Copper isotope fractionation during surface adsorption and intracellular incorporation by bacteria

Abstract

Copper isotopes may prove to be a useful tool for investigating bacteria–metal interactions recorded in natural waters, soils, and rocks. However, experimental data which attempt to constrain Cu isotope fractionation in biologic systems are limited and unclear. In this study, we utilized Cu isotopes (δ65Cu) to investigate Cu–bacteria interactions, including surface adsorption and intracellular incorporation. Experiments were conducted with individual representative species of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, as well as with wild-type consortia of microorganisms from several natural environments. Ph-dependent adsorption experiments were conducted with live and dead cells over the pH range 2.5–6. Surface adsorption experiments of Cu onto live bacterial cells resulted in apparent separation factors (Δ65Cusolution–solid = δ65Cusolution – δ65Cusolid) ranging from +0.3‰ to +1.4‰ for B. subtilis and +0.2‰ to +2.6‰ for E. coli. However, because heat-killed bacterial cells did not exhibit this behavior, the preference of the lighter Cu isotope by the cells is probably not related to reversible surface adsorption, but instead is a metabolically-driven phenomenon. Adsorption experiments with heat-killed cells yielded apparent separation factors ranging from +0.3‰ to –0.69‰ which likely reflects fractionation from complexation with organic acid surface functional group sites. For intracellular incorporation experiments the lab strains and natural consortia preferentially incorporated the lighter Cu isotope with an apparent Δ65Cusolution–solid ranging from ~+1.0‰ to +4.4‰. Our results indicate that live bacterial cells preferentially sequester the lighter Cu isotope regardless of the experimental conditions. The fractionation mechanisms involved are likely related to active cellular transport and regulation, including the reduction of Cu(II) to Cu(I). Because similar intracellular Cu machinery is shared by fungi, plants, and higher organisms, the influence of biological processes on the δ65Cu of natural waters and soils is probably considerable.

Cu isotopic compositions in Elsholtzia splendens: Influence of soil condition and growth period on Cu isotopic fractionation in plant tissue

“…Assuming that (i)~70% of total Cu in the present E. splendens specimens is bound to the root cell wall (Peng et al, 2005), (ii) adsorption occurs at a ratio of 10% Cu(0) and 90% Cu(II)-complex as found in devitalized bacteria samples (González et al, 2016), (iii) similar Cu isotopic compositions occur in Cu(0) as in Cu(II)-complex as found in Cu(0) and precursor Cu(II) mineral in previous research (Markl et al, 2006), and (iv) the isotopic fractionation between Cu adsorbed on root tissue and soil solution is 0.49‰, as found for Cu adsorbed on heat-killed bacteria at pH 5.1 (pH field soil = 6.3; Navarret et al, 2011), then δ 65 Cu taken up by root should be − 4.13‰ based on Eq. (5) (i.e., Cu taken up by whole plant − phytoavailable Cu in soil solution in the field-grown plant is expected to be −4.33‰.…”

“…Biomolecular responses to oxidative stress are therefore likely in yellow soil. Also present in bacterial surfaces, humic acid and natural organic matter, trace Cu(0) is expected to exist in the rhizosphere of E. splendens at the soil-root interface (Fulda et al, 2013;González et al, 2016;Manceau et al, 2008;Navarret et al, 2011). Cu(II) mainly complexes to carboxylic groups (Cu(II)-O/N), and adsorbed on root cell walls in E. splendens (Liu et al, 2014;Shi et al, 2004).…”

The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans

“…Samples were incubated at 37°C with 5% CO 2 until they reached midlogarithmic phase (OD 600 of 0.4 to 0.6). Following incubation, cells were harvested by centrifugation at 3,800 ϫ g. To minimize the probability of measuring membrane-associated copper cations, cells were washed twice with 0.01 M NaClO 4 , an experimental electrolyte previously shown to remove residual metal from medium and bacterial surfaces (54). Cells were resuspended in ice-cold 1ϫ PBS and filtered through a 0.22-mpore-size nitrocellulose filter.…”

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403

“…Up to 0.3 mM copper, the observed reductase activity was low, but increased strongly at higher copper concentrations. The cell walls of Gram-positive bacteria are known to avidly bind copper, which probably reduced the available copper at low copper concentrations (Navarrete et al, 2011). The reductase activity reached a maximum of 1.57±0.03 nmol min 21 (10 8 c.f.u.)…”