F. J. de Bruijn

The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids — a review ☆

The properties of transposon Tn5 that render it useful for in vivo mutagenesis of cloned DNA sequences are reviewed. Transposition frequency, insertional specificity, polarity and stability of Tn5 insertion mutations are among the topics discussed. Examples are cited from the published literature which illustrate the applications of Tn5 mutagenesis to the analysis of cloned prokaryotic and eukaryotic genes. A methods section is included which outlines precisely how to carry out transposon Tn5 mutagenesis analysis of cloned DNA segments.

Model Legumes Get the Nod

Fu, H., Kim, S.Y., and Park, W.D. (1995). High-leve1 tuber expression and sucrose inducibility of a potato Sus4 sucrose synthase gene require 5 ’ and 3’ flanking sequences and the leader intron. Plant Cell7, 1387-1 394. 11 83-1 200. Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1 987). GUS fusions: p-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6,3901-3907.

Characterization of a novel Azorhizobium caulinodans ORS571 two-component regulatory system, NtrY/NtrX, involved in nitrogen fixation and metabolism

SummaryAzorhizobium caulinodans ORS571 nifA regulation is partially mediated by the nitrogen regulatory gene ntrC. However, the residual nifA expression in ntrC mutant strains is still modulated by the cellular nitrogen and oxygen status. A second ntrC-homologous region, linked to ntrC, was identified and characterized by site-directed insertion mutagenesis and DNA sequencing. Tn5 insertions in this region cause pleiotropic defects in nitrogen metabolism and affect free-living as well as symbiotic nitrogen fixation. DNA sequencing and complementation studies revealed the existence of a bicistronic operon (ntrYX). NtrY is likely to represent the transmembrane ‘sensor’ protein element in a two-component regulatory system. NtrX shares a high degree of homology with NtrC proteins of other organisms and probably constitutes the regulator protein element. The regulation of the ntrYX and ntrC loci and the effects of ntrYX, ntrY and ntrX mutations on nifA expression were examined using β-galactosidase gene fusions. NtrY/NtrX were found to modulate nifA expression and ntrYX transcription was shown to be partially under the control of NtrC.

Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

SummaryA cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.

Mini-Mulac transposons with broad-host-range origins of conjugal transfer and replication designed for gene regulation studies in Rhizobiaceae

Novel mini-Mu derivatives were constructed, carrying a truncated lacZYA operon fused to the terminal 117 bp of the Mu S-end, for the isolation of translational lac fusions by mini-Mu-mediated insertion mutagenesis. Different selectable markers (chloramphenicol resistance; gentamycin resistance) were introduced to allow selection for mini-Mu insertions in different replicons and bacterial strains. A mini-Mulac derivative carrying the site for conjugal transfer of plasmid RP4 (oriT) and the origin of replication of the Agrobacterium rhizogenes Ri plasmid (oriRiHRI) was constructed to enable one-step lac-fusion mutagenesis of cloned (plasmid-borne) regions in Escherichia coli and efficient conjugal transfer of gene fusions to to a variety of Gram-negative bacteria. The conjugation frequency, stability and copy number of replicons carrying mini-Mulac derivatives with oriT and oriRiHRI in members of the Rhizobiaceae such as Rhizobium meliloti, Azorhizobium caulinodans ORS571 and Agrobacterium tumefaciens C58 was examined.

The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: Identification of glnR, a gene involved in the regulation of the nif and hut operons

SummaryA 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA- mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut+ and Nif+ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA- mutations and the GlnR- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA- mutations but not the GlnR- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA- strain of E. coli and a glnA- GlnR- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA- mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR- phenotype, while glnA- complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR).

The cloning and characterization of the glnF (ntrA) gene of Klebsiella pneumoniae: Role of glnF (ntrA) in the regulation of nitrogen fixation (nif) and other nitrogen assimilation genes

SummaryA multicopy plasmid carrying the Klebsiella pneumoniae glnF(ntrA) gene was constructed by selecting for plasmids capable of complementing the GlnA− and Ntr− phenotypes of a glnF− (ntrA−#x2212;) strain of Escherichia coli. One GlnF+ (NtrA+) plasmid (pFB71) contained a 2.0 kb ClaI insert into the single ClaI site of pACYC184. In order to construct a correlated physical and genetic map of the cloned glnF(ntrA) region, plasmid pFB71 was mutagenized with transposon Tn5. This analysis revealed that almost the entire 2.0 kb ClaI fragment contains essential glnF(nitrA) sequences. The glnF(ntrA) gene product was identified as an ∼84,000 dalton polypeptide by examining the polypeptide products of pFB71 and a series of pFB71::Tn5 mutants in the Maxicell system. From the Maxicell data, we also determined the direction of transcription of glnF(ntrA) and the approximate location of the presumptive glnF(ntrA) promoter (operator) region. Utilizing pFB71 in genetic complementation tests, we demonstrated directly that the glnF(ntrA) product is essential for the positive regulation of a number of nitrogen assimilation genes, including the nitrogen fixation (nif) gene cluster. Our data indicate that there is no significant transcriptional regulation of the plasmid borne glnF(ntrA) gene in response to fluctuating levels of ammonia in the cell.

Regulation of plant genes specifically induced in nitrogen-fixing nodules: role of cis-acting elements and trans-acting factors in leghemoglobin gene expression

Transgenic alfalfa plants harboring a gene fusion between the soybean leghemoglobin (lbc3) promoter region and the chloramphenicol acetyl transferase (cat) gene were used to determine the influence of rhizobial mutants on lb gene expression in nodules. The promoter region of the Sesbania rostrata glb3 (Srglb3) leghemoglobin gene was examined for the presence of conserved motifs homologous to binding site 1 and 2 of the soybean lbc3 promoter region, found to interact with a trans-acting factor present in soybean nodule nuclear extracts (Jensen EO, Marcker KA, Schell J, de Bruijn FJ, EMBO J 7: 1265–1271, 1988). Subfragments of the S. rostrata glb3 (Srglb3) promoter region were examined for binding to trans-acting factors from nodule nuclear extracts. In addition to the binding sites previously identified (Metz BA, Welters P, Hoffmann HJ, Jensen EO, Schell J, de Bruijn FJ, Mol Gen Genet 214: 181–191), several other sites were found to interact with trans-acting factors. In most cases the same trans-acting factor(s) were shown to be involved. One fragment (202) was found to bind specifically to a different factor (protein) which was extremely heat-resistant (100°C). The appearance of this factor was shown to be developmentally regulated since the expected protein-DNA complexes were first observed around 12 days after infection, concomitant with the production of leghemoglobin proteins. Fragments of the Srglb3 5′ upstream region were fused to the β-glucuronidase reporter gene with its own CAAT and TATA box region or those of the cauliflower mosaic virus 35S and nopaline synthase (nos) promoters. These constructs were used to generate transgenic Lotus corniculatus plants and their expression was measured in different plant tissues. The Srglb3 CAAT and TATA box region was found to be required for nodule-specific expression and several upstream enhancer-type regions were identified.

The ntrC gene of Agrobacterium tumefaciens C58 controls glutamine synthetase (GSII) activity, growth on nitrate and chromosomal but not Ti-encoded arginine catabolism pathways

SummaryThe ntrC locus of Agrobacterium tumefaciens C58 has been cloned using the Azorhizobium sesbaniae ORSS571 ntrC gene as a DNA hybridization probe. Transposon Tn5 mutagenesis of the cloned ntrC locus was carried out and one Tn5 insertion within the region of highest DNA homology with A. sesbaniae ORS571 ntrC was used for gene replacement of the wild-type C58 ntrC gene. The A. tumefaciens ntrC::Tn5 mutant was found to be unable to grow on nitrate as sole nitrogen (N) source, to lack glutamine synthetase (GSII) activity and to be unable to use arginine (or ornithine) as sole N source, unless the Ti-encoded arginine catabolism pathway was induced with small amounts of nopaline. Thus the A. tumefaciens ntrC regulatory gene is essential for (transcriptional) activation of the GSII and nitrate reductase genes, as well as for the chromosomal but not the Ti-borne arginine catabolism pathways.

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

SummaryRandom Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm-). Both Asm- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm- phenotype of E. coli and ORS571 GOGAT- strains. The ORS571 GOGAT- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.