Positive regulation of phenolic catabolism in Agrobacterium tumefaciens by the pcaQ gene in response to beta-carboxy-cis,cis-muconate

Abstract

An Escherichia coli system for generating a commercially unavailable catabolite in vivo was developed and was used to facilitate molecular genetic studies of phenolic catabolism. Introduction of the plasmid-borne Acinetobacter pcaHG genes, encoding the 3,4-dioxygenase which acts on protocatechuate, into E. coli resulted in bioconversion of exogenously supplied protocatechuate into beta-carboxy-cis,cis-muconate. This compound has been shown to be an inducer of the protocatechuate (pca) genes required for catabolism of protocatechuate to tricarboxylic acid cycle intermediates in Rhizobium leguminosarum biovar trifolii. The E. coli bioconversion system was used to explore regulation of the pca genes in a related bacterium, Agrobacterium tumefaciens. The pcaD gene, which encodes beta-ketoadipate enol-lactone hydrolase, from A. tumefaciens A348 was cloned and was shown to be adjacent to a regulatory region which responds strongly to beta-carboxy-cis,cis-muconate in E. coli. Site-specific insertional mutagenesis of the regulatory region eliminated expression of the pcaD gene in E. coli. When the mutation was incorporated into the A. tumefaciens chromosome, it eliminated expression of the pcaD gene and at least three other pca genes as well. The regulatory region was shown to activate gene expression in trans. The novel regulatory gene was termed pcaQ to differentiate it from pca regulatory genes identified in other microbes, which bind different metabolites.

The LysR-type PcaQ protein regulates expression of a protocatechuate-inducible ABC-type transport system in Sinorhizobium meliloti

“…In Acinetobacter baylyi, pcaHG form part of a large operon (pcaIJFBDKCHG) that is regulated by the IclR-type regulatory protein PcaU (Gerischer et al, 1998). In contrast, expression of pcaDCHGB is modulated by PcaQ in S. meliloti and A. tumefaciens (Parke, 1993;MacLean et al, 2006), and we now offer in silico evidence that PcaQ may also regulate the expression of these genes in certain members of the b-and c-proteobacteria. We note that the binding sites identified in the b-proteobacteria atypically contain a 7 nt spacer between the conserved left-and right-hand arms of the binding site; all other predicted sites encode 8 nt.…”

“…The LysR-type protein PcaQ participates in the regulation of pcaD expression in S. meliloti and A. tumefaciens (Parke, 1993(Parke, , 1996MacLean et al, 2006MacLean et al, , 2008, and we here report that expression of a gene encoding a component of an Inoculum was from washed LBmc-grown cells and cultures were incubated with shaking at 30 6C for 57.5 h; growth of each strain was assayed in triplicate cultures and the mean OD 600 is shown; error bars, SD. (e) Expression of pcaI : : lacZ was assayed in wild-type (RmP110) and transporter mutant backgrounds (RmP1710 and RmP1712) in minimal medium with carbon sources as indicated; Pca, protocatechuate.…”

“…PcaQ is a member of the LysR family of regulatory proteins, and requires the co-effectors b-carboxy-cis,cismuconate and c-carboxymuconolactone to induce expression of pcaDCHGB in Agrobacterium tumefaciens and S. meliloti (Parke, 1993(Parke, , 1996MacLean et al, 2006MacLean et al, , 2008. LysR-type transcriptional regulators participate in the regulation of a wide variety of physiological processes, including carbon and nitrogen metabolism, aromatic and amino acid degradation, virulence in plant and animal pathogens, quorum sensing, and the oxidative stress response (Maddocks & Oyston, 2008).…”

Binding Site Determinants for the LysR-Type Transcriptional Regulator PcaQ in the Legume Endosymbiont Sinorhizobium meliloti

“…Enzymes involved in the protocatechuate branch of the ␤-ketoadipate pathway in S. meliloti are encoded within the pcaDCHGB and pcaIJF operons, which are subject to regulation by products encoded by pcaQ and pcaR, respectively (23). The regulator encoded by pcaQ is a member of the LysR-type transcriptional regulator (LTTR) superfamily, and PcaQ homologues are present in many species of ␣-proteobacteria (3,4,7,23,34,36,37).LysR-type regulators comprise one of the largest groups of prokaryotic transcriptional regulators characterized to date; these proteins regulate a diverse range of regulons, including genes whose products are involved in nitrogen and carbon fixation, biofilm formation, the oxidative stress response, bacterial virulence, and the catabolism of various compounds, including aromatic acids (10,16,19,22,23,28,34,46,47,51,55,57). LTTR proteins consist of a conserved helix-turn-helix DNA binding motif located in the N-terminal portion of the polypeptide, whereas the C terminus includes an inducer binding site.…”

Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358 The GenBank/EMBL/DDBJ accession numbers for the pcaR, pobC-pobA and pcaHG sequences reported in this paper are AJ252090, AJ251792 and AJ295623, respectively.

“…Several regulatory genes and proteins sensing and responding to such aromatic compounds have been identified and characterized (Kok et al, 1998 ;Parke, 1993 ;Romero-Steiner et al, 1994). In Acinetobacter sp.…”