Heavy and transition metal(loid)s (“metals” hereafter), such as copper (Cu), silver (Ag), arsenic (As), and mercury (Hg), have been used as antimicrobial agents for centuries in agriculture, medicine, and aquaculture. (1) Nowadays, the release of metals is greatly accelerated by intensive anthropogenic activities such as mining, burning of fossil fuels, and worldwide use of metal-containing pesticides, fertilizers, and growth stimulants. (2−4) Elevated metals persist in the environment and pose a long-standing selective pressure for the evolution of bacterial metal resistance. (5) As a side effect, environmental metal pollution contributes to the selection and spread of antibiotic resistance via coselection, (6−8) causing great threats to public health. Among various metals, cadmium (Cd), lead (Pb), zinc (Zn), chromium (Cr), Hg, and Cu have gained global attention since they are the most prevalent pollutants threatening human, plant, and environmental health. (9−13)

Microorganisms play an irreplaceable role in the biogeochemical cycling of metals. (14−17) Metals, such as Cu, Zn, cobalt (Co), and nickel (Ni), are essential micronutrients for microbial metabolism. These metals function as cofactors, involved in central bioenergetic and biogeochemical processes (e.g., respiration, photosynthesis, antioxidant defense, and nitrogen cycling). (18−20) However, due to the high affinity to thiol groups and/or oxidation properties, metals can be toxic and target multiple cellular processes through attacking iron–sulfur (Fe–S) clusters, replacing essential metals, dysfunction of proteins, oxidation of lipid and nucleic acid, and interfering nutrient assimilation. (1,18) To balance the metabolic function demand and metal toxicity, microbes have evolved sophisticated homeostatic mechanisms to maintain essential metals at a sufficient level. (19) These mechanisms are categorized as efflux, transformation, sequestration, precipitation, and reduced uptake. Among these, efflux pumps are the most expedient mechanism for bacteria to cope with excess metal ions, which is mainly mediated by three types of transport systems: the P-type ATPase, the Cation Diffusion Facilitator (CDF) efflux transporter, and the multicomponent Resistance-Nodulation-Division (RND) transport system. (21) Enzyme-mediated metal biotransformations alter metal speciation through redox and (de)methylation. (18,22,23) Metallothionein or chaperone proteins sequester intracellular metal ions to alleviate toxicity. (24) For instance, Cu chaperone CopZ can bind excess cytoplasmic Cu in Enterococcus hirae, (23) and metallothionein SmtA confers Cd resistance in Cyanobacteria. (25) In addition, various high- and low-affinity uptake systems contribute to the import of essential metals and are of importance for bacterial metal homeostasis. (26) These mechanisms protect bacteria from metal toxicity and meanwhile drive the metal biogeochemical cycling.

To date, the genetic basis for bacterial metal resistance and related biochemical pathways have been elaborated in considerable detail by pure culture-based studies. (27−29) However, the resistance systems identified in several isolates could not provide comprehensive insights into the scenario of resistance strategies in complex environments, since only a tiny fraction of microbes is culturable. Up to now, profiling the metal resistance determinants at the community level is still a great challenge, partially due to lack of comprehensive and targeted tools for gene quantification. Although metagenomic and metatranscriptomic approaches have been used to identify the gene profile underlying microbial resistance to metals, (30−33) they are still facing the challenge of rapid detection and quantification. (34,35) HT-qPCR based quantification approaches overcome the obstacle of meta-omic approaches for cost-effective and accurate quantification, which has been widely used to describe and interpret the microbial functional structure in various ecosystems. (36−40) However, the primer sets which are currently available for detecting metal resistance determinants are restricted to specific types of metals, resistance mechanisms, and bacterial species. (5) Some primer sets target sequences with a length of over 500 bp, which are too long and not suitable for the qPCR assay. Thus, the lack of reliable primer sets hinders the assessment of the metal resistome and microbially mediated metal biogeochemical cycling.

Here, a novel HT-qPCR-based chip, termed the MRG chip, was developed for the comprehensive profiling of known prokaryotic metal resistance genes (MRGs) involved in the homeostasis of 9 metals...

Figure 1. Flowchart of major steps for the development of the MRG chip, including literature review, database construction, primer design, experimental validation, in silico validation, and data processing.