title: Isolation, characterization, and utilization of a temperature-sensitive allele of a Pseudomonas replicon.
abstract: In order to facilitate genetic study of the opportunistic bacterial pathogen Pseudomonas aeruginosa, we isolated a conditional, temperature-sensitive plasmid origin of replication. We mutagenized the popular Pseudomonas stabilizing fragment from pRO1610 in vitro using the Taq thermostable DNA polymerase in a polymerase chain reaction (PCR). Out of approximately 23,000 potential clones, 48 temperature-sensitive mutants were isolated. One mutant was further characterized and the origin of replication was designated as mSF(ts1). The mutations that resulted in a temperature-sensitive phenotype in mSF(ts1) were localized to the 1.2 kb of minimum sequence required for replication in P. aeruginosa. The DNA sequence analysis revealed two mutations within the coding sequence of the Replication control (Rep) protein. Growth of P. aeruginosa carrying the temperature-sensitive plasmid at the non-permissive temperature of 42 degrees C resulted in loss of the plasmid by greater than 99.9999% of the cells after 16 h of growth. In order to facilitate its utilization, the mSF(ts1) was converted into a genetic cassette flanked by mirrored restriction endonuclease digestion sites of a pUC1918 derivative. We demonstrate utilization of the mSF(ts1) for genetic studies involving complementation and regeneration of a mutant in P. aeruginosa research.
authors.affiliation: Department of Biological Sciences, 101 Life Sciences Building, Auburn University, AL 36849, United States.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Silo-Suh
authors.forename: Brett
authors.initials: B
authors.lastname: Elmore
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Ohman
authors.forename: Sang-Jin
authors.initials: SJ
authors.lastname: Suh
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004265:DNA Helicases / Q000235:genetics; D005816:Genetic Complementation Test; D005827:Genetics, Microbial / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D020125:Mutation, Missense; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000235:genetics*; D011995:Recombination, Genetic; D013696:Temperature; D015534:Trans-Activators / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases