Wastewater bacteria remediating the pharmaceutical metformin: Genomes, plasmids and products

Abstract

Metformin is used globally to treat type II diabetes, has demonstrated anti-ageing and COVID mitigation effects and is a major anthropogenic pollutant to be bioremediated by wastewater treatment plants (WWTPs). Metformin is not adsorbed well by activated carbon and toxic N-chloro derivatives can form in chlorinated water. Most earlier studies on metformin biodegradation have used wastewater consortia and details of the genomes, relevant genes, metabolic products, and potential for horizontal gene transfer are lacking. Here, two metformin-biodegrading bacteria from a WWTP were isolated and their biodegradation characterized. Aminobacter sp. MET metabolized metformin stoichiometrically to guanylurea, an intermediate known to accumulate in some environments including WWTPs. Pseudomonasmendocina MET completely metabolized metformin and utilized all the nitrogen atoms for growth. Pseudomonas mendocina MET also metabolized metformin breakdown products sometimes observed in WWTPs: 1-N-methylbiguanide, biguanide, guanylurea, and guanidine. The genome of each bacterium was obtained. Genes involved in the transport of guanylurea in Aminobacter sp. MET were expressed heterologously and shown to serve as an antiporter to expel the toxic guanidinium compound. A novel guanylurea hydrolase enzyme was identified in Pseudomonas mendocina MET, purified, and characterized. The Aminobacter and Pseudomonas each contained one plasmid of 160 kb and 90 kb, respectively. In total, these studies are significant for the bioremediation of a major pollutant in WWTPs today.

Metformin hydrolase is a recently evolved, nickel-dependent, heteromeric ureohydrolase

“…10). Mobilization of MefH on plasmids and the probable horizontal transfer to Pseudomonas strains has been described before 24 . Currently, it is unclear why Aminobacter strains and possibly other bacterial taxa have developed a speci c enzyme system for the degradation of dimethylguanidine, as this compound has not yet been identi ed as a guanidine compound present in biological systems.…”

“…The molecular mechanism of metformin degradation to guanylurea remains unknown. However, recently three independent studies isolated Aminobacter and Pseudomonas strains from sewage sludge that were able to grow on metformin as sole nitrogen and carbon source [22][23][24] . All three teams identi ed a group of genes shared among the isolated strains that likely encodes an enzyme system catalyzing the hydrolysis of metformin to guanylurea and dimethylamine.…”

“…Further genes in the chromosomal Aminobacter operon are related to urea transport and metabolism and directly downstream of the four putative metformin degradation genes, a homolog of the nickel uptake transporter HupE is located. We heterologously expressed the candidate proteins of Aminobacter niigataensis MD1 22 and Pseudomonas mendocina MET 24 and puri ed an active hydrolase that e ciently and speci cally converted metformin to guanylurea and dimethylamine. We solved the crystal structure of the metformin hydrolase from A. niigataensis MD1 that revealed the formation of a heterohexamer with α 2 β 4 stoichiometry in which only the α-subunits contained catalytically active di-nickel metal centers.…”

Insights into the action of the pharmaceutical metformin: Targeted inhibition of the gut microbial enzyme agmatinase

“…which, in brief, used a C18 column and an isocratic mobile phase of 75:25 (v/v) acetonitrile:10 mM potassium phosphate buffer pH 6.6. 71 , 72 To determine if metformin could be transformed by enzymes EcAGM, CbAGM, GpuA, AGMAT, ARG1 and ARG2, 1 mM metformin was incubated with 10 μg of purified enzyme in 20 mM CHES pH 9 overnight and then assayed by HPLC. No transformation of metformin was seen for any of the enzymes tested.…”

Transport of metformin metabolites by guanidinium exporters of the small multidrug resistance family

“…In some cases, metformin may act as a co-selective agent, enhancing the survival of antibiotic-resistant bacteria in the presence of antibiotics ( Wei et al, 2022 ). However, other recent studies have isolated bacteria that utilize metformin as a nitrogen and/or carbon source ( Chaignaud et al, 2022 ; Li et al, 2023 ; Martinez-Vaz et al, 2022 ), suggesting that biodegradation of metformin and guanylurea may be a viable strategy for remediation of these compounds.…”