Processing of Nonconjugative Resistance Plasmids by Conjugation Nicking Enzyme of Staphylococci.

UNLABELLED: Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-like oriT. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids. IMPORTANCE: Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variant oriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase in trans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution.

Structure and Inhibition of Microbiome ß-Glucuronidases Essential to the Alleviation of Cancer Drug Toxicity.

The selective inhibition of bacterial ß-glucuronidases was recently shown to alleviate drug-induced gastrointestinal toxicity in mice, including the damage caused by the widely used anticancer drug irinotecan. Here, we report crystal structures of representative ß-glucuronidases from the Firmicutes Streptococcus agalactiae and Clostridium perfringens and the Proteobacterium Escherichia coli, and the characterization of a ß-glucuronidase from the Bacteroidetes Bacteroides fragilis. While largely similar in structure, these enzymes exhibit marked differences in catalytic properties and propensities for inhibition, indicating that the microbiome maintains functional diversity in orthologous enzymes. Small changes in the structure of designed inhibitors can induce significant conformational changes in the ß-glucuronidase active site. Finally, we establish that ß-glucuronidase inhibition does not alter the serum pharmacokinetics of irinotecan or its metabolites in mice. Together, the data presented advance our in vitro and in vivo understanding of the microbial ß-glucuronidases, a promising new set of targets for controlling drug-induced gastrointestinal toxicity.

Evaluation of redox indicators for determining sulfate-reducing and dechlorinating conditions.

An in situ methodology based on covalently bonded redox indicators has been developed for determining when sulfate-reducing conditions exist in environmental samples. Three immobilized redox indicators [thionine (Thi, formal potential at pH 7 (E(0')7) equals 52 mV), cresyl violet (CV, E(0')7 = -81 mV), and phenosafranine (PSaf, E(0')7 = -267 mV)] were tested for their response to sulfide in synthetic solutions and under sulfate-reducing conditions in wastewater slurries. The byproduct of the sulfate-reducing process, sulfide, was found to couple well to CV in the concentration range of 1-100 microM total sulfide ([S(-II)]) and the pH range of 6-8. Thi, the indicator with the highest formal potential, reacts rapidly with sulfide at levels well below 1 microM while PSaf, the indicator with the lowest formal potential, does not couple to sulfide at levels in excess of 100 microM [S(-II)]. The degree of reduction of the indicators (i.e., the fraction of cresyl violet oxidized) in contact with a given level of sulfide can be modeled qualitatively with an equilibrium expression for [S(-II)]-indicator based on the Nernst equation assuming that rhombic sulfur is the product of sulfide oxidation. In a groundwater sample with dechlorinating microbes, reduction of Thi and partial reduction of CV correlated with dechlorination of TCE to cis-DCE.

The SLIM spectrometer.

A new spectrometer, here denoted the SLIM (simple, low-power, inexpensive, microcontroller-based) spectrometer, was developed that exploits the small size and low cost of solid-state electronic devices. In this device, light-emitting diodes (LED), single-chip integrated circuit photodetectors, embedded microcontrollers, and batteries replace traditional optoelectronic components, computers, and power supplies. This approach results in complete customizable spectrometers that are considerably less expensive and smaller than traditional instrumentation. The performance of the SLIM spectrometer, configured with a flow cell, was evaluated and compared to that of a commercial spectrophotometer. Thionine was the analyte, and the detection limit was approximately 0.2 microM with a 1.5-mm-path length flow cell. Nonlinearity due to the broad emission profile of the LED light sources is discussed.