Partial identification and copper tolerance of soil bacteria from copper-impregnated bog and from a salt marsh

Abstract

Bacteria from two areas in Wales have been extracted and partially identified. The areas are a copper impregnated bog in the Coed-Y-Brenin Forest with low pH and high carbon and copper content, and the Morfa Harlech salt marshes which have neutral pH and lower copper and carbon. Bacillus spp. was the predominant bacterium in both soils, but there was increased diversity of bacteria in the salt marsh soils. Bacteria from the Cu-rich, Coed-Y-Brenin soils showed higher growth densities in nutrient agar plates containing 10, 30 and 100 μg cm^?3 Cu than bacteria from the salt marsh soils, indicating that bacteria from the Coed-Y-Brenin soils could tolerate higher copper concentrations. Pseudomonas spp. from both study areas grew better in media with added Cu at low levels. The possibility is discussed that the bacteria play a part in the bioavailability of Cu particularly in the copper-rich Coed-Y-Brenin area.     

Efficacy of multiple metals against copper-resistant bacterial strains

Aims: The antibacterial efficacy of zeolites containing copper (Cu) or silver (Ag) ions or a combination was assessed against several reported copper‐resistant (Cu^R) bacterial strains. Methods and Results: Comparison strains were obtained from the American Type Culture Collection that had no documented metal resistance. Reductions in bacterial populations were determined after exposure time intervals of 3, 6 and 24 h. All three Cu^R strains of Salmonella enterica exhibited resistance to Cu, Ag and Cu/Ag after three and 6 h of exposure. Both the Cu^R and comparison strain of Enterococcus faecium were resistant to both metals and the metal combination. Cu^RPseudomonas putida was significantly reduced by all zeolites within 3 h. The Cu^REscherichia coli strain was more sensitive to Cu, but more resistant to Ag than the comparison strain; however, significant reductions were achieved within 3 h with both Cu and Cu/Ag, and within 24 h with Ag. Conclusions: Some strains with reported resistance to Cu were also resistant to Ag, suggestive of a shared resistance mechanism such as an indiscriminate Cu efflux pump. Ent. faecium appears to have innate resistance to both metals. In general, Ent. faecium was the most resistant species to the individual metals and the combination of metals, Ps. putida the least resistant, and the Salmonella strains were more resistant than E. coli. Significance and Impact of the Study: Several of the comparison strains with no reported copper resistance were resistant to one or both metals. This may call into question the methods for determining bacterial metal resistance, which typically use nutrient‐rich media containing metals to assess the ability of the bacteria to grow in comparison with a wild‐type strain. Nevertheless, all the Cu^R strains evaluated in this study, with the exception of Ent. faecium, were reduced using the Cu and Ag zeolite combination.     

Inhibitory effects of copper on bacteria related to the free ion concentration

Cu²⁺ ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO₄.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10^–8–10^–6 M Cu²⁺ ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu²⁺ ion concentrations exist at low copper loads, there was competition of Cu²⁺ for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO₄, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu²⁺, up to 10^–3 M.     

Chemiluminescence assay for Listeria monocytogenes based on Cu/Co/Ni ternary nanocatalyst coupled with penicillin as generic capturing agent

Bacterial pathogen control is important in seafood production. In this study, a Cu/Co/Ni ternary nanoalloy (Cu/Co/Ni TNA) was synthesized using the oleylamine reducing method. It was found that Cu/Co/Ni TNA greatly enhanced the chemiluminescence (CL) signal of the hydroxylamine‐O‐sulfonic acid (HOSA)–luminol system. The CL properties of Cu/Co/Ni TNA were investigated systemically. The possible CL mechanism also was intensively investigated. Based on the enhanced CL phenomenon of Cu/Co/Ni TNA, a Cu/Co/Ni TNA, penicillin, and anti‐L. monocytogenes (Listeria monocytogenes) antibody‐based sandwich complex assay for detection of L. monocytogenes was established. In this sandwich CL assay, penicillin was employed to capture and enrich pathogenic bacteria with penicillin‐binding proteins (PBPs) while anti‐L. monocytogenes antibody was adopted as the specific recognition molecule to recognize L. monocytogenes. L. monocytogenes was detected sensitively based on this new Cu/Co/Ni TNA–HOSA–luminol CL system. The CL intensity was proportional to the L. monocytogenes concentration ranging from 2.0 × 10² CFU ml^?1 to 3.0 × 10⁷ CFU ml^?1 and the limit of detection wa 70 CFU ml^?1. The reliability and potential applications of our method was verified by comparison with official methods and recovery tests in environment and food samples.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO₃ precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ ¹³C and δ ¹⁸ O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ ¹³C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ ¹³C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria.     

The role of bacteria on heavy-metal extraction and uptake by plants growing on multi-metal-contaminated soils

Four bacterial isolates were examined for their ability to increase the availability of water soluble Cu, Cr, Pb and Zn in soils and for their effect on metals uptake by Zea mays and Sorghum bicolor. Random Amplified Polymorphic DNA (RAPD) analysis was used to show that the bacterial cultures were genetically diverse. Bacterial isolates S3, S28, S22 and S29 had 16S rRNA gene sequences that were most similar to Bacillus subtilis, Bacillus pumilus, Pseudomonas pseudoalcaligenes and Brevibacterium halotolerans based on 100% similarity in their 16S rDNA gene sequence, respectively. Filtrate liquid media that had supported B. pumilus and B. subtilis growth significantly increased Cr and Cu extraction from soil polluted with tannery effluent and from Cu-rich soil, respectively, compared to axenic media. The highest concentrations of Pb (0.2 g kg⁻¹), Zn (4 g kg⁻¹) and Cu (2 g kg⁻¹) were accumulated in shoots of Z. mays grown on Cu-rich soil inoculated with Br. halotolerans. The highest concentration of Cr (5 g kg⁻¹) was accumulated in S. bicolor roots grown in tannery-effluent-polluted soil inoculated with a mixed inoculum of bacterial strains. These results show that bacteria play an important role in increasing metal availability in soil, thus enhancing Cr, Pb, Zn and Cu accumulation by Z. mays and S. bicolor.     

Antibacterial activity of colloidal copper nanoparticles against Gram-negative (Escherichia coli and Proteus vulgaris) bacteria

Antibacterial activities of as-synthesized nanoparticles have gained attention in past few years due to rapid phylogenesis of pathogens developing multi-drug resistance (MDR). Antibacterial activity of copper nanoparticles (CuNPs) on surrogate pathogenic Gram-negative bacteria Escherichia coli (MTCC no. 739) and Proteus vulgaris (MTCC no. 426) was evaluated under culture conditions. Three sets of colloidal CuNPs were synthesized by chemical reduction method with per batch yield of 0·2, 0·3 and 0·4 g. As-synthesized CuNPs possess identical plasmonic properties and have similar hydrodynamic particle sizes (11–14 nm). Antibacterial activities of CuNPs were evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests, cytoplasmic leakage and reactive oxygen species (ROS) assays. MIC and MBC tests revealed dose dependence bactericidal action. Growth curves of E. coli show faster growth inhibition along with higher cytoplasmic leakage than that of P. vulgaris. This might be because of increased membrane permeability of E. coli. CuNP–microorganism interaction induces oxidative stress generated by ROS. Leakage of cytoplasmic components, loss of membrane permeability and ROS generation are the primary causes of CuNP-induced bacterial cell death. As-synthesized CuNPs exhibiting promising antibacterial activities and could be a promising candidate for novel antibacterial agents.     

One pot synthesis and anti-biofilm potential of copper nanoparticles (CuNPs) against clinical strains of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen frequently associated with nosocomial infections, is emerging as a serious threat due to its resistance to broad spectrum antimicrobials. The biofilm mode of growth confers resistance to antibiotics and novel anti-biofilm agents are urgently needed. Nanoparticle based treatments and therapies have been of recent interest because of their versatile applications. This study investigates the anti-biofilm activity of copper nanoparticles (CuNPs) synthesized by the one pot method against P. aeruginosa. Standard physical techniques including UV–visible and Fourier transform infrared spectroscopy, X-ray diffraction and transmission electron microscopy were used to characterize the synthesized CuNPs. CuNP treatments at 100 ng ml^?1 resulted in a 94, 89 and 92% reduction in biofilm, cell surface hydrophobicity and exopolysaccharides respectively, without bactericidal activity. Evidence of biofilm inhibition was also seen with light and confocal microscope analysis. This study highlights the anti-biofilm potential of CuNPs, which could be utilized as coating agents on surgical devices and medical implants to manage biofilm associated infections.     

Oxidative stress conditions increase the frequency of de novo formation of the yeast [PSI+] prion

Prions are self‐perpetuating amyloid protein aggregates which underlie various neurodegenerative diseases in mammals and heritable traits in yeast. The molecular basis of how yeast and mammalian prions form spontaneously into infectious amyloid‐like structures is poorly understood. We have explored the hypothesis that oxidative stress is a general trigger for prion formation using the yeast [PSI⁺] prion, which is the altered conformation of the Sup35 translation termination factor. We show that the frequency of [PSI⁺] prion formation is elevated under conditions of oxidative stress and in mutants lacking key antioxidants. We detect increased oxidation of Sup35 methionine residues in antioxidant mutants and show that overexpression of methionine sulphoxide reductase abrogates both the oxidation of Sup35 and its conversion to the [PSI⁺] prion. [PSI⁺] prion formation is particularly elevated in a mutant lacking the Sod1 Cu,Zn‐superoxide dismutase. We have used fluorescence microscopy to show that the de novo appearance of [PSI⁺] is both rapid and increased in frequency in this mutant. Finally, electron microscopy analysis of native Sup35 reveals that similar fibrillar structures are formed in both the wild‐type and antioxidant mutants. Together, our data indicate that oxidative stress is a general trigger of [PSI⁺] formation, which can be alleviated by antioxidant defenses.     

Iron–Manganese Concretions Sustaining Microbial Life in the Baltic Sea: The Structure of the Bacterial Community and Enrichments in Metal-Oxidizing Conditions

The abundant deposits of spherical iron-manganese concretions in the Gulf of Finland are colonized by bacteria in vast numbers. Communities on the surface and in the porous interior have formed two separate clusters, in accordance with their genetic differences. The overall bacterial community in the concretions was highly diverse, representing 12 phyla. Half of the bacteria were affiliated with the most common classes of Proteobacteria, while a third of the bacteria were unclassified. Cloned 16S rRNA-gene sequences of the concretion bacteria showed high scores for similarity to the sequences obtained from sea sediments, metal-rich environments, and ocean crust. The clone library of native concretions was not dominated by known Fe- and Mn-oxidizing species. Known Mn-oxidizing bacteria Sphingomonas, Pseudomonas, and Bacillus were enriched in experiments with Mn²⁺-containing liquid media, whereas Prosthecobacter (Verrucomicrobia) and Rheinheimera were enriched in semisolid media possibly better simulating the natural conditions in the concretions. In a corresponding experiment, the Fe²⁺-oxygen gradient favored the enrichment of Shewanella baltica and Thalassolituus oleivorans, which are known to reduce Fe and to degrade petroleum hydrocarbons, respectively. An individual spherical concretion forms a microcosm for a diverse microbial community having potential to oxidize Fe and Mn as shown in cultivation experiments. Therefore, bacteria may significantly affect the formation of the concretions in the Gulf of Finland.     

Copper mobility and toxicity of soil percolation water to bacteria in a metal polluted forest soil

In order to assess the success of in situ remediation of coniferous forest soil polluted by a Cu–Ni smelter, the total Cu concentration in soil percolation water, the fluxes of Cu down through the soil profile, and the toxicity of soil percolation water to soil bacteria were studied. Total Cu in percolation water was also fractionated into free ionic and complexed forms. The toxicity of the percolation water was measured by the [³H]-thymidine incorporation method, which measures bacterial growth rates. Soil percolation water was collected during one growing season by zero tension lysimeters inserted at depths of 0.2 and 0.4 m in the soil. The treatments consisted of a control, mulch application to the forest floor (M) and mulch application after removing the polluted organic soil layer (MR). The mulch consisted of a mixture of compost and woodchips (1/1; vol/vol). Analysis of Cu species and dissolved organic carbon (DOC) indicated that DOC leached from the mulch and complexed Cu into forms that were less toxic to soil bacteria. At 0.2 m depth percolation water toxicity was 19% lower in the M and 42% lower in the MR treatment than in the control. Toxicity correlated with the Cu²⁺ concentration, which was 61 and 84% lower in the M and MR treatments, respectively, compared to the control. However, there were signs that total Cu had leached down through the soil profile, the leaching being more pronounced in the MR treatment.     

Enantiospecific equilibrium adsorption and chemistry of d-/l-proline mixtures on chiral and achiral Cu surfaces

A fundamental understanding of the enantiospecific interactions between chiral adsorbates and understanding of their interactions with chiral surfaces is key to unlocking the origins of enantiospecific surface chemistry. Herein, the adsorption and decomposition of the amino acid proline (Pro) have been studied on the achiral Cu(110) and Cu(111) surfaces and on the chiral Cu(643)^R&S surfaces. Isotopically labelled 1-¹³C-l- Pro has been used to probe the Pro decomposition mechanism and to allow mass spectrometric discrimination of d -Pro and 1-¹³C-l -Pro when adsorbed as mixtures. On the Cu(111) surface, X-ray photoelectron spectroscopy reveals that Pro adsorbs as an anionic species in the monolayer. On the chiral Cu(643)^R&S surface, adsorbed Pro enantiomers decompose with non-enantiospecific kinetics. However, the decomposition kinetics were found to be different on the terraces versus the kinked steps. Exposure of the chiral Cu(643)^R&S surfaces to a racemic gas phase mixture of d -Pro and 1-¹³C-l -Pro resulted in the adsorption of a racemic mixture; i.e., adsorption is not enantiospecific. However, exposure to non-racemic mixtures of d -Pro and 1-¹³C-l -Pro resulted in amplification of enantiomeric excess on the surface, indicative of homochiral aggregation of adsorbed Pro. During co-adsorption, this amplification is observed even at very low coverages, quite distinct from the behavior of other amino acids, which begin to exhibit homochiral aggregation only after reaching monolayer coverages. The equilibrium adsorption of d -Pro and 1-¹³C-l -Pro mixtures on achiral Cu(110) did not display any aggregation, consistent with prior scanning tunneling microscopy (STM) observations of dl -Pro/Cu(110). This demonstrates convergence between findings from equilibrium adsorption methods and STM experiments and corroborates formation of a 2D random solid solution.     

Metabolism of Glutamate in Purple Nonsulfur Bacteria Participation of Vitamin B12

Purple nonsulfur bacteria, Rhodospirillum rubrum and Rhodopseudomonas spheroides were found to possess coenzyme B₁₂-dependent glutamate mutase activity. Cell-free extracts of these bacteria grown on Co²⁺-containing media catalyzed the conversion of glutamate to β-methylaspartate and further to mesaconate. The activity of the cell-free extracts of these organisms cultivated on Co²⁺-deficient media was markedly lower than that of the normal cells. Addition of coenzyme B₁₂ to the former reaction mixture enhanced the mesaconate formation via β-methylaspartate. These results indicate the involvement of coenzyme Independent glutamate mutase of these bacteria in the dissimilation of glutamate to acetyl-CoA and pyruvate through the following pathway.

glutamate→β→methylaspartate→mesaconate→citramalate→→acetyl-CoA, pyruvate On the other hand, a greater part of glutamate was converted to α-hydroxyglutarate and succinate with the cell-free extracts of these photosynthetic bacteria. This fact, taking account of the presence of propionyl-CoA carboxylase in these bacteria, implies the participation of coenzyme B₁₂-dependent (R)-methylmalonyl-CoA mutase in the formation of succinate via the following route.

glutamate→α-ketoglutarate→α-hydroxyglutarate→propionate→propionyl-CoA→(S)-methylmalonyl-CoA→(R)-methylmalonyl-CoA→succinyl-CoA     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Molecular characterization of early colonizer bacteria from wastes in a steel plant

Aims: Forty‐nine bacteria isolated from four newly‐produced waste samples of a steel industry, which had a high content of CaO, MgO, Cr and P₂O₅, were characterized molecularly and phenotypically by susceptibility testing against heavy metals. Methods and Results: Phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to nine genera, Pseudomonas, Micrococcus, Acinetobacter, Bacillus, Dietzia, Kocuria, Diaphorobacter, Staphylococcus and Brevibacillus. Besides, some isolates could be affiliated to species: M. luteus, Ac. junii, Ac. schindleri, B. cereus, K. marina, D. nitroreducens and Staph. warneri. The bacteria that were characterized are taxonomically diverse, and Pseudomonas and Micrococcus predominated. Fingerprinting BOX‐PCR revealed high genomic heterogeneity among the isolates. Among the heavy metal compounds Zn, Ni, Pb and Cu were least toxic to the bacterial isolates, whereas Ag inhibited all isolates at 0·001 mmol l^?1. Conclusions: Heterotrophic bacteria, affiliated with several phylogentic groups, were able to colonize different wastes of a steel industry. Significance and Impact of the Study: This study extends our knowledge of the early colonizers bacteria populating siderurgic environments. Some of these bacteria could have potential for recycling siderurgic waste for steel production.     

The formation of germtubes by candida albicans,when grown with Staphylococcus pyogene,Escherichia coli,Klebsiella pneumoniae,Lactobacilius acidophilus and Proteus vulgaris

The formation of germtubes by twelve clinical isolates of C. albicans was studied in human serum containing per millilitre 10³ to 10⁹ organisms as: Staphylococcus pyegene, Escherichia coli, Klebsiella pneumoniae, Lactobacillus acidophilus and Proteus vulgaris. All the five bacteria inhibited formation of germtubes by C. albicans at all concentrations and the percent germtube formed diminished with increasing concentration of the bacteria. Lactobacillus acidophilus inhibited the formation of germtubes maximally followed by Staphylococcus pyogene, Escherichia coli and Klebsiella pneumoniae. Proteus vulgaris in the concentrations of 10³ to 10⁷ bacteria per millilitre produced only insignificant inhibition of formation of germtubes by C. albicans. Since germtubes of C. albicans are invasive, it is suggested that inhibition of blastospo-regermtube transformation may be significantly responsible for prevention of infection by C. albicans by coexisting bacterial flora.     

Biofilm formation by bacterial associations under various salinities and copper ion stress

Abstract

The study addresses the effect of abiotic (medium salinity and copper ions) and biotic (interactions between populations) factors on the formation of structured communities by binary associations consisting of halotolerant bacteria (Alcaligenes sp. 1‐1 or Acinetobacter sp. 1‐19) and a wild-type B. subtilis 2335 strain or a transgenic strain. The results showed that 250 mg l^?1 of copper ions inhibit formation of biofilms by monocultures of the tested strains. Binary associations of the strains were more resistant to high concentrations (250 mg l^?1) of copper ions. At the lowest NaCl concentration (0.05% and 2.5%) and in the presence of copper ions, bacilli seemed to help halotolerant bacteria survive. Under increased salinity and in the presence of copper ions, structured communities developed due to halotolerant bacteria. Coexistence under stressful conditions was beneficial for the both groups of bacteria.