Operationalising water safety plans for Melbourne - a large city case study.

A wholesale/retail model delivers drinking water to over 5 million residents in metropolitan Melbourne (Australia). Water Safety Plans were implemented in 1999 ahead of being regulatory mandated in 2003. With over 20 years of WSP application, this collaborative paper by the wholesaler and retailer utilities presents practical examples of drinking water quality risk management in challenging operational environments, highlighting lessons learnt, improvements made and outcomes achieved. Melbourne's supply comprises multiple sources, necessitating different tailored treatment configurations. Source waters range from open catchment with multiple treatment barriers, to protected catchment source waters requiring solely disinfection treatment (unfiltered) with gravity driven supply. Potable supply is a combination of unfiltered, filtered, desalinated and blended supplies. This makes for diversity in case studies brought to this paper, and a range of lessons likely to be of interest to the global WSP community. The Melbourne utility experience highlights the importance of developing and continually improving control measures for ongoing (adaptive) risk reduction. A robust emergency management plan is also fundamental to ensure preparedness for complex and unpredictable events. Furthermore, leveraging learnings from audits and incidents has been valuable for process improvement. WSP implementation has also facilitated timely communication with consumers and other stakeholders.

Genetic and Biochemical Analysis of CodY-Mediated Cell Aggregation in Staphylococcus aureus Reveals an Interaction between Extracellular DNA and Polysaccharide in the Extracellular Matrix.

The global regulator CodY links nutrient availability to the regulation of virulence factor gene expression in Staphylococcus aureus, including many genes whose products affect biofilm formation. Antithetical phenotypes of both biofilm deficiency and accumulation have been reported for codY-null mutants; thus, the role of CodY in biofilm development remains unclear. codY mutant cells of a strain producing a robust biofilm elaborate proaggregation surface-associated features not present on codY mutant cells that do not produce a robust biofilm. Biochemical analysis of the clinical isolate SA564, which aggregates when deficient for CodY, revealed that these features are sensitive to nuclease treatment and are resistant to protease exposure. Genetic analyses revealed that disrupting lgt (the diacylglycerol transferase gene) in codY mutant cells severely weakened aggregation, indicating a role for lipoproteins in the attachment of the biofilm matrix to the cell surface. An additional and critical role of IcaB in producing functional poly-N-acetylglucosamine (PIA) polysaccharide in extracellular DNA (eDNA)-dependent biofilm formation was shown. Moreover, overproducing PIA is sufficient to promote aggregation in a DNA-dependent manner regardless of source of nucleic acids. Taken together, our results point to PIA synthesis as the primary determinant of biofilm formation when CodY activity is reduced and suggest a modified electrostatic net model for matrix attachment whereby PIA associates with eDNA, which interacts with the cell surface via covalently attached membrane lipoproteins. This work counters the prevailing view that polysaccharide- and eDNA/protein-based biofilms are mutually exclusive. Rather, we demonstrate that eDNA and PIA can work synergistically to form a biofilm.IMPORTANCEStaphylococcus aureus remains a global health concern and exemplifies the ability of an opportunistic pathogen to adapt and persist within multiple environments, including host tissue. Not only does biofilm contribute to persistence and immune evasion in the host environment, it also may aid in the transition to invasive disease. Thus, understanding how biofilms form is critical for developing strategies for dispersing biofilms and improving biofilm disease-related outcomes. Using biochemical, genetic, and cell biology approaches, we reveal a synergistic interaction between PIA and eDNA that promotes cell aggregation and biofilm formation in a CodY-dependent manner in S. aureus We also reveal that envelope-associated lipoproteins mediate attachment of the biofilm matrix to the cell surface.

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations.

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-electron microscopy (cryo-EM) densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities using experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in, e.g., cryo-EM derived membrane protein structures surrounded by an array of ancillary lipid densities.