Multiple levels of transcriptional regulation control glycolate metabolism inParacoccus denitrificans

Abstract

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described β-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. While the reaction sequence of the BHAC was elucidated inParacoccus denitrificans, little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism inP. denitrificansis surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, as well as the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis inP. denitrificans. Together, these regulators control glycolate metabolism inP. denitrificans, allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.

In vitrotranscription-based biosensing of glycolate for prototyping of a complex enzyme cascade

“…Thus, we turned our attention to another aTF from Paracoccus denitrificans that was recently reported to regulate glycolate assimilation in the β-hydroxyaspartate cycle (BHAC) 28 . This GntR family transcriptional repressor, named GlcR, is encoded by pden4400, binds the intergenic sequence pden4399-4400 and was shown to unbind in the presence of glycolate 29 , which made the protein an interesting candidate for our envisioned IVT-based biosensor.…”

“…MBP-GlcR was produced and purified as previously described by Schada von Borzyskowski et al 29 . T7 RNA polymerase was produced in an E. coli M15 strain harboring plasmid pQE30-T7 RNAP 55 (strain sAP94).…”