Synthesis and Antibacterial Activity of Metal(loid) Nanostructures by Environmental Multi-Metal(loid) Resistant Bacteria and Metal(loid)-Reducing Flavoproteins

Abstract

Microbes are suitable candidates to recover and decontaminate different environments from soluble metal ions, either via reduction or precipitation to generate insoluble, non-toxic derivatives. In general, microorganisms reduce toxic metal ions generating nanostructures (NS), which display great applicability in biotechnological processes. Since the molecular bases of bacterial reduction are still unknown, the search for new -environmentally safe and less expensive- methods to synthesize NS have made biological systems attractive candidates. Here, 47 microorganisms isolated from a number of environmental samples were analyzed for their tolerance or sensitivity to 19 metal(loid)s. Ten of them were highly tolerant to some of them and were assessed for their ability to reduce these toxicants in vitro. All isolates were analyzed by 16S rRNA gene sequencing, fatty acids composition, biochemical tests and electron microscopy. Results showed that they belong to the Enterobacter, Staphylococcus, Acinetobacter, and Exiguobacterium genera. Most strains displayed metal(loid)-reducing activity using either NADH or NADPH as cofactor. While Acinetobacter schindleri showed the highest tellurite (TeO32-) and tetrachloro aurate (AuCl4-) reducing activity, Staphylococcus sciuri and Exiguobacterium acetylicum exhibited selenite (SeO32-) and silver (Ag+) reducing activity, respectively. Based on these results, we used these bacteria to synthetize, in vivo and in vitro Te, Se, Au, and Ag-containing nanostructures. On the other hand, we also used purified E. cloacae glutathione reductase to synthesize in vitro Te-, Ag-, and Se-containing NS, whose morphology, size, composition, and chemical composition were evaluated. Finally, we assessed the putative anti-bacterial activity exhibited by the in vitro synthesized NS: Te-containing NS were more effective than Au-NS in inhibiting Escherichia coli and Listeria monocytogenes growth. Aerobically synthesized TeNS using MF09 crude extracts showed MICs of 45- and 66- μg/ml for E. coli and L. monocytogenes, respectively. Similar MIC values (40 and 82 μg/ml, respectively) were observed for TeNS generated using crude extracts from gorA-overexpressing E. coli. In turn, AuNS MICs for E. coli and L. monocytogenes were 64- and 68- μg/ml, respectively.

Understanding gold toxicity in aerobically-grown Escherichia coli

“…Finally, one of the putative mechanisms of bacterial response to metal(loid)s is their reduction to the respective elemental state, which has been widely studied [44][45][46][47]. Given that E. coli crude extracts were able of gold(III) reduction generating a characteristic red precipitate (not shown), the possibility of synthesizing AuNS in vivo and in vitro was explored (Fig.…”

In vitro biosynthesis of Ag, Au and Te-containing nanostructures by Exiguobacterium cell-free extracts

“…Exiguobacterium strains used in this research were previously isolated from different regions in Chile that display a combination of extreme environments such as high salinity, desiccation, high and low temperatures, and volcanic interventions [38]. Most of these factors are responsible for generating oxidative stress to microorganisms, a situation that also occurs often upon bacterial exposure to metal(loid)s. This is corroborated, because in Chile it has been identified and characterized strains of Exiguobacterium obtained from the Salar del Huasco which have arsenic resistance [1,2].…”

“…Bacterial strains Exiguobacterium acetylicum MF03, E. aurantiacum MF06 and E. profundum MF08 used in this study were previously characterized [38]. Cells were grown in the presence or absence of oxygen at 37°C in Luria-Bertani culture medium (LB) with constant shaking (150 rpm) from 1% inoculum of a preculture grown overnight (approx 10 9 CFU/ml).…”

In vitro biosynthesis of Ag, Au and Te-containing nanostructures by Exiguobacterium cell-free extracts

“…Exiguobacterium strains used in this research were previously isolated from different regions in Chile that display a combination of extreme environments such as high salinity, desiccation, high and low temperatures, and volcanic interventions [48]. Most of these factors are responsible for generating oxidative stress to microorganisms, a situation that also occurs often upon bacterial exposure to metal(loid)s. This is corroborated, because in Chile it has been identified and characterized strains of Exiguobacterium obtained from the Salar del Huasco which have arsenic resistance [1,2].…”

“…Bacterial strains Exiguobacterium acetylicum MF03, E. aurantiacum MF06 and E. profundum MF08 used in this study were previously characterized [48]. Cells were grown in the presence or absence of oxygen at 37° C in Luria-Bertani culture medium (LB) with constant shaking (150 rpm) from 1% inoculum of a preculture grown overnight (approx 10 9 CFU/ml).…”