Characterization of Pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of Pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal DNA fragment able to substitute for xylS in activation of the TOL lower-pathway promoter

Abstract

Mutants of Pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). The latter mutants, represents by strain PP0201, were shown to lack benzoate diol dehydrogenase (benD) activity. Mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dioxygenase (benABC) or the regulatory gene (benR) or to contain multiple mutations. Previous work in this laboratory suggested that benR can substitute for the TOL plasmid-encoded xylS regulatory gene, which promotes gene expression from the OP2 region of the lower or meta pathway operon. Accordingly, structural and regulatory gene mutations were distinguished by the ability of benzoate-grown mutant strains to induce expression from OP2 without xylS by using the TOL plasmid xylE gene (encoding catechol 2,3-dioxygenase) as a reporter. A cloned 12-kb BamHI chromosomal DNA fragment from the P. aeruginosa PAO1 chromosome complemented all of the mutations, as shown by restoration of growth on benzoate minimal medium. Subcloning and deletion analyses allowed identification of DNA fragments carrying benD, benABC, and the region possessing xylS substitution activity, benR. Expression of these genes was examined in a strain devoid of benzoate-utilizing ability, Pseudomonas fluorescens PFO15. The disappearance of benzoate and the production of catechol were determined by chromatographic analysis of supernatants from cultures grown with casamino acids. When P. fluorescens PFO15 was transformed with plasmids containing only benABCD, no loss of benzoate was observed. When either benR or xylS was cloned into plasmids compatible with those plasmids containing only the benABCD regions, benzoate was removed from the medium and catechol was produced. Regulation of expression of the chromosomal structural genes by benR and xylS was quantified by benzoate diol dehydrogenase enzyme assays. The results obtained when xylS was substituted for benR strongly suggest an isofunctional regulatory mechanism between the TOL plasmid lower-pathway genes (via the OP2 promoter) and chromosomal benABC. Southern hybridizations demonstrated that DNA encoding the benzoate dioxygenase structural genes showed homology to DNA encoding toluate dioxygenase from the TOL plasmid pWW0, but benR did not show homology to xylS. Evolutionary relationships between the regulatory systems of chromosomal and plasmid-encoded genes for the catabolism of benzoate and related compounds are suggested.

Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2

“…Ibu-2 performs similar reactions with other arylacetic acids, including PAA, 2-phenylpropionic acid, 3-and 4-tolylacetic acids, and 2-(4-tolyl)propionic acid, converting them to the corresponding catechol (or methylcatechol). Although this is somewhat reminiscent of the removal of the carboxyl moiety from benzoate (Eaton, 1996;Fetzner et al, 1992;Jeffrey et al, 1992;Reiner, 1971), it differs from the previously characterized PAA pathways of other bacteria, which do not involve catecholic intermediates (Fernández et al, 2006;Ismail et al, 2003;Martínez-Blanco et al, 1990;El-Said Mohamed, 2000;Rost et al, 2002;Teufel et al, 2010).…”

Cross talk between catabolic pathways in Pseudomonas putida: XylS-dependent and -independent activation of the TOL meta operon requires the same cis-acting sequences within the Pm promoter

“…Furthermore, a DNA segment from Pseudomonas aeruginosa which complemented the activity of the benR mutation in P. putida also restored XylS-independent expression of Pm (12). The same authors (12) provided indirect evidence that XylS restores the inducibility by benzoate of the benABCD genes in the absence of benR.…”

“…Recent observations from different laboratories (1,12,13) have shown that, in the absence of XylS, the Pm promoter is still responsive to benzoate (but not to 3-methylbenzoate), perhaps by recruiting the regulator of the chromosomal benABCD pathway, BenR (12). Since xylS and benR do not share a high degree of homology, at least as determined by DNA-DNA hybridization (12), Pm would be a type of promoter which can be activated by either of two different regulators. However, an alternative explanation could be that the same DNA region which contains the Pm promoter elements recognized by XylS includes also a set of sequence motifs recognized by an alternative regulator.…”

“…Jeffrey et al (12) found that benR mutants of P. putida, i.e., mutants lacking the regulator of the benABCD cluster for conversion of benzoate to catechol, did not show any XylSunrelated activity of Pm. Furthermore, a DNA segment from Pseudomonas aeruginosa which complemented the activity of the benR mutation in P. putida also restored XylS-independent expression of Pm (12).…”

A Comparison of the Multiple Alleles of xylS Carried by TOL Plasmids pWW53 and pDK1 and its Implications for their Evolutionary Relationship

“…However, we have as yet been unable to demonstrate that either of the cloned xylS2 genes can function as benzoate-or toluate-stimulated activators in an expression system. Furthermore, a recent paper has demonstrated that a gene in the P. putida chromosome, which is probably benR, can substitute for xylS in such an expression system although only with unsubstituted benzoate as inducer (Jeffrey et al, 1992). It is possible that the inducibility of the meta pathway genes on pWW53-1, pWW53-2 and pDKMl could be due to this chromosomal regulator gene.…”