MOLECULAR APPROACH TO STUDY THE PATHOGENICITY OF ERWINIA CAROTOVORA SUBSP. CAROTOVORA
Abstract
The bacteriophage Mud1(Ap('r) lac cts62) obtained from an Escherichia coli double lysogen carrying the defective Mu d1 phage and a Mu-P1 hybrid phage was utilized as a vector for phage mutagenesis of Erwinia carotovora subsp. carotovora. Among ampicillin-resistant transductants, 1.4% were auxotrophs. The wide range of different auxotrophic phenotypes recovered indicated that the insertion of Mud1 is random in the E. carotovora genome. Using His auxotrophs I showed that the expression of the lac Z gene is under the control the promoter-regulatory region of the his operon, thus demonstrating that Mud1 mutagenesis can be used to obtain gene fusions in E. carotovora. Using Mud1 mutagenesis I isolated three classes of apparently nonpathogenic mutants. (I) Mutants blocked in biosynthesis of pyrimidines or purines. (II) Mutants unable to utilize various carbon sources (pleiotrophic). (III) Mutants impaired in their ability to secrete pectolytic and proteolytic enzymes. Class I mutants were nonpathogenic due to the lack of purine and pyrimidine at the cut surface of potato tuber, since application of supplements to potato tuber restored the pathogenic phenotype of mutants. The class II mutant was biochemically characterized as galU. This mutation would result in accumulation of gal-l-P, UDPGal and UDPG which are toxic to bacterial growth. Bacteriophage PIKMclr100 was transferred to Erwinia carotovora subsp. carotovora. P1 was stably maintained as detected by hybridization and transfer of kanamycin resistance. Lysogens of Erwinia carotovora failed to produce any viable P1 phage. Although total DNA from P1-lysogens of Erwinia carotovora hybridized to ('32)P-labeled P1 probe, I was not able to detect P1 DNA as an extrachromosomal element. The broad host-range Inc-P plasmids RP4, R751 and pMO850 were transferred by conjugation from Escherichia coli and Pseudomonas aeruginosa to Erwinia carotovora. These plasmids were stably maintained and their antibiotic resistances were expressed in E. carotovora. Transconjugants of E. carotovora were as efficient donors of plasmids as E. coli and P. aeruginosa. Acquisition of Inc-P plasmid had no effect on pathogenic behavior of E. carotovora. Inc-P plasmid did not mobilize chromosomal markers in E. carotovora. (Abstract shortened with permission of author.)
Degree
Ph.D.
Subject Area
Botany
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