Plasmid pRO1957, which contains a 26.5-kb fragment from the chromosome of Pseudomonas pickettii PKO1, allows P. aeruginosa PAO1 to grow on toluene or benzene as a sole carbon and energy source. A subclone of pRO1957, designated pRO1966, when present in P. aeruginosa PAO1 grown in lactate-toluene medium, accumulates m-cresol in the medium, indicating that m-cresol is an intermediate of toluene catabolism. Moreover, incubation of such cells in the presence of 1802 followed by gas chromatography-mass spectrometry analysis of m-cresol extracts showed that the oxygen in m-cresol was derived from molecular oxygen.Accordingly, this suggests that toluene-3-monooxygenation is the first step in the degradative pathway. Toluene-3-monooxygenase activity is positively regulated from a locus designated tbuT. Induction of the toluene-3-monooxygenase is mediated by either toluene, benzene, ethylbenzene, or m-cresol. Moreover, toluene-3-monooxygenase activity induced by these eflectors also metabolizes benzene and ethylbenzene to phenol and 3-ethylphenol, respectively, and also after induction, o-xylene, m-xylene, and p-xylene are metabolized to 3,4-dimethylphenol, 2,4-dimethylphenol, and 2,5-dimethylphenol, respectively, although the xylene substrates are not effectors. Styrene and phenylacetylene are transformed into more polar products.The degradation of toluene by bacteria incorporating molecular oxygen into toluene has been studied by several laboratories, resulting in the characterization of four pathways in pseudomonads. The best characterized of these pathways is the TOL plasmid pathway of Pseudomonas putida mt-2 (25). The TOL plasmid pathway converts toluene to benzyl alcohol, benzaldehyde, benzoate, and finally to catechol, which undergoes meta cleavage. The structural and regulatory genes of this pathway have been mapped and consist of two regulons. The upper pathway operon, xylCMAB, encodes enzymes for the metabolism of toluene to benzoate and is positively regulated byxylR (7,11,24) together with the sigma factor NtrA (5). The lower pathway enzymes for the metabolism of catechol are encoded by the xylXYZLTEGFJQKIH operon (9) and regulated by xylS (6,10,19,24). Transcription of xylS is also mediated by xylR and the NtrA sigma factor, which results in overproduction of xylS and subsequent transcription of the meta-cleavage pathway operon in the absence of meta-cleavage intermediates (19). The enzymes of the upper pathway also exhibit broad substrate specificity, transforming not only toluene and xylene but also ethyl-, methyl-and chloro-substituted toluene (1).P. putida Fl also metabolizes toluene; however, the first step in the pathway is the transformation of toluene to cis-toluene dihydrodiol, followed by conversion to 3-methylcatechol, which undergoes meta cleavage. The genetic organization (29, 30) and biochemistry (8,20,27) of the enzymes responsible for the catabolism of toluene to 3-methylcatechol have been studied extensively. The toluene 2,3-dioxygenase of P. putida Fl metabolizes a wide range of hydrocarbon...

Several distinctive properties of PRD1, an icosahedral plasmid-dependent phage, are described. The drug-resistance plasmid-dependent host range of PRD1 extends beyond the P incompatibility group and includes gram-negative bacteria containing plasmids of incompatibility groups N and W. PRD1 phage will infect pseudomonads and Enterobacteriaceae containing either a P or W incompatibility group plasmid. PRD1 adsorbs to the cell wall of R + bacteria and thus its infectivity indicates cell wall alterations by these drug-resistance plasmid groups. PRD1 nucleic acid is duplex DNA with an estimated molecular weight of 24 × 10 6 . The appearance of PRD1 in electron micrographs is suggestive of lipid content in addition to its buoyant density of 1.348 in CsCl and its sensitivity to chloroform. The latent period of PRD1 varies with the R + host bacterial strain used for growth of the phage.

No 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.

Plasmid pJP4 enables Alcaligenes eutrophus JMP134 to degrade 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (TFD). Plasmid pRO101 is a derivative of pJP4 obtained by insertion of Tn1721 into a nonessential region of pJP4. Plasmid pRO101 was transferred by conjugation to several Pseudomonas strains and to A. eutrophus AEO106, a cured isolate of JMP134. AEO106(pRO101) and some Pseudomonas transconjugants grew on TFD. Transconjugants with a chromosomally encoded phenol hydroxylase also degraded phenoxyacetic acid (PAA) in the presence of an inducer of the TFD pathway, namely, TFD or 3-chlorobenzoate. A mutant of one such phenol-degrading strain, Pseudomonas putida PPO300(pRO101), grew on PAA as the sole carbon source in the absence of inducer. This isolate carried a mutant plasmid, designated pRO103, derived from pRO101 through the deletion of a 3.9-kilobase DNA fragment. Plasmid pRO103 constitutively expressed the TFD pathway, and this allowed the metabolism of PAA in the absence of the inducer, TFD. Complementation of pRO103 in trans by a DNA fragment corresponding to the fragment deleted in pRO101 indicates that a negative control-regulatory gene (tfdR) is located on the BamHI E fragment of pRO101. Other subcloning experiments resulted in the cloning of the tfdA monooxygenase gene on a 3.5-kilobase fragment derived from pRO101. This subclone, in the absence of other pRO101 DNA, constitutively expressed the tfdA gene and allowed PPO300 to grow on PAA. Preliminary evidence suggests that the monooxygenase activity encoded by this DNA fragment is feedback-inhibited by phenols.