Directed

In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nif D gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome . Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif - phenotype. Four Nif - mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance g enecartridge mutagenesis combined with electrotransformation.


INTRODUCTION
Azospirillum brasilense can grow diazotrophically using a molybdenum-dependent nitrogenase.Nitrogenase, the enzyme that catalyses biological nitrogen fixation, consists of two protein components: iron and molybdenum iron.Native Fe protein is a homodimer of approximately 68-kDa subunits, while the MoFe protein is a tetrameric complex with four [4Fe-4S] centers and two iron-molybdenum cofactors (Dixon, 1984;Haaker and Veeger, 1984).In most N 2 -fixing organisms, the nitrogenase structural genes are organized in a single operon and transcribed in the order nifH-D-K (Arnold et al., 1988;Jacobson et al., 1989;Willison et al., 1993).The nifH gene codes for subunits of the Fe protein and for the MoFe protein nifD, and nifK genes code for α and β subunits, respectively.Transcription of these genes, in general, is repressed by both NH 4 + and O 2 , and occurs only under nitrogen-limiting conditions (Nelson and Knowles, 1978;Postgate and Cannon, 1981).
Azospirillum spp.fix nitrogen under free-living conditions and in association with grasses.The nif structural genes from A. brasilense have been sequenced and present the same sequential organization found in other nitrogenfixing bacteria (Passaglia et al., 1991).Analysis of the molecular genetics of nitrogen fixation in A. brasilense revealed a 45-kb DNA region, comprised of the nifENXO RF3ORF5ORF6Q, ORF2nifUSVORF4 and fixABC operons located, 3, 11, and 15 kb, respectively, downstream from the nifHDKORF1Y operon (Passaglia, L., Frazzon, J. and Vedoy, C., unpublished results).
After the report of Elmerich and Franche (1982) on Tn5-induced auxotroph mutants in A. brasilense, different protocols have been suggested for increasing the efficiency of transposon mutagenesis in this organism.The suicide plas-mid pSUP202 was used successfully to deliver Tn5 into the Azospirillum genome (Singh and Klingmüller, 1986;Abdel-Salam and Klingmüller, 1987;Faure et al., 1994).Plasmids of the P incompatibility group can also be transferred to A. brasilense, and plasmid pRK290 has been used as a vector to isolate Nifmutants of A. brasilense (Jara et al., 1983).
We established an efficient gene disruption system for inducing site-specific mutations in A. brasilense, and then used it to isolate NifD mutants.

Bacterial strains, growth conditions and nitrogenase derepression
Several E. coli strains and one A. brasilense strain were used (Tables I and II).Growth of E. coli strains carrying either hybrid nif-containing plasmids or vectors, and preparation, restriction enzyme digestion, and ligation of hybrid plasmid DNAs were performed as described previously (Sambrook et al., 1989).The wild-type and mutant strains of A. brasilense were cultured in Nfb medium (Ditta et al., 1980) supplemented with ammonium chloride to a final concentration of 40 mM, when a fixed source of nitrogen was introduced into the medium.For nitrogenase synthesis induction, all cultures were grown in nitrogenfree Nfb medium for 24 h.In vivo nitrogenase activity was measured by C 2 H 2 reduction in nitrogen-free, semi-solid Nfb medium (0.175% agar) (Nelson and Knowles, 1978).donor cells were grown at 37 o C overnight in LB broth, diluted 10-fold, and grown for another 2 h to exponential phase.Samples of donor and acceptor cells (1:3 ratio) were passed through nitrocellulose filters, placed on LB agar plates, and incubated for 16 h at 30 o C. Filters containing either donor or recipient cells were incubated as controls.Conjugation and control growth patches were resuspended in 0.85% NaCl solution, washed several times, diluted, and spread on selective plates.The pRK290X derivative pRKm6X was mobilized into A. brasilense by triparental mating, as described by Ditta et al. (1980).Mutants, in which the wild-type region is to be replaced with the mutated DNA fragment, should be obtained by introducing the IncP1 plasmid pPH1J1 into the pKRm6X-carrying A. brasilense strain, followed by selection on both kanamycin and gentamycin.Km r exconjugants were selected on minimal Nfb medium supplemented with NH 4 + and 30 µg/ ml of kanamycin.
Electroporation of A. brasilense was carried out using the Gene Pulser Apparatus (Bio-Rad).The procedure used was based on the method described by Vande Broek et al. (1989).The pSUP202 suicide derivative pSUP6 was electrotransferred to A. brasilense and transformants were selected for kanamycin resistance.Plasmid pRK290X was used as control.
Hybridization procedures 32 P-labelled probes were prepared by nick translation (Sambrook et al., 1989).Southern hybridization was carried out at 68°C under conditions described previously (Singh and Klingmüller, 1986).To verify results of marker exchange, total DNA was isolated from putative mutants, digested with the appropriate restriction enzyme and hybridized against labelled DNA fragments.

Plasmid constructions
A. brasilense nitrogenase structural genes have already been isolated, and their complete nucleotide sequence has been determined.These genes are clustered and arranged in the nifHDKORF1Y operon.To construct pKm6, DNA from the pAbc6 plasmid was digested with PstI and the 2.4kb nifD DNA fragment was replaced with a 1.9-kb PstI DNA fragment originating from partially digested Tn5 DNA (Beck et al., 1982).The 6.0-kb EcoRI DNA fragment, carrying the Km r -cartridge from the pKm6 (nifHKm r K) plasmid, was subcloned into either EcoRI-digested pRK290X or pSUP202 plasmid vectors, generating plasmids pRKm6X and pSUP6, respectively.

Isolation of A. brasilense nif mutants
The A. brasilense nitrogenase structural genes are clustered and arranged as follows: promoter-nifH-nifD-nifK in a 6.5-kb EcoRI DNA fragment (Figure 1).This DNA fragment was originally cloned into an EcoRI-digested pACYC184 vector yielding pAbc6 (Araújo et al., 1988).Since pACYC184 has no PstI sites, it was possible to replace the nifD gene with a Km r -cartridge from Tn5 (Figure 1).In the resulting pKm6 plasmid, the Km r -cartridge is flanked by 1.7-kb and 2.3-kb DNA fragments from the A. brasilense nifHDK cluster.The size of these fragments should be sufficient to direct homologous recombination into the A. brasilense genome, yielding Nifmutants with a deleted nifD gene.To date, mutagenized plasmids have been transferred to A. brasilense only by conjugation (Elmerich and Franche, 1982;Singh and Klingmüller, 1985;Vanstockem et al., 1987). However, Vande Broek et al. (1989) developed an electroporation protocol for DNA delivery into A. brasilense.We used both conjugation and electroporation techniques to isolate Nifmutants from A. brasilense.
Typical mating conditions are outlined in Material and Methods.The A. brasilense strain used as DNA acceptor is resistant to ampicillin up to 200 µg/ml and very sensitive to kanamycin (less than 5 µg/ml).Spontaneous mutants resistant to kanamycin (25 µg/ml) were undetected in control experiments.In order to generate A. brasilense NifDmutants we used two different plasmids: pRKm6X and pSUP6.In the first experiment, nifHKm r K mutagenized genes were transferred to the wild-type strain and the stable replicating vector (pRK290X) was removed using another plasmid belonging to the same incompatibility group (pPH1), thus making it easier to detect the mutagenized phenotype.Azospirillum exconjugants bearing pRKm6 were isolated on Nfb medium containing 30 µg/ml kanamycin at maximum frequencies of 10 -8 per recipient cell.
Several experiments of triparental mating were carried out to remove the replicating vector and to generate Nifmutants. A. brasilense Sp7 carrying pRKm6X was used as acceptor and E. coli strains JA221, carrying pPH1 (Gm r ), and JA221, carrying pRK2013, were used as donors.Approximately 60,000 colonies were screened for Km r and Gm r transconjugants, all of which were also resistant to tetracycline (Tc r ), indicating that pRKm6X had integrated into the A. brasilense chromosome (cointegrate formation).Singh and Klingmüller (1986) have also reported a failed attempt to isolate Nifmutants using the stable replicative plasmid vector pRK290.In addition, we used vectors which were unable to replicate in the recipient bacterium and which were lost after transfer into the recipient cell.
The narrow host range plasmid pSUP202, a mobilizable derivative of pBR325, was transferred to A. brasilense using the special mobilizing E. coli strain S17.1 (Simon et al., 1983).This strain has the RP4 plasmid integrated into its chromosome and hence can mobilize plasmids having the same mob site of IncP plasmids as pSUP202 (Simon et al., 1983).After mating E. coli S17.1 (bearing pSUP6) with A. brasilense, Km r plus Ap r exconjugants were selected on Nfb minimum medium.Although a transfer frequency of 10 -7 per recipient cell was achieved, we were unable to isolate any transconjugant sensitive to tetracycline (Tc s ).The results obtained with pSUP6 also indicated its integration into the A. brasilense chromosome.
Successful mutagenesis of A. brasilense using conjugation methods has been reported previously (Elmerich and Franche, 1982;Abdel-Salam and Klingmüller, 1987;Singh and Klingmüller, 1986;Vanstockem et al., 1987), and has been achieved using either stable replicative or suicide vectors and transposon Tn5 as the mutagenic element.We failed to isolate mutants from A. brasilense using this method.However, we then used a resistance gene-cartridge mutagenesis technique instead of transposon Tn5, which involved the cloning of a DNA fragment containing an antibiotic resistance gene into the genes to be mutagenized.Since the Km r -cartridge is unable to transpose, no interference occurs due to further transposition in the recipient cell genome.
Transformation of Azospirillum by electroporation has proven to be an efficient method for DNA transfer in this bacterium (Vande Broek et al., 1989), and the feasibility of electrotransformation of A. brasilense Sp7 with pSUP6 was analyzed as outlined in Material and Methods, using the broad host range plasmid pRK290X as control throughout the study.Under these conditions 12 Ap r plus Km r colonies were obtained.To determine the integration of the Km r -cartridge into the A. brasilense chromosome, we assayed the colonies for tetracycline resistance.Four out of 12 colonies tested did not acquire pSUP202-encoded tetracycline resistance, confirming the absence of the vector DNA which was further confirmed by the absence of hybridization between total DNA from the transformants and 32 P-labelled pSUP202 plasmid DNA.The remaining eight colonies were Tc r , indicating that, since this marker is coded by a non-transposable gene present in the pSUP202 vector, pSUP202 had been integrated into the genome.This was confirmed by the absence of free plasmid DNA corresponding in size to pSUP202 and by positive hybridization on a Southern blot of total DNA from transformants probed with the vector (data not shown).
When the 12 potential Nifmutants isolated were assayed for acetylene-reducing activity they proved to be completely deficient (0.02-0.4% of wild-type total activity), confirming a Nifphenotype for all isolated transformants.

Physical analysis of A. brasilense mutants
To further characterize the mutagenized 6.5-kb EcoRI A. brasilense genomic region, total DNA from the 12 Ap r and Km r mutants isolated after electroporation was analyzed by Southern blot.Total DNA was digested with EcoRI and hybridized against the 32 P-labelled 6.5-kb EcoRI fragment isolated from the wild-type A. brasilense nifHDK DNA region.Hybridization was detected only in genomes of the four Ap r , Km r and Tc s mutants (data not shown).Due to lack of hybridization of the remaining eight Nifmutants, we further analyzed only the four mutants that did not show vector integration.Therefore, total DNA from Sp7Nif -9, Sp7Nif -10, Sp7Nif -11, and Sp7Nif -13 was digested with EcoRI and hybridized against the 6.0-kb EcoRI fragment isolated from the pKm6 plasmid (Figure 1).Only one band of 4.0 kb was visualized in strain Sp7Nif -10 (Figure 2A, lane 2), showing that after the recombination event nifHKm r K genes were no longer intact in this mutant.With wild-type DNA a 6.5-kb band representing the nifHDK genes was visualized (Figure 2A, lane 1).In the other three recombinants only faint bands were visualized.
To determine the presence of Tn5 in all 12 mutants, total DNA was digested with PstI and hybridized against the 0.9-and 1.0-kb PstI fragments of Tn5.No hybridization was detected (data not shown), suggesting that the mutants had lost the Tn5 DNA fragment, a fact probably explaining the weak hybridization signal obtained when the 6.0-kb EcoRI fragment isolated from the pKm6 plasmid was used as probe.
To further analyze the recombination events that occurred in the Sp7Nif -10 mutant, total DNA was digested with PstI and hybridized against four alternative probes (spanning nifHDK, nifH, nifD, or nifK).All four probes hybridized against the same 3.0-kb PstI DNA fragment (Figure 2B), suggesting that several recombination events had occurred.Thus, the Sp7Nif -10 mutant partially lost both the nifH, nifD, and nifK genes and the entire kanamycin resistance gene.
Narrow host range plasmids, based on pACYC184 and pBR325 replicons, such a pSUP202 plasmid, can be transferred to A. brasilense but are unable to replicate in this bacterium.Such plasmids have been used as suicide vehicles to deliver transposons in Azospirillum and the transposon mutagenesis technique has been efficient in generating different Nifmutants in this bacterium (Elmerich and Franche, 1982;Abdel-Salam and Klingmüller, 1987;Singh and Klingmüller, 1986;Vanstockem et al., 1987).Attempts to use a combination of nif-gene deletion and resistance genecartridge mutagenesis of A. brasilense genes have also been successful.However, all mutants have been obtained by transferring the plasmids using conjugation methods, which are problematic because counterselection of the donor strain requires time-consuming rounds of single-colony purification.Moreover, since attempts to transfer plasmid DNA between Azospirillum strains have so far failed, many manipulations involve passage through E. coli cells.
We have used a novel combination of nifD-gene deletion with kanamycin gene-cartridge mutagenesis and electrotransformation.The method proved to be useful in isolating a number of A. brasilense Nifmutants, all of which were completely defective in nitrogen fixation.However, the technique should be studied in more detail since other genome regions were also lost.Recombination events in A. brasilense are still poorly understood.We suggest that gene disruption combined with electrotransformation is the major cause of genome rearrangements by illegitimate recombination, producing deletions not only of the target gene but also of adjacent regions.

Figure 1 -
Figure 1 -Strategy for constructing the gene replacement cartridge.A) Physical map of the Azospirillum brasilense nif structural gene cluster.B) Partial physical map of the Tn5 transposon.C) Physical map of the pKm6 plasmid containing the nifD gene replaced with the km r -cartridge.

Table II -
Plasmids used in the present study.

Table I -
Bacterial strains used in the present study.