Construction of a recF deletion mutant of Azotobacter vinelandiiand its characterization

Hema Badran, Rashmi Sohoni, T.V. Venkatesh, H.K. Das *Genetic Engineering Unit and Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110 067, India

Received 15 November 1998; received in revised form 19 February 1999; accepted 28 February 1999

Abstract

A deletion was engineered in the cloned recF gene by digestion with suitable restriction endonucleases and a tetracyclineresistance gene cartridge was inserted. The mutation was subsequently transferred to the Azotobacter vinelandii chromosome bydouble cross-over under pressure of tetracycline selection. A recF recA mutant was also constructed in a similar manner. Themutations were found to be stable and mutation of the wild-type recF gene was confirmed by Southern blot hybridization. Boththe mutants were UV sensitive and recombination deficient. Mutations in genes involved in nitrogen fixation in A. vinelandiiare rather frequent and obtained comparatively easily despite of the presence of multiple identical chromosomes in A.vinelandii. It has been speculated that some kind of `homogenotization' process operates which is responsible for the`transmission' of mutation from one chromosome to all the chromosomes. This process is not affected by a mutation in recF orrecA or in both recF and recA. z 1999 Federation of European Microbiological Societies. Published by Elsevier ScienceB.V. All rights reserved.

Keywords: Azotobacter vinelandii ; Recombination; recF recA ; Deletion mutant; ``Homogenotization''

1. Introduction

A novel feature of the Gram-negative bacteriumAzotobacter vinelandii is that it may contain 40^80copies of identical chromosomes when cultured inthe laboratory in supplemented medium [1^3]. Thisview has been con¢rmed by the actual determinationof the number of copies in individual cells by £owcytometry after propidium iodide staining [4],though it was interpreted di¡erently from earlier ge-netic studies [5]. Despite the presence of a large num-ber of chromosomes, at least under certain growth

conditions, it is not di¤cult to isolate mutant strainsin which cassette insertion mutations are introducedto replace the corresponding wild-type genes [6^14].It has been concluded that the mutation ¢rst takesplace only on a single chromosome, which is then`transmitted' to the other copies of the chromosomeby a process of `homogenotization' catalyzed by ahitherto uncharacterized homologous non-reciprocalenzymatic recombination system [14]. The likelihoodof this `homogenotization' happening by simple seg-regation has been inferred as remote because of thevery low probability given by (1/2)n31, where n is thenumber of chromosomes per cell [14].

RecF is known to be involved in the post-replica-tion repair in wild-type E. coli [15]. The RecF path-

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 4 6 - 9

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* Corresponding author. Tel. : +91 (011) 610-1044;Fax: +91 (011) 616-5886; E-mail: hirendas@hotmail.com

FEMS Microbiology Letters 174 (1999) 363^369

way is also thought to be the main pathway of re-combination in a recBC sbcBC background in E. coli[16,17]. These functions are operative in addition tothe ability of RecF to induce the SOS response [18].

Takahashi et al. [19] have described a phenomen-on in E. coli which they have termed `non-conserva-tive' recombination, the end result of which may besimilar to that of `non-reciprocal' recombination. Anactive RecF pathway has been found to lead to thismode of recombination. The cloning and molecularcharacterization of the A. vinelandii recF gene hasbeen reported [20]. Some similarity has been ob-served between the recF genes of A. vinelandii andE. coli.

We report here on the construction of a recF dele-tion mutant of A. vinelandii and also a recF-recAdouble mutant. Some properties of these mutants

have been characterized but these mutants do notseem to be de¢cient in the `homogenotization' activ-ity.

2. Materials and methods

2.1. Bacterial strains and plasmids

The bacterial strains and plasmids used in thiswork are listed in Table 1.

2.2. Techniques of cloning and restriction analysis

Procedures for plasmid DNA isolation, digestionwith restriction endonucleases, analysis of restrictionfragments by electrophoresis on an agarose gel and

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Table 1Bacterial strains and plasmids used

Bacteria/plasmids

Relevant genotype or phenotype Source/reference

A. vinelandiiUW Wild-type (non-gummy derivative) [30]VK21 recA deletion mutant of A. vinelandii UW [31]HB14 recF deletion mutant of A. vinelandii UW This workHB2 recF deletion mutant of A. vinelandii VK21 This workPlasmidspHC79 AmpR TetR 6.4-kb cosmid [32]pUC19-Cmr Cmr, derivative of the multi copy plasmid pUC19 Gary Ditta (personal

communication)pRK404 Tetr, broad host range 10.6-kb cloning vector based on the RK2 replicon [33]pBX404.7 Tetr, recombination probe vector with two truncated versions of the neomycin

phosphotransferase gene[23]

pHB404.7 Ampr, ampicillin resistant derivative of pBX404.7 This workpCDK3 Ampr, Cmr, plasmid containing the 20-kb BamHI fragment containing the thyA, argA, recB,

recC and recD genes of E. coli[28]

pUK100 Cmr, 4.6-kb KpnI fragment from A. vinelandii DNA containing the complete nifL and a partof nifA cloned in pUC19-Cmr

[34]

pUK110 pUK100 digested with BglII and self-ligated, leading to a 0.6-kb deletion Umesh K. Bageshwar(unpublished work)

pUK110: :6Kan

Interposon 6Km as 2.0-kb BamHI fragment from pHP456Km (Fellay et al. 1987)inserted into the BglII site of pUK110, Cmr, Kanr

Umesh K. Bageshwar(unpublished work)

pUK121 Cmr, 3.2-kb SalI fragment from pUK100 cloned in pUC19-Cmr [34]pMK1 pUC19 with a 6.5-kb HindIII fragment containing the recF gene and adjacent regions from

A. vinelandii[20]

pMK5 pUC19 with a 2.1-kb KpnI fragment from A. vinelandii containing the complete recF geneplus about 1 kb of the region in its 5P end

[20]

pHB12 Complete recF gene of A. vinelandii in pUC19 obtained by deleting a 0.67-kb EcoRIfragment from pMK5

[20]

pHB22 pMK1 derivative with a deletion of 0.8 kb at the 3P end of the recF gene and inserting a1.3-kb Tet cartridge there

This work

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cloning were essentially as described by Sambrook etal. [21].

2.3. Growth conditions for bacterial strains

All the A. vinelandii strains were grown in Burk'snitrogen-free medium [22] supplemented with 0.11%ammonium acetate and appropriate antibiotics (¢nalconcentration: ampicillin (Amp) = 50 Wg ml31,chloramphenicol (Cm) = 180 Wg ml31, kanamycin(Kan) = 2 Wg ml31 and tetracycline(Tet) = 5 Wg ml31).

2.4. Determination of the recombination pro¢ciency

The plasmid pBX404.7 [23] is a wide host rangeplasmid containing two inactive versions (truncatedbut overlapping) of the neomycin phosphotransfer-ase gene cloned in the vector pRK404. The di¡erentA. vinelandii strains were transformed with an ampi-cilline resistant (Ampr) derivative of this plasmid(pHB404). As a result of the recombination activity,there would be unequal crossing-over, leading to therestoration of a functional neomycin phosphotrans-ferase gene and giving rise to resistance to Kan.Thus, the frequency of recombination can be ap-proximated from the ratio of Kanr Tetr colonies toTetr colonies using pBX404.7 or the ratio of Kanr

Ampr colonies to Ampr colonies using pHB404.7.

2.5. Transformation of A. vinelandii

The protocol used was adapted from Page andSado¡ [11] and Dennis Dean's (personal communi-cation) protocols. The cells were grown in BNF-Fe-Mo at 170 rpm on a gyratory shaker for 24 h. To200 Wl of resuspended cells, 3^4 Wg of DNA wasadded along with 32 mM MgCl2 and 10 mMMOPS and incubated at 30³C for 30 min. This wasthen overgrown in 1 ml of BNF-ammonium acetateor in BNF-LB (20% LB) for 24 h and then, its dilu-tions were plated on appropriate agar medium.

2.6. DNA hybridization studies

Restriction fragments of genomic DNA were sep-arated by electrophoresis in a 0.8% agarose gel,transferred to a nitrocellulose membrane and hybrid-ized to the appropriate fragment, labelled with K-

[32P]dCTP by the random priming technique [24].The dried blots were then autoradiographed on X-ray ¢lm [25].

3. Results and discussion

3.1. Construction of the recF deletion mutant

A 0.8-kb fragment was deleted from the clonedrecF gene [20] by digesting the plasmid pMK1 withPstI and KpnI and a Tet cartridge was inserted in itsplace. This construct, which would be unstable in A.vinelandii because of its ColE1 replicon, was desig-nated pHB22. The Tet cartridge was removed frompHC79, as a 1.3-kb EcoRI-AvaI fragment, and therestriction sites PstI and KpnI were introduced bysuccessive cloning in vectors containing di¡erentpolylinkers. A. vinelandii wild-type (UW) was trans-formed with pHB22 followed by selection on BNF-Tet plates. Colonies that were sensitive to Cm werechosen to eliminate the single point cross-over re-combinants. To verify that the resulting mutantsthus isolated were the result of homologous recom-bination, due to a double cross-over events, leadingto gene replacement, Southern blotting [25] and hy-bridization were performed. Chromosomal DNAs,isolated from the wild-type and the putative recFmutant (HB14) were digested with KpnI. The South-ern blot was hybridized with the labelled 1.5-kbEcoRI-HindIII fragment from pHB12 which con-tained the complete recF gene. In the case of A.vinelandii UW, a 2.1-kb band was found to hybrid-ize, while in case of the mutant, a 2.6-kb band hy-bridized. The excision of the 0.8-kb fragment and itsreplacement by a 1.3-kb fragment carrying the Tetcartridge resulted in the 2.6-kb band in place of the2.1-kb band, thereby con¢rming the creation of themutant (Fig. 1).

3.2. Construction of the recF recA mutant

A recA deletion mutant of A. vinelandii UW,VK21, was constructed and characterized earlier[31]. The recombination frequency in VK21, deter-mined by using the recombination probe plasmidpBX404.7 [23], was less than 1037, while that of A.vinelandii UW was 3.7U1034 [31]. In case of the

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recA mutant, no recombinant colony could be de-tected when 107 cells were plated. By plating approx-imately 1010 cells of A.vinelandii VK 21 (pHB404.7),we have now determined a recombination frequencyof 1.23U1038 for a recA mutant. The constructpHB404.7 is an Ampr derivative of pBX404.7.Thus, there was some residual homologous recombi-nation activity, though low, even in the recA deletionmutant of A. vinelandii. This residual activity al-lowed construction of a recF recA double mutantby transformation of A. vinelandii VK21 [31] withpHB22 and plating 1011 cells on each BNF-Tet plate.As usual, colonies that were sensitive to Cm werechosen. Replacement of the wild-type recF gene bythe recF deletion/insertion mutation in A. vinelandiiVK21 was con¢rmed by Southern blotting of oneisolate, named HB2. Here, also a 2.6-kb band wasfound in place of the usual 2.1-kb band (Fig. 1).

3.3. Recombination de¢ciency and UV sensitivity ofthe recF deletion mutants

The recF deletion mutants of A. vinelandii UW(HB14) as well as of A. vinelandii VK21 (HB2)were characterized for the recombination pro¢ciencyas well as the UV sensitivity. The A. vinelandii strainsUW, VK21, HB14 and HB2 were grown in BNFsupplemented with 0.11% ammonium acetate,streaked on a BNF-ammonium acetate agar, exposedto UV radiation for di¡erent lengths of time (0 s^40s) and subsequently grown in the dark for 48 h. Theresult is shown in Fig. 2. A mutation in recF didrender A. vinelandii sensitive to UV radiation butto a lesser extent than a mutation in recA. The

recF recA double mutant was slightly more sensitivethan the recA mutant. To test their recombinationpro¢ciency, the recombination probe vectorpBX404.7 [23] was used with some modi¢cations.Since the recF mutants were Tetr, pBX404.7 couldnot be used as such. Hence, its Tetr gene was inacti-vated and replaced by the L-lactamase gene, confer-ring resistance to Amp. The gene conferring resist-ance to Tet in the plasmid pBX404.7 has a HindIIIsite in its promoter region. So, the plasmid was par-tially digested with HindIII and ligated into the Hin-dIII fragment containing 6Amp [26]. The resultingrecombinant plasmids were tested for Ampr andTets. The A. vinelandii strains UW, VK21, HB14and HB2 were transformed with pHB404.7 (themodi¢ed pBX404.7) and the ratio of Kanr to Ampr

colonies was determined. The recF mutation de-creased the normal recombination frequency almostas much as the recA mutation, by factors of5.5U1034 and 2.82U1034, respectively, but resulted

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Table 2Recombination pro¢ciency of A. vinelandii strains

A. vinelandii strains Frequency of homologousrecombinationa

UW (wild-type) 4.36U1034

VK21 (recA mutant) 1.23U1038

HB14 (recF mutant) 2.41U1037

HB2 (recA recF double mutant) 8.39U1039

aThe A. vinelandii strains were transformed with the plasmidpHB404.7 and plated separately on agar medium containingAmp and Kan. The ratio of the number of colonies that appearedin the presence of Kan to that in the presence of Amp was taken asthe frequency of recombination.

Fig. 1. Agarose gel electrophoresis of the KpnI digest of genomicDNA of di¡erent strains of A. vinelandii and the correspondingautoradiogram of the Southern blot. The 32P-labelled probe usedwas the 1.5-kb EcoRI-HindIII fragment from pHB12, containingthe recF gene [20]. A, ethidium bromide-stained DNA. B, autora-diogram of the Southern blot. Lanes a and 1, HindIII-digestedVDNA; lanes b and 2, A. vinelandii UW; lanes c and 3, A. vine-landii HB14; lanes d and 4, A. vinelandii HB2.

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in only a slight further decrease in the recombinationfrequency in the recA recF double mutant (Table 2).

3.4. Test for homogenotization

As already stated, we were looking for a functionof RecF which could be responsible for `transmittng'the mutation to all the copies of the chromosome inA. vinelandii. We considered it to be due to somekind of non-reciprocal recombination. In order totest it, the strategy developed was to transform theA. vinelandii strains UW, VK21, HB14 and HB2with a nifLA deletion mutant clone in pUC8 contain-ing an inserted Kan interposon [26] (pUK110: :6Kan), which should undergo a double point cross-over with the chromosome rendering the wild-typestrain a nifLA (Nif3) mutant. The nifLA mutantclone was chosen for analysis as nif mutants areeasy to obtain in A. vinelandii. It would, of course,be important to remember that the pro¢ciency ofhomologous recombination in the recA or recF mu-tants is low (Table 2), but nevertheless not zero, andhence, the test can be done by screening a su¤cientlylarge number of cells. If either RecA or RecF orboth were involved in `homogenotization', we wouldexpect that only one or a small number of copies ofthe chromosome of transformants would have a mu-

tated nifLA gene, while others would retain the wild-type. If this occurred, the cells would express thewild-type phenotype, i.e. be Nif�. The wild-typeand mutant strains of A. vinelandii were transformedwith pUK110: :6Kan which was unstable in A. vine-landii because of its narrow host range Col E1 repli-con. Kanr, Amp sensitive (Amps) colonies were se-lected to score recombinants arising from a doublecross-over (Kanr, Ampr colonies would be the resultof single point cross-over events). In all the four A.vinelandii strains used, about 1010 cells were plated toselect for transformants on BNF+ammonium aceta-te+Kan plates. From each of the above set of trans-formants, about 200 colonies were patched on

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Fig. 3. Autoradiogram of the Southern blot after agarose gelelectrophoresis of SalI-digested genomic DNA of E. coli and dif-ferent strains of A. vinelandii. The 32P-labelled probe used wasthe 3.2-kb SalI fragment from pUK121 containing the whole ofnifL and a part of nifA gene. Lane 1, E. coli DH5K ; lane 2, A.vinelandii UW; lane 3, the 3.2-kb SalI fragment from pUK121containing the nifLA gene of A. vinelandii ; lane 4, the 4.6-kbSalI fragment from pUK110: :6Kan containing the nifLA geneof A. vinelandii with insertion of the 2.0-kb interposon 6Kanafter the 0.6-kb deletion in the nifLA ; lane 5, A. vinelandii UWwith a genomic mutation in nifLA (0.6-kb deletion plus insertionof the 2.0-kb interposon 6Kan), the strain was cultured in thepresence of kanamycin (2 Wg ml31) ; lane 6, the same as in lane 5but the strain was cultured in the absence of kanamycin; lane 7,A. vinelandii VK21 (recA deletion mutant) with a genomic muta-tion in nifLA as described for lane 5, the strain was cultured inthe presence of kanamycin (2 Wg ml31) ; lane 8, the same as inlane 7 but the strain was cultured in the absence of any kanamy-cin; lane 9, A. vinelandii HB14 (recF deletion mutant) with a ge-nomic mutation in nifLA as described for lane 5, the strain wascultured in the presence of kanamycin (2 Wg ml31) ; lane 10, thesame as in lane 9 but the strain was cultured in the absence ofkanamycin. No wild-type band (like in lanes 2 and 3) could beseen in lanes 4^10 even on a very long exposure of the X-ray¢lm.

Fig. 2. Sensitivity of di¡erent strains of A. vinelandii towardsUV-ray, as determined by the UV streak test [35]. Topmoststreak, A. vinelandii UW; streak second from the top, A. vinelan-dii VK21; streak third from the top, A. vinelandii HB14; bottomstreak, A. vinelandii HB2. Exposure to UV-ray was for 5, 10, 20,30 or 40 s.

H. Badran et al. / FEMS Microbiology Letters 174 (1999) 363^369 367

BNF+Amp+ammonium acetate and BNF+Kan+ammonium acetate plates to check for coloniesof cells derived from double point crossovers(Amps, Kanr). In all the three mutant strains, thefrequency of double crossovers was about 50% andabout 65% in the wild-type strain UW tested. About100 colonies of cells derived from a double cross-overs in each strain were tested for growth onBNF plates containing no ammonium acetate andall were Nif3. This made us conclude that the nifLAdeletion was `transmitted' to all copies of the chro-mosome in A. vinelandii and therefore neither therecA gene nor the recF gene was responsible for `ho-mogenotization' of the mutation. We con¢rmed bySouthern blotting of Nif3 derivatives that no wild-type nifLA was left in any of the chromosome copiesand all the copies of the chromosome had acquiredmutated nifLA (Fig. 3) despite the deletions in therecF or recA genes. No antibiotic pressure was nec-essary during culturing of the cells for the `homoge-notization'.

3.5. Attempts to clone recBCD genes of A. vinelandiiwith a view to obtaining the respective mutants

The RecBCD pathway is an important pathway ofrecombination in E. coli and other bacterial species[27]. The E. coli recBCD gene probe was isolatedfrom the plasmid pCDK3 [28] and A. vinelandiiDNA was probed by Southern blotting [25]. No sig-nal could be obtained even under low stringencyconditions and prolonged autoradiography. At-tempts were made to complement the recBCD dele-tion strain, E. coli V186, with a cosmid library of A.vinelandii genes. More than 50 000 exconjugants werescreened for mitomycin C (0.3 Wg ml31) resistance,though just about 500 colonies should have repre-sented the whole genome at a more than 99% prob-ability [29]. Similarly, more than 40 000 exconjugantswere screened for resistance to UV. No resistant col-ony could be detected by either procedure.

One can imagine various reasons for the failure ofcloning the recBCD genes of A. vinelandii. One ofthese could be that A. vinelandii does not have aRecBCD pathway. A. vinelandii can be transformedwith linear DNA e¤ciently, but E. coli cannot betransformed with linear DNA, as the exonucleaseV, encoded by recBCD, degrades it. That the trans-

formation frequency of A. vinelandii with linearizedplasmid DNA is at least a 4-fold higher than withintact plasmid DNA [14] is consistent with this idea.

Acknowledgments

We are thankful to G. Ditta and P.F. Larquin forplasmid constructs. The study was supported by theDepartment of Biotechnology, Government of India.

References

[1] Sado¡, H.L., Shimei, B. and Ellis, S. (1979) Characterizationof Azotobacter vinelandii deoxyribonucleic acid and foldedchromosome. J. Bacteriol. 138, 871^877.

[2] Nagpal, P., Jafri, S., Reddy, M.A. and Das, H.K. (1989)Multiple chromosomes of Azotobacter vinelandii. J. Bacteriol.171, 3133^3138.

[3] Manna, A.C. and Das, H.K. (1993) Determination of the sizeof the Azotobacter vinelandii chromosome. Mol. Gen. Genet.241, 719^722.

[4] Maldonado, R., Jimenez, J. and Casadesus, J. (1994) Changesin ploidy during Azotobacter vinelandii growth cycle. J. Bac-teriol. 176, 3911^3919.

[5] Maldonado, R., Garzon, A., Dean, D.R. and Casadesus, J.(1992) Gene dosage analysis in Azotobacter vinelandii. Genet-ics 132, 869^878.

[6] Kelly, M.J., Poole, R.K., Yates, M.G. and Kennedy, C.(1990) Cloning and mutagenesis of genes encoding the cyto-chrome bd terminal oxidase complex in Azotobacter vinelan-dii : mutants de¢cient in the cytochrome d complex are unableto ¢x nitrogen in air. J. Bacteriol. 172, 6010^6019.

[7] Ng, T.C., Laheri, A.N. and Maier, R.J. (1995) Cloning, se-quencing and mutagenesis of the cytochrome c4 gene fromAzotobacter vinelandii : characterization of the mutant strainand a proposed new branch in the respiratory chain. Biochim.Biophys. Acta 1230, 119^129.

[8] Mouncey, N.J., Mitchenall, L.A. and Pau, R.N. (1995) Muta-tional analysis of genes of the mod locus involved in molyb-denum transport, homeostasis and processing in Azotobactervinelandii. J. Bacteriol. 177, 5294^5302.

[9] Gutierrez, J. C., Ramos, F., Ortner, L. and Tortolero, M.(1995) nasST, two genes involved in the induction of the as-similatory nitrite-nitrate reductase operon (nasAB) of Azoto-bacter vinelandii. Mol. Microbiol. 18, 579^591.

[10] Gavini, N., Hausman, B.S., Pulakat, L., Schreiner, R.P. andWilliamson, J.A. (1997) Identi¢cation and mutational analysisof rfbG, the gene encoding CDP-D- glucose-4. 6-dehydratase,isolated from free living soil bacterium Azotobacter vinelandii.Biochem. Biophys. Res. Commun. 240, 153^161.

[11] Ray, L. and Maier, R.J. (1997) Cytochrome c terminal oxi-dase pathways of Azotobacter vinelandii : analysis of cyto-

FEMSLE 8741 29-4-99

H. Badran et al. / FEMS Microbiology Letters 174 (1999) 363^369368

chrome c4 and c5 mutants and up-regulation of cytochrome c-dependent pathways with N2 ¢xation. J. Bacteriol. 179, 7191^7196.

[12] Zheng, L. Cash, V.L., Flint, D.H. and Dean, D.R. (1998)Assembly of iron-sulfur clusters. Identi¢cation of an isc-SUA-hscBA-fdx gene cluster from Azotobacter vinelandii.J. Biol. Chem. 273, 13264^13272.

[13] Meletzus, D., Rudnick, P., Doetsch, N., Green, A. and Ken-nedy, C. (1998) Characterization of the glnK-amtB operon ofAzotobacter vinelandii. J. Bacteriol. 180, 3260^3264.

[14] Manna, A.C. and Das, H.K. (1997) Characterization and mu-tagenesis of the leucine biosynthetic genes of Azotobacter vine-landii : an analysis of the rarity of amino acid auxotrophs.Mol. Gen. Genet. 254, 207^217.

[15] Rothman, R.H. and Clark, A.J. (1977) The dependence ofpost replication repair on uvrB in a recF mutant of Escherichiacoli K-12. Mol. Gen. Genet. 155, 279^286.

[16] Horli, Z. and Clark, A.J. (1973) Genetic analysis of the RecFpathway of genetic recombination in Escherichia coli K-12.Isolation and characterization of mutants. J. Mol. Biol. 80,327^344.

[17] James, A.A., Morrison, P.T. and Kolodner, R.D. (1982) Ge-netic recombination of bacterial plasmids: analysis of the ef-fect of recombination-de¢cient mutations on plasmid recom-bination. J. Mol. Biol. 160, 411^430.

[18] Sandler, S.J., Chackerian, B., Li, J.T. and Clark, A.J. (1992)Sequence and complementation analysis of recF gene se-quences from Escherichia coli, Salmonella typhimurium, Pseu-domonas putida and Bacillus subtilis : evidence for an essentialphosphate binding loop. Nucleic Acids Res. 20, 839^845.

[19] Takahashi, N.K., Yamamoto, K., Kitamura, Y., Luo, S.-Q.,Yoshikura, H. and Kobayashi, I. (1992) Nonconservative re-combination in Escherichia coli. Proc. Natl. Acad. Sci. USA89, 5912^5916.

[20] Badran, H., Venkatesh, T.V., Kunnimalaiyaan, M., Sharma,N. and Das, H.K. (1995) Molecular characterization of theAzotobacter vinelandii recF gene. Gene 162, 47^51.

[21] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) MolecularCloning: a Laboratory Manual, 2nd edn., Cold Spring Har-bor Laboratory Press, Cold Spring Harbor, New York, USA.

[22] Strandberg, G.W. and Wilson, P.W. (1968) Formation of ni-trogen ¢xing enzymes in Azotobacter vinelandii. Can. J. Micro-biol. 14, 25^31.

[23] Xu, B., Pastzy, C. and Larquin, P.F. (1988) A plasmid-basedmethod to quantitate homologous recombination frequenciesin Gram-negative bacteria. Biotechniques 6, 752^760.

[24] Feinberg, A.P. and Vogelstein, B. (1983) A technique for ra-diolabelling DNA restriction endonuclease fragments to highspeci¢c activity. Anal. Biochem. 132, 6^13.

[25] Southern, E.M. (1975) Detection of speci¢c sequences amongDNA fragments separated by gel electrophoresis. J. Mol. Biol.98, 503^517.

[26] Fellay, R., Frey, J. and Krisch, H. (1987) Interposon muta-genesis of soil and water bacteria: a family of DNA fragmentsdesigned for in vitro insertional mutagenesis of Gram-negativebacteria. Gene 52, 147^154.

[27] Telander-Muskavitch, M. and Linn, S. (1981) RecBC like en-zymes: The exonuclease V deoxyribonucleases. In: The En-zymes (Boyer, P.D., Ed.), Vol. XIV, pp. 233^250, AcademicPress, New York, USA.

[28] Dykstra, C., Prashar, D. and Kushner, R.S. (1984) Physicaland biochemical analysis of the cloned recB and recC genes ofEscherichia coli K-12. J. Bacteriol. 151, 21^27.

[29] Clarke, L. and Carbon, J. (1976) A colony bank containingsynthetic Col E1 hybrid plasmids representative of the entireEscherichia coli genome. Cell 9, 91^99.

[30] Bishop, P.E. and Brill, W.J. (1977) Genetic analysis of Azoto-bacter vinelandii mutant strains unable to ¢x nitrogen. J. Bac-teriol. 130, 954^956.

[31] Venkatesh, T.V., Reddy, M.A. and Das, H.K. (1990) Cloningand characterization of the Azotobacter vinelandii recA geneand construction of a recA deletion mutant. Mol. Gen. Genet.224, 482^486.

[32] Hohn, B. and Collins, J. (1980) A small cosmid for e¤cientcloning of large DNA fragments. Gene 11, 291^298.

[33] Ditta, G., Schmidhauser, T., Yakobson, E., Lu, P., Lian,X.-W., Finlay, D.R., Guiney, D. and Helinski, D.R. (1985)Plasmids related to the broad host range vector, pRK 290,useful for gene cloning and for monitoring gene expression.Plasmid 13, 149^153.

[34] Raina, R., Bageshwar, U.K. and Das, H.K. (1993) The Azo-tobacter vinelandii nifL-like gene: nucleotide sequence analysisand regulation of expression. Mol. Gen. Genet. 237, 400^406.

[35] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) MolecularCloning: a Laboratory Manual, Cold Spring Harbor Labora-tory, Cold Spring Harbor, New York, USA.

FEMSLE 8741 29-4-99

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