Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the<jats:named-content content-type="genus-species">E. coli</jats:named-content>maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong<jats:italic>in vivo</jats:italic>phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this<jats:named-content content-type="genus-species">E. coli</jats:named-content>strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions<jats:italic>in vivo</jats:italic>.</jats:p>

Unprecedented Silver Resistance in Clinically Isolated Enterobacteriaceae: Major Implications for Burn and Wound Management

“…This finding is similar to previous reports of blackening pigmentation of bacteria exposed to high ionic silver concentrations, including MBP-AgBP2p and P. stutzeri AG259 (9,26,32,33). This color is proposed to be a result of silver particles building up on the surface of the cell after reduction of Ag(I) to the less toxic nanocrystalline silver species Ag(0).…”

“…More recent work has provided details concerning metal binding mechanisms. Cytochromes, periplasmic polypeptides extending through the cell surface, have been shown to be the reducing agent driving electron transfer with heavy metals and other extracellular "oxidizers" (32,34,35). However, how the sequestering and reduction of silver relate to the observed increased resistance remains unclear.…”

“…However, how the sequestering and reduction of silver relate to the observed increased resistance remains unclear. Simple sequestering of silver in EPS may be too parsimonious, and the silver resistance mechanism is more complex than initially proposed (32). It is important to note that while endogenous and exogenous mechanisms accounting for silver resistance have been put forth, detailed characterizations of this phenomenon remain unclear across Gram-positive and Gram-negative bacteria.…”

Disinfection and removal performance for Escherichia coli and heavy metals by silver-modified zeolite in a fixed bed column

“…In order to develop an understanding of the E. coli-metal interactions occurring within the bed, the morphology of E. coli cells before and after treatment was examined using SEM as shown in In extracellular synthesis, the bacterial cells release extracellular polymeric substances which bind to the toxic Ag + ions reducing them to less reactive metallic particles Ag 0 [8]. Intracellular synthesis can occur within the reductive environment in the cytoplasm [9].…”

“…Silver is an effective antibacterial agent, which has been immobilized on different inorganic support materials for a wide range of antimicrobial applications including water and wastewater disinfection [7][8][9][10][11][12][13][14][15]. Zeolites have been used as support materials for silver ions with studies demonstrating the effective elimination of microorganisms from aqueous media by silvermodified zeolites [1,[16][17][18][19][20][21].…”