Graduate Thesis Or Dissertation
 

Physical properties and genetic studies of cryptic plasmids discovered in plant pathogenic pseudomonads

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  • Six plant pathogenic isolates of Pseudomonas syringae pv. glycinea, the causal agent of bacterial blight of Glycine max (L.) Merrill, and one isolate of P. syringae pv. phaseolicola, the causal agent of halo blight of Phaseolus vulgaris L., have been examined for the presence of circular duplex deoxyribonucleic acid (DNA). Extrachromosomal DNA from each strain was first observed by cesium chloride-ethidium bromide dye-buoyant density gradient centrifugation. The size and number of plasmids of four different isolates were initially determined by neutral sucrose gradient centrifugation. Subsequently, plasmid DNA from each of the seven isolates was obtained by a cleared lysate technique and analyzed by agarose gel electrophoresis. Each of the isolates of P. syringae pv. glycinea harbor a different complex array of plasmids. The plasmids ranged from 3 to 109 megadaltons (Mdal) in size and there were from two to seven discrete plasmids per isolate. The isolate of P. syringae pv. phaseolicola contained a single plasmid which had a molecular weight of 98 Mdal. Endonuclease-derived fingerprints of purified total plasmid DNA from each isolate of P. syringae pv. glycinea served as one criterion for measuring plasmid relatedness among these pathovars of varying geographical origin. Similar-sized fragments were present in the digests of some of the isolates, but no single fragment was observed to be present in the digests of all isolates. Evidence for relatedness among the plasmids of these strains was obtained by hybridization of nick-translated total plasmid DNA from one strain to membrane blots of fingerprints of plasmid DNA from other strains. Fifty-five to seventy-five percent of the plasmid fragments from five strains hybridized with radio-labeled plasmid probe prepared from a sixth strain. In reciprocal hybridizations, radio-labeled probe prepared from each of four strains hybridized to a specific fragment of pMC10 plasmid and to three fragments of pMC11 plasmid, which were present in the digests of the sixth strain. Other fragments of pMC10 and pMC11 showed hybridization to labeled probe from some of the strains, but not with others. The plasmid, pMC7105, of a mutant of P. syringae pv. phaseolicola integrated into the bacterial chromosome. Biochemical evidence for integration was obtained by demonstrating that all of the plasmid sequence was present in a plasmidless isolate when membrane blots of total restriction endonuclease-cleaved DNA was probed with radio-labeled pMC7105 DNA. Furthermore, new autonomous plasmids have been detected in three strains which have resulted from partial excision of pMC7105. The probable insertion locus of p1/iC7105 and regions of excision for one of the derived plasmids were surmised from fingerprint analysis of plasmid DNA. The wide host-range antibiotic- resistance - plasmid RP4 was transferred by conjugation from Escherichia coli to strains of both pathovars. The RP4-containing strains of P. syringae pv. glycinea produced RP4-less colonies of which about 1.4% remained resistant to ampicillin, indicating transposition of Tn1 from RP4 onto DNA of the host. Ampicillin resistance was stably inherited by the Tn1 containing strains and could be mobilized in matings with nearly isogenic strains. However, the stability of Tn1 among the transconjugants was greatly reduced. RP4 was stably inherited by two strains of P. syringae pv. phaseolicola. Restriction endonuclease-cleavage patterns of plasmid DNA from an isolate of one of these strains reveal evidence for the insertion of a sequence into the pMC7105 plasmid. Its restriction pattern and molecular weight were consistant with results expected by insertion of Tn1. The competency of this strain to serve as a donor of RP4 or the newly derived plasmid, pMC7105: :Tn1, were greatly reduced when compared with identical strains which do not have Tn1 transposed onto pMC7105.
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