Molecular and Genetic Relationships of Aromatic Metabolic Plasmids in Pseudomonads

145 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.This thesis elucidates the similarities among the pseudomonad metabolic plasmids required for growth on the substrates naphthalene, NAH, salicylate, SAL, xylene and toluene, XYL and TOL. Naphthalene and xylene dissimilation pass through salicylate and toluate as intermediates to converge at catechol or its methylated derivative. Catechol is further dissimilated, through the (alpha)-keto acid pathway (meta cleavage), to acetaldehyde and pyruvate which enter the tricarboxylic acid cycle. The plasmids are large, 40-75 megadaltons (Md) {60-107 kilobases (Kb)}, and encode, in addition to the aromatic growth phenotypes, self-fertility for conjugal transfer and SAL and TOL also for the mobilization of chromosomal DNA. The plasmids belong to the P-9 incompatability group.NAH (naphthalene) plasmids were isolated from three wild type strains. They were classed as P. putida, biotype A and B, and an unnamed intermediate between P. putida and P. fluorescens. The biotype B strains are more nutritionally versatile than the A including growth on the L-enantiomers of tryptophan and kynurenine. A similar 42 Md plasmid termed NAH-7 was isolated from each wild type strain. The biotype B strain also contained two smaller plasmids of 10.1 and 4.6 Md. The 42 Md plasmids had identical restriction patterns with six Type II endonucleases and show complete cross-hybridization, thus suggesting that a single plasmid has been conjugally spread among the pseudomonads and other receptive hosts.The SAL-1 plasmid, isolated from one wild type strain is 45 Md ((TURN)67.5 kilobases) but upon transfer to Pseudomonas aeruginosa strains yields a plasmid, SAL-1a, with a deletion of 2.4 Md of DNA and a new restriction endonuclease digest. Upon transformation of SAL-1a DNA back into the P. putida host, the 42 Md, SAL-lap plasmid is isolated with the identical restriction patterns as SAL-1a.The Type II restriction endonuclease pattern of SAL-1 was identical to NAH-7 except for an extra 3.4 Md segment. This additional segment with Sma I digests was recovered as two 1.7 Md fragments. The insertion may thus turn off the gene expression of an important initial component of the naphthalene pathway, thus leaving SAL-1 unable to complete conversion of napthalene.The TOL plasmid from the wild type P. arvilla mt-2 dissociates to two plasmids, TOL('*) and TOL(DELTA) on transfer to P. aeruginosa or in P. putida when grown on benzoate. TOL('*) encodes at least part of the enzymes for growth on xylenes and toluene and TOL(DELTA) for fertility and they cross hybridize to show (TURN)50% homology.XYL plasmid, required also for xylene metabolism, does not show fertility and can be isolated as a plasmid only when cointegrated with K, a pseudomonad sex factor.The TOL and XYL-K plasmids with the SAL and NAH plasmids show cross restriction hybridization, thus the four aromatic (ARO) plasmids are related.Genetic techniques of mutation, conjugation, transduction and transformation along with physical techniques such as electron microscopy, electrophoresis density banding and melting temperature were used to elucidate the relatedness of the four plasmids.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD