JOURNAL OF BACTERIOLOGY, Jan. 1981, p. 348-3570021-9193/81/010348-10$02.00/0

Vol. 145, No. 1

Regulation of Nitrogen Fixation in Klebsiella pneumoniae:Isolation and Characterization of Strains with nif-lac Fusions

DOUGLAS MAcNEIL,t JLABI ZHU, AND WINSTON J. BRILL*Department ofBacteriology and Center for Studies of Nitrogen Fixation, University of Wisconsin, Madison,

Wisconsin 53706

Strains with lac fused to each of the seven nif operons were isolated by twodifferent methods. Repressing conditions prevented expression of all nifoperons,whereas derepressing conditions led to the expression of all nif operons. Nodifferences in Nif regulation were observed between Escherichia coli and Kleb-siella pneumoniae with the same nif-lac fusions. Most derivatives of nif-lacfusion strains selected on lactose and NH4' contained nif operator mutations.Some derivatives contained deletions, which establishes that the direction oftranscription of all seven nif operons is toward his.

Sixteen nif (nitrogen fixation) genes in Kleb-siella pneumoniae and the site of ammoniumregulation, arranged in seven operons, have beenidentified by complementation analysis (6, 12-14), fine-structure mapping experiments (12,13),and analysis of polarity effects of point, inser-tion, and deletion mutations (6, 12-14, 16, 17).The protein products and functions of most ofthe nif gene products have been identified (17,18).High levels of NH4' (17), certain amino acids

(20), and oxygen (7, 19) prevent synthesis ofnitrogenase in K. pneumoniae. Glutamine syn-thetase or factors that regulate glutamine syn-thetase (21, 22), the nifA product (17, 18), andthe nifL product (17) are involved in nifregula-tion. However, nothing is known about how theseven nif operons are individually regulated inresponse to various N sources. One approach toinvestigating the control of the nif operonswould be to assay activities of enzymes coded bya gene in each operon. However, enzyme assayshave not been developed for the products ofmost nif operons.Gene fusion, especially the fusion of the lacZ

gene to other operons, has proved to be a valu-able tool in studying the regulation of severalsystems (4, 5). Two general methods were de-veloped by M. Casadaban for the fusion of thelacZ gene to other operons. The method devel-oped first uses a Mu prophage to direct theintegration of a A lac phage into a specific gene(4). Upon deletion of the Mu prophage, the lacZgene can become fused to nearby operons (4). Asecond method utilizes a defective Mu prophage,Mu dl, which carries the lacZ gene. When Mudl inserts into an operon in the correct orienta-

t Present address: D,evelopmental Microbiology, Merck,Sharp & Dohme Research Laboratories, Rahway, NJ 07065.

tion, the lacZ gene is controlled by that operon(5). We have used both methods to isolatestrains with nif-lac fusions.

MATERIALS AND METHODSChemicals and media. LC, a rich medium (11), K,

a nitrogen-free medium with sucrose (11), and KN, aminimal medium containing NH4' (12), have beendescribed. CPN medium is CP medium (8) with 0.2%amnmonium acetate; CA medium is LC with carbeni-cillin (800 pg/ml) and ampicillin (60 pg/ml); KLN isKN in which lactose is substituted for sucrose; KX isK containing serine (100 pg/ml) and X-gal (5-bromo-4-chloro-3-indoyl-16-D-galactoside) (30 pg/ml); andKNX is KN with X-gal (30 pg/ml). For growing Esch-erichia coli, glucose (4 mg/ml) and thiamine (1 pg/ml)replaced sucrose in K medium; in KX medium, nitrate(100 pg/ml) was substituted for serine. KB buffer (8)and Z buffer (15) have been described. Where needed,amino acids were added to 20 pg/ml. Tetracycline (20pg/ml) was obtained from Sigma Chemical Co., St.Louis, Mo.; 6-cyanopurine (110 pg/ml) was obtainedfrom Burroughs Welicome Co., Research TrianglePark, N.C.; X-gal was obtained from Bachem Inc.,Torrance, Calif.; and diethyl sulfate (DES) was ob-tained from Eastman Kodak Co., Rochester, N.Y.

Bacterial strains. UN is wild-type K. pneumoniaeM5al. UQ6, a derivative of CSH26 (ara thi A(pro-lac)), was used as the parental E. coli strain becauseit expresses the K. pneumoniae nif genes well, isdeleted for lac, and is A sensitive. Mu dl insertionswere isolated in UN2979, which, unlike the parentalK. pneumoniae strain UN209 (hisD4226), is resistantto lysis in the presence of lactose and contains a lacZmutation (induced by DES and which reverts at lessthan 10-9). Plasmids pTM4010 (his' nir) and otherpTM plasmids (his+ nif) are derivatives ofpRD1 (12).Other bacterial strains are described in Table 1.

Phages. P1 kc hl (12) is an extended-host-rangemutant of P1 kc. P1CM clrlOO, Mu cts62 hP15, whichhas the host range of P1, A c b2, and A vir wereobtained from M. M. Howe. Mu dl (cts62 AmprlacZ+Y+), X1(209), and A123(209) were obtained from

348

nif-lac FUSIONS 349VOL. 145, 1981

TABLE 1. Bacterial strains

Strain Genotype Source or referenceE. coliJC5466 trp his recA56 rpsE (pRD1) R. DixonMAL103 araB::Mu cts araD139 (Mu dl (Ampr lac)) 5MH812 hsdM hsdR thr leu met lac supE M. HoweQD5003 pro mel supF M. HoweSB1801 Ahis-750 ara-14 galK2 maLAI xyl-5 mtl-l rpsL P. HartmanUQ6 A(pro-lac) ara thi rpsE hisD Mu resistant 10UQ8 pro mel supF Mu resistant 10UQ129 pro mel supF Mu resistant (Mu cts62 hP15) (Mu dl)

K. pneumoniaeUN Wild type, galactose sensitive 12UN209 hisD4226 12UN976 pro-4001 A(his-nif) (pTM4010) 12UN2374 hisD4226 galactose resistantUN2979 hisD4226 lacZ4()1 galactose resistantUN4102 hisD4226 lacZ4001 recA56 srl-300::TnlO galactose resistantUN4482 hisD4226 lacZ4()1 galactose resistant (Mu cts62 hP15) (Mu dl)

M. Casadaban (4, 5), and the latter two were renamedA placl and A plac2. A placl, A plac2, and Mu dlcontain the lacZ+Y+ genes preceded by some trpDNA, but the lac genes are not expressed in a lysogendue to the absence of the lac control sequences (4, 5).Phage methods. Lysates of P1 kc hl and P1CM

clrlOO were prepared, and transductions were per-formed as previously described (12). Lysates of Aphages were prepared by infection of QD5003 (10). Alac-Mu dilysogens were isolated as previously de-scribed (5, 10). Mu dl is defective and requires a helperphage. Since K. pneumoniae strains are Mu resistant(2), Mu cts62 hP15 was used as a helper for growingMu dl and isolating lysogens in K. pneumoniae. Mudl lysates were prepared from UN4482 by heat induc-tion (2).

Bacterial matings. Transfers of pTM plasmidsand the mapping of nif mutations were performed aspreviously described (12).

,-Galactosidase activity. Cultures to be assayedwere grown in KN overnight and diluted 1:50 into Kmedium with various nitrogen sources. After 18 h, the,B-galactosidase activity was determined (15). Enzymeactivity is reported as

1,000 x OD420OD60o x (time of assay in minutes)

x (volume of culture assayed)

where OD420 and OD600 are optical densities at 420 and600 nm, respectively.Mutants which grow on lactose and ammo-

niium. DES-treated (12) or untreated cultures ofstrains with nif-lac fusions were grown overnight inKN and spread onto KLN medium. After 5 days ofanaerobic incubation at 300C, Lac' clones on NH4'were picked and purified. These clones were tested forAmp', Nif, the ability to complement nif mutations(12), and expression of NifA on NH4+ using CPNmedium (9).

Radioactive labeling, two-dimensional poly-acrylamide gel electrophoresis, and autoradiog-raphy. Cultures were derepressed for Nif in 0.15%

histidine, labeled, and visualized on two-dimensionalpolyacrylamide gels (17, 18).

RESULTS

Isolation of strains with nif-lkc fusions inE. coli One procedure (4) for constructingstrains with operon fusions used A lac phage.Because K. pneumoniae is A resistant, nif-lacfusions were isolated in a A-sensitive derivativeof E. coli strain UQ6 which contained deriva-tives of nif plasmid pTM4010. The phages Aplacl and A plac2 carry the wild-type lacZ andlacY genes, but these genes are not expressedbecause the phages do not contain the lacOPregion (4). The A lac phages also carry DNAfrom one end of Mu; A placl has the Mu c end,and A plac2 has the Mu S end. These phages aredeficient in A-mediated integration, but they canintegrate by homologous recombination be-tween the Mu DNA of a A lac phage and a Mucts prophage to form a dilysogen with A lac andMu adjacent. Subsequent heat treatment in-duces the Mu cts prophage and kills most cells.Among the survivors will be strains with a dele-tion ofthe Mu prophage. Some ofthese deletionswill fuse the lacZ and lacY genes to nearbypromoters. Steps involved in isolating strainswith nif-lac fusions are illustrated in Fig. 1.

Several hundred Mu cts insertions in nifwereisolated in K. pneumoniae (12), and 11 weretransduced onto pTM plasmids. They weretransferred to E. coli strain UQ6 (his A(pro-lac)). The E. coli strains containing nif::Muwere lysogenized by A placl and A plac2 aspreviously described with A pMu (10). Dilyso-gens, identified as A c b2 resistant and A virsensitive, arose at frequencies between 10'- and10-4 per cell. In approximately 10% of the dily-

350 MACNEIL ET AL.

lc 2A

his nif Mu nifQ N US Ac E H

X integration J

his nif X loc2 lac Mu rifO N US N J YZ US Ac E H

deletion and

fusion

his nif X loc2 lac nifO N US N J YZ E H

nit-loc fusion

FIG. 1. Formation ofnif-lac fusions. Letters belowthe lines represent nif, Mu, or A genes, and arrowsabove lac and nifindicate the directions oftranscrip-tion of these genes. The Mu cts prophage directs theintegration of a A plac by recombination betweenhomologous Mu DNA sequences. The resulting Aplac-Mu dilysogens are subjected to a heat treatmentwhich induces theMuprophage. Among the survivorsare strains with a deletion oftheMu prophage. Someofthese deletions fuse the lac genes to nifoperons. Asshown, a Muprophage in one orientation directs theintegration of A plac2, which results in the nif andlac genes being oriented in the same direction. (A Muprophage in the other orientation would direct theintegration of A placl, which contains the Mu c endrather than the Mu S end on A plac2. Such a Mu-Aplacl dilysogen would also contain the lac and nifgenes in the same orientation.) As shown, a deletionofMu DNA between lac and nif results in a nif-lacfusion.

sogens, A apparently integrated into trp, sincethe strains were Trp-. Both A placl and X plac2contained some trp DNA between the lac andthe Mu DNA (4). Dilysogens with A integratedinto nif were identified as strains that couldcotransfer the properties of A release, Murelease, and His' via a pTM plasmid to SB1801(His- A resistant). The A lac-Mu dilysogens andtheir parental Mu lysogens are described in Ta-ble 2.

Strains with nif-lac fusions were isolatedamong the survivors of heat-treated cultures ofthe A lac-Mu dilysogens. Lac' strains were se-lected on KL medium (K medium with lactosesubstituted for sucrose) or identified on KXplates as blue colonies. After 2 days of anaerobicincubation for the KX plates and 5 days ofanaerobic incubation for the KL plates, Lac'clones appeared at frequencies between 10-4 and

J. BACTERIOL.

10-2 per survivor. A total of 1,000 heat selectionswere performed with the 21 A lac-Mu dilysogens.Approximately 5,000 Lac' clones were purifiedand tested for LacZ expression by spottingwashed overnight KN cultures on KNX and KXplates. After anaerobic incubation, eight inde-pendently isolated strains containing nif-lac fu-sions were isolated which were repressed forLacZ expression on KNX but not KX medium(Table 2). The extent of nifDNA remaining inthe nif-lac fusion strains was determined bymapping and complementation analysis. Sincethe nif-lac fusions were isolated on derivativesof pTM4010, they could be tested for nif dele-tions by mating them into K. pneumoniaestrains with mutations in each nif gene. Onlyfusion (niJ-lacZ)6220 contained a detectable nifdeletion adjacent to the site of the parental Muinertion. This deletion did not extend out ofnifJ, the gene which contained the parental Muinsertion.We were unsuccessful in a search for lac fu-

sions to nif genes not repressed by NH4'. Astrain with a nif-lac fusion not repressed byNH4' could be identified as a Lac' strain with anif deletion adjacent to the original Mu inser-tion. We tested 600 of the 5,000 purified Lac'isolates for deletions within nif, but none wasfound. Approximately 40% of the Lac' isolatescontained deletions with the one endpoint at thesite ofthe A-Mu insertion and the other endpointoutside either nifQ or nifJ. These strains appar-ently contain fusions to operons near nif. Themajority of the strains in which LacZ is notrepressed by ammonia had no detectable dele-tion and might contain fusions to Mu operons.Isolation of strains with nif-lac fusions in

K pneumoniae A second procedure (4) wasused to isolate K. pneumoniae strains with ad-ditional nif-lac fusions. This procedure utilizeda defective Mu phage, Mu dl, which containsthe lacZ-lacY genes (but not the lacOP region)and a nontransposable Ampr gene. These genesreplace DNA at the S end of Mu. However, Mudl still has sufficient Mu DNA at its ends sothat it integrates randomly into other DNAs.When Mu dl integrates into a nifoperon in theproper orientation, the lac genes will be con-trolled by that operon's promoter because tran-scription would continue from nif into the lacgenes of Mu dl.UN2979 (hisD4226 Lacr lacZ400l) was in-

fected with Mu dl lysates prepared by heatinduction of UN4482, and Ampr clones wereselected on CA medium (5). The confluentgrowth on the CA plates was suspended in KBand plated on KX and K media to yield 200 to1,000 single colonies; then the plates were incu-bated anaerobically for 2 days. Fifteen percent

nif-lac FUSIONS 351

TABLE 2. nif-lac fusions derived from A plac-Mu dilysogens in E. coliA plac-Mu dilysogenUQ194(X placl)UQ195(X plac2)UQ196(X placl)UQ197(X plac2)UQ198(X placl)UQ199(X plac2)UQ202(X plac2)

UQ203(X plac 1)UQ204(X plac2)UQ205(X placl)UQ206(X plac2)UQ207(X placl)UQ208(X plac2)UQ209(X placl)UQ210(X plac2)UQ215(X placl)UQ216(X plac2)UQ217(X placl)UQ218(A plac2)UQ219(X placl)UQ220(X plac2)

Strain with nif-lac fusion

UQ223 (0(nifB-lacZ)6214)

UQ225 ('(nifV-lacZ)6216)UQ226 (O(nifV-lacZ)6217)

UQ227 (4(nifU-lacZ)6218)

UQ228 (4(nifE-lacZ)6219)

UQ229 (4(nifJ-lacZ)6220)UQ230 (O(nifJ-lacZ)6221)

UQ231 (O(nifJ-lacZ)6=222)a Mu lysogens are derivatives of UQ6 containing pTM plasmids (his' nif::Mu cts6l) (10).

of the colonies on KX medium were Lac', andin 5% of the Lac' isolates Lac was repressed byNH4'. About 75% of the NH4+-repressible iso-lates contained insertions which mapped in nif.About 0.5% of the colonies on K medium wereNif- (identified as small colonies). Approxi-mately half of the Nif- strains were Lac+, andall of these were repressed by NH4'. This resultis consistent with the ability of Mu dl to inte-grate in either of two orientations, one fused toa nifoperon (Lac') and the other not (Lac-). Allputative nif-lac fusion strains were tested forlinkage of the Mu dl insertion to hisD (hisD is39 to 72% linked to nif [12]). Twenty-one of 100independent strains in which ammonia re-pressed Lac and in which mapping and comple-mentation analysis indicated there was an inser-tion in nif are described in Table 3. Also, sixpTM plasmids containing nif-lac fusions iso-lated in E. coli (Table 2) were transferred toUN4102 (His- LacZ- RecAk Nift), selecting forHis' and scoring for NH4' repression of Lac.These nif-lac fusion strains also are described inTable 3.Response of strains with nif-lac fusions

to various N sources. Nif is repressed by am-monium (7) and certain amino acids (20). Theeffect of N sources on expression of each nifoperon was investigated. E. coli and K. pneu-moniae strains containing nif-lac fusions werederepressed overnight in minimal media con-taining variousN sources. TheN sources includethose which at 2 mg/ml promote good growth

TABLE 3. nif-lac fusions in K. pneumoniaeStrain Fusion allele

Fusion formed by Mu dl lysogenyUN4520 (nifB-lacZ)5960UN4533 (nifB-lacZ)5973UN4837 (nifB-lacZ)6125UN4847 (nifA-lacZ)6135UN4493 (nifL-lacZ)5933UN4523 (nifF-lacZ)5963UN4489 (nifM-lacZ)5929UN4846 (nifM-lacZ)6134UN4897 (nifM-lacZ)6185UN4903 (nifS-lacZ)6191UN4499 (nifN-lacZ)5939UN4508 (nifN-lacZ)5948UN4579 (nifN-lacZ)66019UN4487 (nifE-lacZ)5927UN4507 (nifE-lacZ)5947UN4512 (nifE-lacZ)5952UN4484 (nifK-lacZ)5924UN4495 (nifK-lacZ)5935UN4842 (nifK-lacZ)6130UN4515 (nifD-lacZ)5955UN4504 (nifJ-lacZ)5944

Transfer of fusions isolated in E. coli to UN4102UN5052 (nifB-lacZ)6214UN5053 (nifV-lacZ)6216UN5055 (nifU-lacZ) 6218UN5056 (nifE-lacZ)6219UN5057 (nifJ-lacZ)6220UN5058 (nifJ-lacZ)6222

(an increase overnight in OD6e from 0.2 togreater than 2; ammonium, serine, asparagine,glutamine, and Casamino Acids) N sources

Mu lysogenaUQ109

UQ110

UQlll

UQ190

UQ115'

UQ116

UQ117

UQ118

UQ120

UQ121

UQ192

Mu inaertion

nifB4444

nifB44O8

nifA4422

nifV4436

nifU4438

nifN4411

nifE4420

nifK4452

nifJ4441

nifJ4434

nifJ4456

VOL. 145, 1981

352 MAcNEIL ET AL.

which at 2 mg/ml promoted poorer growth (anincrease overnight in OD6oo from 0.2 to less than0.5; glutamate, arginine, nitrate, histidine, pro-

line, and urea), and N sources which at 100 ,ug/ml limit growth (Casamino Acids, serine, andnitrate). By assaying f-galactosidase in a set ofstrains with nif-lac fusions in each nif operon,

the expression of each nif operon could be de-termined. All of the nif operons were repressedby ammonium, serine, glutamine, and aspara-

gine (Table 4). The level of repression varied.Each fusion showed lower,8-galactosidase levels

in E. coli than K. pneumoniae. Also, the second-

ary trp promoter on A plac2 (4) appeared to

contribute to the expression of ,6-galactosidasein most fusions derived from A plac2. Strains

with fusions 6216, 6217, 6218, and 6222 had

higher repressed levels of fl-galactosidase than

fusions derived from A placl. Furthermore, somestrains with nif-lac fusions were repressed more

by NHFL than by asparagine, serine, or gluta-

mine. Different amounts of 46-galactosidase wereobserved when the same fusion strain was de-

repressed on different poor orlimiting N sources.

TABLE 4. ,-Galactosidase activities ofstains with nif-lac fUsionsa

Good N sources' Poor N sourcesCnif-lacZ fusion(allele)bo

NH4+ Asparagine AAcids Arginine Histidine Limiting N

E. coliA lac fusionB6214 0.6 1.4 280 580 310 1,130V6216 7.0 9.4 150 270 95 360V6217 8.8 10.4 560 960 300 1,100U6218 8.4 12.0 250 665 360 360E6219 0.8 0.6 160 340 140 290J6220 0.8 2.0 2,300 2,300 360 1,600J6222 9.6 10.8 300 470 120 190J6221 3.4 6.8 49 93 40 40

K. pneumoniae:CA lac fusionB6214 1.8 2.0 180 150 120 640V6216 21 24 120 300 160 69U6218 20 52 75 970 320 700E6219 1.8 12 170 380 140 390J6220 4.0 2.4 640 1,700 520 2,400J6222 21 28 410 400 180 980

Mu dl fusionB5960 5.0 30 NIDd ND 225 800A6135 6.0 20 ND ND 300 240L5933 7.6 17 ND ND 250 470F5963 6.8 6.2 ND ND 100 190M6134 24 30 ND ND 520 910S6191 8.6 9.6 ND ND 460 1,200N6019 2.8 4.4 ND ND 260 550E5947 10.8 10.0 ND ND 410 300K5924 6.6 9.4 ND ND 500 1,200D5955 5.4 4.0 ND ND 900 1,800J5944 7.8 8.2 ND ND 280 900

a NHii-grown cultures were washed and diluted into K medium with 2 mg of the indicated N source per mland incubated anaerobically for 18 h at 30°C. 8-Galactosidase activity is expressed as:

1,000 x OD42oOD.oo x (time of assay) x (volume assayed)

b E. coli values are the averages of five or more experiments with each N source; K. pneumoniae results arefrom three experiments.'The quality of the N source was determined by the amount of growth after incubation anaerobically for 18

h. Good N sources resulted in cultures with ODsoo greater than 2.0. Poor N sources resulted in cultures withODooo less than 0.5. Limiting N source was 100 pg of serine, Casamino Acids, or nitrate per ml Glutamine andserine yielded the same results as asparagine. Glutamate, nitrate, and urea yielded the same results as arginine.Proline yielded the same results as histidine.

d ND, Not determined.

J. BACTERIOL.

nif-lac FUSIONS 353

This may indicate a partial repression of Nif, orit may only indicate how N starved the cellswere. Under conditions of severe N starvation,protein synthesis, and hence derepression of thenif-lac fusion, may be reduced.

Repression and derepression results similar tothose enumerated in Table 4 were found whenall fusion strains in Table 3 were spot tested onKX media containing variousN sources at 2 mg/ml. Serine, glutamine, asparagine, and ammoniarepressed all the nif operons. Histidine, proline,lysine, glutamate, arginine, ornithine, urea, me-thionine, and glycine did not repress any of thenif operons.

All nif-lac fusions derived from insertion ofMu dl synthesize a wild-type,B-galactosidaseprotein. Hence, the levels of B-galactosidase syn-thesized in strains with lac fusions to differentnif operons should be indicative of the relativetranscription of each nif operon. However, nif-lac fusions derived from A plac-Mu dilysogensoften result in protein fusions (4). These hybridproteins will have different specific activities,and this will limit any comparison among fusionsto different operons. Evidence for fusion strainswhich synthesize ,-galactosidase with differentactivities can be found by comparing the level of,B-galactosidase assayed in the three strains withnifJ-lac fusions 6220, 6222 and 6221. Under de-repressing conditions, f,-galactosidase activitiesvaried up to 40-fold.Effect of oxygen on strains with nif-lac

fulsions. Oxygen prevents synthesis ofthe nitro-genase polypeptides (7, 19), but little is knownabout the effect of oxygen on other nif-codedpolypeptides. E. coli strains with nif-lac fusionswere grown anaerobically with ammonium andderepressed in low-N, aerobic minimal medium,and the ,B-galactosidase activity was determined9 h later. All strains were still repressed; fl-ga-lactosidase activities were between 0.8 and 16U-similar to the NH4+-repressed level of 0.5 to10 U. In K. pneumoniae, repressed levels of ,B-galactosidase (6.0 to 20 U) were obtained withmost fusion strains. Only strain UN4493, with anifL-lacZ fusion, had higher activity (81 U).Another strain with a fusion in the nifRLAoperon, UN4847 ((nifA-lacZ)6135), had only 16U on oxygen.Mutants insensitive to NHE repression

ofLac. Strains with altered control of Nif mightbe isolated among the mutants of nif-lac fusionstrains which overcome NH4+ repression andgrow on lactose and ammonium. The NH4+-in-sensitive strains might contain mutations ingenes that regulate all the nifgenes, such as nif-specific genes (nifA-nifL; 17, 18) or other genesinvolved in nifregulation (1, 20,21). Also, strains

with mutations in nif regulatory sites, such asnif promoters and operators, might be isolatedwhich express a single nif operon on NH4+-con-taining media. Mutants which grow on lactoseand ammonium anaerobically were isolated fromstrains with nif-lac fusions at 10' per cell. Over1,000 independent mutants were isolated fromnif-lac fusion derivatives of UQ6, UN4102, andUN2979. None of these appeared to containmutations leading to the expression of all nifgenes on ammonia, since they all failed to de-velop color on CPN, an indicator of NifA expres-sion (8, 9). About 10% of the NH4+-insensitivemutants contained deletions past nifJ. Presum-ably these deletions resulted in fusions of lac toother nearby operons.The majority of the mutants insensitive to

NH4' repression isolated from fusion derivativesof UN2979 appeared to contain nif operatormutations. Twenty-one strains, Lac' on NH4',derived from strains with nif-lac fusions werechosen to test the linkage of their nifmutationsto his. These 21 strains did not contain detect-able nif deletions. Phage P1 was grown on thesestrains and used to transduce UN209 (hisD4226)to His+. Transductants were screened for fl-ga-lactosidase activity on KX (Nif derepressed) andKNX (Nif repressed) media. All strains showeda separation of the fusion mutations from themutation leading to f8-galactosidase expressionon ammonia. Each mutation causing cells to beLac+ on NH4+ was located as distal to his andthe parental fusion. This is consistent with themutations being located at each operon's oper-ator-promoter region, since all seven nifoperonsare transcribed toward his (see below). Repre-sentative linkage data for derivatives from fu-sions in each nif operon are presented in Table5. The dominance of the mutations leading toLac' on NH4' was tested by mating in pRD1(his+ nit), selecting Nif, and scoring exc6nju-gants on KX and KNX media. The mutationsin all 21 strains tested were dominant, since thediploids were Lac' on both media. This suggeststhat the mutants contain operator mutations.The effect of the putative operator mutations onexpression of LacZ in several fusion strains isshown in Table 6. NH4' still reduced the ,B-galactosidase levels, but only by 50 to 80% of thelevel on limiting N. This may indicate that thenifA product still has some effect on limiting N,or it may indicate that nifmRNA is unstable inthe presence of NH4+ (G. Roberts and W. J.Brill, manuscript in preparation).To further demonstrate that the mutations

causing cells to be Lac' on ammonium are op-erator mutations specific for one nifoperon, thesynthesis of nif-coded polypeptides was com-

VOL. 145, 1981

354 MAcNEIL ET AL.

TABLE 5. Cotransduction ofnif-lac fusion andoperator mutations with his

Donor ~~~% CotranaductionDonor to hisD4226a

Operatoral- FusionFusion allele lele Fusion and op-

erator

(nifB-lacZ)6125 nif-6245 16.3 9.4(nifL-lacZ)5933 nif-6267 14.6 8.9(nifA-lacZ)6135 nif-6256 14.1 8.3(nifF-lacZ)5963 nif-6272 12.4 6.4(nifM-lacZ)6134 nif-6226 12.2 5.7(nifN-lacZ)6019 nif-6246 10.1 6.1(nifK-lacZ)5924 nif-6262 8.5 6.7(nifJ-lacZ)5944 nif-6264 7.3 5.1

a Pl kc hl was grown on the fusion donor (Lac' onNH4+) strain and used to tranaduce UN209 (hisD4226)to His+. More than 200 His' transductants were scoredfor cotransduction of the nif-lac fusion on Nif-dere-pressing medium (KX) and for cotransduction of boththe nif-lac fusion mutation and putative operator mu-tation on Nif-repressing medium (KNX).

TABLE 6. Effect ofputative operator mutations on/B-galactosidase synthesis in nif-lac fusion strains

f?-Galactosid-

Operator ase activityStrain Fusion allele allele Lil-Wtalee Limit- With

ing N NH4+

UN4493 (nifL-lacZ)5933 200 9.0UN5156 (nifL-lacZ)5933 nif-6270 300 140UN4846 (nifM-lacZ)6134 290 3.0UN5115 (nifM-lacZ)6134 nif-6229 400 60UN4579 (nifN-lacZ)6019 250 1.0UN5140 (nifN-IacZ)6019 nif-6254 380 100UN4484 (nifK-lacZ)5924 470 4.0UN5149 (nifK-lacZ)5924 nif-6263 420 150

pared in fusion strains with and without putativeoperator mutations. Two NH4+-derepressedLac' strains containing fusions in multicistronicoperons were chosen for further studies. Onestrain contained a fusion in the nifUJSVMoperon('1(nifM-lacZ)6134), and the other contained a

fusion in he nifHDK operon (Q(nifK-lacZ)-5924). These strains were grown on Nif-repress-ing and -derepressing media, and the polypep-tides synthesized were visualized on two-dimen-sional polyacrylamide gels. Under derepressingconditions, all identified nifproducts (except theproduct of the gene containing the insertion)were synthesized. However, under repressingconditions only nif products encoded by theoperon containing the fusion were synthesized.Most notably, the first gene product from eachoperon was synthesized. For example, the nifH

product (component II) was synthesized in anifK fusion strain that was Lac' on NH4', butthe nifJ product was absent when the strain wasgrown on NH4' (Fig. 2). When this strain wasderepressed for nifexpression (on limiting nitro-gen), both the nifH and nifJ polypeptides wereproduced.Direction of transcription of the nif op-

erons. In all nif-lac fusions, the direction oftranscription of the lac and nif genes must bethe same. Thus, if the orientation of the lacgenes is determined, the direction of transcrip-tion of the nif operons containing that fusionwill be established. Some of the NH4+-dere-pressed Lac' derivatives contained deletions be-yond nifJ4057, a nifJ mutation located in themost his-distal nif deletion interval (12). Thesedeletion derivatives were isolated from strainswith fusions to all nif operons. No derivativescontained deletions past the his-proximal nifgene, nifQ. Presumably, the deletions result infusions of lac to nearby operons. The directionof the deletion establishes that the lac genes onMu dl lysogens are transcribed toward his.Thus, all seven nif operons must be transcribedtoward his.

DISCUSSIONStrains containing nif-lac fusions were used to

evaluate the effects of various N sources onexpression of the nif operons in K. pneumoniae.N sources which promote good growth (NH4',serine, asparagine, and glutamine) repress theseven nif operons, whereas poorer N sources(histidine, proline, urea, arginine, and gluta-mate) allow the expression of all nif operons.The only good N source which allowed nif de-repression was Casamino Acids. Except for onefusion strain, oxygen prevented derepression ofall nifoperons. This coordinate regulation of thenif operon is consistent with the proposal thatthe nifA product is the sole positive regulator ofthe other six nif operons (9). However, the ob-served derepression of the strain with the (nifA-lacZ)6135 fusion indicates that the nifA productis not required for expression of its own operon,nifRLA. Thus, it is likely that regulatory factors(gin factors) (9, 21) may mediate amino acidand ammonium repression of nif by modulatingnifRLA transcription, presumably at nifR (9,17).Oxygen repression of nif expression may also

involve nifand non-niffactors. The nifL productis involved in rapid turn-off of nif by oxygen(Roberts and Brill, manuscript in preparation).However, UN4493 (nifL-lacZ fusion), which hasno nifL product, is still partially repressed byoxygen (80 U on oxygen versus 470 U on limiting

J. BACTERIOL.

nif-lac FUSIONS 355

_. 4

_A*

BGG

9, *, ,~

* 9# w

Iei:-ow: '>'.. /*~~~~~~~A

4 0

L...

FIG. 2. Two-dimensional polyacrylamide gel of a nif-lac fusion strain derepressed for lac expression onNH4'. Strain UN5149 [0(nifK-lacZ)5924 nif-6263] was grown with excess NH4'. Under these conditions, theparent strain (UN4484) produces no known nif-encoded proteins or,8-galactosidase. J is the normalpositionof the nifJ-coded protein (18), H is the position of the nifH-coded protein, and BG is /B-galactosidase.

nitrogen), suggesting that other factors also mayregulate nif expression by oxygen. Recently, R.Dixon (personal communication) isolated nif-lacfusions and also observed coordinate regulationof the nif operons by NH4' and oxygen. Inaddition, he observed that a nifA mutation pre-vented derepression of /?-galactosidase in strainswith lac fused to nifoperons other than nifRLA.The strains containing nif-lac fusions were

isolated by two different methods (4, 5). A mul-tistep process was used to isolate E. coli strainswith nif-lac fusions in four of the seven nifoperons. Although X lac-Mu dilysogens werereadily isolated, only a small proportion (ap-proximately 0.1%) of the Lac' isolates containedfusions to the nif operons, and these were iso-lated from only 8 of 21 A lac-Mu dilysogens. Incontrast, strains with ara-lac fusions comprised10 to 90% of the Lac' derivatives of ara-lacdilysogens (4). The difference in frequencies maybe due to our use of strains containing deriva-tives of pTM4010. This nif plasmid is unstable(11). In addition to operon fusions, Lac' fusionsgenerated by this method may be accompaniedby a deletion resulting in a fused protein product(4). The f8-galactosidases synthesized from thethree E. coli strains with different nifJ-lacZfusions demonstrate this. These fusions pro-duced a range of derepressed ,B-galactosidaseactivities from 40 to 1,600 U, probably reflectingthe synthesis ofnifJ-lacZ-encoded proteins withdiffering specific activities. The effect of a sec-

ondary trp promoter carried on A plac2 (4) isevident in four of the five fusions derived fromA plac2-Mu dilysogens. On NH4', E. coli strainswith fusions derived from A plac2 had higherfi-galactosidase activities (7.0 to 9.6 U) thanother fusion strains (0.6 to 3.4 U). The otherfusion strain derived from a A plac2-Mu dilyso-gen, 4(nifJ-lacZ)6220, contained a detectablenif deletion which may also have deleted thesecondary tip promoter. The fusions derivedfrom A plac2 have even higher levels of 8-galac-tosidase on NH4' and amino acids in K. pneu-moniae (20 to 52 U), suggesting that this sec-ondary promoter functions more efficiently in K.pneumoniae than in E. coli.A much more efficient method for isolating

nif-lac fusions was used to isolate K. pneumo-niae strains with fusions to all seven nifoperons.Using phage Mu dl (5), approximately half ofthe Nif Mu dl lysogens contained nif-lac fu-sions. Under derepressing conditions with lim-iting N, fi-galactosidase activities between 800and 1,800 U were observed for Mu dl strainswith fusions to nifBQ, nifUVSM, nifHDK, andnifJ operons. Lower levels of f8-galactosidase(190 to 300 U) were found in Mu dl strains withfusions to nifRLA, nifWF, and nifNE. Underrepressive conditions (NH4' or certain aminoacids), a /i-galactosidase activity of 2.5 to 20 Uwas obtained. A comparison of strains with thesame nif-lacZ fusion in E. coli and K. pneumo-niae indicated no substantial differences in nif

VOL. 145, 1981

356 MACNEIL ET AL.

regulation. Thus, the E. coli and K. pneumoniaefactors which regulate the nifRLA operon ap-pear to be functionally similar. Also, the nifproducts inactivated by the fusions in nifE, -U,- V, -E, and -J do not appear to be important forthe regulation of their own operons. The E. colistrains were haploid for nif, whereas the K.pneumoniae strains had nif-lac fusions on aplasmid and a nif+ chromosome, yet no differ-ences in nif regulation were observed.Among the derivatives of nif-lac fusion strains

selected for growth on lactose-ammonium me-dium, strains with deletions resulting in newfusions and strains with apparent nif operatormutations were found. The direction of the dele-tions in strains Lac' on ammonia indicates thatall nif operons are transcribed from promoterstoward his. This confirms the direction of tran-scription deduced from polarity studies in fivemulticistronic nif operons (12, 13) and estab-lishes the direction of transcription for the nifJand nifWF operons. The majority of the NH4+-derepressed Lac' strains without new deletionsappear to contain nif operator mutations. Map-ping and dominance studies are consistent withthe mutations being in nifoperators. In addition,an analysis of the polypeptides synthesized intwo mutants insensitive to NH4' repression,grown on NH4', showed only nif polypeptidesencoded by genes between the site of the nif-lacfusion and the nifoperator region of that operon.The synthesis of the nifU polypeptide on am-monium in a derivative from fusion (nifM-lacZ)6134 confirms our previous genetic analysisthat nifSVM forms a single operon (12). Thisrefutes the other proposals for the organizationof this region (13,14). Mutations designated nifS(12) have been subdivided into two genes, nifSand nifU (17). All NH4+-derepressed Lac' fusionstrains tested had less than one-half the ,8-galac-tosidase activity on NH4' as on limiting nitrogen.This is consistent with a proposal that nifmRNA is unstable in the presence of ammonia.Alternatively, it may indicate that the nifA prod-uct still interacts under N-limiting conditions toincrease nif transcription. The operator muta-tions isolated from strains with nif-lac fusionswill be useful for defining nif regulatory se-quences when the nif control regions are se-quenced from purified nifDNA (10, 16).

ACKNOWLEDGMENTSThis research was supported by the College of Agricultural

and Life Sciences, University of Wisconsin, Madison, and byNational Science Foundation grant PCM-7624271. J.Z. wassupported in part by funds from The People's Republic ofChina.We thank L. Silver for preparation of the polyacrylamide

gels.

UlTERATURE CITED

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