Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu1+ to the less toxic Cu2+. Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb3-type cytochrome oxidase (cbb3-Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb3-Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF.

Sequestration and efflux largely account for Cd and Cu resistance in the deep sea Epsilonproteobacterium Nitratiruptor sp. SB155 2

In deep sea hydrothermal vent environments, metal- and metalloid-enriched fluids and sediments abound, making these habitats ideal to study metal resistance in prokaryotes. In this investigation, the architecture of the Epsilonproteobacterium, Nitratiruptor sp. SB155-2 transcriptome in combination with sub-cellular analysis using scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy (STEM-EDX) was examined to better understand mechanisms of tolerance for cadmium (Cd) and copper (Cu) at stress-inducing concentrations. Transcriptomic expression profiles were remarkably different in the presence of these two metals, displaying 385 (19%) and 629 (31%) genes differentially expressed (DE) in the presence of Cd and Cu, respectively, while only 7% of DE genes were shared, with genes for non-specific metal transporters and genes involved in oxidative stress-response predominating. The principal metal-specific DE pathways under Cu stress, including those involving sulfur, cysteine, and methionine, are likely required for high-affinity efflux systems, while stress and flagella formation and chemotaxis were over-represented under Cd stress. Consistent with these differences, STEM-EDX analysis revealed that polyphosphate-like granules (pPLG), the formation of Cd-S particles, and the periplasmic space may be crucial for Cd sequestration. Overall, this study provides new insights regarding metal-specific adaptations of Epsilonproteobacteria to deep sea hydrothermal vent environments.

Heavy Metal Resistance in Salmonella Typhimurium and Its Association With Disinfectant and Antibiotic Resistance

Metals are widely used in animal feed for their growth-stimulating and antimicrobial effects, yet their use may potentially promote the proliferation of antibiotic resistance through co-selection. We studied the prevalence and associations of metal, antibiotic, and disinfectant resistances of 300 Salmonella Typhimurium isolates from pig meat, pig manure, chicken meat, poultry manure, and human stool from Sichuan, China. Seventy four percent of the 300 Salmonella Typhimurium isolates were considered resistant to Cu, almost 50% to Zn and Cr, over 25% to Mn and Cd, and almost 10% to Co. Most of the isolates carried at least one heavy metal resistance gene (HMRG). The Cr-Zn-Cd-resistance gene czcD was carried by 254 isolates and the Cu-resistance genes pcoR and pcoC by 196 and 179 isolates, respectively. Most of the isolates were resistant to at least one antibiotic and almost 80% were multidrug-resistant. The prevalence of resistance to six antibiotics was higher among the pig meat and manure isolates than among other isolates, and that of streptomycin and ampicillin were highest among the pig meat isolates and that of ciprofloxacin and ofloxacin among the pig manure isolates. From 55 to 79% of the isolates were considered resistant to disinfectants triclosan, trichloroisocyanuric acid, or benzalkonium chloride. The metal resistances and HMRGs were associated with resistance to antibiotics and disinfectants. Especially, Cu-resistance genes were associated with resistance to several antibiotics and disinfectants. The transfer of the Cr-Zn-Cd-resistance gene czcD, Cu-resistance gene pcoC, and Co-Ni-resistance gene cnrA into Escherichia coli and the increased Cu-resistance of the transconjugants implied that the resistance genes were located on conjugative plasmids. Thus, the excessive use of metals and disinfectants as feed additives and in animal care may have the potential to promote antibiotic resistance through co-selection and maintain and promote antibiotic resistance even in the absence of antibiotics.

High-throughput screening and selection of PCB-bioelectrocholeaching, electrogenic microbial communities using single chamber microbial fuel cells based on 96-well plate array.

We demonstrate a single chamber, 96-well plate based Microbial Fuel Cells (MFCs). This invention is aimed at robust selection of electrogenic microbial community under specific conditions, (pH, external resistance, inoculum) that can be altered within the 96 well plate array. Using this device, we selected and multiplicated electrogenic microbial communities fed with acetate and lactate that can operate under different pH and produce current densities up to 19.4 A/m3 (0.6 A/m2) within 5 days past inoculation. Moreover, studies shown that Cu mobilization through PCB bioleaching occurred, thus each community was able to withstand presence of Cu2+ ions up to 600 mg/L. Metagenome analysis reveals high abundance of Dietzia spp., previously characterized in MFCs, but not reported to grow at pH 4, as well as novel species, closely related to Actinotalea ferrariae, not yet associated with electrogenicity. Microscopic observations (combined SEM and EDS) reveal that some of the species present in the anodic biofilm were adsorbing copper on their surface, probably due to the presence of metalloprotein complexes on their outer membranes. Taxonomy analysis indicated that similar consortia populate anodes, cathodes and OCP controls, although total abundances of aforementioned species are different among those groups. Annotated metagenomes showed high presence of multicopper oxidases and Cu-resistance genes, as well as genes encoding aliphatic and aromatic hydrocarbon-degrading enzymes. Comparison between annotated and binned metagenomes from pH 4 and 7 anodes, as well as their OCP controls revealed unique genes present in all of them, with majority of unique genes present in pH 7 anode, where novel Actinotalea spp. was present.

Characteristics of the copper‐induced viable‐but‐non‐culturable state in bacteria

The antimicrobial applications of copper (Cu) are exploited in several industries, such as agriculture and healthcare settings. While Cu is capable of efficiently killing microorganisms, sub-lethal doses can induce a viable-but-non-culturable (VBNC) state in bacteria of many distinct clades. VBNC cells cannot be detected by standard culture-based detection methods, and can become a threat to plants and animals as they often retain virulent traits upon resuscitation. Here we discuss the putative mechanisms of the Cu-induced VBNC state. Common observations in Cu-induced VBNC cells include a cellular response to reactive oxygen species, the exhaustion of energy reserves, and a reconfiguration of the proteome. While showing partial overlap with other VBNC state-inducing stressors, these changes seem to be part of an adaptive response to Cu toxicity. Furthermore, we argue that Cu resistance mechanisms such as P-type ATPases and multicopper oxidases may ward off entry into the VBNC state to some extent. The spread of these mechanisms across multi-species populations could increase population-level resistance to Cu antimicrobials. As Cu resistance mechanisms are often co-selected with antibiotic resistance mechanisms, this threat is exacerbated.

Impact of added copper, alone or in combination with chlortetracycline, on growth performance and antimicrobial resistance of fecal enterococci of weaned piglets

Studies suggest a link between added copper (Cu) and co-selection of antimicrobial resistance (AMR) in Enterococcus spp., but data are inconsistent. This study aimed to assess the impact of added Cu, alone or with a feed-grade antimicrobial, on growth performance, transferable Cu resistance gene (tcrB) prevalence, abundance of tcrB in fecal community DNA, and AMR in fecal enterococci in weaned piglets. A total of 320 barrows (DNA 200 × 400, DNA Genetics) weaned at approximately 21 d of age with 7.4 kg (7.4 ± 0.06 kg) BW were used in a 28-d study. Piglets were fed a common non-medicated diet for 7 d of acclimation. Treatments were arranged in a 2 × 2 factorial design with main effects of added Cu (0 vs. 200 mg/kg Cu from Cu sulfate) and chlortetracycline (0 vs. 440 mg/kg CTC). Growth performance and fecal samples were obtained on days 0, 14, and 28. There was no evidence (P > 0.05) for Cu and CTC interaction in growth performance. Pigs fed diets with added Cu had increased (P < 0.05) ADG and ADFI from days 0 to 14, with no evidence for differences (P > 0.05) from days 15 to 28 and 0 to 28. Pigs fed diets with CTC had improved (P < 0.01) ADG, ADFI, and G:F from days 0 to 28. Prevalence of tcrB-positive enterococci was not affected by the addition of Cu and/or CTC (P > 0.05). Prevalence of tcrB-positive enterococci was higher on day 14 than other sampling days (P = 0.002). Prevalence of tetracycline resistance gene [tet(M)]-positive enterococci was not affected by treatments or day (P > 0.05). Prevalence of macrolide resistance gene [erm(B)]-positive enterococci had a significant treatment and sampling day interaction (P = 0.021). The abundance of the tcrB gene in feces, quantified by PCR, was not affected by Cu treatment. The median Cu minimum inhibitory concentrations (MIC) of tcrB-negative and -positive isolates were 3 and 20 mM, respectively (P < 0.001). For day 0 and day 28, all Enterococcus isolates were susceptible to gentamicin, kanamycin, streptomycin, daptomycin, and tigecycline, with a majority of isolates resistant to chloramphenicol, erythromycin, lincomycin, linezolid, tetracycline, tylosin tartrate, and Synercid. In conclusion, 200 mg/kg added Cu or 440 mg/kg CTC in nursery diets improved growth performance of nursery pigs. Added Cu, with or without a selection pressure of CTC, did not increase Cu-resistant enterococci and did not co-select resistance to antibiotics.

Gene expression profiling of copper-resistant Caco-2 clones

Multiple Caco-2 clones resistant to CuSO4 and Cu proteinate were characterised to evaluate transcriptomic expression patterns associated with intestinal Cu-resistance

8-Hydroxyquinolines Are Boosting Agents of Copper-Related Toxicity in Mycobacterium tuberculosis

ABSTRACTCopper (Cu) ions are likely the most important immunological metal-related toxin utilized in controlling bacterial infections. Impairment of bacterial Cu resistance reduces viability within the host. Thus, pharmacological enhancement of Cu-mediated antibacterial toxicity may lead to novel strategies in drug discovery and development. Screening for Cu toxicity-enhancing antibacterial molecules identified 8-hydroxyquinoline (8HQ) to be a potent Cu-dependent bactericidal inhibitor ofMycobacterium tuberculosis. The MIC of 8HQ in the presence of Cu was 0.16 μM for replicating and nonreplicatingM. tuberculosiscells. We found 8HQ's activity to be dependent on the presence of extracellular Cu and to be related to an increase in cell-associated labile Cu ions. Both findings are consistent with 8HQ acting as a Cu ionophore. Accordingly, we identified the 1:1 complex of 8HQ and Cu to be its active form, with Zn, Fe, or Mn neither enhancing nor reducing its Cu-specific action. This is remarkable, considering that the respective metal complexes have nearly identical structures and geometries. Finally, we found 8HQ to killM. tuberculosisselectively within infected primary macrophages. Given the stark Cu-dependent nature of 8HQ activity, this is the first piece of evidence that Cu ions within macrophages may bestow antibacterial properties to a Cu-dependent inhibitor ofM. tuberculosis. In conclusion, our findings highlight the metal-binding ability of the 8-hydroxyquinoline scaffold to be a potential focus for future medicinal chemistry and highlight the potential of innate immunity-inspired screening platforms to reveal molecules with novel modes of action againstM. tuberculosis.

The Copper-Responsive RicR Regulon Contributes to Mycobacterium tuberculosis Virulence

ABSTRACTAs with most life on Earth, the transition metal copper (Cu) is essential for the viability of the human pathogenMycobacterium tuberculosis. However, infected hosts can also use Cu to control microbial growth. Several Cu-responsive pathways are present inM. tuberculosis, including the regulated in copper repressor (RicR) regulon, which is unique to pathogenic mycobacteria. In this work, we describe the contribution of each RicR-regulated gene to Cu resistancein vitroand to virulence in animals. We found that the deletion or disruption of individual RicR-regulated genes had no impact on virulence in mice, although several mutants had Cu hypersensitivity. In contrast, a mutant unable to activate the RicR regulon was not only highly susceptible to Cu but also attenuated in mice. Thus, these data suggest that several genes of the RicR regulon are required simultaneously to combat Cu toxicityin vivoor that this regulon is also important for resistance against Cu-independent mechanisms of host defense.IMPORTANCEMycobacterium tuberculosisis the causative agent of tuberculosis, killing millions of people every year. Therefore, understanding the biology ofM. tuberculosisis crucial for the development of new therapies to treat this devastating disease. Our studies reveal that although host-supplied Cu can suppress bacterial growth,M. tuberculosishas a unique pathway, the RicR regulon, to defend against Cu toxicity. These findings suggest that Cu homeostasis pathways in both the host and the pathogen could be exploited for the treatment of tuberculosis.