Characterization of the β-Ketoadipate Pathway inSinorhizobium meliloti

Abstract

Aromatic compounds represent an important source of energy for soil-dwelling organisms. The ␤-ketoadipate pathway is a key metabolic pathway involved in the catabolism of the aromatic compounds protocatechuate and catechol, and here we show through enzymatic analysis and mutant analysis that genes required for growth and catabolism of protocatechuate in the soil-dwelling bacterium Sinorhizobium meliloti are organized on the pSymB megaplasmid in two transcriptional units designated pcaDCHGB and pcaIJF. The pcaD promoter was mapped by primer extension, and expression from this promoter is demonstrated to be regulated by the LysR-type protein PcaQ. ␤-Ketoadipate succinyl-coenzyme A (CoA) transferase activity in S. meliloti was shown to be encoded by SMb20587 and SMb20588, and these genes have been renamed pcaI and pcaJ, respectively. These genes are organized in an operon with a putative ␤-ketoadipyl-CoA thiolase gene (pcaF), and expression of the pcaIJF operon is shown to be regulated by an IclR-type transcriptional regulator, SMb20586, which we have named pcaR. We show that pcaR transcription is negatively autoregulated and that PcaR is a positive regulator of pcaIJF expression and is required for growth of S. meliloti on protocatechuate as the carbon source. The characterization of the protocatechuate catabolic pathway in S. meliloti offers an opportunity for comparison with related species, including Agrobacterium tumefaciens. Differences observed between S. meliloti and A. tumefaciens pcaIJ offer the first evidence of pca genes that may have been acquired after speciation in these closely related species.

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

“…b-Galactosidase enzyme assays were performed as previously described (MacLean et al, 2006) upon S. meliloti strains subcultured into M9 minimal medium with glycerol and/or protocatechuate as carbon sources. Gfp fluorescence was assayed as previously described .…”

“…The Gram-negative legume microsymbiont Sinorhizobium meliloti encodes the protocatechuate branch of the b-ketoadipate pathway on the pSymB megaplasmid, and S. meliloti can utilize protocatechuate as a sole source of carbon (MacLean et al, 2006). Protocatechuate catabolic genes are organized into two operons (pcaDCHGB and pcaIJF), whose expression is regulated by the transcriptional regulators PcaQ and PcaR, respectively (MacLean et al, 2006.…”

“…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).…”

“…The Gram-negative legume microsymbiont Sinorhizobium meliloti encodes the protocatechuate branch of the b-ketoadipate pathway on the pSymB megaplasmid, and S. meliloti can utilize protocatechuate as a sole source of carbon (MacLean et al, 2006). Protocatechuate catabolic genes are organized into two operons (pcaDCHGB and pcaIJF), whose expression is regulated by the transcriptional regulators PcaQ and PcaR, respectively (MacLean et al, 2006. While systems involved in the active transport of aromatic acids such as protocatechuate have been described in many species, including Pseudomonas putida (Harwood et al, 1994;Nichols & Harwood, 1997), a protocatechuate transport system has yet to be identified in S. meliloti or any other member of the a-proteobacteria.…”

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

“…Acknowledging the vast numbers of transcriptional activators found among LTTR members 22 21,[33][34][35][36] . In this proof-of-principle study, we selected the four candidates based on a minimal set of information, including knowledge about operator sequences, experimental evidence for ligand-inducible control of target operons, and their mode of action in the native chassis (i.e., activation; Supplementary Fig.…”