Premise Plumbing Pipe Materials and In-Building Disinfectants Shape the Potential for Proliferation of Pathogens and Antibiotic Resistance Genes.

In-building disinfectants are commonly applied to control the growth of pathogens in plumbing, particularly in facilities such as hospitals that house vulnerable populations. However, their application has not been well optimized, especially with respect to interactive effects with pipe materials and potential unintended effects, such as enrichment of antibiotic resistance genes (ARGs) across the microbial community. Here, we used triplicate convectively mixed pipe reactors consisting of three pipe materials (PVC, copper, and iron) for replicated simulation of the distal reaches of premise plumbing and evaluated the effects of incrementally increased doses of chlorine, chloramine, chlorine dioxide, and copper-silver disinfectants. We used shotgun metagenomic sequencing to characterize the resulting succession of the corresponding microbiomes over the course of 37 weeks. We found that both disinfectants and pipe material affected ARG and microbial community taxonomic composition both independently and interactively. Water quality and total bacterial numbers were not found to be predictive of pathogenic species markers. One result of particular concern was the tendency of disinfectants, especially monochloramine, to enrich ARGs. Metagenome assembly indicated that many ARGs were enriched specifically among the pathogenic species. Functional gene analysis was indicative of a response of the microbes to oxidative stress, which is known to co/cross-select for antibiotic resistance. These findings emphasize the need for a holistic evaluation of pathogen control strategies for plumbing.

Long-range electrostatic interactions significantly modulate the affinity of dynein for microtubules.

The dynein family of microtubule minus-end-directed motor proteins drives diverse functions in eukaryotic cells, including cell division, intracellular transport, and flagellar beating. Motor protein processivity, which characterizes how far a motor walks before detaching from its filament, depends on the interaction between its microtubule-binding domain (MTBD) and the microtubule. Dynein's MTBD switches between high- and low-binding affinity states as it steps. Significant structural and functional data show that specific salt bridges within the MTBD and between the MTBD and the microtubule govern these affinity state shifts. However, recent computational work suggests that nonspecific, long-range electrostatic interactions between the MTBD and the microtubule may also play an important role in the processivity of dynein. To investigate this hypothesis, we mutated negatively charged amino acids remote from the dynein MTBD-microtubule-binding interface to neutral residues and measured the binding affinity using microscale thermophoresis and optical tweezers. We found a significant increase in the binding affinity of the mutated MTBDs for microtubules. Furthermore, we found that charge screening by free ions in solution differentially affected the binding and unbinding rates of MTBDs to microtubules. Together, these results demonstrate a significant role for long-range electrostatic interactions in regulating dynein-microtubule affinity. Moreover, these results provide insight into the principles that potentially underlie the biophysical differences between molecular motors with various processivities and protein-protein interactions more generally.

Identifying weak interdomain interactions that stabilize the supertertiary structure of the N-terminal tandem PDZ domains of PSD-95.

Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.