Graduate Thesis Or Dissertation
 

Genetic recombination of the integrative plasmid pMC7105 of Pseudomonas syringae pv. phaseolicola involves repetitive sequences

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  • A strain of the phytopathogenic bacterium Pseudomonas syringae pv. phaseolicola, a pathogen of common bean Phaseolus vulgaris, harbors a 150 kilobase pair (kb) plasmid pMC7105. In one strain, LR719, pMC7105 is stably integrated in the bacterial chromosome. Imprecise excision of the pMC7105 replicon has been shown to result in the formation of stable excision plasmids. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb, and three plasmids contained from 90 to 130 kb of chromosomal sequences flanking the integration site. These eight excision plasmids can be arranged into five classes based on the sites at which excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of the adjacent fragments 1 and 10, is present in all excision plasmids and regarded to contain the origin of replication. Blot hybridization analyses were used to identify the two plasmid-chromosome juncture fragments formed by integration of pMC7105. The site of integration on pMC7105 was shown to lie within a 2.6 kb region of BamHI fragment 8. Moreover, this fragment contained a repetitive sequence (RS-II) which showed homology with five BamHI fragments of pMC7105 and more than 20 EcoRI fragments from the chromosome. Two additional repetitive sequences from pMC7105 were identified, one (RS-I) of which shows no homology to chromosomal sequences. In every case examined, the BamHI fragments which were involved in excision of the integrated replicon each contained a homologous repetitive sequence. The frequency of excision involving these three repetitive elements varied, with only one of eight events involving RS-I and four of eight involving RS-II. These results indicate that integration and excision of the pMC7105 replicon occurs through general recombination at homologous repetitive sequences.
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