We have developed a new vector strategy for the insertion of foreign genes into the genomes of gram negative bacteria not closely related to Escherichia coli. The system consists of two components: special E. coli donor strains and derivatives of E. coli vector plasmids. The donor strains (called mobilizing strains) carry the transfer genes of the broad host range IncP–type plasmid RP4 integrated in their chromosomes. They can utilize any gram negative bacterium as a recipient for conjugative DNA transfer. The vector plasmids contain the P–type specific recognition site for mobilization (Mob site) and can be mobilized with high frequency from the donor strains. The mobilizable vectors are derived from the commonly used E. coli vectors pACYC184, pACYC177, and pBR325, and are unable to replicate in strains outside the enteric bacterial group. Therefore, they are widely applicable as transposon carrier replicons for random transposon insertion mutagenesis in any strain into which they can be mobilized but not stably maintained. The vectors are especially useful for site–directed transposon mutagenesis and for site–specific gene transfer in a wide variety of gram negative organisms.

A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria

A DNA fragment of the broad host range plasmid RP4 carrying the cis-acting DNA recognition site for conjugative DNA transfer between bacterial cells (Mobsite) was cloned into the kanamycin-neomycin resistance transposon Tn5. Using conventrional transposon mutagenesis techniques the new transposon, called Tn5-Mob, can easily be inserted into the host DNA of gram-negative bacteria. A host replicon carrying Tn5-Mob is then mobilizable into any other gram-negative species if the transfer functions of plasmid RP4 are provided in trans. The potential of Tn5-Mob was demonstrated by mobilizing Rhizobium meliloti plasmids as well as the E. coli chromosome at high frequencies.

High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon.

Publisher Summary This chapter describes the experimental application of a vector system for Rhizobium that enables efficient transposon mutagenesis, site-specific insertion of selectable markers, creation of deletions in predetermined DNA regions, complementation studies, and high-frequency transfer of any DNA molecule between different strains. The essential ingredients of the system are the broad host range transfer and mobilization functions of plasmid RP4, transposable elements, mainly Tn5, and usual Escherichia coli specific vector plasmids. The major area of research in Rhizobium genetics is the identification, characterization, and manipulation of symbiotic genes. Modern recombinant DNA techniques have been applied to the analysis of cloned rhizobial genes in their parental environment by the construction of broad host range vector plasmids. The antibiotic resistance plasmid RP4 is known to be self-transmissible to any Gram-negative bacterium. The advantages of the use of antibiotic resistance transposons as mutagenic agents are insertion of a transposon into a gene leads to a complete loss of its function, transposon insertion mutation can be directly selected so that the yield of a transposon mutagenesis experiment is 100%, and the antibiotic resistance marker carried by the transposon acts as a probe for the genetic and physical location of the affected gene.

Plasmid vectors for the genetic analysis and manipulation of rhizobia and other gram-negative bacteria.

Summary: Transposon mutagenesis in a tomato isolate of Pseudomonas solanacearum (strain Kourou) is reported, using Tn7 and Tn5 inserted in suicide conjugative plasmids. Whereas Tn7 integrates at high frequency in a particular site of the the genome, Tn5 appears to transpose much more randomly, allowing isolation of auxotrophic mutants with a frequency of 035%. The mutants showed a wide range of nutritional requirements. Following Tn5 mutagenesis, screening of 8250 clones on axenic tomato seedlings led to the isolation of 12 avirulent mutants. Southern blot analysis revealed that, for avirulent mutants, insertion of Tn5 occurred in at least 10 different EcoRI restriction fragments. Additional independent insertions of IS50 were also detected in four of these mutants. For each mutant, transformation experiments demonstrated that the Tn5-encoded kanamycin resistance and the avirulent phenotype are linked. Based on their ability or inability to induce a collapse of tobacco leaf parenchyma, and on the timing of reaction of the plant, avirulent mutants have been divided in to two and possibly three groups.

Transposon Mutagenesis of Pseudomonas solanacearum: Isolation of Tn5-Induced Avirulent Mutants

Production of the chlorosis-inducing phytotoxin coronatine in the Pseudomonas syringae pathovars atropurpurea, glycinea, maculicola, morsprunorum, and tomato has been previously reported. DNA hybridization studies previously indicated that the coronatine biosynthetic gene cluster is highly conserved among P. syringae strains which produce the toxin. In the present study, two 17-bp oligonucleotide primers derived from the coronatine biosynthetic gene cluster of P. syringae pv. glycinea PG4180 were investigated for their ability to detect coronatine-producing P. syringae strains by PCR analysis. The primer set amplified diagnostic 0.65-kb PCR products from genomic DNAs of five different coronatine-producing pathovars of P. syringae. The 0.65-kb products were not detected when PCR experiments utilized nucleic acids of nonproducers of coronatine or those of bacteria not previously investigated for coronatine production. When the 0.65-kb PCR products were digested with ClaI, PstI, and SmaI, fragments of identical size were obtained for the five different pathovars of P. syringae. A restriction fragment length polymorphism was detected in the amplified region of P. syringae pv. atropurpurea, since this pathovar lacked a conserved PvuI site which was detected in the PCR products of the other four pathovars. The 0.65-kb PCR products from six strains comprising five different pathovars of P. syringae were cloned and sequenced. The PCR products from two different P. syringae pv. glycinea strains contained identical DNA sequences, and these showed relatedness to the sequence obtained for the pathovar morsprunorum. The PCR products obtained from the pathovars maculicola and tomato were the most similar to each other, which supports the hypothesis that these two pathovars are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and relatedness of coronatine-producing Pseudomonas syringae pathovars by PCR analysis and sequence determination of the amplification products.

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements.

In planta Transfer of the Gene (s) for Virulence between Isolates of Pseudomonas syringae pv. atropurpurea

Strains of Pseudomonas syringae pv. mori, the causal agent of bacterial blight of mulberry, contain 2 to 4 indigenous plasmids ranging from approximately 4.5 to 70mdal and were classified into 9 groups according to their characteristic plasmid patterns. A survey of 17 isolates did not reveal apparent correlations between plasmid pattern and toxigenicity, phage sensitivity or other phenotypes tested. All strains tested accepted freely the non-conjugative plasmid RSF1010 through mobilization by naturally occurring conjugative plasmid pBPW1 of P. s. pv. tabaci BR2, but re-transferred RSF1010 into Esherichia coli at very low frequency. Thus, it seems to be indicated that they possess an indigenous fertility system which is either naturally repressed or mobilizes RSF1010 with low efficiency.

Indigenous Plasmids of Pseudomonas syringae pv. mori, the Causal Agent of Bacterial Blight of Mulberry

The conjugational proficiency and replicative ability of plasmid pBPW1, found in Pseudomonas syringae pv. tabaci BR2, in P. syringae pathovars mori, atropurpurea, tabaci, eriobotryae and glycinea were examined. The plasmid was transferred and maintained as an autonomous replicon in ca. 40% of the strains at frequencies ranging from ca. 10-6 to 100% per recipient. These strains also expressed donor ability for pBPW1, with transfer frequencies ranging from 10-6 to 100%. In other strains the plasmid was apparently transferred but unable to replicate. This behaviour suggests that pBPW1 is potentially useful as a suicidal carrier for transposon mutagenesis in these strains. With one exception, the bacteriological properties of transconjugants were identical to those of the wild type recipient strains. The exception was pv. atropurpurea NIAES 1309 transconjugants, the majority of which aggregated in King's B broth and had altered serological properties. These findings suggest that pBPW1 carries a gene(s) responsible for cell aggregation and antigen alteration. However, the gene expression depends on the host's genetic background and does not occur in all hosts.

https://www.jstage.jst.go.jp/article/jjphytopath1918/51/4/51_4_426/_pdf

Conjugational Transmission and Replicative Properties of Plasmid pBPW1, and Antigen Alteration Induced by the Plasmid in Pathovars of Pseudomonas syringae

Pseudomonas syringae pv. atropurpurea (abbrev. pv. atropurpurea) is the causal agent of halo blight of Italian ryegrass (Lolium multiflorum Lam). The bacterium produces a phytotoxin designated as coronatine responsible for developing symptoms of halc blight1.2). Coronatine producing ability of the bacterium is associated with 58 Mdal plasmid named pCORl3). Here, we report the experimental result that pCORI plasmid is excluded (curing) from the host bacterium pv. atropurpurea at a high frequency, accom panying with loss of bacterial virulence, when the bacterium was inoculated to the plants of tobacco or Italian ryegrass. Bacterial strains used in this experiment were pv. atropurpurea NIAES 1309 wild type

High Frequency Exclusion of pCOR1 Plasmid from Pseudomonas syringae pv. atropurpurea in Plants

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