Molec. gen. Genet. 176, 17 24 (1979) © by Springer-Verlag 1979

Cloning of the Entire Region for Nitrogen Fixation from Klebsiella pneumoniae on a Multicopy Plasmid Vehicle in Escherichia coli A. Piihler, H.J. Burkardt, and W. Klipp Institut ffir Mikrobiologie und Biochemier Lehrstuhl Mikrobiologie, Universitfit Erlangen-Nfirnberg, Egerlandstr. 7, D-8520 Erlangen, Federal Republic of Germany

Summary. Two deletion mutants pAP1 (MW 82 Mdals) and pAP2 (MW 64 Mdals) were isolated by P1 transduction of the plasmid pRD1 (MW 101 (Mdals). These plasmid mutants still carry the his-nif region of K. pneumoniae. They are selftransmissible and mediate resistance to ampicillm, kanamycin and tetracycline. Comparing the HindIII maps of pRD1, pAPl and pAP2 showed that pAPll was derived from pRD 1 by an 8 jam deletion and pAP2 by two deletions the same 8 gm deletion and a further 9 gm deletion. The plasmids pAP1 and pAP2 helped us to locate the his-nif region of pRD1 on 3 adjacent HindIII fragments (number 5, 4 and 3 according to gelelectrophoresis). The molecular weights of these fragments were 8.2, 10 and 15 Mdals. These 3 fragments were cloned separately on the multicopy plasmid vehicle pWL625 giving rise to the hybrid plasmids pWK1 (pWL625+HindIII fragment 4), pWK2 (pWL625 + HindIII fragment 3) and pWK4 (pWL625+HindIII fragment 5). None of these hybrid plasmids conferred nitrogen fixation capacity on E. colt C cells. By combining HindIII fragment 4 and 3 in the same alignment as in pRD1 and cloning them together on pWL625 the hybrid plasmid pWK120 (pLW625 +HindIII fragraents 4 and 3) was found to carry the entire mf regJon. An E. colt C strain harbouring the plasmid pWK 120 grew on nitrogen free medium and reduced acetylene. The plasmid pWK 120 had a contourlength of 17 lain, a buoyant density of 1.715 g/ml and a copy number up to 65.

from Klebsiella pneumoniae. It was constructed by Dixon et al. (1976) using in vivo genetic engineering techniques. Beside the n/f region further markers like rfb +, his + and shiA + are located on this plasmid. Since the pRD1 plasmid with a molecular weight of 101 Mdals is unstable in recA + hosts (Ptihler et al., 1979) it is not the ideal plasmid for the study of n/f-genes from K. pneumoniae. Cloning of the nif region on appropriate plasmid vehicles should circumvent these difficulties. The first cloning experiments of mf-genes were reported by Ausubel et al. (1977) and Cannon et al. (1977) but only parts of the mf region were cloned. We now report the cloning of the entire ntf-region of K. pneumoniae on a multicopy plasmid vehicle in Escherichia colt. Prior to these experiments we analyzed more precisely the genetical and physical organization of the pRD1 plasmid. We isolated deletion mutants which helped us to localize the n/f genes on the HindIII map of plasmid pRD1. Parts of this work were reported during the EMBO workshop on "Plasmids and other extrachromosomal genetic elements", (Berlin, Germany, April 1978) and during the Steenbock Kettering Symposium on "Nitrogen Fixation" (Madison, Wisconsin, USA, June 1978). Material and Methods Bacterial Strains, Phages and Plasmids. These are listed in Table 1. The pRD1 plasmid originally present in J62 1 was transferred to F122 and F127 by conjugation. Media. The media used in this study were described by Ptihler et al. (1979).

Introduction

The plasmid pRD1 which belongs to the P1 incompatibility group carries the tT~f(nitrogen fixation) region For qffprints contact: A. Ptihler

P1CMts- Transduction Procedure. For transduction experiments the donor strain was first lysogenized by P1CMts, the P1CMts lysogen was then heat induced and the resulting P1CMts-lysate was used for transduction experiments. For lysogenization the P1CMts stock and the donor culture were mixed at a multiplicity of m - 1. CaClx was added at a final

0026-8925/79/0176/0017/$01.60

18

A. Piihler et al. : Nitrogen Fixation from Klebsiella pneumoniae

Table 1. Bacterial strains, phages and plasmids Genetic marker

Source or references

E. coli strains E. coli K12 J62-1

pro, trp, his, lac, nal-r

Cannon et al. (1976)

E. coli K12 $52.12

ara, zx (lac, pro), str A, thi, (~2380dlacI), his: :Muc ÷, rec A

his: : Muc + mutation of CSH52

E. coli C F122

prototrophic, str-r

Strain collection, Lehrstuhl Mikrobiologie, Universit~t Erlangen

E. coli C F127

his, nal-r

(Miller, 1972)

phages P1CMts

Cm, heat inducible prophage

Rosner (1972)

plasmids pWL625

Km, Ap

Goebel et al. (1977)

pRD1

Kin, Tc, Ap, Tra +, Inc P1, Gnd, His, Nif

Dixon et al. (1976)

(formerly RP41)

Shi A

Genetic symbols for chromosomal and plasmid markers are those of Bachman et al. (1976) and Novick et al. (1976) respectively

concentration of 5mM. After incubation at 32 ° C for 30 min dilutions of the mixture were plated on chloramphenicol (100 ~tg/ml) containing agar The plates were incubated at 32 o C. The P1CMts lysogen was grown in Luria Broth containing i0mM MgC12 to a density of 2 x 108 cells/ml. The P1CMts prophage was then heat induced at 40 ° C for 30 min and the maturation of the P1CMts phage was carried out with slow shaking at 37 ° C. When lysis occurred chloroform was added and the debris was pelleted by centrifugation. The clear supernatant was removed and again sterilized by a few drops of chloroform. The P1CMts lysate was titered using LB plates and LB soft agar containing 2 and 5 m M CaC12 respectively. For the transduction experiment the recipient strain grown to 2 x l0 B cells/ml in LB containing 10 m M CaCI2 was mixed with the P1CMts lysate, containing 10 m M MgC12, at a multiplicity of m = 1 and m = 0 . 1 and incubated at 32 ° C for 30 rain. The mixture was centrifuged, the pellet resuspended and the cells plated on selective agar.

sary, slab gels were calibrated using D N A fragments of known sizes present in an EcoRI/HindlII double digest of 2 ci857S7 D N A isolated from the E. coli strain CSH45 (Miller, 1972).

Cloning Experiments. Prior to ligation pWL625 and pRD1 D N A isolated from E. coli C were digested with the restriction endonuclease HindIII. The endonuclease was inactivated by heat. fl-mercaptoethanol and ATP was added to give a final concentration of ligation buffer containing 10 m M Tris-HC1 (pH 7.5), 10 m M MgC12, 50raM NaC1, 1 0 m M ~-mercaptoethanol and 0.1 m M ATP. Appropriate quantities of T4 polynucleotide ligase were added and the solution incubated at 5 ° C overnight. Cells of E. coli C (F122) grown in Luria Broth were made competent for the uptake of D N A by the CaC12 shock procedure (Cohen et al., 1972).

Agarose Gel Electrophoresis of Crude Lysates. Starting from overTest.for Nitrogenase Activity of Nif + Strains. The acetylene reduction method was used to measure the nitrogenase activity as described by Piihler et al.. (1979).

Isolation, Buoyant Density Determination and Electron Microscopy of Plasmid DNA. All these methods were described by Pfihler et al. (1979).

HindIII-Digestion and Gel Electrophoresis. DNAs at concentrations in the range of 100 250 ~tg/ml were digested at 37 ° C, using appropriate quantities of restriction endonuclease HindIII. The digestions were carried out in 10 m M Tris-HC1 (pH 7.5), 10 mM MgC12 and 50 m M NaC1. The enzyme was inactivated by heating to 70 ° C for 10 min. The DNA-fragments were separated by electrophoresis in 1% (w/v) agarose gel in 40 m M Tris, 10 m M Na-acetate (pH 7.8) and 1 m M EDTA. The samples containing 0.5-1 gg D N A mixed with 10 ~tl 50% glycerol containing 0.025% bromophenol-blue were applied to wells in the gel. After electrophoresis the gels were stained for 30 rain in ethidiumbromide (1 gg/m) and photographed under UV-light on Polaroid-positive-negative-film. When neces-

night cultures the cells were lysed by lysozyme (5 mg/ml) for 10 rain at 0 ° C and by SDS treatment with a final concentration of 1% SDS and 0.25 M EDTA pH 8.0. The proteins were extracted by phenol and this crude lysate was directly used for gel electrophoresis in 0.7% (w/v) agarose gel in 90 m M Tris, 90 m M boric acid (pH 8.2) and 2.5 m M EDTA. Under these conditions plasmid DNA bands according to its size at different positions in the gel.

Results A. Isolation and Characterization o f Deletion M u t a n t s o f P l a s m i d p R D 1 D u e t o its h i g h m o l e c u l a r w e i g h t o f 101 M d a l s c o r r e s p o n d i n g t o 49 ~tm l e n g t h ( P t i h l e r e t al., 1979) t h e p R D 1 p l a s m i d is d i f f i c u l t t o u s e f o r t h e s t u d y o f

19

A. Pfihler et al. : Nitrogen Fixation from K,!ebsiellapneumon&e Table 2. Molecular properties of plasmids pRDI, pAP1 and pAP2" Plasmid

Length in gm

Density in g/cm 3

pRD1 b pAP1 pAP2

49.4_+ 1.4 (28 molecules) 41 +_ 1.4 (20 molecules) 32 + 1.4 (I9 molecules)

1.715 1.718 1.718

All plasmids are: Ap, Km, Tc, Tra +, His + , Nif + b The molecular properties of plasmid pRD1 were reported by Piihler et al. (1979)

nif-genes from K. pneumoniae. We therefore tried to isolate deletion mutants of pRD1 by the P1 transduction method. Phage P1 capable of generalized transduction is not able to package the whole pRD1 plasmid. But deletion mutants of' pRD1 which are approximately the size of the P1 genome should be packaged. Using the phage PlCMts (Rosner, 1972) we first lysogenized the strain J62-1 (pRD1), induced the prophage by raising the temperature and harvested the P1CMts-lysate. Strain J62-1 was then transduced by this lysate and His + transductants were selected by plating the mixture on minimal agar supplemented with proline and tryptophane. Since we did not use deletion inducing methods the transduction rate was rather low. Two transductants which showed ampicillin, kanamycin and tetracycline resistance and were able to reduce acetylene were Chosen for further studies. Furthermore all these markers (amp-r, kan-r, tet-r, his +, nif +) could be transferred as a unit by conjugation to strain $52.12 showing that these markers are located on a plasmid. We isolated the plasmid DNA of the two transconjugants and by electron microscopy we found that one plasmid was 41 gm and the other 32 gm long. The longer plasmid was designated pAPl, the shorter' one pAP2. In a CsC1 gradient of an analytical ultracentrifuge both plasmids show a buoyant density of 1.718 g/cm 3 indicating that plasmid pAPl as well as plasmid pAP2 lacks a DNA region of low buoyant density which is present in the plasmid pRD1 'with a density of 1.715 g/cm 3 (Pfihler et al., 1979). Tile molecular properties of plasmids pRD1, pAP1 and pAP2 are summarized in Table 2.

B. HindlII-Restriction l~laps of the Plasmids pRD1, pAP1 and pAP2 To determine the extent of deletions in pAP1 and pAP2 we examined the restriction fragments of pRD1, pAP1 and pAP2 using the endonucleases EcoRI and HindIII. Digestion of plasmid DNA by HindIII gave the answer. The HindIII fragments of pRD1, pAP1 and pAP2 separated by agarose gel elec-

Fig. 1. Agarose gel electrophoresis of HindIll-fragments of the plasmids pRD1, pAP1 and pAP2. DNA of the plasmids pRDI, pAP1 and pAP2 was digested by the endonuclease HindIII and the fragments were separated by agarose gel electrophoresis. The large HindIII-fragments ofpRD1 (A), pAP1 (B) and pAP2 (C) are numerated. Using an EcoRI-HindIII-double digest of 2-DNA as marker system the following molecular weights for the HindIII fragments of pRD1 were calculated Frag. Frag. Frag. Frag. Frag. Frag.

1 2 3 4 5 6

24 Mdals 20 Mdals 15 Mdals 10 Mdals 8.2 Mdals 8.2 Mdals

Frag. Frag. Frag. Frag. Frag. Frag. Frag.

7 8 9 10 11 12 13

4.4 Mdals 3.1 Mdals 2.0 Mdals 2.0 Mdals 1.6 Mdals 1.54 Mdals 1.5 Mdals

trophoresis are shown in Fig. 1. By comparing the restriction patterns of the different plasmids the following results were obtained: -

-

The first 6 HindIII fragments of pRD1 (1, 2, 3, 4, 5 and 6) and pAPl (1% 2% 3', 4% 5' and 6') correspond in size. The following smaller HindIII fragments of pRD1 are lacking in the HindIII digest of pAP1 with the exception of fragment 10.

A. P~ihler et al. : Nitrogen Fixation from Klebsiella pneumoniae

20

pRD1 i

'

1

SEGMENT

X

5

i

4

6

3

10 V v

pAP1 5'

2'

4'

31

T

|

'i

6'

V

V

10' vv

i

DELETION

A

i

pAP2 1"

2"

5" ,r~/v%/-~-~v~_,

ae~/'X./,

INTEGRATED RP4 PLASMID

V

i

i

i

!

DELETIONA

4" V

1" V i

HIS-NIF GENES

=! DELE T ION B

Fig. 2. The HindIII restriction m a p s of pRD1, pAP1 and pAP2. The HindIII restriction maps of the plasmids pRD1, pAP1 and pAP2 are given in a linear mode. The pRD1 m a p is opened at the HindIII site in order to start with the largest HindIII fragment. The fragment n u m b e r s defined in Fig. 1 are used to m a r k the different HindIII fragments on the map. Segment x of the p R D l - m a p contains 8 additional HindIII sites. The probable location of the deletions A and B, of RP4 and of the his-nil genes are depicted in the maps. v HindIII restriction site

-

pAP2 consists of only 4 HindIII fragments called 1", 2", 4" and 5". Fragment 1" seems to be longer than HindIII fragment 1' of pAP1. Fragments 2", 4" and 5" band at similar positions to fragments 2', 4' and 5' of pAP1. - Fragments 3', 6' and 10' of pAP1 do not appear in the HindIII digest of pAP2.

To learn more about the deleted DNA segments of pRD1 we analyzed more precisely the HindIII and the EcoRI fragments of pRD1 and found 16 HindIII and 20 EcoRI fragments with molecular weights > 0.15 Mdals. By combining the results of different methods we were able to construct the EcoRI and HindIII restriction map of the plasmid pRD1. Since this construction is not straight forward we do not report the different experiments which finally helped us to establish and to test the map. The complete EcoRI and HindIII restriction map will be published elsewhere. Part of the HindIfI restriction map is shown in Fig. 2. The HindlII fragments of pRD1 are arranged in the following order: 5, 4, 3, 6, 10, 1 and 2. Between the HindIII fragments 2 and 5 there is a DNA segment containing 8 additional HindIII sites. From the order of HindIII fragments in pRD1 the restriction maps of pAP1 and pAP2 were determined. Plasmid pAP1 can be derived from plasmid pRD1 by deleting the DNA-segment with the additional 8 HindIII sites. This deletion is called A and is depicted in Fig. 2. The remaining DNA still possesses 7 Hind

III sites the positions of which are in agreement with the HindIII pattern of plasmid pRD1 (Fig. 1). Plasmid pAP2 can be derived from plasmid pAP1 by an additional deletion, called B, which comprises the HindIII fragments 6' and 10' and parts of the HindIII fragments 3' and 1'. There are 4 HindIII sites left which after HindIII digestion generate the fragment pattern of plasmid pAP2 shown in Fig. 1. The length of the deletions, A and B can be calculated from the contour length measurements of the plasmids pRD1, pAP1 and pAP2 (Table 1). These data show that deletion A is 8 lam and deletion B 9 gm long. The exact starting and end points of the deletions A and B cannot be derived from the HindIII restriction patterns of the plasmids pRD1, pAP1 and pAP2. But it is assumed that deletion A starts at the right end and deletion B at the left end of the integrated plasmid RP4 (Fig. 2). The position of the RP4 plasmid integrated into the pRD1 plasmid can be determined as follows: Since the plasmid RP4 is 19.1 gm long (Burkardt et al., 1978) and shows only 1 HindIII restriction site (De Picker et al., 1977) it has to cover most of the HindIII fragments 1 and 2 of plasmid pRD1 (Fig. 2).

C. Cloning of HindlII Fragments of p R D l on a Multicopy Plasmid Vehicle Plasmids pRD1, pAPl and pAP2 all contain the entire RP4 plasmid and the his-nif-region of K. pneumoniae.

21

A. Ptihler et al. : Nitrogen Fixation from Klebsiella pneumoniae Table 3. Properties of the piasmids pWL625, pWKI, pWK2 or pWK120 and their hoststrain E. coli C Plasmid

pWL625 pWK1 pWK2 pWK120

Length of the plasmid in gm

Properties of E. coli C harboring the plasmid amp-r

kan-r

Growth in Acetylen mtrogenreduction free medium

+ + + +

+ -

+

4.6_+ 1.2 (19 molecules) 9.6+ 1.7 (17 molecules) 11.6+ 1.9 (14 molecules) I6.7_+2.3(11 molecules)

+

pWK1 =pWL625+HindIII fragment 4 pWK2 =pWL625+HindIII fragment 3 pWK 120= pWL625+ HindIII fragment 4 and 3 Since the RP4 plasmid covers the HindIII fragments 1 and 2 one can deduce from Fig. 2 that the his-n/f-region is located on the HindlII fragments 4 and 5 and on a part of the HindIII fragment 3. In order to get the whole mf-region of K. pneumoniae on a multicopy plasmid we first started to clone the HindIII fragments 3, 4 and 5 separately. As cloning vehicle we used the multicopy plasmid pWL625 which carries genes for ampicillin and kanamycin resistance (Goebel et al., 1977). pWL625 possesses only 1 HindIII site which is located directly in the kan-r gene. Cloning HindIII fragments into this HindIII site inactivates the kan-r gene. Hybrid plasmids are therefore indicated by ampicillin resistance and kanamycin sensitivity of transformed cells. For the cloning experiment we digested pRD1 D N A completely by HindIII. The same was done with pWL625 DNA. The completeness of the digestion was checked by agarose gel electrophoresis. Both DNA-fractions were then mixed and ligated over night by T4 ligase. CaC12 treated cells of E. coli C were then transformed with the ligation mixture. Single colonies of the transformed cells were selected on ampicillin containing agar. These colonies were replica plated on kanamycin containing agar in order to detect colonies harboring hybrid plasmids. Such colonies were isolated and the sizes of their hybrid plasmids were determined by agarose gel electrophoresis of crude lysates. We were able do detect colonies containing hybrid plasmids of high molecular weight. We isolated the D N A of such plasmids and after digestion with HindlII the fragments were compared with a HindIII digestion of pRD1 DNA. In this way we were able to show that we had cloned the HindIIII fragments 3, 4, 5 and 6 of the plasmid pRD1. Hybrid plasmids containing the HindIIII fragments 1 and 2 of p R D 1 could not be detected. In addition many hybrid plasmids with small HindIII fragments could be found but their properties are not reported here.

The hybrid plasmids which contained HindIII fragments 3, 4 and 5 were called p W K l = p W L 6 2 5 + HindIII fragment 4, p W K 2 - - pWL625 + HindIII fragment 3, and p W K 4 = pWL625 + HindIII fragment 5. None of these plasmids conferred nitrogen fixing ability on an E. coli C strain. The plasmid pWK4 complemented the his-mutation of strain F127. The HindIII restriction patterns of pWK1 and pWK2 which prove the hybrid nature of the plasmids are shown in Fig. 4. The contour lengths of the plasmids pWK1 and pWK2 are reported in Table 3.

D. Cloning of the n/f-Region of K. pneumoniae on a Multicopy Plasmid Vehicle

Our strategy to clone the whole n/f-region of K. pneumoniae was the following : A combination of the Hind III fragments 5, 4 and 3 of the plasmid pRD1 should give the functional n/f-region. Since there was some information that the HindIII fragment 5 of pRD1 does not carry nif-genes, we first designed an experiment to connect the HindIII fragments 4 and 3 in the same alignment as in plasmid pRD1 and to clone them in the plasmid vehicle pWL625. The cloning scheme is shown in Fig. 3. By an incomplete HindIII digestion we opened pWK1 and inserted the HindIII fragment 3 which we obtained by complete digestion of pWK2. If the resulting plasmid carries the whole n/f-region of K. pneumoniae it should be found by the following procedure: E. coli C is transformed by the ligation mixture. Transformants are enriched by subculturing the transformation mixture in ampicillin containing medium. After this step the transformants are transferred to nitrogen free medium. A significant increase in optical density should indicate Nz-fixing E. coli ceils, carrying a Nif + plasmid. The critical point in the above procedure is the transformation step. A plasmid carrying HindIII frag-

22

A. Piihler et al. : Nitrogen Fixation from Klebsiella pneumoniae pWL 6 2 5 pWL625

.... .,.

/,o Ill ......

H I N D Ill

HIND DIGESTION

DIGESTION pWL625

I LIGATION

@

pWL625

Fig. 3. The cloning scheme for the nif region from K. pneumoniae. Starting from the hybrid plasmids pWK 1 and pWK2 which contain the HindIII fragment 4 and 3 the plasmid pWK 120 was constructed by ligation of an incomplete digest of pWK1 and an complete digest of pWK2

ments3 and 4 would be 17gin or 34Mdals (pWL625:4.6 gm (Table 3), HindIII fragment 4: 5 gm and HindIII fragment 3:7.5 gm). This means that its transformation probability would be rather low. By following this procedure we finally got growth of transformants in nitrogen free medium. We isolated single colonies and found that they were ampicillin resistant, kanamycin sensitive and could reduce acetylene (Table 3). Plasmid DNA purified from such colonies was HindlII digested and analyzed by agarose gel electrophoresis (Fig. 4). A comparison of the HindIII fragments of the plasmids pWL625, pWK1, pWK2 and pAP1 with the HindIII fragments of the newly isolated plasmid (pWK120) showed that it contained the linearized pWL625 and the HindIII fragments 4 and 3. Since E. coli C (pWK120) is able to fix nitrogen we assume that pWK120 carries all essential genes which are necessary for nitrogen fixation. In addition we were able to show by EcoRI digestion experiments that pWK120 contains the HindIII fragments 4 and

Fig. 4.A-E Agarosegel electrophoresis of HindIII fragments of the plasmids pWL625, pWK1, pWK2, pWK120 and pAP1. Plasmid DNA was digested with the restriction endonuclease HindIII and the HindIII fragments were separated by agarose gel electrophoresis A pWL625 HindIII digested; B pWKI HindIII digested; C pWK2 HindIII digested; D pWK120 HindIII digested; E pAP1 HindIII digested

B

1

C

J

Fig. 5.A-C Copy number determination of plasmid pWK120. A crude lysate of E. coli C (pWKI20) cells was centrifuged in a CsC1 gradient of an analytical ultracentrifuge. The resultant DNA profile is shown. A chromosomal DNA of E. coli C (1.710 g/cm3); B pWK120 DNA (1.715 g/cm3) ; C reference DNA of M. lysodeicticus (1.7308 g/cm 3)

3 in the same alignment like in pRD1. This experiment will be reported elsewhere in connection with the EcoRI and HindIII restriction map of pRD1. We further characterized the plasmids pWL625, pWK1, pWK2 and pWK120 by contourlength measurements in the electron microscope. The results are given in Table 3. From these results one can calculate that the HindIII fragments 4 and 3 are 5 gm and 7 Ixm long respectively. These lengths are in good agreement with the molecular weight determinations of the HindIII fragments 4 and 3 obtained by agarose gel electrophoresis.

A. Pfihleretal. : Nitrogen Fixationfrom Klebsiellapneumoniae The plasmid vehicle pWL625 is a multicopy plasmid (Goebel et al., 1977). To determine the copy number of our hybrid plasmid pWK120, we grew E. coli C (pWK120) in nitrogen free medium, harvested cells and lysed them by the sarcosyl method. The crude lysate was then analyzed in a CsCl-gradient of an analytical ultracentrifuge. The resulting DNA profile is shown in Fig. 5. Beside the chromosomal DNA of E. coli (1.71 g/cm 3) there is a second peak of 1.715 g/cm 3 density. This peak represents pWK120 DNA. Since the plasmid peak is higher than the chromosomal peak a high copy number for the plasmid pWK120 is indicated. By comparing these two peaks and taking into account the lengths of the E. coli chromosome (1.100 gm) and of the plasmid pWK 120 (17 gin) we calculated a copy number of approximately 65.

Discussion

The aim of this work was the cloning of the entire mf-region of K. pneumoniae on a multicopy plasmid vehicle in E. coli. As starting material we used the plasmid pRD1, which consists of the plasmid RP4 and chromosomal DNA carrying the genetic markers, rfb, his, nil and shiA (Dixon et al., 1976). We first tried to isolate deletion mutants of plasmid pRD1 in order to get information about the location of the n/f genes on the plasmid. This was done by P1 transduction of the plasmid pRD1 and selection for His +. Two plasmids were isolated which are selftransmissible and carry both the resistance markers of the RP4 plasmid and the his-nif region of pRD1. Other chromosomal markers were not tested. These two plasmids, called pAP1 and pAP2 with contour lengths of 41 ~tm and 32 gm are shorter than the pRD1 plasmid with a contour length of 49 gm (Table 2). pAP1 and pAP2 show a buoyant density of 1.718 g/cm 3 (Table 2) which is higher than that of pRD1 (1.715 g/ cm3). Hence pAP1 and pAP2 lack a DNA region of high AT content. Such a DNA region of high AT content was found in the pRD1 plasmid after construction of the AT map from partial denatured pRD1 molecules (Piihler et al., 1979). Further information about the DNA segments deleted in pAP1 and pAP2 was obtained by gel electrophoresis of HindIII digested DNA of the plasmids pRD1, pAP1 and pAP2 (Fig. 1). From the HindlII restriction map of pRD1 the deleted segments coulld be determined (Fig. 2). Plasmid pAP1 is derived from pRD1 by a single deletion (A) whereas plasmid pAP2 shows a further deletion (B) in addition to deletion A. Deletion A seems to be the DNA segment with the high AT content. It is interesting to speculate about the

23 deletion forming process. Deletion A and deletion B border the integrated RP4 plasmid. We assume that the RP4 plasmid is flanked by two insertion (IS)elements. Since IS-elements are capable of deletion formation, they may have produced deletions A and B. Also the proposed IS-elements could have played a role in the formation of pRD1 by fusion of the plasmids FN68 and RP4 (Dixon et al., 1976) and they also could be responsible for the segregation of RP4 like plasmids from the pRD1 plasmid in recA ÷ hosts (Ptihler et al., 1979). From the HindIII map of plasmid pAP2 (Fig. 2) one can deduce that the his-nif region of pRD1 is located on the HindIII fragments 5, 4 and 3. We first cloned these single HindIII fragments separately in the multicopy vehicle pWL625, which is derived from the copy mutant R1 drd-19B2, now called pKN 102 (Nordstr6m et al., 1972). pWL625 carries resistance genes against ampicillin and kanamycin. The ampicillin resistance gene is located on the transposon Tn3. The kanamycin resistance gene was introduced by in vitro insertion of the kan-fragment from pML21 (Hershfield et al., 1976) into Tn3. In this way an enlarged transposon Tn3 carrying amp-r and kan-r was constructed. The plasmid pWL625 possesses only one HindIII site which maps in the kan-r gene. By cloning the HindIII fragments number 5, 4 and 3 of pRD1 separately into this HindIII site, the cloned HindIII fragments are also located on enlarged Tn3 transposons which can be transposed to other replicons (Goebel et al., 1977). The separately cloned HindIII fragments number 5, 4 and 3 on the hybrid plasmids pWK4, pWK1 and pWK2 were not able to confer nitrogen fixation capacity on E. coli C. But we showed that the cloned HindIII fragment number 5 could complement the his" mutation of an E. coli C strain. Therefore at least part of the his operon of plasmid pRD1 is located on the HindIII fragment number 5. Part of the his operon of plasmid pRD1 was also cloned by Cannon et al., (1977) using EcoRI-fragments of pRD1 and pMB9 as cloning vehicle. One of the hybrid plasmids, called pCRA37, containing 4 adjacent EcoRI-fragments, shows one HindIII site in the cloned region (Cannon, personal communication). This site should be identical with one end of our cloned HindIII fragment 5 in plasmid pWK4. In order to clone the entire mf-region we developed a procedure which allows the combining of two HindIII fragments in one plasmid vehicle (Fig. 3). With this procedure we constructed the hybrid plasmid pWK120 which carries the entire nif region of K. pneumoniae, since E. coli C cells harboring pWK120 are able to fix nitrogen and to reduce acetylen (Table 3). Part of the ntf-region (n/f B, mf F) was cloned on pCRA37 by Cannon et al., (1977). In the meantime

24

a hybrid plasmid, called pCM1, was isolated which carries most of the mf-region. Plasmid pCMI contains a HindIII fragment of pRD1 with a molecular weight of 11.4 Mdals (Cannon et al., 1979). This fragment closely resembles our cloned HindIII fragment 4 with a molecular weight of ~ 10 Mdals. nif D, H and J are not on pCM1 (Cannon et al., 1979). These genes should map on our cloned HindIII fragment 3. Since the HindIII fragment 3 shows a molecular weight of ~ 15 Mdals most of its coding capacity is probably not used for n/f. This means that the DNA on pRD1 which carries the mfregion is shorter than the cloned DNA (HindlII fragments 4 and 3) on pWK120. The copy number of plasmid pWK120 in E. coli C cells grown in nitrogen free medium is approximately 65 (Fig. 5). This means that a large quantity of K. pneumoniae nif DNA is present in E. coli cells. Isolation of plasmid DNA from such cells is much easier than from pRD1 carrying E. coli cells. Since pWK120 with a length of 17 gm (Table 3) is also much shorter than pRD1 with a length of 49 gm further molecular experiments to analyze the nif region of K. pneumoniae are now possible. In addition the high copy number of pWK120 enables one to study the transcription and translation of the n/f region in minicells of E. coll. The single copy plasmid pRD1 which is very unstable in recA + cells (Ptihler et al., 1978) is unsuitable for such experiments (Alden and Rogers, 1977). Acknowledgements. We thank Mrs. A. Simon for excellent technical assistance, Dr. F.C. Cannon for critical reading the manuscript, Mrs. K. Bauer for photographic aid and Mrs. A. Hofmann for typing. This work was supported by a grant from Deutsche Forschungsgemeinschaft (Pu 28/7). This paper is dedicated to Dr. W. Heumann, head of the Institute of Microbiology and leader of the nitrogen fixation group at the University of Erlangen who will celebrate this year his 65 Lh birthday.

References Alden, M., Rogers, S. : Segregation of genes for nitrogen fixation into minicells of Escherichia coli. Proc. Soc. Exp. Biol. Med. 155, 357-360 (1977) Ausubel, F.M., Cannon, F.C., Riedel, R.: Cloning of his and nifgenes from Klebsiellapneurnoniae. In : Recent developments in

A. Pfihler et al. : Nitrogen Fixation from Klebsiella pneumoniae nitrogen fixation, pp. 357-364. New York: Academic Press 1977 Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K12. Bacteriol. Rev. 40, 116-167 (1976) Burkardt, H.J., Mattes, R., Pfihler, A., Heumann, W.: Electron microscopy and computerized evaluation of some partially denatured group P resistance plasmids. J. Gen. Microbiol. 105, 51-62 (1978) Cannon, F.C., Dixon, R.A., Postgate, J.R. : Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumonieae. J. Gen. Microbiol. 93, 111 125 (1976) Cannon, F.C., Riedel, G.E., Ausubel, F.M. : Recombinant plasmid that carries part of the nitrogen fixation (nif) gene cluster of Klebsiella pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 74, 7. 2963 2967 (1977) Cannon, F.C., Riedel, G.E., Ausubel, F.M. : Overlapping sequences of Klebsiella pneumoniae n(f DNA cloned and characterized. Mol. Gen. Genet., submitted for publication (1979) Cohen, S.N., Chang, A.C.Y., Hsu, L.: Non chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coil by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69, 2110 2114 (1972) DePicker, A., Van Montague, M., Schell0 J.: Physical map of RP4. In: DNA insertions elements, plasmids and episomes. (A.I. Bukhari, J.A. Shapiro, and S.L. Adhaya, eds.), p. 678, New York: Cold Spring Harbor Laboratory 1977 Dixon, R., Cannon, F.C., Kondorosi, A.: Construction of a P plasmid carrying nitrogen fixation genes from Klebsiella pneumoniae. Nature 260, 5548, 268-271 (1976) Goebel, W., Lindenmaier, W., Pfeiffer, F., Schrempf, H., Schelle, B. : Transposition and insertion of intact, deleted and enlarged ampicillin transposon Tn3 from mini-R1 (Rsc) plasmids into transfer factors. Mol. Gen. Genet. 157, 119 129 (1977) Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A., Helinski, D.R.: Plasmid ColE1 as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U.S.A. 71, 9, 3455-3459 (1974) Miller, J.H. : Experiments in molecular genetics. New York: Cold Spring Harbor Laboratory 1972 Nordstr6m, K., Ingram, L.C., Lundbfick, A. : Mutations in R-factors of Escherichia coli causing an increased number of R-factor copies per chromosome. J. Bacteriol. 110, 562-569 (1972) Novick, R.P., Clowes, R.C., Cohen, S.N., Curtiss III, R., Datta, N., Falkow, S. : Uniform nomenclature for bacterial plasmids : a proposal. Bacteriol. Rev. 40, 168 189 (1976) Pfihler, A., Burkardt, H.J., Cannon, F.C., Wohlleben, W. : Spontaneous degradation of pRD1 DNA into unique size classes is recA dependent. Mol. Gen. Genet. 171, 1-6 (1979) Rosner, J.L.: Formation, induction and curing of bacteriophage P1 lysogens. Virology 49, 679-689 (1972)

Communicated by F. Kaudewitz Received June 5, 1979