The biological synthesis of metal nanoparticles has been examined in a wide range of organisms, due to increased interest in green synthesis and environmental remediation applications involving heavy metal ion contamination. Deinococcus radiodurans is particularly attractive for environmental remediation involving metal reduction, due to its high levels of resistance to radiation and other environmental stresses. However, few studies have thoroughly examined the relationships between environmental stresses and the resulting effects on nanoparticle biosynthesis. In this work, we demonstrate cell-free nanoparticle production and study the effects of metal stressor concentrations and identity, temperature, pH, and oxygenation on the production of extracellular silver nanoparticles by D. radiodurans R1. We also report the synthesis of bimetallic silver and gold nanoparticles following the addition of a metal stressor (silver or gold), highlighting how production of these particles is enabled through the application of environmental stresses. Additionally, we found that both the morphology and size of monometallic and bimetallic nanoparticles were dependent on the environmental stresses imposed on the cells. The nanoparticles produced by D. radiodurans exhibited antimicrobial activity comparable to that of pure silver nanoparticles and displayed catalytic activity comparable to that of pure gold nanoparticles. Overall, we demonstrate that biosynthesized nanoparticle properties can be partially controlled through the tuning of applied environmental stresses, and we provide insight into how their application may affect nanoparticle production in D. radiodurans during bioremediation.IMPORTANCE Biosynthetic production of nanoparticles has recently gained prominence as a solution to rising concerns regarding increased bacterial resistance to antibiotics and a desire for environmentally friendly methods of bioremediation and chemical synthesis. To date, a range of organisms have been utilized for nanoparticle formation. The extremophile D. radiodurans, which can withstand significant environmental stresses and therefore is more robust for metal reduction applications, has yet to be exploited for this purpose. Thus, this work improves our understanding of the impact of environmental stresses on biogenic nanoparticle morphology and composition during metal reduction processes in this organism. This work also contributes to enhancing the controlled synthesis of nanoparticles with specific attributes and functions using biological systems.KEYWORDS Deinococcus, environmental stress, extremophile, nanoparticles M ining and other industrial processes produce significant amounts of metal-ioncontaminated waste streams, necessitating water remediation efforts (1, 2). One attractive method for remediation is the use of microorganisms with the ability to degrade organic pollutants into less toxic species or to reduce metal ions to their solid metal forms via nanoparticle (NP) formation, as these organisms can be genetically e...

The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans. IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.R ecent work regarding the biological engineering of extremophiles has increased interest in their use as a robust chassis for metabolic engineering. The appeal of using extremophiles in various applications is largely due to their ability to survive conditions toxic to traditional engineering strains. One extremophile considered attractive is Deinococcus radiodurans, a Gram-positive bacteria known for its tolerance to ionizing radiation, heavy metal exposure, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens (1-3). In the context of its applications in bioremediation,

Genetic engineering of nanoparticle biosynthesis in bacteria could help facilitate the production of nanoparticles with enhanced or desired properties. However, this process remains limited due to the lack of mechanistic knowledge regarding specific enzymes and other key biological factors. Herein, we report on the ability of small noncoding RNAs (sRNAs) to affect silver nanoparticle (AgNP) biosynthesis using the supernatant from the bacterium Deinococcus radiodurans. Deletion strains of 12 sRNAs potentially involved in the oxidative stress response were constructed, and the supernatants from these strains were screened for their effect on AgNP biosynthesis. We identified several sRNA deletions that drastically decreased AgNP yield compared to the wild-type (WT) strain, suggesting the importance of these sRNAs in AgNP biosynthesis. Furthermore, AgNPs biosynthesized using the supernatants from three of these sRNA deletion strains demonstrated significantly enhanced antimicrobial and catalytic activities against environmentally relevant dyes and bacteria relative to AgNPs biosynthesized using the WT strain. Characterization of these AgNPs using electron microscopy (EM), energy-dispersive X-ray spectroscopy (EDX), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) revealed that the deletion of these small RNAs led to changes within the supernatant composition that altered AgNP properties such as the surface chemistry, surface potential, and overall composition. Taken together, our results demonstrate that modulating specific sRNA levels can affect the composition of supernatants used to biosynthesize AgNPs, resulting in AgNPs with unique material properties and improved functionality; as such, we introduce sRNAs as a new platform for genetically engineering the biosynthesis of metal nanoparticles using bacteria. Many of the sRNAs examined in this work have potential regulatory roles in oxidative stress responses; further studies into their targets could help provide insight into the specific molecular mechanisms underlying bacterial biosynthesis and metal reduction, enabling the production of nanoparticles with enhanced properties.

There is rising interest in nanoparticle biosynthesis using bacteria due to the potential for applications in bioremediation, catalysis, or as antimicrobials. However, biosynthesis remains limited by the inability to control nanoparticle morphology and size due to the lack of knowledge regarding explicit molecular mechanisms. Due to their importance in nanoparticle biosynthesis and as antimicrobials, we focus our discussion on silver, gold, and copper nanoparticles. We discuss recent efforts to elucidate reduction mechanisms that have identified generic enzymes and metal resistance genes as strong candidates to facilitate nanoparticle biosynthesis. Although it is known that these enzymes and genes play significant roles in maintaining bacterial homeostasis, there are few reports discussing this topic. Thus, we discuss examples of how metal resistance genes are conserved across bacteria and have been shown to be important for both nanoparticle biosynthesis and processes such as virulence or oxidative stress responses. Overall, this review highlights biological connections between nanoparticle biosynthesis and stress responses by examining the role of reductases and metal resistance genes in both processes. This understanding provides a greater role for nanoparticle biosynthesis in bacteria and could enable a systems biology level of control over nanoparticle biosynthesis.