Copper Resistance Determinants Research Articles

A gold speciation that adds a second layer to synergistic gold-copper toxicity in Cupriavidus metallidurans.

The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.

Genomic insights into a Pseudomonas amygdali isolate from Hibiscus rosa-sinensis

The taxonomy of Pseudomonas has been extensively studied, yet the determination of species is currently difficult because of recent taxonomic changes and the lack of complete genomic sequence data. We isolated a bacterium causing a leaf spot disease on hibiscus (Hibiscus rosa-sinensis). Whole genome sequencing revealed similarity to Pseudomonas amygdali pv. tabaci and pv. lachrymans. The genome of this isolate (referred to as P. amygdali 35–1) shared 4987 genes with P. amygdali pv. hibisci, but possessed 204 unique genes and contained gene clusters encoding putative secondary metabolites and copper resistance determinants. We predicted this isolate's type III secretion effector (T3SE) repertoire and identified 64 putative T3SEs, some of which are present in other P. amygdali pv. hibisci strains. Assays showed that the isolate was resistant to copper at a concentration of 1.6 mM. This study provides an improved understanding of the genomic relatedness and diversity of the P. amygdali species.

A Copper-Responsive Two-Component System Governs Lipoprotein Remodeling in Listeria monocytogenes.

Bacterial lipoproteins are membrane-associated proteins with a characteristic acylated N-terminal cysteine residue anchoring C-terminal globular domains to the membrane surface. While all lipoproteins are modified with acyl chains, the number, length, and position can vary depending on host. The acylation pattern also alters ligand recognition by the Toll-like receptor 2 (TLR2) protein family, a signaling system that is central to bacterial surveillance and innate immunity. In select Listeria monocytogenes isolates carrying certain plasmids, copper exposure converts the lipoprotein chemotype into a weak TLR2 ligand through expression of the enzyme lipoprotein intramolecular acyltransferase (Lit). In this study, we identify the response regulator (CopR) from a heavy metal-sensing two-component system as the transcription factor that integrates external copper levels with lipoprotein structural modifications. We show that phosphorylated CopR controls the expression of three distinct transcripts within the plasmid cassette encoding Lit2, prolipoprotein diacylglyceryl transferase (Lgt2), putative copper resistance determinants, and itself (the CopRS two-component system). CopR recognizes a direct repeat half-site consensus motif (TCTACACA) separated by 3 bp that overlaps the -35 promoter element. Target gene expression and lipoprotein conversion were not observed in the absence of the response regulator, indicating that CopR phosphorylation is the dominant mechanism of regulation. IMPORTANCE Copper is a frontline antimicrobial used to limit bacterial growth in multiple settings. Here, we demonstrate how the response regulator CopR from a plasmid-borne two-component system in the opportunistic pathogen L. monocytogenes directly induces lipoprotein remodeling in tandem with copper resistance genes due to extracellular copper stress. Activation of CopR by phosphorylation converts the lipoprotein chemotype from a high- to low-immunostimulatory TLR2 ligand. The two-component system-mediated coregulation of copper resistance determinants, in tandem with lipoprotein biosynthesis demonstrated here in L. monocytogenes, may be a common feature of transmissible copper resistance cassettes found in other Firmicutes.

Molecular Insights into the Copper-Sensitive Operon Repressor in Acidithiobacillus caldus.

The copper-sensitive operon repressor (CsoR) family, which is the main Cu(I)-sensing family, is widely distributed and regulates regulons involved in detoxification in response to extreme copper stress (a general range of ≥3 g/liter copper ions). Here, we identified CsoR in hyper-copper-resistant Acidithiobacillus caldus (CsoRAc), an organism used in the bioleaching process of copper ores. CsoRAc possesses highly conserved Cu(I) ligands and structures within the CsoR family members. Transcriptional analysis assays indicated that the promoter (PIII) of csoR was active but weakly responsive to copper in Escherichia coli. Copper titration assays gave a stoichiometry of 0.8 mol Cu(I) per apo-CsoRAc monomer in vitro combined with atomic absorption spectroscopy analysis. CuI-CsoRAc and apo-CsoRAc share essentially identical secondary structures and assembly states, as demonstrated by circular dichroism spectra and size exclusion chromatography profiles. The average dissociation constants (KD = 2.26 × 10-18 M and 0.53 × 10-15 M) and Cu(I) binding affinity of apo-CsoRAc were estimated by bathocuproine disulfonate (BCS) and bicinchoninic acid (BCA) competition assays, respectively. Site-directed mutations of conserved Cu(I) ligands in CsoRAc did not significantly alter the secondary structure or assembly state. Competition assays showed that mutants had the same order of magnitude of Cu(I) binding affinity as apo-CsoRAc. Moreover, apo-CsoRAc could bind to the DNA fragment P08430 in vitro, although with low affinity. Finally, a working model was developed to illustrate putative copper resistance mechanisms in A. caldus. IMPORTANCE Research on copper resistance among various species has attracted considerable interest. However, due to the lack of effective and reproducible genetic tools, few studies regarding copper resistance have been reported for A. caldus. Here, we characterized a major Cu(I)-sensing family protein, CsoRAc, which binds Cu(I) with an attomolar affinity higher than that of the Cu(I)-specific chelator bathocuproine disulfonate. In particular, CsoR family proteins were identified only in A. caldus, rather than A. ferrooxidans and A. thiooxidans, which are both used for bioleaching. Meanwhile, A. caldus harbored more copper resistance determinants and a relatively full-scale regulatory system involved in copper homeostasis. These observations suggested that A. caldus may play an essential role in the application of engineered strains with higher copper resistance in the near future.

Presence of Colistin- and Tigecycline-Resistant Klebsiella pneumoniae ST29 in Municipal Wastewater Influents in Japan.

The aim of this study was to investigate the presence of colistin- and/or tigecycline-resistant Klebsiella spp. in influents from four wastewater treatment plants (WWTPs), which partly reflect the gut microbiome of human populations. Colistin- and tigecycline-resistant Klebsiella pneumoniae isolates (K30/ST29) were detected four times from the WWTP A during a period of 3 months. Disruptions of the mgrB and ramR genes by ISEc68 and ISKpn21, respectively, were identified in those four isolates. They also shared the IncL/M 86,197-bp plasmids carrying a blaCTX-M-3 and Tn1548-associated armA [IS26-IntI1-dfrA12-gucF-aadA2-qacEΔ1-sul1-ISCR1-ISEc28-armA-ISEc29-msr(E)-mph(E)-IS26]. Those isolates formed a distinct cluster within wgMLST clusters of ST29 K30 public reference strains of human origin and were unique due to harboring of Tn21-like mercury resistance operon transposons in addition to silver, copper, and arsenic resistance determinants. Five K. pneumoniae strains with different STs and 1 Klebsiella quasipneumoniae strain, exhibiting colistin resistance, were detected in WWTPs B, C, and D. For these isolates, disruptions of mgrB by ISEc68 (three isolates) or ISEcl1 (one isolate), insertion of IS2 in the mgrB promoter region (one isolate), and inactivation of MgrB by a nonsense mutation (one isolate) were identified. Close monitoring of these mcr-negative colistin- and/or tigecycline-resistant bacteria in wastewater influents is imperative to avoid further limiting of treatment options.

A Large Tn7-like Transposon Confers Hyper-Resistance to Copper in Pseudomonas syringae pv. syringae.

Copper resistance mechanisms provide an important adaptive advantage to plant pathogenic bacteria under exposure to copper treatments. Copper resistance determinants have been described in Pseudomonas syringae pv. syringae (Pss) strains isolated from mango intimately associated with 62 kb plasmids belonging to the pPT23A family (PFP). It has been previously described that the indiscriminate use of copper-based compounds promotes the selection of copper resistant bacterial strains and constitutes a selective pressure in the evolution of copper resistance determinants. Hence, we have explored in this study the copper resistance evolution and the distribution of specific genetic determinants in two different Pss mango populations isolated from the same geographical regions, mainly from southern Spain with an average of 20 years of difference. The total content of plasmids, in particular the 62 kb plasmids, and the number of copper resistant Pss strains were maintained at similar levels over the time. Interestingly, the phylogenetic analysis indicated the presence of a phylogenetic subgroup (PSG) in the Pss mango phylotype, mostly composed of the recent Pss population analyzed in this study that was strongly associated with a hyper-resistant phenotype to copper. Genome sequencing of two selected Pss strains from this PSG revealed the presence of a large Tn7-like transposon of chromosomal location, which harbored putative copper and arsenic resistance genes (COARS Tn7-like). Transformation of the copper sensitive Pss UMAF0158 strain with some putative copper resistance genes and RT-qPCR experiments brought into light the role of COARS Tn7-like transposon in the hyper-resistant phenotype to copper in Pss.IMPORTANCECopper compounds have traditionally been used as standard bactericides in agriculture in the past few decades. However, the extensive use of copper has fostered the evolution of bacterial copper resistance mechanisms. Pseudomonas syringae is a plant pathogenic bacterium used worldwide as a model to study plant-pathogen interactions. The adaption of P. syringae to plant surface environment is the most important step prior to an infection. In this scenario, copper resistance mechanisms could play a key role in improving its epiphytic survival. In this work, a novel Tn7-like transposon of chromosomal location was detected in P. syringae pv. syringae strains isolated from mango. This transposon conferred the highest resistance to copper sulfate described to date for this bacterial phytopathogen. Understanding in depth the copper resistance mechanisms and their evolution are important steps to the agricultural industry to get a better improvement of disease management strategies.

High copper concentration reduces biofilm formation in Acidithiobacillus ferrooxidans by decreasing production of extracellular polymeric substances and its adherence to elemental sulfur

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms. SignificanceCopper is a metal frequently found in high concentrations at mining environments inhabitated by acidophilic microorganisms. Copper resistance determinants of A. ferrooxidans have been previously studied in planktonic cells. Although biofilms are recurrent in these types of environments, the effect of copper on their formation has not been studied so far. The results obtained indicate that high concentrations of copper reduce the capacity of A. ferrooxidans ATCC 23270 to form biofilms on sulfur. These findings may be relevant to consider for a bacterium widely used in copper bioleaching processes.

Resistance to Metals Used in Agricultural Production.

Metals and metalloids have been used alongside antibiotics in livestock production for a long time. The potential and acute negative impact on the environment and human health of these livestock feed supplements has prompted lawmakers to ban or discourage the use of some or all of these supplements. This article provides an overview of current use in the European Union and the United States, detected metal resistance determinants, and the proteins and mechanisms responsible for conferring copper and zinc resistance in bacteria. A detailed description of the most common copper and zinc metal resistance determinants is given to illustrate not only the potential danger of coselecting antibiotic resistance genes but also the potential to generate bacterial strains with an increased potential to be pathogenic to humans. For example, the presence of a 20-gene copper pathogenicity island is highlighted since bacteria containing this gene cluster could be readily isolated from copper-fed pigs, and many pathogenic strains, including Escherichia coli O104:H4, contain this potential virulence factor, suggesting a potential link between copper supplements in livestock and the evolution of pathogens.

Bacterial resistance to arsenic protects against protist killing

Protists kill their bacterial prey using toxic metals such as copper. Here we hypothesize that the metalloid arsenic has a similar role. To test this hypothesis, we examined intracellular survival of Escherichia coli (E. coli) in the amoeba Dictyostelium discoideum (D. discoideum). Deletion of the E. coli ars operon led to significantly lower intracellular survival compared to wild type E. coli. This suggests that protists use arsenic to poison bacterial cells in the phagosome, similar to their use of copper. In response to copper and arsenic poisoning by protists, there is selection for acquisition of arsenic and copper resistance genes in the bacterial prey to avoid killing. In agreement with this hypothesis, both copper and arsenic resistance determinants are widespread in many bacterial taxa and environments, and they are often found together on plasmids. A role for heavy metals and arsenic in the ancient predator-prey relationship between protists and bacteria could explain the widespread presence of metal resistance determinants in pristine environments.

A role for copper in protozoan grazing - two billion years selecting for bacterial copper resistance.

The Great Oxidation Event resulted in integration of soft metals in a wide range of biochemical processes including, in our opinion, killing of bacteria by protozoa. Compared to pressure from anthropologic copper contamination, little is known on impacts of protozoan predation on maintenance of copper resistance determinants in bacteria. To evaluate the role of copper and other soft metals in predatory mechanisms of protozoa, we examined survival of bacteria mutated in different transition metal efflux or uptake systems in the social amoeba Dictyostelium discoideum. Our data demonstrated a strong correlation between the presence of copper/zinc efflux as well as iron/manganese uptake, and bacterial survival in amoebae. The growth of protozoa, in turn, was dependent on bacterial copper sensitivity. The phagocytosis of bacteria induced upregulation of Dictyostelium genes encoding the copper uptake transporter p80 and a triad of Cu(I)-translocating PIB -type ATPases. Accumulated Cu(I) in Dictyostelium was monitored using a copper biosensor bacterial strain. Altogether, our data demonstrate that Cu(I) is ultimately involved in protozoan predation of bacteria, supporting our hypothesis that protozoan grazing selected for the presence of copper resistance determinants for about two billion years.

Cytoplasmic CopZ-Like Protein and Periplasmic Rusticyanin and AcoP Proteins as Possible Copper Resistance Determinants in Acidithiobacillus ferrooxidans ATCC 23270.

Acidophilic organisms, such as Acidithiobacillus ferrooxidans, possess high-level resistance to copper and other metals. A. ferrooxidans contains canonical copper resistance determinants present in other bacteria, such as CopA ATPases and RND efflux pumps, but these components do not entirely explain its high metal tolerance. The aim of this study was to find other possible copper resistance determinants in this bacterium. Transcriptional expression of A. ferrooxidans genes coding for a cytoplasmic CopZ-like copper-binding chaperone and the periplasmic copper-binding proteins rusticyanin and AcoP, which form part of an iron-oxidizing supercomplex, was found to increase when the microorganism was grown in the presence of copper. All of these proteins conferred more resistance to copper when expressed heterologously in a copper-sensitive Escherichia coli strain. This effect was absent when site-directed-mutation mutants of these proteins with altered copper-binding sites were used in this metal sensitivity assay. These results strongly suggest that the three copper-binding proteins analyzed here are copper resistance determinants in this extremophile and contribute to the high-level metal resistance of this industrially important biomining bacterium.

The CrdRS two-component system in Helicobacter pylori responds to nitrosative stress.

Helicobacter pylori inhabits the gastric mucosa where it senses and responds to various stresses via a two-component systems (TCSs) that enable its persistent colonization. The aim of this study was to investigate whether any of the three paired TCSs (ArsRS, FleRS and CrdRS) in H. pylori respond to nitrosative stress. The results showed that the expression of crdS was significantly increased upon exposure to nitric oxide (NO). crdS-knockout (ΔcrdS) and crdR/crdS-knockout (ΔcrdRS) H. pylori, but not arsS-knockout (ΔarsS) or fleS-knockout (ΔfleS) H. pylori, showed a significant loss of viability upon exposure to NO compared with wild-type strain. Knockin crdS (ΔcrdS-in) significantly restored viability in the presence of NO. Global transcriptional profiling analysis of wild-type and ΔcrdS H. pylori in the presence or absence of NO showed that 101 genes were differentially expressed, including copper resistance determinant A (crdA), transport, binding and envelope proteins. The CrdR binding motifs were investigated by competitive electrophoretic mobility shift assay, which revealed that the two AC-rich regions in the crdA promoter region are required for binding. These results demonstrate that CrdR-crdA interaction enables H. pylori to survive under nitrosative stress.

Survival in amoeba—a major selection pressure on the presence of bacterial copper and zinc resistance determinants? Identification of a “copper pathogenicity island”

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs.

Characterization and structure prediction of partial length protein sequences of pcoA, pcoR and chrB genes from heavy metal resistant bacteria from the Klip River, South Africa.

The Klip River has suffered from severe anthropogenic effects from industrial activities such as mining. Long-term exposure to heavy metal pollution has led to the development of heavy metal resistant strains of Pseudomonas sp. KR23, Lysinibacillus sp. KR25, and E. coli KR29. The objectives of this study were to characterize the genetics of copper and chromate resistance of the isolates. Copper and chromate resistance determinants were cloned and sequenced. Open reading frames (ORFs) related to the genes CopA and CopR were identified in E. coli KR29, PcoA in Lysinibacillus sp. KR25 and none related to chromate resistance were detected. The 3D-models predicted by I-TASSER disclose that the PcoA proteins consist of β-sheets, which form a part of the cupredoxin domain of the CopA copper resistance family of genes. The model for PcoR_29 revealed the presence of a helix turn helix; this forms part of a DNA binding protein, which is part of a heavy metal transcriptional regulator. The bacterial strains were cured using ethidium bromide. The genes encoding for heavy metal resistance and antibiotic resistance were found to be located on the chromosome for both Pseudomonas sp. (KR23) and E. coli (KR29). For Lysinibacillus (KR25) the heavy metal resistance determinants are suspected to be located on a mobile genetic element, which was not detected using gel electrophoresis.

Molecular analysis of copper resistance determinant (copA) in copper oxide nanoparticles resistant Pseudomonas fluorescens CuO-2 isolated from soil

Molecular analysis of copper resistance determinant (copA) in copper oxide nanoparticles resistant Pseudomonas fluorescens CuO-2 isolated from soil

New Copper Resistance Determinants in the ExtremophileAcidithiobacillus ferrooxidans: A Quantitative Proteomic Analysis

Acidithiobacillus ferrooxidans is an extremophilic bacterium used in biomining processes to recover metals. The presence in A. ferrooxidans ATCC 23270 of canonical copper resistance determinants does not entirely explain the extremely high copper concentrations this microorganism is able to stand, suggesting the existence of other efficient copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE, real time PCR (qRT-PCR) and quantitative proteomics with isotope-coded protein labeling (ICPL). A total of 594 proteins were identified of which 120 had altered levels in cells grown in the presence of copper. Of this group of proteins, 76 were up-regulated and 44 down-regulated. The up-regulation of RND-type Cus systems and different RND-type efflux pumps was observed in response to copper, suggesting that these proteins may be involved in copper resistance. An overexpression of most of the genes involved in histidine synthesis and several of those annotated as encoding for cysteine production was observed in the presence of copper, suggesting a possible direct role for these metal-binding amino acids in detoxification. Furthermore, the up-regulation of putative periplasmic disulfide isomerases was also seen in the presence of copper, suggesting that they restore copper-damaged disulfide bonds to allow cell survival. Finally, the down-regulation of the major outer membrane porin and some ionic transporters was seen in A. ferrooxidans grown in the presence of copper, indicating a general decrease in the influx of the metal and other cations into the cell. Thus, A. ferrooxidans most likely uses additional copper resistance strategies in which cell envelope proteins are key components. This knowledge will not only help to understand the mechanism of copper resistance in this extreme acidophile but may help also to select the best fit members of the biomining community to attain more efficient industrial metal leaching processes.

Novel Cell Envelope Proteins Related to Copper Resistance in <i>Acidithiobacillus ferrooxidans</i>

The presence in At. ferrooxidans of canonical copper resistance determinants does not explain the extremely high copper concentrations this microorganism is able to tolerate. This suggests that At. ferrooxidans may have additional copper resistance mechanisms. New possible copper resistance determinants were searched by using 2D-PAGE and real time PCR (qRT-PCR). Results showed the up-regulation of RND-type Cus systems and different RND-type efflux pumps in At. ferrooxidans grown in the presence of copper, suggesting that these proteins may be implied in resistance to this metal. Furthermore, the up-regulation of putative periplasmatic disulfide isomerases was also seen in the presence of copper. These proteins are most likely involved in the formation and rearrangement of disulfide bonds in proteins in the periplasm. Copper ions catalyze the formation of incorrect disulfide bonds in proteins. However, the up-regulated disulfide isomerases found could restore native disufide bonds allowing cell survival. In conclusion, At. ferrooxidans may resist high copper concentrations by using additional copper resistance strategies in which cell envelope proteins are very important. This knowledge could be used to select the best fit members of the bioleaching community to attain more efficient industrial biomining processes.

Novel erm (T)-Carrying Multiresistance Plasmids from Porcine and Human Isolates of Methicillin-Resistant Staphylococcus aureus ST398 That Also Harbor Cadmium and Copper Resistance Determinants

This study describes three novel erm(T)-carrying multiresistance plasmids that also harbor cadmium and copper resistance determinants. The plasmids, designated pUR1902, pUR2940, and pUR2941, were obtained from porcine and human methicillin-resistant Staphylococcus aureus (MRSA) of the clonal lineage ST398. In addition to the macrolide-lincosamide-streptogramin B (MLSB) resistance gene erm(T), all three plasmids also carry the tetracycline resistance gene tet(L). Furthermore, plasmid pUR2940 harbors the trimethoprim resistance gene dfrK and the MLSB resistance gene erm(C), while plasmids pUR1902 and pUR2941 possess the kanamycin/neomycin resistance gene aadD. Sequence analysis of approximately 18.1 kb of the erm(T)-flanking region from pUR1902, 20.0 kb from pUR2940, and 20.8 kb from pUR2941 revealed the presence of several copies of the recently described insertion sequence ISSau10, which is probably involved in the evolution of the respective plasmids. All plasmids carried a functional cadmium resistance operon with the genes cadD and cadX, in addition to the multicopper oxidase gene mco and the ATPase copper transport gene copA, which are involved in copper resistance. The comparative analysis of S. aureus RN4220 and the three S. aureus RN4220 transformants carrying plasmid pUR1902, pUR2940, or pUR2941 revealed an 8-fold increase in CdSO4 and a 2-fold increase in CuSO4 MICs. The emergence of multidrug resistance plasmids that also carry heavy metal resistance genes is alarming and requires further surveillance. The colocalization of antimicrobial resistance genes and genes that confer resistance to heavy metals may facilitate their persistence, coselection, and dissemination.

Copper resistance genes from different xanthomonads and citrus epiphytic bacteria confer resistance to Xanthomonas citri subsp. citri

Genetic exchange is considered to be an important process in the selective adaptation of microorganisms to shifting and challenging environmental conditions. As a consequence of the copious use of copper bactericides, many species of plant pathogenic bacteria, including Xanthomonas citri subsp. citri (Xcc), have developed resistance to copper. This study assesses whether copper resistant (CuR) strains of other Xanthomonas species and citrus epiphytic bacteria pose a risk for the development of copper resistance in Xcc. CuR epiphytic bacteria were isolated on MGY agar from citrus leaves collected in two citrus groves treated with copper bactericides in Florida. Horizontal gene transfer of copper resistance genes was investigated within different Xanthomonas species and from citrus epiphytic bacteria to Xanthomonas. CuR epiphytic bacteria from citrus were screened for the presence of copper resistance genes homologous to copL, copA and copB genes from Xcc and characterized regarding tolerance to copper. Copper resistance determinants from a citrus epiphytic strain of Stenotrophomonas maltophilia (Stm) were cloned and expressed in Xcc and other Xanthomonas strains. Copper resistance genes in Xcc were determined to be present on a large (~300 kb) conjugative plasmid. Cu resistance was transferred via conjugation from two copper resistant citrus strains, Xcc and X. alfalfae subsp. citrumelonis (Xac), and two tomato pathogens, X. euvesicatoria (Xe) and X. perforans (Xp), to Xcc. PCR analysis revealed that two CuR strains from citrus, an epiphytic Xanthomonas ssp. and a strain of Stm, harboured homologs of the copper resistance genes found in CuR Xcc. The introduction of copLAB gene cluster from Stm into different xanthomonads conferred copper resistance to sensitive strains of Xcc, Xac, Xe and Xp. Based on these results there is a low, but significant, likelihood of horizontal gene transfer of copper resistance genes from other xanthomonads or epiphytic bacteria to Xcc in nature.

A genomic island provides Acidithiobacillus ferrooxidans ATCC 53993 additional copper resistance: a possible competitive advantage

There is great interest in understanding how extremophilic biomining bacteria adapt to exceptionally high copper concentrations in their environment. Acidithiobacillus ferrooxidans ATCC 53993 genome possesses the same copper resistance determinants as strain ATCC 23270. However, the former strain contains in its genome a 160-kb genomic island (GI), which is absent in ATCC 23270. This GI contains, amongst other genes, several genes coding for an additional putative copper ATPase and a Cus system. A. ferrooxidans ATCC 53993 showed a much higher resistance to CuSO(4) (>100 mM) than that of strain ATCC 23270 (<25 mM). When a similar number of bacteria from each strain were mixed and allowed to grow in the absence of copper, their respective final numbers remained approximately equal. However, in the presence of copper, there was a clear overgrowth of strain ATCC 53993 compared to ATCC 23270. This behavior is most likely explained by the presence of the additional copper-resistance genes in the GI of strain ATCC 53993. As determined by qRT-PCR, it was demonstrated that these genes are upregulated when A. ferrooxidans ATCC 53993 is grown in the presence of copper and were shown to be functional when expressed in copper-sensitive Escherichia coli mutants. Thus, the reason for resistance to copper of two strains of the same acidophilic microorganism could be determined by slight differences in their genomes, which may not only lead to changes in their capacities to adapt to their environment, but may also help to select the more fit microorganisms for industrial biomining operations.