Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome

Abstract

A host-vector system for Pseudomonas aeruginosa PAO was developed. Scattered regions of the strain PAO chromosome were cloned by direct selection for complementation of auxotrophs or from a DNA gene bank which contains over 1,000 independently isolated chromosome-vector recombinant plasmids. The use of partially digested chromosomal DNA facilitated the selection of a variety of strain PAO chromosomal markers. The progenitor of the vector was a small, multicopy plasmid, pRO1600, found in a PAO strain which had acquired RP1 in a mating experiment. The bacterial host range that could be determined by transformation of vectors produced from pRO1600 resembles that for plasmid RP1. Two derivative plasmids were formed: pRO1613, for cloning DNA cleaved with restriction endonuclease PstI, and pRO1614, which was formed by deleting part of pRO1613 and fusion with plasmid pBR322. Plasmid pRO1614 utilizes known cloning sites within the tetracycline resistance region of pBR322.

Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production

“…All subclones were first generated in the ColEI plasmids pUC18 or pUC19. However, ColEI plasmids do not replicate stably in P. aeruginosa (Olson et al, 1982). Thus, the constructs were then subcloned into the P. aeruginosa vector pKT230, and the resulting recombinant plasmids were transferred to PAO1 by triparental mating (Ditta et al, 1980) or electroporation (Smith and Iglewski, 1989).…”

“…The 740 bp of toxA upstream region plus the region coding for the exotoxin A leader peptide and the first 30 amino acids of the mature toxin were fused inframe with the lacZ gene. In order to integrate pSW226 into the chromosome of PAO1, the 1.8 kb Pst I fragment, which is required for the stable replication of ColEI plasmids in P. aeruginosa (Olson et al, 1982), was deleted from pSW226. The resulting plasmid was introduced into PAO1 by electroporation and transformants were selected on carbenicillin plates.…”

The Pseudomonas aeruginosa pilK gene encodes a chemotactic methyltransferase (CheR) homologue that is translationally regulated

“…Transformation of E. coti and the Introduction of recombinant piasmids into P. aeruginosa from E coli by triparental matings were performed as previously described (Darzins, 1993). P. aeruginosa cells were transformed by the method of Olsen et al (1982). Preparation of phage and phage-sensitivity assays were performed as described previously (Darzins, 1994).…”

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

“…Bacterial strains and plasmids are listed in Table 1. Pseudomonas putida PF015 was previously identified as Pseudomonas fluorescens [24]. However, characteristics obtained using API 20 NE (Analytab Products, Bio Merieux SA, France) * and confirmed by gas chromatography-fatty acid methyl esterase analysis (performed by Microbe Inotech Laborato-* Mention of trade names or commercial products does not constitute endorsement or recommendation for use.…”