechococcus MoaC ep esen s a MoaB o a Mog domain. In Synechocys is sp. s ain PCC 6803, ORFs showing simila i y o moaA (sl 0901) and moaC (sl 0902) a e also clus e ed oge he (19). In his case, moaC would be used o mobA (a gene equi ed in a la e s ep o MGD biosyn hesis), bu he ac ual assignmen s o hese genes o he co esponding ORFs awai s expe imen al con i - ma ion. The moaDE genes o E. coli encode he wo subuni s o he so-called con e ing ac o o MPT syn hase ha adds di hiolene sul u s o p ecu so Z, hus gene a ing MPT (37). The Synechococcus moa ope on also con ains moaDE ho- mologs (Table 2 and Fig. 2), as is also he case o he Syn- echocys is genome (19). The pu a i e cyanobac e ial MoaD polypep ides a e peculia in ha hey show app eciable iden- i y o E. coli MoaD only in he 30 C- e minal amino acids; no ably, howe e , hese include he C- e minal Gly-Gly se- quence ha is hough o be essen ial o MoaD unc ion (39). On he o he hand, he moeB gene, which in E. coli encodes MPT syn hase sul u ylase ha ca alyzes he ans e o sul u o he MoaD subuni o MPT syn hase, is no p esen in he Synechococcus moeA-moa gene clus e . Finally, he MoeA p o- ein om s ain PCC 7942 would be simila o MoeA om E. coli ha has ecen ly been sugges ed o be in ol ed in ac i a ion o molybda e (13). The ac ha Synechococcus s ains bea ing mu a ions in he moeA-moa gene clus e a e de oid o ni a e educ ase ac i i y indica es ha his gene clus e is in ol ed in he syn hesis o he Mo co ac o o ni a e educ ase. In pa icula , ou o he genes in he clus e (moeA, moaC,moaE, and moaA) ha e been inac i a ed, he pheno- ype o he co esponding mu an s showing he in ol emen o hese genes in p oduc ion o an ac i e ni a e educ ase. I should be no ed ha bo h he lacZ-C.K3 gene casse e and ansposon Tn901 allow ansc ip ion o genes loca ed down- s eam om hem in a ansc ip ional uni (30, 46, 50). Resul s o RT-PCR p esen ed in his wo k show ha Syn- echococcus sp. s ain PCC 7942 syn hesizes mRNA molecules con aining a message o bo h he moaC and moaA genes. This inding indica es ha he moa genes in he iden i ied gene clus e can be exp essed as a single mRNA molecule om a p omo e loca ed ups eam om moaC, hus cons i u ing an ope on. On he o he hand, moeA, which is loca ed in he complemen a y DNA s and, would be exp essed indepen- den ly. In Synechococcus sp. s ain PCC 7942, s uc u al genes o ni a e assimila ion p o eins including ni i e educ ase, he componen s o he ni a e/ni i e anspo sys em, and ni a e educ ase, which cons i u e he ni ope on, a e subjec ed o ep ession by ammonium. Resul s p esen ed he e on he ex- p ession o he moeA gene and he moa ope on, compa ed o ha o he ni ope on, using b-galac osidase as a ansc ip- ional epo e indica e ha he exp ession o hese Mo co ac- o biosyn hesis genes is no egula ed by he ni ogen sou ce o he same ex en as he ni ope on is. This esembles he si ua ion wi h he moa genes in E. coli, whose exp ession is no a ec ed by he egula o y elemen Na L, a ni a e- esponsi e ac i a o o he syn hesis o ni a e educ ase, o by high le els o ni a e in he g ow h medium (4). 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