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.

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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.

R1822, a plasmid specifying multiple drug resistances, has been transferred to a variety of species representative of related and unrelated genera. The host range of the plasmid includes Enterobacteriaceae, soil saprophytes, Neisseria perflava, and photosynthetic bacteria. With the acquisition of drug resistance(s), these strains became sensitive to a small, ribonuclease-sensitive bacteriophage, designated PRR1, isolated by enrichment from sewage. ' Received as PAT 2; spontaneous streptomycin-resistant mutant (1 mg/ml). d Received as PA0602; PAL(R1822) x PA0602. e Auxotrophic mutant of P71 isolated from milk by J. J. Jezeski, Univ. of Minnesota. ' Department of Microbiology, Univ. of Michigan. ' Spontaneous streptomycin-resistant mutant (1 mg/ml). hDepartment of Biochemistry, Univ. of Michigan. colonies from the primary isolation medium (always containing carbenicillin) to TGE agar medium containing tetracycline or neomycin at 50 ug/ml. Mating. R+ strains used in subsequent quantitative estimates of transfer frequencies were constructed by mixed inoculation of TGE broth medium I 773

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl-and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18 O 2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.

Burkholderia pickettii PKO1 metabolizes toluene and benzene via a chromosomally encoded toluene-3-monooxygenase pathway. Expression of the toluene-3-monooxygenase operon (tbuA1UBVA2C) is activated by the regulator, TbuT, in the presence of toluene. We have identified the TbuT coding region downstream of the toluene-3-monooxygenase structural genes by nucleotide sequence analysis and have shown that although TbuT is similar to XylR and DmpR, two members of the NtrC family of transcriptional activators which control toluene-xylene and (methyl)phenol catabolism, respectively, it is significantly different in the domain associated with effector specificity. Using a tbuA1-lacZ fusion reporter system, we determined that TbuT is activated not only by aromatic effectors but also the chlorinated aliphatic hydrocarbon trichloroethylene. Expression of tbuT and that of the tbuA1UBVA2C operon were found to be linked by readthrough transcription of tbuT from the toluene-3-monooxygenase promoter. As a result, transcription of tbuT is low when the toluene-3-monooxygenase operon is uninduced and high when expression of tbuA1UBVA2C is induced by toluene. Thus, the toluene-3-monooxygenase promoter drives the cascade expression of both the toluene-3-monooxygenase operon and tbuT, resulting in a positive feedback circuit. Examination of the nucleotide sequence upstream of the toluene-3-monooxygenase operon for promoter-like sequences revealed a ؊24 TGGC, ؊12 TTGC sequence, characteristic of 54 (rpoN)-dependent promoters. Primer extension and tbuA1-lacZ fusion analyses demonstrated that this ؊24, ؊12 promoter sequence, referred to as PtbuA1, was the toluene-3-monooxygenase promoter. Upstream of PtbuA1, a DNA region with dyad symmetry exhibited homology with the XylR-binding site present upstream of the Pu promoter. Deletions within this DNA sequence resulted in complete loss of expression from PtbuA1, suggesting that this region may serve as the TbuT-binding site.Burkholderia (formerly Pseudomonas) pickettii PKO1 metabolizes benzene and toluene via a toluene-3-monooxygenase pathway (36). The initial step of this pathway involves the hydroxylation of toluene and benzene to m-cresol and phenol, respectively, by toluene-3-monooxygenase (36). The phenolic intermediates (phenol and m-cresol) are then further hydroxylated to catechol and methylcatechol, respectively, by a phenol hydroxylase, prior to ring cleavage by a meta-fission dioxygenase (25-27). We recently reported on the ability of B. pickettii PKO1 to degrade trichloroethylene (TCE) and provided evidence suggesting that this oxidation reaction was catalyzed by this same pathway (29). The toluene-3-monooxygenase is encoded by six tightly clustered chromosomal genes, tbuA1UBVA2C (6). On the basis of functional and sequence data, the toluene-3-monooxygenase is similar to the toluene-4-monooxygenase from Pseudomonas mendocina KR1 (57, 58) and the toluene/ benzene-2-monooxygenase from Pseudomonas sp. strain JS150 (24). These enzyme systems together with the toluene-2-monooxygenase fro...