Two RNA binding proteins, ADAD2 and RNF17, interact to form a heterogeneous population of novel meiotic germ cell granules with developmentally dependent organelle association.

Mammalian male germ cell differentiation relies on complex RNA biogenesis events, many of which occur in non-membrane bound organelles termed RNA germ cell granules that are rich in RNA binding proteins (RBPs). Though known to be required for male germ cell differentiation, we understand little of the relationships between the numerous granule subtypes. ADAD2, a testis specific RBP, is required for normal male fertility and forms a poorly characterized granule in meiotic germ cells. This work aimed to understand the role of ADAD2 granules in male germ cell differentiation by clearly defining their molecular composition and relationship to other granules. Biochemical analyses identified RNF17, a testis specific RBP that forms meiotic male germ cell granules, as an ADAD2-interacting protein. Phenotypic analysis of Adad2 and Rnf17 mutants identified a rare post-meiotic chromatin defect, suggesting shared biological roles. ADAD2 and RNF17 were found to be dependent on one another for granularization and together form a previously unstudied set of germ cell granules. Based on co-localization studies with well-characterized granule RBPs and organelle-specific markers, a subset of the ADAD2-RNF17 granules are found to be associated with the intermitochondrial cement and piRNA biogenesis. In contrast, a second, morphologically distinct population of ADAD2-RNF17 granules co-localized with the translation regulators NANOS1 and PUM1, along with the molecular chaperone PDI. These large granules form a unique funnel-shaped structure that displays distinct protein subdomains and is tightly associated with the endoplasmic reticulum. Developmental studies suggest the different granule populations represent different phases of a granule maturation process. Lastly, a double Adad2-Rnf17 mutant model suggests the interaction between ADAD2 and RNF17, as opposed to loss of either, is the likely driver of the Adad2 and Rnf17 mutant phenotypes. These findings shed light on the relationship between germ cell granule pools and define new genetic approaches to their study.

Source-to-tap investigation of the occurrence of nontuberculous mycobacteria in a full-scale chloraminated drinking water system.

Nontuberculous mycobacteria (NTM) in drinking water are a significant public health concern. However, an incomplete understanding of the factors that influence the occurrence of NTM in drinking water limits our ability to characterize risk and prevent infection. This study sought to evaluate the influence of season and water treatment, distribution, and stagnation on NTM in drinking water. Samples were collected source-to-tap in a full-scale, chloraminated drinking water system approximately monthly from December 2019 to November 2020. NTM were characterized using culture-dependent (plate culture with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry [MALDI-TOF MS] isolate analysis) and culture-independent methods (quantitative PCR and genome-resolved metagenomics). Sampling locations included source waters, three locations within the treatment plant, and five buildings receiving water from the distribution system. Building plumbing samples consisted of first draw, 5-min flush, and full flush cold-water samples. As the study took place during the COVID-19 pandemic, the influence of reduced water usage in three of the five buildings was also investigated. The highest NTM densities source-to-tap were found in the summer first draw building water samples (107 gene copies/L), which also had the lowest monochloramine concentrations. Flushing was found to be effective for reducing NTM and restoring disinfectant residuals, though flush times necessary to improve water quality varied by building. Clinically relevant NTM species, including Mycobacterium avium, were recovered via plate culture, with increased occurrence observed in buildings with higher water age. Four of five NTM metagenome-assembled genomes were identified to the species level and matched identified isolates.IMPORTANCENTM infections are increasing in prevalence, difficult to treat, and associated with high morbidity and mortality rates. Our lack of understanding of the factors that influence NTM occurrence in drinking water limits our ability to prevent infections, accurately characterize risk, and focus remediation efforts. In this study, we comprehensively evaluated NTM in a full-scale drinking water system, showing that various steps in treatment and distribution influence NTM presence. Stagnant building water contained the highest NTM densities source-to-tap and was associated with low disinfectant residuals. We illustrated the differences in NTM detection and characterization obtained from culture-based and culture-independent methods, highlighting the complementarity between these approaches. We demonstrated that focusing NTM mitigation efforts in building plumbing systems, which have the highest NTM densities source-to-tap, has potential for immediate positive effects. We also identified steps during treatment that increase NTM levels, which provides beneficial information for utilities seeking to reduce NTM in finished water.

ADAD1 is required for normal translation of nuclear pore and transport protein transcripts in spermatids of Mus musculus†.

ADAD1 is a testis-specific RNA-binding protein expressed in post-meiotic spermatids whose loss leads to defective sperm and male infertility. However, the drivers of the Adad1 phenotype remain unclear. Morphological and functional analysis of Adad1 mutant sperm showed defective DNA compaction, abnormal head shaping, and reduced motility. Mutant testes demonstrated minimal transcriptome changes; however, ribosome association of many transcripts was reduced, suggesting ADAD1 may be required for their translational activation. Further, immunofluorescence of proteins encoded by select transcripts showed delayed protein accumulation. Additional analyses demonstrated impaired subcellular localization of multiple proteins, suggesting protein transport is also abnormal in Adad1 mutants. To clarify the mechanism giving rise to this, the manchette, a protein transport microtubule network, and the LINC (linker of nucleoskeleton and cytoskeleton) complex, which connects the manchette to the nuclear lamin, were assessed across spermatid development. Proteins of both displayed delayed translation and/or localization in mutant spermatids implicating ADAD1 in their regulation, even in the absence of altered ribosome association. Finally, ADAD1's impact on the NPC (nuclear pore complex), a regulator of both the manchette and the LINC complex, was examined. Reduced ribosome association of NPC encoding transcripts and reduced NPC protein abundance along with abnormal localization in Adad1 mutants confirmed ADAD1's impact on translation is required for a NPC in post-meiotic germ cells. Together, these studies lead to a model whereby ADAD1's influence on nuclear transport leads to deregulation of the LINC complex and the manchette, ultimately generating the range of physiological defects observed in the Adad1 phenotype.

KAT2A and KAT2B prevent double-stranded RNA accumulation and interferon signaling to maintain intestinal stem cell renewal.

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and sequencing of immunoprecipitated double-stranded RNA were used to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions and maintaining intestinal health.

KAT2 paralogs prevent dsRNA accumulation and interferon signaling to maintain intestinal stem cells.

Histone acetyltransferases KAT2A and KAT2B are paralogs highly expressed in the intestinal epithelium, but their functions are not well understood. In this study, double knockout of murine Kat2 genes in the intestinal epithelium was lethal, resulting in robust activation of interferon signaling and interferon-associated phenotypes including the loss of intestinal stem cells. Use of pharmacological agents and sterile organoid cultures indicated a cell-intrinsic double-stranded RNA trigger for interferon signaling. Acetyl-proteomics and dsRIP-seq were employed to interrogate the mechanism behind this response, which identified mitochondria-encoded double-stranded RNA as the source of intrinsic interferon signaling. Kat2a and Kat2b therefore play an essential role in regulating mitochondrial functions as well as maintaining intestinal health.

Niche Preference of Escherichia coli in a Peri-Urban Pond Ecosystem.

Escherichia coli comprises diverse strains with a large accessory genome, indicating functional diversity and the ability to adapt to a range of niches. Specific strains would display greatest fitness in niches matching their combination of phenotypic traits. Given this hypothesis, we sought to determine whether E. coli in a peri-urban pond and associated cattle pasture display niche preference. Samples were collected from water, sediment, aquatic plants, water snails associated with the pond, as well as bovine feces from cattle in an adjacent pasture. Isolates (120) were obtained after plating on Membrane Lactose Glucuronide Agar (MLGA). We used the uidA and mutS sequences for all isolates to determine phylogeny by maximum likelihood, and population structure through gene flow analysis. PCR was used to allocate isolates to phylogroups and to determine the presence of pathogenicity/virulence genes (stxI, stxII, eaeA, hlyA, ST, and LT). Antimicrobial resistance was determined using a disk diffusion assay for Tetracycline, Gentamicin, Ciprofloxacin, Meropenem, Ceftriaxone, and Azithromycin. Our results showed that isolates from water, sediment, and water plants were similar by phylogroup distribution, virulence gene distribution, and antibiotic resistance while both snail and feces populations were significantly different. Few of the feces isolates were significantly similar to aquatic ones, and most of the snail isolates were also different. Population structure analysis indicated three genetic backgrounds associated with bovine, snail, and aquatic environments. Collectively these data support niche preference of E. coli isolates occurring in this ecosystem.

Microbial Nitrogen Metabolism in Chloraminated Drinking Water Reservoirs.

Ammonia availability due to chloramination can promote the growth of nitrifying organisms, which can deplete chloramine residuals and result in operational problems for drinking water utilities. In this study, we used a metagenomic approach to determine the identity and functional potential of microorganisms involved in nitrogen biotransformation within chloraminated drinking water reservoirs. Spatial changes in the nitrogen species included an increase in nitrate concentrations accompanied by a decrease in ammonium concentrations with increasing distance from the site of chloramination. This nitrifying activity was likely driven by canonical ammonia-oxidizing bacteria (i.e., Nitrosomonas) and nitrite-oxidizing bacteria (i.e., Nitrospira) as well as by complete-ammonia-oxidizing (i.e., comammox) Nitrospira-like bacteria. Functional annotation was used to evaluate genes associated with nitrogen metabolism, and the community gene catalogue contained mostly genes involved in nitrification, nitrate and nitrite reduction, and nitric oxide reduction. Furthermore, we assembled 47 high-quality metagenome-assembled genomes (MAGs) representing a highly diverse assemblage of bacteria. Of these, five MAGs showed high coverage across all samples, which included two Nitrosomonas, Nitrospira, Sphingomonas, and Rhizobiales-like MAGs. Systematic genome-level analyses of these MAGs in relation to nitrogen metabolism suggest that under ammonia-limited conditions, nitrate may be also reduced back to ammonia for assimilation. Alternatively, nitrate may be reduced to nitric oxide and may potentially play a role in regulating biofilm formation. Overall, this study provides insight into the microbial communities and their nitrogen metabolism and, together with the water chemistry data, improves our understanding of nitrogen biotransformation in chloraminated drinking water distribution systems.IMPORTANCE Chloramines are often used as a secondary disinfectant when free chlorine residuals are difficult to maintain. However, chloramination is often associated with the undesirable effect of nitrification, which results in operational problems for many drinking water utilities. The introduction of ammonia during chloramination provides a potential source of nitrogen either through the addition of excess ammonia or through chloramine decay. This promotes the growth of nitrifying microorganisms and provides a nitrogen source (i.e., nitrate) for the growth for other organisms. While the roles of canonical ammonia-oxidizing and nitrite-oxidizing bacteria in chloraminated drinking water systems have been extensively investigated, those studies have largely adopted a targeted gene-centered approach. Further, little is known about the potential long-term cooccurrence of complete-ammonia-oxidizing (i.e., comammox) bacteria and the potential metabolic synergies of nitrifying organisms with their heterotrophic counterparts that are capable of denitrification and nitrogen assimilation. This study leveraged data obtained for genome-resolved metagenomics over a time series to show that while nitrifying bacteria are dominant and likely to play a major role in nitrification, their cooccurrence with heterotrophic organisms suggests that nitric oxide production and nitrate reduction to ammonia may also occur in chloraminated drinking water systems.

Long-term spatial and temporal microbial community dynamics in a large-scale drinking water distribution system with multiple disinfectant regimes.

Long-term spatial-temporal investigations of microbial dynamics in full-scale drinking water distribution systems are scarce. These investigations can reveal the process, infrastructure, and environmental factors that influence the microbial community, offering opportunities to re-think microbial management in drinking water systems. Often, these insights are missed or are unreliable in short-term studies, which are impacted by stochastic variabilities inherent to large full-scale systems. In this two-year study, we investigated the spatial and temporal dynamics of the microbial community in a large, full scale South African drinking water distribution system that uses three successive disinfection strategies (i.e. chlorination, chloramination and hypochlorination). Monthly bulk water samples were collected from the outlet of the treatment plant and from 17 points in the distribution system spanning nearly 150 km and the bacterial community composition was characterised by Illumina MiSeq sequencing of the V4 hypervariable region of the 16S rRNA gene. Like previous studies, Alpha- and Betaproteobacteria dominated the drinking water bacterial communities, with an increase in Betaproteobacteria post-chloramination. In contrast with previous reports, the observed richness, diversity, and evenness of the bacterial communities were higher in the winter months as opposed to the summer months in this study. In addition to temperature effects, the seasonal variations were also likely to be influenced by changes in average water age in the distribution system and corresponding changes in disinfectant residual concentrations. Spatial dynamics of the bacterial communities indicated distance decay, with bacterial communities becoming increasingly dissimilar with increasing distance between sampling locations. These spatial effects dampened the temporal changes in the bulk water community and were the dominant factor when considering the entire distribution system. However, temporal variations were consistently stronger as compared to spatial changes at individual sampling locations and demonstrated seasonality. This study emphasises the need for long-term studies to comprehensively understand the temporal patterns that would otherwise be missed in short-term investigations. Furthermore, systematic long-term investigations are particularly critical towards determining the impact of changes in source water quality, environmental conditions, and process operations on the changes in microbial community composition in the drinking water distribution system.