Five copper homeostasis gene clusters encode the Cu-efflux resistome of the highly copper-tolerant Methylorubrum extorquens AM1

Abstract

Background In the last decade, the use of copper has reemerged as a potential strategy to limit healthcare-associated infections and to control the spread of multidrug-resistant pathogens. Numerous environmental studies have proposed that most opportunistic pathogens have acquired antimicrobial resistance in their nonclinical primary habitat. Thus, it can be presumed that copper-resistant bacteria inhabiting a primary commensal niche might potentially colonize clinical environments and negatively affect the bactericidal efficacy of Cu-based treatments. The use of copper in agricultural fields is one of the most important sources of Cu pollution that may exert selection pressure for the increase of copper resistance in soil and plant-associated bacteria. To assess the emergence of copper-resistant bacteria in natural habitats, we surveyed a laboratory collection of bacterial strains belonging to the order Rhizobiales. This study proposes that Methylorubrum extorquens AM1 is an environmental isolate well adapted to thrive in copper-rich environments that could act as a reservoir of copper resistance genes. Methods The minimal inhibitory concentrations (MICs) of CuCl2 were used to estimate the copper tolerance of eight plant-associated facultative diazotrophs (PAFD) and five pink-pigmented facultative methylotrophs (PPFM) belonging to the order Rhizobiales presumed to come from nonclinical and nonmetal-polluted natural habitats based on their reported source of isolation. Their sequenced genomes were used to infer the occurrence and diversity of Cu-ATPases and the copper efflux resistome of Mr. extorquens AM1. Results These bacteria exhibited minimal inhibitory concentrations (MICs) of CuCl2 ranging between 0.020 and 1.9 mM. The presence of multiple and quite divergent Cu-ATPases per genome was a prevalent characteristic. The highest copper tolerance exhibited by Mr. extorquens AM1 (highest MIC of 1.9 mM) was similar to that found in the multimetal-resistant model bacterium Cupriavidus metallidurans CH34 and in clinical isolates of Acinetobacter baumannii. The genome-predicted copper efflux resistome of Mr. extorquens AM1 consists of five large (6.7 to 25.7 kb) Cu homeostasis gene clusters, three clusters share genes encoding Cu-ATPases, CusAB transporters, numerous CopZ chaperones, and enzymes involved in DNA transfer and persistence. The high copper tolerance and the presence of a complex Cu efflux resistome suggest the presence of relatively high copper tolerance in environmental isolates of Mr. extorquens.

Draft genome sequence of the highly copper-tolerant Methylobacterium radiotolerans MLP1 isolated from the rhizosphere of grasses adjacent to mine tailings

“…To understand how is organized the Cu-efflux resistome in bacteria thriving in Cu-rich environments, soil suspension from the rhizosphere of grasses sampled in the inactive mine “La Prieta” (Hidalgo del Parral, Chihuahua, México, 26° 56´ 27´´N, 105° 39´ 32´´W) ( 1 ) was screened for the presence of highly copper-tolerant bacteria by dilution and spread plate technique ( 2 ). After 72 h of incubation at 30°C, the most copper-tolerant isolate showed full growth on plates of succinate-ammonium mineral medium ( 3 ) supplemented with 4.0 mM CuCl 2 and total growth inhibition in 4.5 mM CuCl 2 (MIC), which falls into the range of tolerance reported for other highly Cu-tolerant species ( 4 , 5 ).…”