Nitrogen Fixation by Klebsiella Is Inhibited by Certain ... · In our studies ofnifgeneregulation,...

Vol. 153, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1983, p. 45-560021-9193/83/010045-12$02.00/0Copyright © 1983, American Society for Microbiology

Nitrogen Fixation by Klebsiella pneumoniae Is Inhibited byCertain Multicopy Hybrid nif Plasmids

GERARD E. RIEDEL,12t* SUSAN E. BROWN,1 AND FREDERICK M. AUSUBEL'Department of Cellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 021381and Division ofPlant Industry, Commonwealth Scientific and Industrial Research Organization, Canberra

City, A.C.T. 2601, Australia2

Received 20 July 1982/Accepted 16 September 1982

In our studies of nifgene regulation, we have observed that certain hybrid nifplasmids drastically inhibit the expression of the chromosomal nif genes ofKlebsiella pneumonia. Wild-type (Nif) K. pneumoniae strains that acquirecertain hybrid nif plasmids also acquire the Nif- phenotype; these strains lose 90to 99% of all detectable nitrogen fixation activity and grow poorly (or not at all) onsolid media with N2 as the sole nitrogen source. We describe experiments whichdefined this inhibition of the Nif+ phenotype by hybrid nif plasmids and identifyand characterize four nifDNA regions associated with this inhibition. We showthat plasmids carrying these nif regions could recombine with, but not comple-ment, nifchromosomal mutations. Our results suggest that inhibition of the Nif+phenotype will provide a useful bioassay for some of the factors that mediate nifgene expression.

Genes specifically required for dinitrogen fix-ation by Klebsiella pneumoniae are clustered onthe chromosome of this organism near the oper-ator end of the histidine biosynthesis (his) oper-on. A total of 15 distinct nitrogen fixation (nif)genes have been identified in this cluster on thebasis of complementation analysis, and thesegenes have been assigned to seven separatetranscription units on the basis of merodiploidanalysis of polar nif mutations (10, 12, 22, 27,28). All of the nil operons are transcribed in thesame direction, toward the his operon (21) (seeFig. 6). Two additional genes, designated niJXand nifY, have also been identified as polypep-tide-encoding regions on hybrid nif plasmidswhich produce proteins in minicells (30). All ofthe his-linked nif genes have been cloned (5, 6,20, 30), and a correlated physical and geneticmap of the nif genes has been constructed (1,32).We report here the observation that some

hybrid nif plasmids inhibit chromosome-en-coded Nif expression in wild-type K. pneumo-niae. Wild-type (Nif+) K. pneumoniae strainsthat are transformed with certain hybrid nifplasmids acquire a Nif- phenotype. These K.pneumoniae strains lose 90 to 99% of all detect-able nitrogen fixation activity, as measured inliquid cell cultures by the acetylene reductionassay, and they grow poorly (or not at all) with

t Present address: The Genetics Institute, Boston, MA02115.

N2 as the sole nitrogen source on solidifiedmedia.We describe experiments which operationally

defined the inhibition of the Nif+ phenotype andwhich identified and characterized several nifDNA fragments associated with this inhibition.Our results suggest that inhibition of the Nif+phenotype will be a useful bioassay for some ofthe factors that mediate nifgene expression.(A summary of this work has been presented

previously [31], and a portion of this work waspresented in partial fulfillment of the Ph.D.degree requirement for G.E.R. at Harvard Uni-versity.)

MATERIALS AND METHODSBacteria and plasmids. The bacteria and plasmids

used are listed in Tables 1 through 3.Media. Rich medium (LB medium) and minimal

medium (BS medium) have been described previously(5) and were solidified with 1.5% agar (Difco Labora-tories, Detroit, Mich.). W medium was identical to BSmedium except that it lacked (NH4)2SO4. Nitrogen-deficient medium (NFDM medium) has been de-scribed previously (6). SNFDM medium was identicalto NFDM medium except that sucrose (2%) wassubstituted for glucose. NFDM and SNFDM mediawere supplemented with either L-aspartic acid, histi-dine, serine, or Casamino Acids (100 ,ug/ml; Difco) andwere solidified with 1.5% Serva ultrapure agar (type11396; Accurate Chemical and Scientific Corp.,Hicksville, N.Y.); other amino acids were used atfinal concentrations of 50 ,ug/ml. Antibiotics were filtersterilized and were added to media at the following

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46 RIEDEL, BROWN, AND AUSUBEL

TABLE 1. Bacterial strains used in this study

Strain Genotype and/or Source orphenotypea reference

Escherichiacoli K-12JC5446 trp his recAS6 rpsE F. CannonHB101 rB- mB leu pro thi H. Boyer

lacY recA str endoIK. pneumoniaeKP1 Wild type (MSA1) 23

Nif+KP5521 KP1 srl-300::TnJO This paper

recAS6KP5525 KP1 recAS6 This paperKP5022 hisD2 hsdRI Nif+ 39KP5614 KP5022 recAS6 This paperKP5058 hisD2 hsdRl nifB213 5

strKP5617 KP5058 recAS6 This paperUN1291 hisD4226 srl-300::TnlO W. Brill

recAS6 (P1 Cm'clrl 00) Nif+

CK3191 hsdRI nifJ C. Kennedya CM,, Chloramphenicol resistance.

final concentrations (micrograms per milliliter): tetra-cycline hydrochloride, 10; chloramphenicol, 25; kana-mycin, 20; ampicillin, 100.K. pneumoniae strain construction. Strain KP5521, a

RecA- Tcr derivative of KP1, was constructed essen-tially by the method of MacNeil et al. (22). StrainKP5525, a tetracycline-sensitive derivative of KPS521,was isolated by a modification (H. Meade, personalcommunication) of the penicillin enrichment proce-dure (18). Strains KP5521 (recAS6 Tc') and KP5525(recA56) grow at the same rate as KP1 on LB medium,BS minimal medium, and NFDM medium. All threestrains utilize citrate and histidine equally well as solecarbon and nitrogen sources, respectively. KP5521,KP5525, and KP1 show identical nitrogen fixationactivities, as measured in whole cell liquid cultures bythe acetylene assay (date not shown). Strain KP5614,

a RecA- derivative of KP5022, was constructed andtested in a similar fashion.K. pneumoniae strains containing hybrid nif plas-

mids were constructed by the CaCl2-heat shock meth-od of Mandel and Higa (24). The presence of a hybridnif plasmid within a K. pneumoniae strain was con-firmed by isolating plasmid DNA from 5-ml cultures ofeach constructed strain, using a small-scale plasmidisolation procedure (17), and by subjecting the isolatedDNA to restriction enzyme cleavage analysis.

Nitrogen fixation assays. (i) Liquid medium assay. A0.2-ml sample of a mid-logarithmic LB medium culturewas added to 5 ml of NFDM or SNFDM mediumcontaining appropriate antibiotics and amino acids,ammonium sulfate (2 mg/ml), and either aspartic acidor Casamino Acids (100 pg/ml) in a 12-ml sterileconical centrifuge tube (W. Sarstedt, Princeton, N.J.).The tubes were capped with serum stoppers (type 03-225-5: Fisher Scientific Co., Pittsburgh, Pa.) and incu-bated overnight at 32°C on a revolving (60 rpm) tissueculture tube roller. The cultures were collected bycentrifugation and suspended in 5 ml of NFDM orSNFDM medium containing appropriate amino acids,antibiotics, and aspartic acid, serine, histidine, orCasamino Acids (100 ,ug/ml). Anaerobiosis was notmaintained during the change of medium. The tubeswere recapped with serum stoppers, and 2 ml of thegas phase was replaced with acetylene. Nitrogen fixa-tion activity was monitored by reduction of acetyleneto ethylene (29). Cultures were incubated at 32°C, andethylene production was measured hourly for 6 to 9 hand at 24 h by injecting 0.5-ml gas samples into aVarian model 940 gas chromatograph fitted with a 45-cm Parapak N (Waters Associates, Inc.) column (in-ternal diameter, 1 mm) at 37°C, using N2 as the carriergas at a flow rate of 7 ml/min. In this liquid assay, thegrowth of cells is not dependent upon nitrogen fixationactivity. We used this assay in our experiments toavoid the possibility that bacterial strains would beidentified as Nif because they grew poorly underthese conditions and not because they were unable toderepress and express nif genes specifically. We spe-cifically added aspartic acid or Casamino Acids to theassay medium to avoid the possibility that bacterialstrains would be identified as Nif because the plas-

TABLE 2. Plasmids used in this study

Plasmid Vector Phenotype and/or genotypea referencepMB9 Tcr 35pBR322 Tcr Apr 4pACYC184 Tcr Cm, 7pACYC177 Apr Kmr 7pCRA13 pMB9 Tcr hisDG 5pCRA37 pMB9 Tcr hisDG nifQBALRFWMVSUXN' 5pSA30 pACYC184 Tcr n#'EYKDH 6pMCl pACYC177 Apr nif'KDHJ M. CannonpMF291 pMF100 His' Gnd+ Tra+ nifJ2591::TnS M. FilserpWK25b pACYC184 Tcr nif'FWMVSUXN' W. KlipppWK250b pACYC184 Tcr nif'FWMVSUXN' W. KlipppWK120 pACYC184 CmT nifQBALRFWMVSUXNEYKDHJ W. Klipp

a A prime after a gene indicates the 3' end of the gene; a prime before a gene indicates the 5' end of the gene.Tcr, Tetracycline resistance; ApT, ampicillin resistance; Cmr, chloramphenicol resistance; Kmr, kanamycinresistance.

b pWK25 and pWK250 differ in the orientation of the cloned nifDNA.

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K. PNEUMONIAE NITROGEN FIXATION 47

mids which they carried were somehow lowering theamino acid pools within the cells.

(ii) Solid medium assay. Cultures were streaked fromsolid LB medium containing appropriate antibioticsonto NFDM or SNFDM solid medium containingappropriate antibiotics, amino acids, and either Casa-mino Acids (100 gxg/ml), aspartic acid, histidine, orserine (100 ,ug/ml). Plates were incubated anaerobical-ly at 30°C for 4 to 6 days in anaerobic chambers(Becton, Dickinson & Co., Cockeysville, Md.).Strains which grew to confluent, dark brown patchesduring this incubation period were scored as Nif+;strains which either grew to very small, pale coloniesor did not grow at all were scored as Nif-.Enzymes and reagents. Restriction endonucleases,

T4 DNA ligase, DNA polymerase I, and DNase I wereused as previously described (32). 5'-a-32P-labeleddeoxynucleoside triphosphates (350 Ci/mmol) wereobtained from Amersham Corp., Arlington Heights,Ill.; nitrocellulose (type BA85) was obtained fromSchleicher & Schuell Co., Keene, N.H.; and Sepha-dex G-50 (medium) was obtained from Pharmacia FineChemicals, Inc., Piscataway, N.J. Phenol was redis-tilled over nitrogen, adjusted to pH 7.0 with NaOH,and equilibrated with 10 mM Tris-hydrochloride (pH8.0).DNA biochemistry. Supercoiled plasmid DNA was

isolated by the cleared lysate procedure (8). Small-scale preparations of plasmid DNA were preparedfrom 5-mi saturated cultures as described previously(17), with the following modification: plasmid DNAwas desalted before enzymatic digestion by passagethrough a 0.4-ml Sephadex G-50 (medium) columnwhich had been equilibrated with water. The condi-tions used for digestion of DNA by restriction en-zymes (32), horizontal agarose gel electrophoresis(32), elution ofDNA fragments from agarose gels (36),ligation (11), nick translation (32), DNA transfer tonitrocellulose filters (38), DNA filter hybridization(32), colony filter hybridization (13), and restrictionenzyme mapping (5) have been described previously.

Plasmid construction methods. (i) Molecular cloningof the nifJ gene. To clone the nifJ gene, we tookadvantage of the fact that Tn5 confers kanamycinresistance to its host and is not cleaved by EcoRI (16).These two properties facilitate the construction ofhybrid plasmids that carry an EcoRI fragment contain-ing TnS, even when the starting source of DNA iscomplex.We first cloned a 20.8-kilobase EcoRI fragment

(pGR120) that carries the ni,2591::TnS allele and usedit as a physical probe to clone a 15-kilobase EcoRIfragment that carries wild-type nifJ sequences(pGR119). The details of the construction of pGR120and pGR119 have been described previously (G. E.Riedel, Ph.D. thesis, Harvard University, Cambridge,Mass., 1980). The data available from the correlatedphysical and genetic map of nif genes (1, 32) suggestvery strongly that pGR119 carries an intact nifJ gene.

(ii) Subcloning of nif DNA fragments derived fromhybrid nif plasmids. In a typical experiment, 1 ,ug ofhybrid nif plasmid DNA was mixed with 1 ,ug of thenew plasmid cloning vector, digested to completionwith a restriction enzyme, and ligated. The new hybridnifplasmids produced by this protocol were character-ized by restriction enzyme analysis.

(iii) Construction of deletions in hybrid nif plasmids

by restriction enzyme digestion. In a typical experi-ment, 1 ,ug of hybrid nif plasmid NDA was digestedeither partially or to completion with an appropriaterestriction enzyme in a volume of 50 ,ul. The digestedDNA was incubated at 65°C for 10 min (to inactivatethe restriction enzyme), diluted, and ligated. The newhybrid nif plasmids constructed by this protocol werecharacterized by restriction enzyme analysis to deter-mine their deletion endpoints.

RESULTS

Some hybrid nifplasmids inhibit Nif expressionin wild-type K. pneumoniae. Some hybrid nifplasmids inhibit Nif expression in wild-type K.pneumoniae, whereas other hybrid nif plasmidsdo not affect Nif expression. An example of thisobservation is presented in Fig. 1, which showsthe effects of pGR112, pGR113, and pACYC184upon Nif expression in wild-type K. pneumoniaestrain KP5525. pGR112 is a hybrid nif plasmidthat contains an 8.0-kilobase nifDNA fragment(nifQBALF') inserted into the EcoRI site ofpACYC184 (Table 3), and pGR113 is a hybrid nifplasmid that contains a 2.1-kilobase nif DNAfragment (rif 'NE') inserted into the EcoRI siteof pACYC184 (Table 3). pGR112, pGR113, andpACYC184 confer tetracycline resistance upontheir hosts; in addition, pACYC814 conferschloramphenicol resistance (7). Figure 1 showsthat the nitrogen fixation activity of a liquidculture of KP5525(pGR112) was less than 1% ofthe activity of wild-type parent strain KP5525,KP5525 (pGR113), or KP5525(pACYC184).When KP5525(pGR112) was cultured in LBbroth nonselectively, bacteria which had sponta-

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FIG. 1. Assay of nitrogen fixation activity. Liquidcultures of K. pneumoniae strains carrying differentplasmids were grown, derepressed, and assayed fornitrogen fixation activity as described in the text. Theonset of derepression coincided with the zero timepoint. Further details of the experiment are discussedin the text.

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neously cured pGR112 appeared in the culturepopulation. One such bacterial strain wasKP6056, which was not tetracycline resistantand contained no detectable plasmid DNAs(data not shown). The level of nitrogen fixationactivity in strain KP6056 was the same as thelevel in parent strain KP5525 (Fig. 1). Anothereffect of pGR112 was that Kp5525(pGR112)grew very poorly (usually not at all) onNFDM medium containing tetracycline, butKP5525(pACYC184) and KP5525(pGR113) grewto confluent, dark brown patches (data notshown).For brevity, we refer to the inhibition of the

Nif+ phenotype as "Nif inhibition" and to thehybrid nif plasmids associated with this inhibi-tion as "inhibitory" plasmids.

Figure 1 shows that Nif inhibition was corre-lated with the presence of pGR112 in cells.Figure 1 also shows that Nif inhibition was notassociated solely with the plasmid cloning vec-tor, pACYC184, or with a pACYC184 plasmidwhich contained an insertion in its EcoRI siteper se. These results suggest that Nif inhibitionis specifically associated with some or all of thenif sequences cloned in pGR112.To investigate further whether cloned nif se-

quences are specifically responsible for Nif inhi-bition and to identify those regions that areresponsible, we constructed 35 hybrid nif plas-mids containing nif insertions of various sizesand examined their effects upon Nif expressionin wild-type K. pneumoniae. Table 3 showssome of the characteristics of the nif plasmidsthat we constructed.

his (R) (W) ni.DG Q B A L F MVSUX NE Y K D H

Inhibitory plasmids can be constructed from atleast three separate regions of the nifgene cluster.We used the two effects described above forpGR112 to determine whether other hybrid nifplasmids were also associated with Nif inhibi-tion. We classified a hybrid nifplasmid as inhibi-tory only if the presence of the hybrid nifplasmid within a wild-type K. pneumoniae strainwas correlated with the loss by that strain ofmore than 90% of wild-type nitrogen fixationactivity in liquid culture assays and with thepoor growth or lack of growth by that strain onsolidified medium when N2 was used as the solenitrogen source.

K. pneumoniae strains KP1, KP5525, andKP5614 were transformed with different hybridnifplasmids, and the presence of each hybrid nifplasmid within each strain was confirmed byanalyzing plasmid DNA from each strain (datanot shown). All strains were assayed for nitro-gen fixation activity in liquid cultures and for theability to grow on solidified nitrogen-free medi-um.

Figure 2 shows the results of experiments inwhich EcoRI fragments spanning the entire his-nifgene cluster were assayed for Nif inhibition.This figure shows that there were at least threeregions of the nifgene cluster which were asso-ciated with Nif inhibition; these were containedin plasmids pGR112, pWK250, and pSA30. Thehybrid nif plasmid pWK120 (30), which con-tained the entire nifgene cluster (and thus all ofthe inhibitory regions), did not inhibit Nifexpression in wild-type K. pneumoniae. Howev-er, the hybrid nif plasmid pCRA37, which con-

J genetic map

physical map

hybrid nif plasmid

L-a pGR114 (4)

L..JpGR111 (4)

I I pGR112 (-

I XJ pWK250 F-)

I I pGR113 (+)

I IpSA30 (-)

.J pGR119 (+)I I pCRAl 3 (+)

I--A pGR103 N+

I___Ak pGR102 (-)

ffi I pCRA37 F-)pWK120 (4)

FIG. 2. Hybrid plasmids which inhibit nitrogen fixation in K. pneumoniae. At the top is a genetic and physicalmap (32) of the K. pneumoniae chromosome. nifR (R) and nfW (W) are enclosed with parentheses to indicatethat these regions may not be protein-encoding regions, but rather may affect nifexpression as sites (33). Thevertical lines indicate EcoRI sites, the open triangles indicate Hindlll sites, and the solid triangles indicateBamHI sites. +, Strains containing this plasmid produce wild-type levels of nitrogen fixation activity; -, strainscontaining this plasmid produce less than 10% of the wild-type levels of nitrogen fixation activity.

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TABLE 4. Nitrogen fixation activities of KP5525 containing different Nif inhibitory plasmidsaRelative nitrogen fixation activities of KP5525 containing:

Assay mediumbpACYC184 pGR112 pWK250 pSA30

NFDM 100 (5,020)c 0.2 8 0.5NFDM + aspartic acid 100 (2,150) 0.1 3 1.1NFDM + histidine 100 (2,870) 0.8 5 2NFDM + serine 100 (2,150) 0.2 4 4NFDM + Casamino Acids 100 (2,150) 1.0 10 4SNFDM 100 (5,380) 0.3 7 3SNFDM + aspartic acid 100 (3,230) 1.0 9 3SNFDM + histidine 100 (2,870) 0.1 8 2SNFDM + serine 100 (3,580) 0.8 14 6SNFDM + Casamino Acids 100 (2,150) 0.7 11 3

a Liquid cultures of K. pneumoniae strain KP5525 carrying different plasmids were grown, derepressed, andassayed for nitrogen fixation activity as described in the text.

b All media contained tetracycline (20 ,ug/ml).c The numbers in parentheses are the nanomoles of ethylene produced per milliliter of culture 24 h after

derepression.

tained at least two inhibitory nif regions, didinhibit Nif expression in wild-type K. pneumo-niae.We investigated whether inhibitory plasmids

affected only nifexpression in K. pneumoniae orwhether these plasmids also affected the expres-sion of other genes. It has been hypothesized(39, 41) that there is a common regulatory sys-tem which governs the expression of K. pneu-moniae genes which code for nitrogen fixationand histidine utilization. We investigated wheth-er Nif inhibitory plasmids affected the ability ofK. pneumoniae to grow in a variety of media andto utilize histidine as a nitrogen source duringcell growth. The rates of growth of KP5525-(pACYC184), KP5525(pGR112), KP5525-(pWK250), and KP5525(pSA30) were similar inseveral different media (LB medium, NFDMmedium supplemented with NH4', SNFDM me-dium supplemented with NH4+). Inhibitory plas-mids did not significantly affect the ability ofKP5525 to grow anaerobically or its growth ratewhen histidine was used as a nitrogen source.These results are consistent with the hypothesisthat Nif inhibition is a phenomenon which spe-cifically affects nif expression, rather than aphenomenon which generally affects K. pneu-moniae nitrogen metabolism.We were also interested in whether inhibitory

plasmids inhibited Nif expression in K. pneumo-niae as a result of the assay conditions which weused. We conducted liquid and plate nitrogenfixation assays of strain KP5525 containingpACYC184, pGR112, pWK250, and pSA30 byusing several different conditions and media.Table 4 shows that Nif inhibition was not aproperty of any particular assay medium in theliquid culture assay. The results with solidifiedmedia paralleled the results of the liquid culture

assays (data not shown). pGR112 was consis-tently more inhibitory than pSA30, which wasmore inhibitory than pWK250.

Fine-structure mapping of nif regions associat-ed with Nif inhibition. We constructed a collec-tion of subclone and deletion derivatives ofpGR112, pWK250, pSA30, and several other nifplasmids to map more precisely the locationsand boundaries of nifDNA fragments associatedwith Nif inhibition. The collection of hybrid nifplasmids which we constructed allowed us toperform a deletion analysis on each of the EcoRInif DNA fragments associated with Nif inhibi-tion. The cloning vectors that we used (pMB9,pBR322, pACYC184, and pACYC177) did notdetectably affect Nif expression in wild-type K.pneumoniae strains KP1, KP5525, and KP5614(data not shown).

Figures 3 through 5 show the results of thedeletion analyses and indicate the location of thesmallest nif DNA fragment in each region towhich we could assign inhibitory properties.Figure 6 summarizes the results of Fig. 3through 5 and shows the locations and bound-aries of the four inhibitory nifregions in relationto the entire nifgene cluster. These four regionsare as follows: region I, a -2,600-base pair (bp)fragment containing the 3' end of the nifA geneand an intact nifB gene and extending into, orincluding, the nifQ gene; region II, a -3,300-bpfragment containing the 3' end of the nifF gene,nilfR, and intact nij'L gene, and the 5' end of thenifA gene; region III, a -830-bp fragment con-taining most or all of the nifU gene and, possi-bly, the nifUSVM promoter (see legend to Fig.6); region IV, a 391-bp fragment (40) containingthe 5' end of the nifH gene and the nifHDKpromoter. The sizes given above represent themaximum length for each inhibitory region; the

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I a I B I A L (R) F

H Xm XmI I

I I ISc Sc Sc Sc

cloned DNA fragments

Xm Xm

Xm XmI I

H

H

genetic map

-, physical map

plasmid

-1 pGR112,115 (-)116

-1 pGR377 (+

pGR397,398 1*1pGR117 (+)

J pGR118 (-)

HuSc Sc Slc *pGR378 lNl

H Sc Sc Sc pGR394 I-HUSc I Sc Sc . pGR379 l+

H 1Sc S15c Sc ' pGR393 I-Ip pGR382 (-)I pGR383 N+Sc 3300bp

Sc 2600bp _Scmaximum sizes of inhibitory fragments

FIG. 3. Hybrid plasmids which inhibit nitrogen fix-ation in K. pneumoniae. At the top is a genetic andphysical map (32) of the nifQBALRF' region of the K.pneumoniae chromosome. The dashed lines betweennUfgenes represent approximate gene boundaries (32).Solid lines indicate gene boundaries which could beassigned with an accuracy of ±150 bp. The location ofn#R (R) was assigned from genetic data only (33). Theunmarked vertical lines indicate EcoRI sites. Sc, SacI; Xm, XmaI; H, HindIII; P, PstI. The asterisksindicate Sacl fragments whose orientations were op-posite their orientations in the chromosome. For fur-ther explanation, see the legend to Fig. 2.

nifDNA fragment responsible for Nif inhibitionin each region may be shorter than the length-which we determined in this work.

Nif Inhibition is caused solely by the nif DNAsequence doned in each Nif Inhibitory plasmid.The results presented above (Fig. 2 through 5)strongly suggest that the inhibition activity ofeach inhibitory plasmid is associated with asequence within the cloned nif DNA and notassociated with junctions of vector and nifDNA. Three lines of evidence lead to this sug-gestion. (i) Cloned DNA fragments that con-tained a particular inhibitory nif region but haddifferent endpoints retained inhibitory activity.(ii) DNA fragments that contained a particularinhibitory nifregion could be cloned in oppositeorientations in the same cloning vector andretain inhibitory activity. (iii) DNA fragmentsthat contained a particular inhibitory nif regioncould be cloned in different multicopy cloningvectors and retain inhibitory activity.These data collectively suggest that Nif inhibi-

tion is caused specifically by nif sequencescloned in the inhibitory nif plasmid and is notcaused by sequences comprised of vector-nifDNA junctions.

Inhibitory plasmids recombine with but do notcomplement nfchromosomal mutations. The ob-

(W)F M V : S U ' X N

genetic map

I W-I I I I I I1 physical mapBS P B P SB PB

cloned DNA fragments

B

plasmid

g pWK25,250 (-)

r--,- pGR104 N.

rl pGR356 (+)B

IB rl pGR357 (I)B B B B

I I pGR154,163 (-)B B

m Ip

mP

I pGR362 (+)p

I | pGR364 (-)p

pGR369 (+)mB S

maximum sizeof inhibitory fragment

830bp

P B

FIG. 4. Hybrid plasmids which inhibit nitrogen fix-ation in K. pneumoniae. At the top is a genetic andphysical map ofthe nif'FWMVSUXN' region ofthe K.pneumoniae chromosome (32). The physical locationof nifX was based upon the data of Klipp and PNhler(30); nifW was located solely on the basis of geneticdata (33). B, BamHI; S, Sall; P, PstI. pGR104 carriesadditional DNA from the K. pneumoniae chromosomewhich is not included in this diagram (see Table 3). Forfurther explanation, see the legends to Fig. 2 and 3.

servation that certain hybrid nifplasmids causedNif inhibition necessitated re-interpretation ofseveral previously published experiments. Can-

E, Y K D H

I ifI I

S HS B Bg

cloned DNA fragment

I IH

genetic map

physical map

plasmid

pSA30 I-)

p BS2 l+l

I , p BS1 (-)H

p BS4 l+lBI I p BS3 (I)B

pGR367 l+l

pGR151 I-)

pGR373 I-)

I IB Bg

BgS-sSs

391bpmaximum size of inhibitory fragment r-I

Sa

FIG. 5. Hybrid plasmids which inhibit nitrogen fix-ation in K. pneumoniae. At the top is a genetic andphysical map of the nif 'EYKDH region of the K.pneumoniae chromosome (32). The location of nifYwas assigned on the basis of the data of Klipp andPtihler (30). Bg, BglII; Sa, Sau3A; H, Hindlll; B,BamHI; S, Safi. pGR367 carries additional DNA fromthe K. pneumoniae chromosome which is not includedin this diagram (see Table 3). For further explanation,see the legends to Fig. 2 and 3.

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4 -4- 4-4 - 4 4 4(R) (W)

Q B A L F M V S U XIN E Y K JDH genetic map

R It Ir R AD physical map

Regions associated with Nif inhibition:

m -I2600bp 11

III_

830bp

IVri

391bp

3300bp

FIG. 6. Regions of the nif gene cluster which are associated with Nif inhibition. The arrows indicate thedirection of transcription and the extent of each nifoperon. nifR (R) is part of the nifAL operon, and nifW (W) ispart of the nfliF operon (33). nijX and nifYhave not yet been assigned to nifoperons by genetic criteria. AlthoughnifMWVSU is depicted as one operon (21, 22), there is evidence which has been interpreted such that this region istwo operons (27, 28). The open triangles indicate EcoRI (R) sites.

non et al. (5, 6) observed that the hybrid nifplasmids pCRA37, pCM1, and pSA30 restoredthe Nif+ phenotype to appropriate K. pneumo-niae nif mutant strains. From these data, wededuced the number of nif genes carried onpCRA37, pCM1, and pSA30. We also concludedthat the nif genes carried on these plasmidscomplemented the nif mutations in the hoststrains. This conclusion is contradicted by theobservations presented here; pCRA37, pSA30,and pCM1 are inhibitory plasmids, and any nifcomplementation of which they are capableshould be masked by the Nif inhibition pheno-type which they confer. We reconcile this con-ffict in the following analysis.There are two differences between the experi-

mental methods used here and the methods usedby Cannon et al. (5, 6). The first difference isthat we used RecAM strains, whereas Rec+strains were used by Cannon et al. When RecA-derivatives of appropriate nif mutants of K.pneumoniae were transformed with eitherpCRA37, pCMI, or pSA30, the transformantswere Nif- regardless of the manner in which theassay for nitrogen fixation activity was per-formed (see below). This result demonstratesthat the previous conclusion that pCRA37,pCM1, and pSA30 were able to complement nifmutations is incorrect.The second difference between the methods

used here and the methods used by Cannon et al.is the manner in which the assays for nitrogenfixation activity were performed. In our experi-ments appropriate antibiotics were used in theassays; no antibiotics were used in the assaysperformed by Cannon et al. The liquid cultureassays performed in one of the studies of Can-non et al. (5) differ further from the liquid cultureassays described here. Cannon et al. allowedappropriate nif mutant K. pneumoniae strainscontaining pCRA37 to grow anaerobically for 12to 16 h in (nitrogen-free) NFDM medium beforethey were assayed for nitrogen fixation activity.

There was no comparable growth period inliquid NFDM medium in the assays describedhere (see above). When appropriate Rec+ nifmutants of K. pneumoniae were transformedwith pCRA37, pCM1, and pSA30 and assayedfor nitrogen fixation activity, the strains wereNif+ only when (i) antibiotics were not includedin solid NFDM medium and (ii) liquid assays(lacking antibiotics) were performed as de-scribed by Cannon et al. (5). When nitrogenfixation assays were performed as describedhere, the strains were Nif.These results led us to hypothesize that

pCRA37, pCM1, and pSA30 could recombinewith appropriate nif mutations in Rec+ hosts.However, it would not be sufficient for inhibi-tory nif plasmids such as pCRA37, pCM1, andpSA30 to recombine with a chromosomal nifmutation in order to restore the Nif+ phenotypeto a Nif- host strain. The recombination eventwould result in cells with wild-type chromo-somal nifgenes, but the cells would still containmany copies of the inhibitory plasmid and there-fore should remain Nif-. We hypothesized thatan additional event after recombination wasnecessary to yield a Nif+ strain, namely, thecuring of the inhibitory plasmid from its host.This hypothesis predicts that if an inhibitory nifplasmid restores the Nif+ phenotype to a Rec+nif mutant host, the Nif+ cell will no longercontain the Nif inhibitory plasmid. We testedthis hypothesis with the following experiment.KP5058 (hisD2 nifB213) and its RecA-

derivative KP5617 were transformed with theinhibitory nif plasmid pGR102 (hisDGnifQBALRFM') ( Fig. 2). The presence ofpGR102 plasmid DNA in the His' transform-ants was verified as described above.KP5617(pGR102) was His' Nif- in all assays fornitrogen fixation activity, whether they wereperformed as described above or as described byCannon et al. (5). KP5058(pGR102) was Nif inthe liquid culture assays described here and

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was usually Nif+ (22 of 25 assays) in theliquid culture assays performed as described byCannon et al. (5). KP5058(pRD1) andKP5617(pRD1) were Nif' under all assay condi-tions. These results are consistent with pGR102having the capacity to recombine with theniJB213 mutation but not the capacity to comple-ment it. To analyze further the Nif+ derivativesof KP5058(pGR102), we grew a culture ofKP5058(pGR102) in BS medium (containingNH4+), verified the presence of pGR102 DNA,and plated this culture onto NFDM medium andNFDM medium containing NH4'. After 6 daysof anaerobic culture, His.' Nif+ colonies ofKP5058(pGR102) appeared at a frequency of 5 x10-4 colonies per His' colony-forming unit. Weprepared DNA from 100 His' Nif+ derivativesof KP5058(pGR102); none contained any detect-able pGR102 DNA. As an additional test, wereplica-plated colonies of KP5058(pGR102) fromNFDM medium containing NH4' onto NFDMmedium and isolated DNA from 10 His' Nif+colonies and their His + parents. All of the His'parent colonies contained pGR102 DNA; noneof the His' Nif+ derivatives contained anydetectable pGR102 DNA. These results supportthe hypothesis that inhibitory nif plasmids re-store the Nif+ phenotype to nif mutant strainsby recombination, followed by curing of theplasmid.

DISCUSSIONThe data presented in this paper show that

certain hybrid nif plasmids cause inhibition ofNif expression in wild-type K. pneumoniae. Nifinhibition is defined operationally by the assaybehavior that wild-type K. pneumoniae strainsshow when they contain certain hybrid n#fplas-mids. (i) The strains exhibit <10%o of the wild-type nitrogen fixation activity in whole cellliquid culture assays, and (ii) the strains growvery poorly (or not at all) on solid media with N2as the sole nitrogen source.Our results show that there are at least four

regions of the njfgene cluster which can be usedto construct inhibitory plasmids (Fig. 6). Thespecific assignment of genes or parts of genesand their regulatory regions to each of the inhibi-tory nif regions depends upon the accuracy ofthe correlated genetic and physical map of thenifgenes (1, 32). In some cases the accuracy ofthis map has been confirmed by DNA sequenc-ing (37, 40) and by an analysis of polypeptide-encoding regions (30). Nevertheless, there areareas of the map, particularly in the nifVSUregion, where the mapping data are insufficientto yield unambiguous gene assignments (for ex-ample, inhibitory region III). It will be necessaryto map each inhibitory region at the nucleotidelevel. There may be additional inhibitory regions

in the nif gene cluster, and the construction ofhybrid nif plasmids which contain nif DNAfragments that overlap noninhibitory regionsmay identify new inhibitory regions.

It is likely that Nif inhibition is a dosage effectand is related to multiple copies of an inhibitorynif plasmid within the cell. This hypothesis issupported by the observation that stringentlyreplicating nif plasmids which contain approxi-mately the same regions of nifDNA contained ininhibitory plasmids pCRA37, pGR102, and pBS3are able to complement nifmutations in Rec- K.pneumoniae strains (22) and thus are not inhibi-tory plasmids. Furthermore, a stringently repli-cating plasmid that contains the same nWf DNAas pGR112 is not inhibitory (D. Ow, unpublisheddata). Finally, when the nif DNA from eitherpGR112 or pSA30 is integrated into the chromo-some of wild-type K. pneumoniae, there is noinhibition effect (D. Ow and V. Sundaresan,unpublished data).The data in this paper suggest strongly that

Nif inhibition is associated only with the njfDNA fragment contained in an inhibitory plas-mid. These data are consistent with the hypothe-sis that Nif inhibition is caused by a mechanismwhich is specific for Nif expression and that Nifinhibition is not caused by a general effect uponcellular metabolism. Our data show that Nifinhibition occurs under a variety of assay condi-tions, and our results show that inhibitory plas-mids can recombine with, but not complement,appropriate nif mutations. Thus, although theearlier conclusions of Cannon et al. (5, 6) werecorrect as to the location of nif genes onpCRA37, pCM1, and pSA30, the conclusion thatthese plasmids complemented nifmutations wasincorrect.There are two simple models for the mecha-

nism of Nif inhibition by hybrid nifplasmids: (i)the inhibitory plasmid makes a polypeptide orRNA product which interferes with Nif expres-sion (product model), and (ii) the inhibitoryplasmid DNA interacts directly with a macro-molecule in the cell, and this interaction inter-feres with Nif expression (site model).One possible product model is that inhibitory

nif plasmids make an excess of a gene product(or part of a gene product) relative to other njfgene products and that this excess inhibits Nifexpression by interfering with transcription,translation, or nif protein interaction. Inhibitoryregion II (Fig. 6) is a promising candidate for theproduct mechanism because our deletion analy-sis (Fig. 3) suggests strongly that an intact nifLgene is necessary for Nif inhibition (comparepGR394 with pGR379). The nifL gene producthas been implicated as a negative effector of nifregulation (15, 33). One possible mechanism,among many, is that Nif inhibitory region II

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VOL. 153, 1983

makes the host cell hypersensitive to oxygenrepression.One possible site model is that an inhibitory

nif plasmid contains a site which is recognizedand bound by a positive regulatory element. Nifinhibition would be caused by a binding by theNif inhibitory plasmid of this element to theextent that the element is no longer present inthe cell in sufficient quantities to perform itsfunction at its usual site(s) in the nifgene cluster.Conceptually, this site model for Nif inhibition isequivalent (but with an opposite effect) to themechanism by which multicopy plasmids con-taining the lac operator confer a Lac constitutivephenotype on their hosts by binding most (or all)of the lac repressor molecules in the cell (3, 14,25). In this example of the site model, Nifinhibitory plasmids bind a hypothetical tran-scription activator in the cell and prevent normalniftranscription of some nifoperons. It is possi-ble that the transcriptional activator is the nifAgene product, because the nifA gene product hasbeen implicated as a positive regulatory elementin Nif expression (9, 19, 21, 33, 34, 42). Nifinhibitory region IV is a promising candidate forthe site type of mechanism, because this regioncontains the niJHDK promoter (37, 40; V. Sun-daresan, personal communication) and only asmall fraction of the nifH coding region. Thisregion is also transcribed heavily during Nifinhibition (31).

Despite the attractiveness of the site andproduct models for certain inhibitory regions,the data presented in this paper do not rigorous-ly distinguish between the site and product mod-els of Nif inhibition for any of the four inhibitorynif regions.Although the four nif regions involved in Nif

inhibition produce the same general phenotype,it is likely that the four nif regions affect Nifexpression by different mechanisms. It will benecessary to determine the mode of action ofeach inhibitory nif region independently. Theelucidation of these modes of action may pro-vide insights into the molecular basis of nifgeneregulation.

ACKNOWLEDGMENTSWe thank W. Brill, M. Cannon, M. Filser, and W. Klipp for

strains and plasmids, D. Ow and V. Sundaresan for communi-cating unpublished data, and the members of the laboratory ofF.M.A. for critical discussion of this work.

This work was supported in part by grant PCM 78-15450from the National Science Foundation to F.M.A. and by theCommonwealth Scientific and Industrial Research Organiza-tion. G.E.R. was supported in part by a fellowship from theHelen Hay Whitney Foundation.

LITERATURE CITED1. Appelbaum, E. R., and R. A. Kramer. 1980. Restriction

mapping of deletions in the nif region of the Klebsiellapneumoniae chromosome. Mol. Gen. Genet. 179:349-354.


2. Ausubel, F. M., S. C. Bird, K. J. Durbin, K. A. Janssen,R. F. Margolskee, and A. P. Peskin. 1979. Glutaminesynthetase mutations which affect th. expression of nitro-gen fixation genes in Klebsiella pneumioniae. J. Bacteriol.140:597-606.

3. Backlman, K., M. Ptashne, and W. Gilbert. 1976. Con-struction of plasmids carrying the cI gene of bacterio-phage X. Proc. Natl. Acad. Sci. U.S.A. 73:4174-4178.

4. Bolvar, F., R. L. Rodriguez, P. J. Greene, M. C. Betlach,H. L. Heyneker, H. W. Boyer, J. H. Crosa, and S. Falkow.1977. Construction and characterization of new cloningvehicles. II. A multipurpose cloning system. Gene 2:95-113.

5. Cannon, F. C., G. E. Riedel, and F. M. Ausubel. 1977.Recombinant plasmid that carries part of the nitrogenfixation (niO cluster of Klebsiella pneumoniae. Proc. Natl.Acad. Sci. U.S.A. 74:2963-2967.

6. Cannon, F. C., G. E. Riedel, and F. M. Ausubel. 1979.Overlapping sequences of K. pneumoniae nifDNA clonedand characterized. Mol. Gen. Genet. 174:59-66.

7. Chang, A. C. Y. C., and S. N. Cohen. 1978. Constructionand characterization of amplifiable multicopy DNA clon-ing vehicles derived from the P15A cryptic miniplasmid. J.Bacteriol. 134:1141-1156.

8. Clewell, D. B. 1972. Nature of Col El plasmid replicationin Escherichia coli in the presence of chloramphenicol. J.Bacteriol. 110:667-676.

9. Dixon, R., R. R. Eady, G. Espin, S. Hill, M. laccarino, D.Kahn, and M. Merrick. 1980. Analysis of regulation ofKlebsiella pneumoniae nitrogen fixation (nij) gene clusterwith gene fusions. Nature (London) 286:128-132.

10. Dixon, R. A., C. Kennedy, A. Kondorosi, V. Krishnapillal,and M. Merrick. 1977. Complementation analysis of Kleb-siella pneumoniae mutants defective in nitrogen fixation.Mol. Gen. Genet. 157:189-198.

11. Dugaiczyk, A., H. W. Boyer, and H. M. Goodman. 1975.Ligation of EcoRI fragments into linear and circularstructures. J. Mol. Biol. 96:171-184.

12. Elmerich, C., J. Houmard, L. Sibold, I. Manheimer, andN. Charpin. 1978. Genetic and biochemical analysis ofmutants induced by bacteriophage Mu DNA integrationinto Klebsiella pneumoniae nitrogen fixation genes. Mol.Gen. Genet. 165:181-189.

13. Grunstein, M., and D. S. Hogness. 1975. Colony hybrid-ization: a method for the isolation of cloned DNAs thatcontain a specific gene. Proc. Natl. Acad. Sci. U.S.A.72:3961-3965.

14. Heyneker, H. L., J. Shine, H. M. Goodman, H. W. Boyer,J. Rosenberg, R. E. Dickerson, S. A. Narang, K. Itakura,S. Lin, and A. D. Riggs. 1976. Synthetic lac operatorDNA is functional in vivo. Nature (London) 263:748-752.

15. Hill, S., C. Kennedy, E. Kavanagh, R. B. Goldberg, andR. Hanau. 1981. Nitrogen fixation gene (nifL) involved inoxygen regulation of nitrogenase synthesis in K. pneumo-niae. Nature (London) 290:424-426.

16. Jorgensen, R. A., S. J. Rothstein, and W. S. Reznlkoff.1979. A restriction enzyme cleavage map of Tn5 andlocation of a region encoding neomycin resistance. Mol.Gen. Genet. 177:65-72.

17. Klein, R. D., E. Selsing, and R. D. Wels. 1980. A rapidmicroscale technique for isolation of recombinant plasmidDNA suitable for restriction enzyme analysis. Plasmid3:88-91.

18. Lederberg, J., and N. Zinder. 1948. Concentration ofbiochemical mutants of bacteria with penicillin. Am.Chem. Soc. J. 70:4267-4269.

19. MacNeil, D., and W. J. Bril. 1980. Mutations in nifgenesthat cause Klebsiella pneumoniae to be derepressed fornitrogenase synthesis in the presence of ammonium. J.Bacteriol. 144:744-751.

20. MacNeil, D., M. Howe, and W. J. Brill. 1980. Isolation andcharacterization of X specialized transducing phages car-rying the Klebsiella pneumoniae nif (nitrogen fixation)genes. J. Bacteriol. 141:1264-1271.

21. MacNei, D., J. Zhu, and W. J. Brill. 1981. Regulation of


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nitrogen fixation in Klebsiella pneumoniae: isolation andcharacterization of strains with nif-lac fusions. J. Bacte-riol. 145:348-357.

22. MacNell, T., D. MacNeil, G. P. Roberta, M. A. Supiano,and W. J. Brill. 1978. Fine structure mapping and comple-mentation analysis of nif (nitrogen fixation) genes inKlebsiella pneumoniae. J. Bacteriol. 136:253-266.

23. Mabl, M. C., P. W. WiUson, M. A. Fife, and W. H. Ewing.1965. Nitrogen fixation by members of the tribe Kiebsiel-leae. J. Bacteriol. 89:1482-1487.

24. Mandel, M., and A. Eip. 1970. Calcium dependent bacte-riophage DNA infection. J. Mol. Biol. 53:159-162.

25. Marlans, K. J., R. Wu, J. Stawinsid, T. Hozumi, and S. A.Narng. 1976. Cloned synthetic lac operator DNA isbiologically active. Nature (London) 263:744-748.

26. Marnur, J. 1961. A procedure for the isolation of deoxyri-bonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218.

27. Merrick, M., M. Filaer, R. Dixon, C. Elmerich, L. Sibold,and J. Houmard. 1980. The use of translocatable geneticelements to construct a fine-structure map of the Klebsiel-la pneumoniae nitrogen fixation (nit) gene cluster. J. Gen.Microbiol. 117:509-520.

28. Merrick, M., M. Flwer, C. Kennedy, and R. Dixon. 1978.Polarity of mutations induced by insertion of transposonsTnO, Tn7, and TnlO into the n(fgene cluster of Klebsiellapneumoniae. Mol. Gen. Genet. 165:103-111.

29. Postse, J. R. 1972. The acetylene reduction test fornitrogen fixation, p. 343-356. In J. R. Norris and D. W.Ribbons (ed.), Methods in microbiology, vol. 6B. Aca-demic Press, Inc., London.

30. PFUIer, A., and W. Klipp. 1981. Fine structure analysis ofthe gene region for N2-fixation (nit) of Klebsiella pneumo-niae, p. 276-286. In H. Bothe and A. Trebst (ed.), Biologyof inorganic nitrogen and sulphur. Springer-Verlag, Ber-lin.

31. Riedel, G. E., and F. M. Auaubel. 1981. Nitrogen fixationin wildtype Klebsiella pneumoniae is inhibited in vivo incertain hybrid n(fplasmids, p. 396. In A. H. Gibson andW. E. Newton (ed.), Current perspectives in nitrogenfixation. Australian Academy of Science, Canberra.

32. Rbedel, G. E., F. M. Aumbel, and F. C. Cannon. 1979.

J. BACTERIOL.

Physical map of chromosomal nitrogen fixation (nif) genesof Klebsiella pneumoniae. Proc. Natl. Acad. Sci. U.S.A.76:2866-2870.

33. Roberta, G. P., and W. J. BrIll. 1980. Gene-productrelationships of the nif (nitrogen fixation) regulon ofKlebsiella pneumoniae. J. Bacteriol. 144:210-216.

34. Roberb, G. P., T. MacNeil, D. MacNeil, and W. J. Brill.1978. Regulation and characterization of protein productscoded by the nif (nitrogen fixation) genes of Klebsiellapneumoniae. J. Bacteriol. 136:267-279.

35. Rodru, R. L., F. Boivar, H. M. Goodman, H. W.Boyer, and M. C. Betach. 1976. Construction and charac-terization of cloning vehicles, p. 471-477. In C. P. Nier-lich, W. S. Rutter, and C. F. Fox (ed.), Molecular mecha-nisms in the control of gene expression. Academic Press,Inc., San Francisco.

36. Sakano, H., J. H. Rogers, K. HIppI, C. Brack, A. Traun-ecker, R. Maki, R. Wall, and S. Tonegawa. 1979. Domainsand the hinge region of an immunoglobulin heavy chainare encoded in separate DNA segments. Nature (London)277:627-633.

37. Scott, K. F., B. G. RoUfe, and J. Shine. 1981. Biologicalnitrogen fixation: primary structure of the Klebsiellapneumoniae nfH and n#! genes. J. Mol. Appl. Genet1:71-81.

38. Southern, E. M. 1975. Detection of specific sequencesamong DNA fragments separated by gel electrophoresis.J. Mol. Biol. 98:503-517.

39. Strdcber, S. L., K. T. Shanmugam, F. M. Auwbel, C.Morandi, and R. B. Goldberg. 1974. Regulation of nitro-gen fixation in Klebsiella pneumoniae: evidence for gluta-mine synthetase as a regulator of nitrogenase synthesis. J.Bacteriol. 120:815-821.

40. nrean, V., and F. M. Awaubel. 1981. Nucleotidesequence of the gene coding for the nitrogenase ironprotein from Klebsiella pneumoniae. J. Biol. Chem.256:2808-2812.

41. Tyler, B. 1978. Regulation of the assimilation of nitrogencompounds. Annu. Rev. Biochem. 47:1127-1162.

42. Zhu, J., and W. J. Brlll. 1981. Temperature sensitivity ofthe regulation of nitrogenase synthesis by Klebsiella pneu-moniae. J. Bacteriol. 145:1116-1118.


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