A patchwork pathway of apparently recent origin enables degradation of the synthetic buffer compound TRIS in bacteria

Abstract

The widely used synthetic chemical 2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS) was long considered to be biologically inert. Herein, we describe a complete degradation pathway for the catabolism of TRIS in bacteria. By serendipity,Pseudomonas hunanensisTeo1 was isolated from sewage sludge and shown to be able to grow with TRIS as only carbon and nitrogen source. Genome sequencing and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be upregulated during growth with TRIS. Conjugational transfer of this plasmid intoP. putidaKT2440 enabled this strain to grow with TRIS confirming that these clusters encode the complete TRIS degradation pathway. Heterologous gene expression inEscherichia colirevealed cluster I to encode a TRIS uptake protein, a TRIS dehydrogenase, and a TRIS aldehyde dehydrogenase, which catalyze the oxidation of TRIS into 2-hydroxymethylserine. Phylogenetic analysis suggests that the TRIS dehydrogenase proteins evolved from choline dehydrogenases. Genes encoded in cluster II encode a methylserine hydroxymethyltransferase and a D-serine dehydratase which could plausibly catalyze conversion of 2-hydroxymethylserine into pyruvate. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from thePseudomonasandShinellagenera which all carried the respective gene clusters in a similar genetic context as strain Teo1.Our data indicate that TRIS degradation evolved by gene assembly and enzyme adaptation from multiple independent metabolic pathways. Genomic database searches showed that TRIS degradation is now globally distributed, confirming its dissemination by horizontal gene transfer.Significance statementTRIS is a synthetic organic chemical that has been mass-produced since the 1950s and is now used in countless applications in industry, medicine, and households world-wide, in parts due to its presumed biological inertness. Our results challenge this common conception, showing that TRIS is biologically degradable by an apparently recently evolved pathway, providing insight into the evolution of metabolic pathways that make such ubiquitous chemicals available as growth substrates for bacteria. Such knowledge is valuable for developing new processes of wastewater purification in times of increasing water scarcity.

Assessing microbial plastic degradation requires robust methods

“…Here are some critical points: Some mineral media contain carbon‐based buffers that can be used by certain microbes as an undesired carbon source, causing misleading growth that is not based on plastic biodegradation. For example, we have noted that Alcanivorax and Marinobacter strains, and recently published Pseudomonas strains (Holert et al., 2024), can grow on Tris. Preferably, inorganic buffers should be used. Negative controls must always be included (e.g., the inoculum without the plastic and non‐inoculated culture controls).…”