TRANSCRIPTIONAL CONTROL OF THE PSEUDOMONAS TOL PLASMID CATABOLIC OPERONS IS ACHIEVED THROUGH AN INTERPLAY OF HOST FACTORS AND PLASMID-ENCODED REGULATORS

Abstract

The xyl genes of Pseudomonas putida TOL plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xylUWCAMBN for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylXYZLTEGFJQKIH, which encodes the enzymes for further conversion of these compounds into Krebs cycle intermediates; and xylS and xylR, which are involved in transcriptional control. The XylS and XylR proteins are members of the XylS/AraC and NtrC families, respectively, of transcriptional regulators. The xylS gene is constitutively expressed at a low level from the Ps2 promoter. The XylS protein is activated by interaction with alkylbenzoates, and this active form stimulates transcription from Pm by sigma70- or sigmaS-containing RNA polymerase (the meta loop). The xylR gene is also expressed constitutively. The XylR protein, which in the absence of effectors binds in a nonactive form to target DNA sequences, is activated by aromatic hydrocarbons and ATP; it subsequently undergoes multimerization and structural changes that result in stimulation of transcription from Pu of the upper operon. This latter process is assisted by the IHF protein and mediated by sigma54-containing RNA polymerase. Once activated, the XylR protein also stimulates transcription from the Ps1 promoter of xylS without interfering with expression from Ps2. This process is assisted by the HU protein and is mediated by sigma54-containing RNA polymerase. As a consequence of hyperexpression of the xylS gene, the XylS protein is hyperproduced and stimulates transcription from Pm even in the absence of effectors (the cascade loop). The two sigma54-dependent promoters are additionally subject to global (catabolite repression) control.

Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

“…1) (25,26). The latter drives transcription of TOL plasmid upper operon for the degradation of toluene (27) and shows the typical modular structure of the 54 -dependent promoters (for review, see Refs. 28 and 29) that consists of: (i) the Ϫ12/Ϫ24 region (consensus: TGGCAC N5 TTGCa/t located between positions Ϫ11 and Ϫ26) (30) recognized by 54 and considered the functional analogue of Ϫ10/Ϫ35 core promoter bound by 70 (31), (ii) DNA enhancer sequences (known as upstream activating sequences or UAS) targets for the activators of the 54 -RNAP, usually located at Ͼ100 bp from the transcription start site, and (iii) an intervening sequence between UAS and Ϫ12/Ϫ24 motif that may contain a target site of the IHF (32), which, by its ability to bind and bend DNA sequences, assists the looping out required to bring closer together the 54 -activator prebound at UAS and 54 -RNAP assembled in a closed complex with Ϫ12/Ϫ24 DNA region.…”

Critical Nucleotides in the Upstream Region of the XylS-dependent TOL meta-Cleavage Pathway Operon Promoter as Deduced from Analysis of Mutants

“…Genes encoding the TOL meta-cleavage pathway are grouped into a single operon, the expression of which is positively regulated at the level of transcription by the xylS gene product, which is activated by benzoate effectors (1)(2)(3)(4). Stimulation of transcription from the Pm promoter requires a DNA sequence extending to about 80 bp 1 upstream of the transcription initiation point (5)(6)(7).…”

Linking genes to microbial growth kinetics—An integrated biochemical systems engineering approach

“…A complete model of the transcriptional regulation of TOL was constructed based on existing biological knowledge of its function (Moreno et al, 2010;Ramos et al, 1997) and specified by various molecular components, which interact to guide the catabolism of m-xylene (Figs. 1A,B).…”

“…The meta operon comprises 13 genes (xylXYZLTEGFJQKIH) consisting one of the largest operons in prokaryotes (Ramos et al, 1997). The transcription originating at the Pm promoter leads to the formation of nine enzymes involved in the meta pathway.…”

“…This strain harbours the TOL plasmid (pWW0), which specifies a pathway for the oxidative catabolism of toluene and m-xylene (Timmis, 2002). The required genetic information for the metabolism of these compounds is encoded by the xyl operons of the plasmid, synthesizing the relevant biocatalytic enzymes for conversion of substrates to Krebs cycle intermediates, while xylS and xylR are involved in transcriptional control (Ramos et al, 1997). The complex interactions between TOL plasmid-encoded regulators, a set of sigma factors and DNA-bending proteins, resulting in expression by the catabolic operons, render the TOL plasmid a paradigm of specific and global regulation (Aranda-Olmero et al, 2005).…”