Date of Award
Doctor of Philosophy
Bonita A. Glatz
A simple and rapid method was developed for the isolation of plasmid DNA from propionibacteria. Effective cellular lysis was achieved in all species when cells were treated with a high concentration of lysozyme. Purification by acid phenol extraction or cesium chloride-density gradient centrifugation was required to remove contaminating chromosomal and open circular DNA and to obtain complete endonuclease digestions. With this method, 119 strains representing all species of the classical propionibacteria were screened for the presence of plasmid DNA. Twenty strains were found to contain plasmids ranging in size from 4.4 to greater than 119 megadaltons (Mdal);Restriction endonucleases and DNA-DNA hybridizations were used to characterize the native plasmids. Seven distinct plasmids have been identified to date, and restriction maps have been constructed for four of these. Eleven strains contained an identical 4.4 Mdal plasmid, pRG01. Two other strains contained an identical 6.3 Mdal plasmid, pRG02, and one of these strains contained a multimeric form of this plasmid. Plasmids pRG04 and pRG06, 30 and 5.6 Mdal respectively, were also found in more than one strain. Plasmids pRG01 and pRG02 shared extensive sequence homology, and both were homologous to pRG07 (6.3 Mdal) and to identical sequences of pRG05 (35 Mdal). Plasmids pRG04 and pRG06 did not share any significant sequence homology with any of the other plasmids. Plasmid pRG03 (25 Mdal) had partial sequence homology only with pRG07. At least four additional unique plasmids have been isolated but have not been characterized to date;Curing of plasmids pRG01, pRG02, pRG03 and pRG05 was possible by treatment with acriflavin. Three traits have been tentatively linked to specific plasmids: clumping phenotype of P. jensenii P38 to plasmid pRG05; fermentation of lactose and pellet formation after culture centrifugation in P. freudenreichii P93 to plasmid pRG03. Plasmid pRG03 was found to code for a constitutive B-galactosidase enzyme. Maximum enzyme activity was observed for late-log cells assayed at 50°C in buffer at pH 7.5. Hybridization of an E. coli lacZ[superscript]' probe to restriction digests of plasmid pRG03 did not identify any regions of homology.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Thomas G. Rehberger
Rehberger, Thomas G., "Isolation and characterization of plasmid DNA from Propionibacterium " (1989). Retrospective Theses and Dissertations. 9079.