Isolation of ntrA-like mutants of Azobacter vinelandii

JOURNAL OF BACTERIOLOGY, May 1986, p. 541-544 Vol. 166, No. 2 0021-9193/86/050541-04$02.00/0 Copy igh C) 1986, Ame ican Socie y o Mic obiology Isola ion o n A-Like Mu an s o Azo obac e inelandii E. SANTERO, F. LUQUE, J. R. MEDINA, AND M. TORTOLERO* Depa men o de Gene ica, Facul ad de Biologia, Uni e sidad de Se illa, 41080 Se ille, Spain Recei ed 1 July 1985/Accep ed 10 Feb ua y 1986 A numbe o chlo a e- esis an mu an s o Azo obac e inelandii a ec ed in a gene al con ol o ni ogen me abolism we e isola ed. These mu an s could no u ilize dini ogen, ni a e, o ni i e as a ni ogen sou ce. The eason o his inabili y is ha hey we e simul aneously de icien in ni ogenase and ni a e and ni i e educ ase ac i i ies. They we e complemen ed by a cosmid ca ying a DNA agmen o A. inelandii able o complemen n A mu an s o Esche ichia coli, so hey seemed o be n A-like mu an s. Ni a e and molecula dini ogen a e con e ed by ni a e and ni i e educ ases and ni ogenase, espec i ely, o am- monia. Bo h p ocesses, ni ogen ixa ion and ni a e assim- ila ion, a e egula ed by he ni ogen sou ce a ailable in he g ow h medium. The egula ion o ni ogen ixa ion has been ex ensi ely s udied in Klebsiella spp. The ni ogen ixa ion gene clus e o Klebsiella spp. con ains a leas 17 ni genes, a anged in se e al ope ons, which a e equi ed o he syn hesis and ac i i y o he enzyme ni ogenase. The exp ession o hese genes is subjec o "ni ogen con ol" a wo le els. The ni LA ope on is esponsible o he p oduc ion o an ac i a- o , he ni A p oduc , and a ep esso , he ni L p oduc , which oge he egula e ansc ip ion om all ni p omo e s excep ha o he ni LA ope on i sel (3). T ansc ip ion o ni LA is in u n con olled by he ni ogen egula o y (n ) genes n A, n B, and n C, which exe a gene al con ol on ope ons subjec o ni ogen egula ion in en e ic bac e ia (9). The n A p oduc is also equi ed o gene ac i a ion by he ni A p oduc (12, 20). The ni LA p omo e is he e o e he p ima y a ge o he egula ion o ni ansc ip ion in esponse o he le el o ixed ni ogen in he g ow h medium. Azo obac e inelandii is a g am-nega i e soil bac e ium ha is able o educe bo h dini ogen and ni a e in ae obic condi ions. The ni ogen ixa ion sys em o Azo obac e spp. has no been as s udied as i has in Klebsiella spp. Ni oge- nase syn hesis is comple ely ep essed by excess ammo- nium. Many Ni mu an s ha e been cha ac e ized biochem- ically (17). The e a e indica ions ha con ol may occu h ough a posi i e e ec o , pe haps analogous o he ni A gene p oduc o Klebsiella pneumoniae. In ac , he ni A gene p oduc o K. pneumoniae can ac i a e he exp ession o ni genes in one mu an o A. inelandii unable o syn hesize any componen o ni ogenase (5). Compa ed o ni ogen ixa ion, a he li le is known abou ni a e assimila ion om bac e ia o he assimila o y ni a e- educing ype (14). Ni a e educ ase ac i i y le els in cell- ee ex ac s om hese bac e ia a e g ea ly in luenced by he ni ogen sou ce in he cul u e medium: ni a e educ ase con en is high in cells g own wi h ni a e o ni i e, hus indica ing he appa en ly inducible na u e o he enzyme (4). The ni a e educ ase o A. inelandii is inducible and binds o la ge enzyme pa icles (2). One chlo a e- esis an (Chl ) mu an lacking ni a e educ ase has been desc ibed (21). A p esen he e a e no epo s abou egula o y ni a e educ ase mu an s in his o ganism. The pu pose o his wo k was o isola e egula o y mu an s * Co esponding au ho . unable o ix dini ogen and o assimila e ni a e. He e we epo he isola ion o mu an s, selec ed on he basis o hei chlo a e esis ance, ha a e a ec ed in a gene al con ol o ni ogen me abolism. MATERIALS AND METHODS Bac e ial s ains. Table 1 lis s he bac e ia used, hei geno ypes o pheno ypes, and hei sou ces. Media and cul u e condi ions. Cul u es o A. inelandii we e g own a 30°C unde ae a ion in modi ied Bu k ni o- gen- ee medium (4). The inal EDTA and i on concen a- ions we e 0.1 mM. To a oid he p ecipi a ion o i on, we mixed neu alized EDTA wi h he FeSO4 solu ion and ae - a ed he mix u e igo ously o e nigh . When necessa y, he ni ogen- ee medium was supplemen ed wi h ammonium ace a e (1 g/li e ), po assium ni a e (0.8 g/li e ), sodium ni i e (0.2 g/li e ), o glu amine (1 g/li e ). Cul u es o Esche ichia coli we e g own a 30°C in Lu ia- Be ani medium (10). Enzyme assays. (i) Ni ogenase. Cul u es g own o he la e loga i hmic phase in ammonium ace a e medium we e cen- i uged, washed, and p eincuba ed in ni ogen- ee medium o 3 o 12 h o allow he de ep ession o ni ogenase syn hesis. Ni ogenase ac i i y was measu ed as ace ylene educ ion by in ac cells as desc ibed p e iously (22). (ii) Ni a e educ ase. Cul u es g own o he la e loga i h- mic phase in ammonium ace a e medium we e cen i uged, washed, and suspended in ni a e medium. To de e mine basal ni a e educ ase ac i i y, we emo ed a sample and incuba ed he emaining cul u e o 5 h o induce ni a e educ ase. Ni a e educ ase ac i i y was measu ed, by a modi ica ion o a p ocedu e desc ibed p e iously (4), as ni i e o med ei he by oluenized o non oluenized cells incuba ed a 30°C o 1 h. The eac ion mix u e con ained, in a inal olume o 1 ml, he ollowing: mo pholine- p opanesul onic acid (MOPS)-KOH (pH 7.0), 100 .Amol; KNO3, 10 ,umol; me hyl iologen, 0.15 ,umol; KCNO 1 ,umol; p o ein, 0.1 o 0.2 mg; and Na2S204 in 95 mM NaHCO3 0.8 mg. Ni i e was es ima ed by adding diazocoupling eagen s as desc ibed p e iously (8). (iii) Ni i e educ ase. Ni i e educ ase ac i i y was as- sayed, by a p ocedu e desc ibed p e iously (24), as he disappea ance o ni i e om in ac cells p e iously induced by ni a e in he same way as o ni a e educ ase. (i ) Glu amine syn he ase. Cells g own in ammonium ace- a e medium o e nigh we e washed and incuba ed o 0 o 60 min in Bu k medium o allow enzyme deadenylyla ion. Glu amine syn he ase was measu ed in cells pe meabilized by an alkyl ime hyl ammonium b omide mix u e by a 541 on July 26, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om 542 SANTERO ET AL. TABLE 1. Bac e ial s ains and plasmids S ain o plasmid Geno ype o pheno ype Sou ce S ains E. coli 1843 P o- Me - Nal /pJB3JI J. E. Be inge 5K esK modK Th - Leu- C. Kennedy onA supE(pCK1 o pCK3) JC5466 T p- ecA His- psE C. Kennedy Lac-/pRK2013 HB101 leuB p oA hi-I S C. Kennedy hsdR hsdM ecA supE lacZ(pLV50 o pLV72) ET9045 bs lacZ::ISI gy A C. Kennedy hu CKA glnF::TnIO A. inelandii UW Wild ype W. B ill UW6 Ni l- Ni ll- W. B ill UW (Ri ) Ri C. Kennedy AS22 and AS23 n A Chl Ri ' This wo k AS24 and AS25 n A Chi Ri ' This wo k AS27 and AS29 n A Chl Ri ' This wo k AS51 and AS52 n A Chi Ri ' This wo k AS54 and AS55 n A Chl Ri This wo k Plasmids pJB3JI T al Cmal Tc Ap J. E. Be inge pCK1 T a- Sm (Km ) ni AKp C. Kennedy p(ni j) Km pCK3 T a- Tc (Km ) ni AKp C. Kennedy p(nijB) Km pRK2013 T a+ Km C. Kennedy pRK2073 T al Spc Sm Tp (Tn7) C. Kennedy PLV50 n C Tc A. Toukda ian pLV72 n A Tc A. Toukda ian ans e ase assay wi h and wi hou 60 mM MgCl2 as p e i- ously desc ibed (6). P o ein de e mina ion. P o ein was es ima ed by a modi i- ca ion o he Low y p ocedu e (11). Isola ion o chlo a e- esis an mu an s. Chlo a e- esis an mu an s we e selec ed, a e mu agenesis o s ain UW (Ri ) wi h ICR 191 o N-me hyl-N'-ni o-N-ni osoguanidine, on solid glu amine o ammonium ace a e medium supplemen ed wi h po assium chlo a e (100 mM). Mu agenesis o pLV72. Cosmid pLV72 was mu agenized wi h TnS by in ec ing HB101 (pLV72) wi h X::TnS (18) and selec ing he ans e ence o kanamycin esis ance by using pRK2073 as a "helpe plasmid" and s ain ET8045 as a ecep o . Gene ic analysis. (i) Complemen a ion. In expe imen s o in agene ic conjuga ion, a de i a i e o R68-45, he Kms plasmid pJB3JI, was used as a ch omosomal ans e ec o . In expe imen s o in e gene ic conjuga ion, plasmids pCK1 and pCK3 and cosmids pLV50 and pLV72 we e mobilized by he T a+ plasmid pRK2013 om E. coli 5K o HB101, espec i ely, o A. inelandii s ains. In all cases, bipa en al o ipa en al ma ings we e pe o med on ammonium ace a e pla es a 30°C o 12 o 15 h. Ni + exconjugan s we e selec ed on solid Bu k ni ogen- ee medium. When necessa y, he dono wild ype was coun e selec ed in medium con aining i ampin (20 ,ug/ml). The ans e o pCK1 and pCK3 was selec ed by s ep omycin (10 ,ug/ml) and e acycline (10 ,ug/ml), espec i ely, on solid ammonium ace a e medium. The exp ession o kanamycin esis ance was de e mined by g ow h in medium supplemen ed wi h 1 ,ug o kanamycin pe ml. The ans e o pLV50 and pLV72 was selec ed by e acycline (10 ,g/ml) on solid ammonium ace a e medium. (ii) T ans o ma ion. T ans o ma ion o ch omosomal ma ke s was done wi h c ude DNA ex ac s as p e iously desc ibed (13). RESULTS Isola ion o mu an s de ec i e in dini ogen, ni a e, and ni i e assimila ion. Chlo a e- esis an colonies, selec ed as desc ibed in Ma e ials and Me hods, we e es ed o g ow h in ni ogen- ee medium and in media supplemen ed wi h ni a e, ni i e, ammonium ace a e, o glu amine. We iso- la ed 20 mu an s unable o use N2, ni a e, o ni i e as a ni ogen sou ce. The equency o hese mu an s among he chlo a e- esis an colonies was 1%. To ule ou double mu an s, we sea ched o spon aneous e e an s and pe - o med ans o ma ion expe imen s wi h wild- ype DNA c ude ex ac s. Re e an s o ans o man s able o g ow in ni ogen- ee medium o in ni a e-supplemen ed medium we e isola ed, and hei abili y o exc e e ni i e when g own in ni a e and hei sensi i i y o chlo a e in ni ogen- ee medium we e es ed. The equency o spon aneous e e - an s was he same in ni ogen- ee medium and in ni a e- supplemen ed medium: abou 7 x 10-8 o 8 x 10-8 in 19 o he 20 mu an s and 7 x 10-9 o 8 x 10-9 in he emaining 1 mu an . The majo i y o mu an s, 15, beha ed as single mu an s, hei e e an s and ans o man s eco e ing he ou wild- ype cha ac e is ic s simul aneously: u iliza ion o dini ogen and ni a e as sou ces o ni ogen, ni i e exc e- ion in ni a e medium, and chlo a e sensi i i y. We used 10 o hese single mu an s o la e s udies: AS22, AS23, AS24, AS25, AS27, AS29, AS51, AS52, AS54, and AS55. Gene complemen a ion. To classi y he 10 mu an s in complemen a ion g oups, we pe o med ma ings among all possible couples as desc ibed in Ma e ials and Me hods. The ch omosomal ans e ec o used was plasmid pJB3JI. This plasmid mobilizes A. inelandii ni genes a equencies anging om 10-4 o 10-5, simila o hose epo ed p e i- ously o R68.45 (23). We ob ained no Ni + exconjugan s om all hese ma ings. We made wo kinds o con ols. On he one hand, we c ossed he wild- ype s ain as he dono wi h all he mu an s as ecep o s, pla ed hem in ni ogen- ee medium, and ob ained Ni + exconjugan s in all cases, hus p o ing ha he 10 independen isola ed mu a ions a e ecessi e. On he o he hand, o e i y whe he mu a ions in di e en ni genes would gi e Ni + exconjugan s, we ma ed he mu an s wi h s ain UW1. Ni + exconjugan s we e ob ained in e e y case. S a ing om hese da a, we as- sumed ha he 10 mu an s we e a ec ed in he same gene, and we chose 5 mu an s o enzyma ic cha ac e iza ion. Enzyma ic cha ac e iza ion o mu an s. We measu ed ni- ogenase, ni a e and ni i e educ ase, and glu amine syn- he ase ac i i ies in i e AS mu an s (Table 2). Ni ogenase was no de ec ed in any o he mu an s. Basal ni a e educ ase ac i i ies we e pa ially educed in he mu an s, whe eas induced ni a e educ ase ac i i ies we e s ongly educed, showing ha he e was no induc ion by ni a e o ni a e educ ase in hese mu an s. In he same way, he e was no induc ion o ni i e educ ase. In con as , he glu amine syn he ase le el was no changed. The ade ylyl- a ed and deadenylyla ed ac i i ies measu ed immedia ely a e ans e o he cells om ammonium ace a e medium o ni ogen- ee medium, as desc ibed in Ma e ials and Me h- ods, we e simila in he mu an s and in he wild ype. The e J. BACTERIOL. on July 26, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om n A-LIKE MUTANTS OF A. VINELANDII 543 TABLE 2. Enzyme ac i i ies in Ch1 mu an s Ac i i y o : Ni a e educ asea Glu amine syn he asea S ain Ni i e educ aseb Ni ogenasea =Oh =5 h ( = 5h) Mn2 Mg2+ Mn2 /Mn2+ + Mg2+ ( =O0h) (= Oh) i-=Oh =l1h UW (Ri ) 75 0.27 4.68 15 190 63.7 2.98 1.13 AS22 NDC 0.02 0.05 2.12 139 53 2.62 1.07 AS23 ND 0.07 0.1 1.68 115.5 43.3 2.67 1.01 AS24 ND 0.03 0.08 0.48 121 39.9 3.03 2.12 AS27 ND 0.02 0.27 0.83 99.7 43.2 2.31 1.4 AS29 ND 0.02 0.05 0.93 138 67.6 2.04 0.74 a Ac i i y is epo ed as nanomoles o p oduc o med pe minu e pe millig am o p o ein. b Ac i i y is epo ed as nanomoles o ni i e disappea ed pe minu e pe millig am o p o ein. c ND, No de ec ed. we e no signi ican di e ences be ween he mu an s and he wild ype in he a io o adenylyla ed o deadenylyla ed ac i i ies, ei he a 0 o 60 min a e he change o medium. Complemen a ion o mu an s by pLV72 and pLV50. To de e mine whe he ou mu an s should be classi ied as gene al egula o y ni ogen me abolism mu an s (N ), we used wo cosmids, cons uc ed by A. Toukda ian and C. Kennedy a he Uni o Ni ogen Fixa ion in B igh on, England, which complemen n mu a ions in E. coli and K. pneumoniae (C. Kennedy, pe sonal communica ion). Cosmid pLV50 con ains A. inelandii DNA which co ec s n C mu a ions, and cosmid pLV72 con ains A. inelandii DNA which co ec s n A mu a ions. Nei he pLV50 no pLV72 is sel - ansmissible, bu each was mobilized by he T a+ plasmid pRK2013. E. coli s ains (HB101 and JC5466) and each o ou 10 mu an s we e ma ed as ios on ammo- nium ace a e aga pla es. The ans e o pLV50 and pLV72 was selec ed as desc ibed in Ma e ials and Me hods. T ans- conjugan s we e eplica ed on ni ogen- ee medium o check he es o a ion o he no mal pheno ype. The 10 mu an s analyzed we e complemen ed by pLV72. No mu- an s we e complemen ed by pLV50. We measu ed ni oge- nase and ni a e and ni i e educ ase ac i i ies in he Ni + ansconjugan s ob ained om he same i e mu an s ana- lyzed in Table 2. The enzyme ac i i ies o he ansconju- gan s we e simila o he wild- ype enzyme ac i i ies o he h ee enzymes assayed (Table 3). To de ini i ely es ablish ha he chlo a e- esis an mu an s we e a ec ed in an n A- like gene, we mu agenized cosmid pLV72 wi h TnS. Fou cosmids ca ying TnS inse ions, ob ained as desc ibed in Ma e ials and Me hods, lacked he abili y o complemen bo h n A mu an s o E. coli and n A-like mu an s o A. inelandii. TABLE 3. Enzyme ac i i ies in Chl mu an s ca ying he pLV72 cosmid Ac i i y o : S ain a Ni a e educ asea Ni i e educ aseb Ni ogenase ( 5h = Oh =5h ( = h) UW (Ri ) 75 0.27 4.68 15 AS22(pLV72) 65.3 0.4 3.09 12.1 AS23(pLV72) 54.8 0.22 3.42 12.9 AS24(pLV72) 61.3 0.55 2.87 12.8 AS27(pLV72) 47.3 0.47 4.49 10.9 AS29(pLV72) 56.7 0.2 4.94 15.1 a See Table 2, oo no e a. b See Table 2, oo no e b. E ec o he ni A p oduc in n A mu an s o A. inelandii. To s udy whe he he n A p oduc is equi ed o he no mal unc ion o he ni A p oduc in A. inelandii, as p e iously desc ibed in o he o ganisms, plasmids pCK1 and pCK3 we e ans e ed o n A A. inelandii mu an s. These wide- hos - ange plasmids ca y he ni A gene o Klebsiella spp. exp essed om a cons i u i e p omo o and a kanamycin esis ance gene exp essed om a ni A-dependen ni l p o- mo o (5). They di e in ha pCK1 is mul icopy, whe eas pCK3 has a low copy numbe . The ans e o pCK1 and pCK3 was selec ed as desc ibed in Ma e ials and Me hods. All ansconjugan s we e Ni - and Kms. As expec ed, he ans e o pCK1 and pCK3 o A. inelandii UW6, a molyb- denum-i on p o ein ni ogenase mu an , gi e Km anscon- jugan s, whe eas he ans e o hese plasmids o A. inelandii UW1, a egula o y ni ogenase mu an , ga e Ni + and Km ansconjugan s. F om hese esul s, we conclude ha in A. inelandii he n A p oduc is equi ed o he ni A p oduc o ac i a e ni genes. DISCUSSION A. inelandii is a e y in e es ing o ganism o s udy because i exhibi s he peculia abili y o ixing dini ogen in nonsymbio ic and obliga o y ae obic condi ions. Howe e , he gene ic analysis o A. inelandii has been hampe ed by he lack o e icien gene ans e sys ems. T ans o ma ion, he i s gene ans e sys em desc ibed in A. inelandii (1, 13), canno be eadily used o la ge-scale analysis. This di icul y has been su moun ed by he conjugal ans e o genes by means o p omiscuous plasmids belonging o he P1 incompa ibili y g oup (23). In A. inelandii i is easy o isola e an ibio ic- and an ime aboli e- esis an mu an s, bu i has p o ed exceed- ingly di icul o isola e ecessi e nonselec able mu an s (17). A possible eason o his di icul y is ha A. inelandii may ha e a minimum o 40 ch omsomes pe cell (19). Ou s a egy has been o sea ch o mu an s wi h al e ed ni ogen me abolism egula ion om among a la ge collec ion o mu an s isola ed by hei esis ance o chlo a e. This ap- p oach is based on he ollowing a ionale. Some o ms o he enzyme ni a e educ ase a e known o educe non oxic chlo a e o oxic chlo i e (16); consequen ly, s ains lacking ni a e educ ase ac i i y a e esis an o chlo a e. I ni a e educ ase syn hesis is subjec o ni ogen me abolism egu- la ion, some o he chlo a e- esis an s ains should be N -de ec i e mu an s. These mu an s would be easily iden- i iable as unable o assimila e ni a e, ni i e, and dini ogen. Ou esul s show ha abou 1% o chlo a e- esis an s ains a e N mu an s. These mu an s a e simul aneously de icien VOL. 166, 1986 on July 26, 2017 by USE/BCTA.GEN UNIVERSITARIAh p://jb.asm.o g/Downloaded om 544 SANTERO ET AL. o ni a e and ni i e educ ase and ni ogenase ac i i ies bu a e no mal o glu amine syn he ase ac i i y. This esul is ha dly su p ising, since he syn hesis o glu amine syn he- ase seems o be cons i u i e in A. inelandii (7). Mo e su p ising is he ac ha all he egula o y mu an s belong o a single complemen a ion g oup, he n A gene. By analogy wi h Klebsiella spp. i would be expec ed ha A. inelandii N mu an s should ep esen a leas wo di e en genes, n A and n C. In ac , he isola ion o Azospi illum b asiliensis mu an s ha ail o g ow ei he on dini ogen o on ni a e and ha a e complemen ed by he K. pneumoniae n C gene has been epo ed (15). Du ing he de elopmen o his wo k, o he s ob ained n C mu an s o A. inelandii (A. Toukda ian and C. Kennedy, EMBO J., in p ess) ha we e Ni . Howe e , we sc eened o a Ni -, ni a e educ ase- nega i e pheno ype, and his may be he eason why we missed he n C class. All egula o y mu an s desc ibed in his wo k exhibi he h ee ollowing cha ac e is ics: hey do no complemen each o he in ou gene ans e p obe; hey a e comple- men ed by an n A gene-ca ying cosmid, and hey ail o exp ess he kanamycin esis ance gene d i en by he ni B p omo e ca ied by he pCK1 and pCK3 plasmids. So, he assignmen o hese mu an s o he n A gene seems o be a sa e conclusion. ACKNOWLEDGMENTS We a e indeb ed o A. Toukda ian and C. Kennedy o p o iding bac e ial s ains and plasmids. We hank J. Casades is o many help ul discussions and A. Paneque and E. Ce da-Olmedo o p o iding acili ies o ca ying ou his wo k. We acknowledge he assis ance o A. Fe nandez-Es e ane. This wo k was suppo ed by Comision Aseso a Cien ica y Tdcnica (g an 3026). LITERATURE CITED 1. Bishop, P. E., and W. J. B ill. 1977. Gene ic analysis o Azo obac e inelandii mu an s ains unable o ix ni ogen. J. Bac e iol. 130:954-956. 2. Bo he, H., and K. P. Hage . 1981. Elec on anspo o assimila- o y ni a e educ ase in Azo obac e inelandii. Z. Na u s o sch. Sec . C 36:1030-1035. 3. Dixon, R., C. Kennedy, and M. Me ick. 1981. Gene ic con ol o ni ogen ixa ion, p. 161-185. In S. W. Glo e and D. A. Hopwood (ed.), Gene ics as a ool in mic obiology. Camb idge Uni e si y P ess, Camb idge. 4. Gue e o, M. G., J. M. Vega, E. 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