Longitudinal and Source-to-Tap New Orleans, LA, U.S.A. Drinking Water Microbiology.

The two municipal drinking water systems of New Orleans, LA, U.S.A. were sampled to compare the microbiology of independent systems that treat the same surface water from the Mississippi River. To better understand temporal trends and sources of microbiology delivered to taps, these treatment plants and distribution systems were subjected to source-to-tap sampling over four years. Both plants employ traditional treatment by chloramination, applied during or after settling, followed by filtration before distribution in a warm, low water age system. Longitudinal samples indicated microbiology to have stability both spatially and temporally, and between treatment plants and distribution systems. Disinfection had the greatest impact on microbial composition, which was further refined by filtration and influenced by distribution and premise plumbing. Actinobacteria spp. exhibited trends with treatment. In particular, Mycobacterium spp., very low in finished waters, occurred idiosyncratically at high levels in some tap waters, indicating distribution and/or premise plumbing as main contributors of mycobacteria. Legionella spp., another genus containing potential opportunistic pathogens, also occurred ubiquitously. Source water microbiology was most divergent from tap water, and each step of treatment brought samples more closely similar to tap waters.

Effect of monochloramine treatment on the microbial ecology of Legionella and associated bacterial populations in a hospital hot water system.

Opportunistic pathogens, including Legionella spp. and non-tuberculous mycobacteria, can thrive in building hot water systems despite municipal and traditional on-site chlorine disinfection. Monochloramine is a relatively new approach to on-site disinfection, but the microbiological impact of on-site chloramine use has not been well studied. We hypothesized that comparison of the microbial ecology associated with monochloramine treatment versus no on-site treatment would yield highly dissimilar bacterial communities. Hot water samples were collected monthly from 7 locations for three months from two buildings in a Pennsylvania hospital complex supplied with common municipal water: (1) a hospital administrative building (no on-site treatment) and (2) an adjacent acute-care hospital treated on-site with monochloramine to control Legionella spp. Water samples were subjected to DNA extraction, rRNA PCR, and 454 pyrosequencing. Stark differences in the microbiome of the chloraminated water and the control were observed. Bacteria in the treated samples were primarily Sphingomonadales and Limnohabitans, whereas Flexibacter and Planctomycetaceae predominated in untreated control samples. Serendipitously, one sampling month coincided with dysfunction of the on-site disinfection system that resulted in a Legionella bloom detected by sequencing and culture. This study also demonstrates the potential utility of high-throughput DNA sequencing to monitor microbial ecology in water systems.

Molecular analysis of point-of-use municipal drinking water microbiology.

Little is known about the nature of the microbiology in tap waters delivered to consumers via public drinking water distribution systems (DWDSs). In order to establish a broader understanding of the microbial complexity of public drinking waters we sampled tap water from seventeen different cities between the headwaters of the Arkansas River and the mouth of the Mississippi River and determined the bacterial compositions by pyrosequencing small subunit rRNA genes. Nearly 98% of sequences observed among all systems fell into only 5 phyla: Proteobacteria (35%), Cyanobacteria (29%, including chloroplasts), Actinobacteria (24%, of which 85% were Mycobacterium spp.), Firmicutes (6%), and Bacteroidetes (3.4%). The genus Mycobacterium was the most abundant taxon in the dataset, detected in 56 of 63 samples (16 of 17 cities). Among the more rare phylotypes, considerable variation was observed between systems, and was sometimes associated with the type of source water, the type of disinfectant, or the concentration of the environmental pollutant nitrate. Abundant taxa (excepting Cyanobacteria and chloroplasts) were generally similar from system to system, however, regardless of source water type or local land use. The observed similarity among the abundant taxa between systems may be a consequence of the selective influence of chlorine-based disinfection and the common local environments of DWDS and premise plumbing pipes.

A cell cycle phosphoproteome of the yeast centrosome.

Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)-directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

Budding yeast centrosome duplication requires stabilization of Spc29 via Mps1-mediated phosphorylation.

Protein phosphorylation plays an important role in the regulation of centrosome duplication. In budding yeast, numerous lines of evidence suggest a requirement for multiple phosphorylation events on individual components of the centrosome to ensure their proper assembly and function. Here, we report the first example of a single phosphorylation event on a component of the yeast centrosome, or spindle pole body (SPB), that is required for SPB duplication and cell viability. This phosphorylation event is on the essential SPB component Spc29 at a conserved Thr residue, Thr(240). Mutation of Thr(240) to Ala is lethal at normal gene dosage, but an increased copy number of this mutant allele results in a conditional phenotype. Phosphorylation of Thr(240) was found to promote the stability of the protein in vivo and is catalyzed in vitro by the Mps1 kinase. Furthermore, the stability of newly synthesized Spc29 is reduced in a mutant strain with reduced Mps1 kinase activity. These results demonstrate the first evidence for a single phosphorylation event on an SPB component that is absolutely required for SPB duplication and suggest that the Mps1 kinase is responsible for this protein-stabilizing phosphorylation.