Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity

Abstract

When organisms are subjected to environmental challenges, including growth inhibitors and toxins, evolution often selects for the duplication of endogenous genes, whose overexpression can provide a selective advantage. Such events occur both in natural environments and in clinical settings. Microbial cells—with their large populations and short generation times—frequently evolve resistance to a range of antimicrobials. While microbial resistance to antibiotic drugs is well documented, less attention has been given to the genetic elements responsible for resistance to metal toxicity. To assess which overexpressed genes can endow gram-negative bacteria with resistance to metal toxicity, we transformed a collection of plasmids overexpressing all E. coli open reading frames (ORFs) into naive cells, and selected for survival in toxic concentrations of six transition metals: Cd, Co, Cu, Ni, Ag, Zn. These selections identified 48 hits. In each of these hits, the overexpression of an endogenous E. coli gene provided a selective advantage in the presence of at least one of the toxic metals. Surprisingly, the majority of these cases (28/48) were not previously known to function in metal resistance or homeostasis. These findings highlight the diverse mechanisms that biological systems can deploy to adapt to environments containing toxic concentrations of metals.

Positive Selection during Niche Adaptation Results in Large-Scale and Irreversible Rearrangement of Chromosomal Gene Order in Bacteria

“…The occurrence of duplications of large and small segments of the bacterial chromosome is common during growth in nonlethal selective environments ( Straus and Hoffmann 1975 ; Sonti and Roth 1989 ; Näsvall et al 2012 ; Hoegler and Hecht 2018 ; Hufnagel et al 2021 ). The ability to utilize a novel carbon source has been shown to be a potential key event in the establishment of a population within a new environmental niche ( Blount et al 2012 ).…”

Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria

“…Indeed, the impact of enhanced gene dosage on adaptation and evolution are well documented across all three kingdoms of life and have been shown to be an important contributor to numerous diseases and drug resistance phenotypes 16–18 . Increased gene copy or overexpression as a genetic tool has a rich history of connecting genes to cellular functions and has been exploited as a versatile screening technique to identify drug targets 16,19,20 , antibiotic and metal resistance genes 17,21,22 , virus resistance genes 23 , genetic suppressors 24,25 , as well as for a number of chemical genomics 8,9 and biotechnology applications 26–28 . Although a number of technologies have been developed for overexpression screens including defined open reading frame (ORF) libraries 6,20,29 and activation modes of recombineering 30,31 , transposon insertions 32 , or CRISPR systems 33 , these strategies are limited, either due to the need for expensive and laborious generation of archived strains or the need for organism-specific genetic tools.…”

Effects of a Four-Week High-Dosage Zinc Oxide Supplemented Diet on Commensal Escherichia coli of Weaned Pigs

“…In this context, the P1b-type ATPase ZntA and the CDF ZitB have been frequently nominated (Hantke, 2005; Ding et al, 2012; Porcheron et al, 2013; Watly et al, 2016; Deus et al, 2017; Ojer-Usoz et al, 2017). In addition, the protein ZraP associated with zinc-resistance, the transcriptional regulatory protein ZraR (synonym: HydG), the serine acetyltransferase CysE and the low-affinity inorganic phosphate transporter PitA have been linked to zinc tolerance (Casewell et al, 2003; Hoegler and Hecht, 2018; Stocks et al, 2019). We were able to provide evidence that genes encoding these and many other factors involved in bacterial zinc hemostasis were present in almost all 179 isolates investigated, indicating that the sheer presence of these factors or even certain combinations do not confer zinc tolerance MICs deviating from the unimodal distribution.…”

“…Therefore, zinc quantities within cells are highly regulated, as zinc deprivation hinders bacterial growth, while an excess of zinc could be toxic (Gielda and DiRita, 2012). Factors reported to increase zinc tolerance levels in E. coli described so far include the cation diffusion facilitator (CDF) ZitB, the P 1 b -type ATPase ZntA and the low-affinity inorganic phosphate transporter Pit (Beard et al, 2000; Grass et al, 2005; Deus et al, 2017; Hoegler and Hecht, 2018).…”