molecular cloning of nif dnafrom azotobacter vinelandiit · pneumoniae nif fragments al, a2, and a3...

Vol. 162, No. 1JOURNAL OF BACTERIOLOGY, Apr. 1985, p. 21-280021-9193/85/040021-08$02.00/0Copyright © 1985, American Society for Microbiology

Molecular Cloning of nifDNA from Azotobacter vinelandiitPAUL E. BISHOP,lt* THOMAS M. RIZZO,' AND KENNETH F. BOTT2

Agricultural Research Service, U.S. Department of Agriculture, and Department of Microbiology, North Carolina StateUniversity, Raleigh, North Carolina 27695,1 and Department of Microbiology, University of North Carolina School of

Medicine, Chapel Hill, North Carolina 275142

Received 3 August 1984/Accepted 27 December 1984

Two clones which contained nif DNA were isolated from a clone bank of total EcoRI-digested Azotobactervinelandii DNA. The clones carrying the recombinant plasmids were identified by use of the 32P-labeled6.2-kilobase (kb) nf insert from pSA30 (which contains the Klebsiella pneumoniae WjK, niD, and nipI genes)as a hybridization probe. Hybridization analysis with fragments derived from the nif insert of pSA30 showedthat the 2.6-kb insert from one of the plasmids (pLB1) contains nifK whereas the 1.4-kb insert from the otherplasmid (pLB3) contains nJID. Marker rescue tests using genetic transformation indicated that the 2.6-kb A.vinelandii nf fragment contains the wild-type alleles for the nif-6 and nif-38 mutations carried by Nif strainsUW6 and UW38. The 1.4-kb insert contains the wild-type allele for the nif-10 mutation carried by Nif strainUW1O.

The enzyme system in Azotobacter vinelandii that isresponsible for the reduction of N2 to ammonia consists ofdinitrogenase, a molybdenum- and iron-containing protein,and dinitrogenase reductase which is an iron-containingprotein. Mutant strains of A. vinelandii which are unable tofix N2 (Nif-) have been isolated and characterized withrespect to electron paramagnetic resonance signals, theiractivities for dinitrogenase and dinitrogenase reductase, andantigenic cross-reactive material (25). The nif mutationscarried by these strains have been genetically mapped withrecombination index values (1). However, fine-structureanalysis of the nif region on the A. vinelandii genome hasbeen hampered by the lack of genetic markers (other than nifmutations) and, with the exception of genetic transformation(17, 18), the lack of gene transfer systems suitable for geneticmapping. To facilitate a genetic analysis of the nif region onthe A. vinelandii genome, we have decided to use a combi-nation of conventional genetic techniques and recombinantDNA techniques. A nif DNA probe (pSA30) consisting ofcloned nifHDK genes (which code for subunits of dinitroge-nase and dinitrogenase reductase) from Klebsiella pneumo-niae has been shown to hybridize to five different EcoRIfragments of A. vinelandii DNA (22). This probe has beenused to identify recombinant cosmids containing nifDNA ina cosmid library of A. vinelandii genomic DNA (13). Clonesbearing nifgenes have been selected from a gene bank of A.chroococcum DNA, using both pSA30 and pLB3 (a plasmiddescribed in this report) as probes (21). In this report wedescribe the isolation and characterization of recombinantplasmids containing the nifK and nifD genes ofA. vinelandii.

(Preliminary results of this study were presented at the83rd Annual Meeting of the American Society for Microbio-logy, New Orleans, La., 3 to 7 March 1983 [Abstr. Annu.Meet. Am. Soc. Microbiol. 1983, K69, K70, p. 188]).

* Corresponding author.t Paper no. 8725 of the Journal Series of the North Carolina

Agricultural Research Service at Raleigh.t Present address: A.R.C. Unit of Nitrogen Fixation, University

of Sussex, Brighton BN1 9RQ, U.K.

MATERIALS AND METHODS

Bacterial strains and media. The A. vinelandii strains usedin this investigation (Table 1) were cultured in modified Burkmedium (28). When it was necessary to include fixed nitro-gen (N) in the medium, ammonium acetate (NH4OAc) wasadded to a concentration of 400 ,ug of N per ml. Solid N-freeBurk medium contained 1.5% purified agar (Difco Labora-tories), whereas solid medium containing NH4OAc con-tained 1.5% Bacto-Agar (Difco). Escherichia coli strainsLE392, HB101 (6), and JM83 (14) were maintained at -20°Cin 50% TYE broth (tryptone [Difco], 15 g; yeast extract, 10g; NaCl, 5 g; per liter of water)-50% glycerol.DNA preparation and restriction endonuclease reactions.

Total DNA was prepared from 200 ml of a late-log-phaseculture of A. vinelandii (strain CA), .using the procedure ofSaito and Miura (24). Plasmid DNA was prepared essentiallyby the method of Norgard (16) from 1-liter cultures of E. colistrain LE392, HB101, or JM83 carrying the plasmid. Restric-tion enzymes were used as described in the 1981 BethesdaResearch Laboratories catalog. Double and triple restrictionenzyme digests were conducted as described by Ruvkun andAusubel (23). DNA fragments were recovered from agarosegels by the procedure of Dretzen et al. (11).

Electrophoresis of DNA and hybridization reactions. Elec-trophoresis of DNA in 0.8% agarose gels and ethidium

TABLE 1. A. vinelandii strainsStrain Genotype Phenotype' Reference

CA Wild type I+ II+ 7CA13 AnifK13 I- II+ This studyUWi nif-l I-I 25UW3 nif-3 I- I 25UW6 nif-6 I- I+ 25UW1o nif-10 I- II+ 25UW38 nif-38 I- Il+ 25UW136 rif-l I+ II+ 2

a I and II represent dinitrogenase and dinitrogenase reductase, respective-ly. Superscripts + and - indicate active and inactive proteins, respectively,as determined by the acetylene reduction method.

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22 BISHOP, RIZZO, AND BOTT

-15.3

-.2

-4.0

-2.7

-1.5

FIG. 1. Agarose gel of EcoRI-digested DNA (-5 jig) from A.vinelandii (left) and autoradiogram of 32P-labeled pSA30 DNAhybridized to this DNA (right). Numbers on the right margin refer tokilobase pairs.

bromide staining were conducted as described previously(15, 19). The transfer of DNA to nitrocellulose filters (Sch-leicher & Schuell, Inc.) was a modification (26) of thetechnique of Southern (27). Filters were treated as describedby Denhardt (10) and hybridized in 6x SSC (0.15 M NaClplus 0.015 M sodium citrate)-0.5% sodium dodecyl

sulfate-0.02% bovine serum albumin-0.02% Ficoll-0.02%polyvinylpyrrolidone overnight at 70°C to DNA probeslabeled with 32P by nick translation (12). Sizes of DNAfragments were determined by reference to known sizes ofDNA fragments from phage lambda or a 1-kilobase (kb)ladder (Bethesda Research Laboratories, Inc.) run on eachgel.

Genetic transformation. Transformation of calcium-treatedE. coli by plasmid DNA was conducted as described byDagert and Ehrlich (9). Transformation of A. vinelandii wasperformed by using a modification of the plate methoddescribed by Page and Sadoff (17). The modification in-volved the use of competent cells generated by Fe starvationas described by Page and Von Tigerstrom (18).

Cloning of A. vinelandii nif DNA. An EcoRI digest of A.vinelandii DNA (2 jig) was incubated at 12°C for 16 h withEcoRI-cleaved pBR325 DNA (0.8 pLg) in the presence of T4ligase as described in the 1981 Bethesda Research Labora-tories catalog. After transformation of E. coli strain LE392with the ligated DNA, the transformation culture was incu-bated in the presence of ampicillin (20 p.g/ml) followed byD-cycloserine (100 ,ug/ml) treatment in the presence of 25 pugof chloramphenicol per ml as described by Bolivar andBackman (5). Greater than 99% of the tetracycline-resistant(Tetr) transformants were chloramphenicol sensitive (Cms),indicating that a majority of the Tetr transformants carriedplasmids containing insert DNA. The colony hybridizationprocedure of Thayer (29) was used to screen Tetr transform-ants for ability to hybridize to the K. pneumoniae nifDNAinsert from pSA30.

Plasmids. Vector pBR325 was obtained from F. Bolivar(4), plasmid pSA30 (which contains the K. pneumoniae nifK,nifD, and nifH genes) was obtained from F. M. Ausubel (8),and vector pUC9 was obtained from Bethesda ResearchLaboratories, Inc. (30).

RESULTS AND DISCUSSION

Hybridization of 32P-labeled pSA30 to wild-type A. vinelan-dii DNA. Ruvkun and Ausubel (22) demonstrated that theinsert DNA carried by pSA30 (consisting of cloned nifHDKgenes from K. pneumoniae) hybridizes with five differentEcoRI fragments from A. vinelandii (strain UW10) DNA. Toensure that we could obtain the same result, pSA30 waslabeled with 32P by nick translation and hybridized toEcoRI-digested DNA from A. vinelandii strain CA. Figure 1shows that the 32P-labeled probe DNA hybridized to fiveDNA fragments with sizes of 15.3, 9.2, 4.0, 2.7, and 1.5 kb.Except for some discrepancy in fragment sizes, our resultswere similar to those obtained by Ruvkun and Ausubel (22).Recombinant plasmids containing A. vinelandii nif DNA.

EcoRI-digested A. vinelandii DNA was ligated into pBR325,followed by transformation of E. coli strain LE392 andD-cycloserine enrichment as described in Materials andMethods. Four clones out of 3,428 Tetr Cms transformantshybridized to the 32P-labeled nif insert from pSA30. Resultsfrom agarose gel electrophoresis of EcoRI-cleaved recombi-nant plasmids showed that, among the four clones, twodifferent sized EcoRI fragments had been cloned. Twoplasmids containing different sized insert DNAs (pLB1 andpLB3) were chosen for further study. Cleavage of pLB1 andpLB3 with EcoRI showed that pLB1 contains a 2.6-kb insertand pLB3 contains a 1.4-kb insert (Fig. 2A and 2B). Frag-ments of identical size which hybridized to the 2.6 and1.4-kb inserts were found in EcoRI digests of A. vinelandiiDNA as seen in the autoradiograms shown in Fig. 2A and B.

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MOLECULAR CLONING OF nif DNA FROM A. VINELANDII

Aa b c a b c d

kb

-41.:. *

:. ::::* . . .: .

q:w, -2.6

kb

-6.9-6.0

-4.1

-2.6

-1.4

FIG. 2. Agarose gels and autoradiograms of EcoRI-digested A. vinelandii DNA (-5 ,ug) and EcoRI-cleaved recombinant plasmid DNA.(A) Lane a contains EcoRI-digested A. vinelandii DNA, lane b does not contain DNA, and lane c contains EcoRI-cleaved pLB1. Theautoradiogram (right) shows hybridization of 32P-labeled pLB1 insert DNA to the DNA shown in the agarose gel (left). (B) Lane a containsEcoRI-digested A. vinelandii DNA, lane b contains EcoRI-cleaved pLB1, lane c does not contain DNA, and lane d contains EcoRI-cleavedpLB3. The autoradiogram (right) shows hybridization of 32P-labeled pLB3 insert DNA to the DNA shown in the agarose gel (left).

.-Al

A3m :A2

In addition to bands corresponding to the 2.6- and 1.4-kbinsert DNAs, there is a faint band which represents a 4.1-kbfragment in EcoRI digests of A. vinelandii DNA that hybrid-ized to both insert DNAs. This fragment was also found inthe EcoRI-digested DNA shown in Fig. 1, where pSA30 wasused as the hybridization probe. Since the size of the 4.1-kbfragment is roughly equal to the sum (4.0 kb) of the lengthsof the cloned 2.6- and 1.4-kb fragments and both of thesefragments hybridized to the 4.1-kb fragment, this fragmentprobably represents a partial digestion product consisting ofthe 2.6- and 1.4-kb fragments.The band corresponding to the vector DNA for pLB3 (Fig.

2B) appears to contain an addition since it is 0.9 kb largerthan pBR325, which is 6.0 kb.

FIG. 3. Agarose gels and autoradiograms of BamHI-HindIIIdouble digests of fragment A from pSA30. (A) Agarose gel of K.pneumoniae nif fragments Al, A2, and A3 (left) and matchingautoradiogram of 32P-labeled pLB1 insert DNA hybridized to theseDNA fragments. (B) Agarose gel of K. pneumoniae nif fragmentsAl, A2, and A3 (left) and matching autoradiogram of 32P-labeledpLB3 insert DNA hybridized to these DNA fragments.

A B

-A1

-A3

:-A2:

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FIG. 4. Restriction map of pLB1 (A) and pLB3A (B). The heavy lines represent the nif insert DNAs.

To further characterize the cloned A. vinelandii nifDNAs,32P-labeled insert DNAs were hybridized to the K. pneumo-niae nifDNA fragments Al, A2 and A3, which result fromBamHI-HindIII double digests of the nif insert from pSA30(22).

AR P P P S S

LS R

pLB1

LiPTMR 10I I pTMR 11

J pTMR 12Li pTMR 13

Li pTMR 14IpTMR 15Li pTMR 16

CR H

I-I Al

Fragment Al contains the NH2-terminal region of the K.pneumoniae niWE gene and the COOH-terminal region of theni,fK gene; the A2 fragment contains the NH2-terminial regionof the nifK gene and the COOH-terminal region of the nifDgene; and the A3 fragment contains the NH2-terminal region

BR Al ILi pTMR17

A RI nLBR3A

I J pTMR 18

LpIpTR19

B Bg RJ pSA30

I 1A2I A3I A3A

I l1kb I IA3BFIG. 5. Physical maps and plasmids derived from cloned nif DNAs. Abbreviations: A, AvaI; B, BamHI; Bg, BgiII; H, HindIll; P, PstI;

R, EcoRI; S, Sall. (A) Physical map of the 2.6-kb insert of pLB1 showing specific fragments ligated to pUC9 to form the pTMR plasmids.(B) Physical map of the 1.4-kb insert of pLB3A showing specific fragments ligated to pUC9 to form the pTMR plasmids. (C) Physical mapof the A fragment of K. pneumoniae carried by pSA30. The sizes of the purified fragments are as follows: Al, 2.9 kb; A2, 1.5 kb; A3A, 1.1kb; A3B, 0.74 kb.

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kb

4.1

3.1

2.0-

a b c d e f g h ii k IA

B

D

U

FIG. 6. Autoradiograms showing hybridization of 32P-labeled K. pneumoniae nifDNA to the pTMR plasmids. Lanes a to 1 contained thefollowing: a, 1.0-kb ladder; b, pUC9; c, pTMR10; d, pTMR11; e, pTMR12; f, pTMR13; g, pTMR14; h, pTMR15; i, pTMR16; j, pTMR17; k,pTMR18; 1, pTMR19. (A) Agarose gel of EcoRI-linearized pTMR plasmids. (B) Autoradiogram showing hybridization of 32P-labeled K.pneumoniae nif fragment Al. (C) Autoradiogram showing hybridization of 32P-labeled K. pneumoniae nif fragment A2. (D) Autoradiogramshowing hybridization of 32P-labeled K. pneumoniae niffragment A3A. The 32P-labeled K. pneumoniae nif fragment A3B did not hybridizeto any of the pTMR plasmids.

of nifjD plus the entire nijH gene (22). The results of theseexperiments are shown in Fig. 3A and B. The 2.6-kb inserthybridized to fragments Al and A2, whereas the 1.4-kbinsert hybridized to fragments A2 and A3. Thus it appearsthat the 2.6-kb insert contains nucleotide sequence homol-ogy to the nifK gene and possibly the niJD gene of K.pneumoniae, whereas the 1.4-kb insert shows homology tothe nipj gene and also possibly to the nifK or the nifH gene.

In Fig. 4A a restriction enzyme map of pLB1 is presented.To avoid possible problems with the 0.9-kb addition locatedon pLB3, the 1.4-kb insert from pLB3 was recloned intopBR325. A restriction map of the resulting plasmid, pLB3A,is shown in Fig. 4B.To examine the homology between the A. vinelandil nif

DNA and that of K. pneumoniae in more detail, small

fragments of the 2.6- and 1.4-kb inserts of pLB1 and pLB3A(ranging in size from 120 to 980 base pairs) were ligated topUC9. The nif DNA inserts of these newly generatedrecombinant plasmids (pTMR10 through pTMR19) togetherrepresent the nifDNA inserts of pLB1 and pLB3A (Fig. 5Aand B).An unconventional ligation was performed in the construc-

tion of pTMR17, pTMR18, and pTMR19. The nif DNAinserts of pTMR17 and pTMR19 originally contained oneAvaI-generated cohesive end whereas that of pTMR18 con-tained two (Fig. SB). However, the physical map of pUC9presented by Vieira and Messing (30) does not show AvaIrestriction sites.

This problem was overcome by ligating AvaI-generatedcohesive ends of the nif DNA fragments to Sall-generated

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ilf DNA of pLBI

A RILI A.v. physical map

Hybridization:Strong

rF. - -o#-ModeratefEZJ:lWeakI EINone

.: ~ ~., ...N..1.V.-2A2A2

iA3A

I- + nifDELZ IA3B

I lIK n

genetic map

I 11kbFIG. 7. DNA homology relationships between the nifDNAs of A. vinelandii (A.v.) and K. pneumoniae (K.p.). By using this hybridization

data, the nifDNA restriction endonuclease map of A. vinelandii can be superimposed on the genetic map of the K. pneumoniae structuralgenes. Since pTMR17 and pTMR19 did not hybridize to K. pneumoniae DNA, it cannot be determined which EcoRI-generated cohesive endof the 1.4-kb nifDNA insert (shown at the right of the A. vinelandii physical map) is joined to the 2.6-kb nifDNA insert.

cohesive ends of pUC9. This was possible since at least twoof the four bases in both cohesive ends matched. Hydrogenbonding of matched base pairs was evidently enough topermit ligation. This fusion of heterogeneous cohesive endsresulted in the loss of the original AvaI and Sall sites (datanot shown).

R PH Pl1

p R

To show that the pTMR plasmids carried the specified nifDNA fragments, each plasmid was cleaved (in most cases)with the enzyme(s) used in its construction. Since the insertsof pTMR17, pTMR18, and pTMR19 could not be releasedfrom the vector DNA with AvaI or Sall, restriction endonu-cleases having sites within the lacZ fragment of pUC9

Hindx

Pst Ickawage

H p p''.53kb 6

Pst I

EcoRIcleavage

R

2.6 - kb insert

1.4 kb

IU;, R2.7 kb I lacZ fragment

Hin ZEcoR I Rcleavage 1 2.7 kb

H

I llkbFIG. 8. Construction of an in vitro deletion in the 2.6-kb nifDNA fragment.

RSII

InoDNAOfPLS3A I

S S P P P R AIllI I I I I

I 11 mmmmd I I

.

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immediately adjacent to the lost restriction endonucleasesites were used. This increased the size of each insert DNAby only a few base pairs. Their predicted sizes (from therestriction endonuclease maps of pLB1 and pLB3A) matchedthose determined from the restriction analysis of the pTMRplasmids (data not shown).To locate specific regions of homology between the nif

DNAs of A. vinelandii and K. pneumoniae, 32P-labeled Al,A2, A3A, and A3B (A3A and A3B were the result ofcleavage of A3 with BglII [Fig. SC]) fragments were hybrid-ized to the pTMR plasmids. It was found that the Alfragment hybridized strongly to pTMR14 and weakly topTMR12 and pTMR15 (Fig. 6). The A2 fragment hybridizedstrongly to pTMR18 and moderately to pTMR10 andpTMR11. The A3A fragment also hybridized strongly topTMR18, whereas the A3B fragment did not hybridize toany of the pTMR plasmids.These homology relationships are diagrammed in Fig. 7.

Since the A2 fragment hybridized to nif DNA carried byboth pLB1 and pLB3A, the 2.6- and 1.4-kb nifDNA insertsare probably very close to each other, if not contiguous, on

the A. vinelandii chromosome as shown in Fig. 7. A more

definitive experiment, however, is necessary to show that

abvv kb

.4a1

-3*6

-2.6

FIG. 9. Autoradiogram showing hybridization of 32P-labeledpLB1 to EcoRI digests of genomic DNA from strain CA (lane a) andstrain CA13 (lane b).

TABLE 2. Marker rescue of nif mutations by transformation withrecombinant plasmids

Donor Recipient A. NifrDonorida vinelandii transformationplasmid' strain frequencybpBR325 UW6 4 x 10-8pLB1 UW6 6 x 1O-5pLB3 UW6 <8.3 x 10-9pBR325 UW1o 2.4 x 10-8pLB1 UW1o 1.7 x 10-8pLB3 UW1o 1.4 x 10-4pBR325 UW38 <2.3 x 10-9pLB1 UW38 6.6 x 10-7pLB3 UW38 9.0 x io-9

aApproximately 3 ,ug of plasmid DNA was used for each cross.bThe NifC transformation frequencies were calculated as the number of

Nif' transformants per milliliter divided by the total number of cells permilliliter.

these fragments are contiguous. If the 4-kb fragment seen inFig. 1 and 2 represents a partial digestion product consistingof the 2.6- and 1.4-kb fragments, then a deletion in the A.vinelandii genome which affects either fragment should alsoresult in a shortened partial digestion product. Thus weremoved the 0.47-kb PstI fragment from the 2.6-kb insertcarried by pLB1 and the deletion plasmid, pTMR20, wasconstructed as diagrammed in Fig. 8. From the hybridizationresults it was predicted that this deletion would result in anifK deletion after insertion into the A. vinelandii genome byhomologous recombination. A deletion strain of A. vinelan-dii was constructed by transforming strain CA with a mix-ture of strain UW136 (0.6 p.g) and pTMR20 (6 ,ug) DNAfollowed by selection of rifampin-resistant (Rif) transform-ants which formed small colonies on Burk medium contain-ing 5 ,ug of N per ml. One of 78 small-colony Rif' transform-ants was found to be Nif- after testing for growth on BurkN-free agar. Consistent with a deletion in niJK, two-dimen-sional gel electrophoresis showed that this isolate (strainCA13) lacked the 13-subunit ofdinitrogenase (data not shown).Marker rescue tests with pLB1 and pLB3A showed that thenif mutation carried by strain CA13 was corrected by pLB1,but not pLB3A (data not shown). Figure 9 shows the resultsof hybridization of pLB1 with EcoRI digests of genomicDNA from strains CA and CA13. It is clear from theseresults that the 4.1-kb fragment is a partial digestion productconsisting of the 2.6- and 1.4-kb fragments since both the4.1- and 2.6-kb fragments appear to be shortened by about0.5 kb in strain CA13. Thus the 2.6- and 1.4-kb fragmentsappear to be contiguous on the A. vinelandii genome.

This conclusion is also in agreement with results of apartial sequence analysis of the nifHDK region from A.vinelandii (D. Dean, personal communication).Marker rescue of nif mutations by transformation with

pLB1 and pLB3. Plasmids pLB1 and pLB3 were tested forability to correct nif mutations carried by the Nif- strainsUW1, UW3, UW6, UW10, and UW38. Neither plasmid wascapable of transforming strains UW1 or UW3 to Nif+;however, pLB1 transformed strains UW6 and UW38 to Nif+and pLB3 transformed strain UW10 to Nif+ (Table 2). Thus,pLB1 carries the wild-type alleles for nif-6 and nif-38, bothof which result in the lack of the 13-subunit of dinitrogenase(3; unpublished data). Plasmid pLB3 contains the wild-typeallele for the nif-10 mutation. Strain UW10, which carriesthe nif-10 mutation, synthesizes both the a- and 1-subunitsof dinitrogenase but is inactive for this nitrogenase compo-nent (3, 25). Thus the results of the marker rescue tests

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provide further evidence that the inserts carried by pLB1and pLB3 contain DNA sequences for at least part of thenifK and niJD genes of A. vinelandii.

ACKNOWLEDGMENTSWe thank G. C. Stewart and M. F. Lampe for helpful discussions

during this study. We also acknowledge the technical assistance ofD. R. Hetherington and J. Kopczynski.

This project was supported by competitive grant 59-2371-670-0from the U.S. Department of Agriculture. These investigations werecooperative investigations of the Agricultural Research Service,U.S. Department of Agriculture, and the North Carolina Agricul-tural Research Service, Raleigh.

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single-stranded DNA bacteriophage M13. Recombinant DNATech. Bull. NIH Publ. 79-99. 2:43-48.

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17. Page, W. J., and H. L. Sadoff. 1976. Physiological factorsaffecting transformation of Azotobacter vinelandii. J. Bacteriol.125:1080-1087.

18. Page, W. J., and M. Von Tigerstrom. 1979. Optimal conditionsfor transformation of Azotobacter vinelandii. J. Bacteriol.139:1058-1061.

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