Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants.

BACKGROUND: Microorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. RESULTS: Here, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter. CONCLUSIONS: Our study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs. Video Abstract.

Improved description of terrestrial habitat types by including microbial communities as indicators.

Soils host diverse communities of microorganisms essential for ecosystem functions and soil health. Despite their importance, microorganisms are not covered by legislation protecting biodiversity or habitats, such as the Habitats Directive. Advances in molecular methods have caused breakthroughs in microbial community analysis, and recent studies have shown that parts of the communities are habitat-specific. If distinct microbial communities are present in the habitat types defined in the Habitats Directive, the Directive may be improved by including these communities. Thus, monitoring and reporting of biodiversity and conservation status of habitat types could be based not only on plant communities but also on microbial communities. In the present study, bacterial and plant communities were examined in six habitat types defined in the Habitats Directive by conducting botanical surveys and collecting soil samples for amplicon sequencing across 19 sites in Denmark. Furthermore, selected physico-chemical properties expected to differ between habitat types and explain variations in community composition of bacteria and vegetation were analysed (pH, electrical conductivity (EC), soil texture, soil water repellency, soil organic carbon content (OC), inorganic nitrogen, and in-situ water content (SWC)). Despite some variations within the same habitat type and overlaps between habitat types, habitat-specific communities were observed for both bacterial and plant communities, but no correlation was observed between the alpha diversity of vegetation and bacteria. PERMANOVA analysis was used to evaluate the variables best able to explain variation in the community composition of vegetation and bacteria. Habitat type alone could explain 46% and 47% of the variation in bacterial and plant communities, respectively. Excluding habitat type as a variable, the best model (pH, SWC, OC, fine silt, and Shannon's diversity index for vegetation) could explain 37% of the variation for bacteria. For vegetation, the best model (pH, EC, ammonium content and Shannon's diversity index for bacteria) could explain 25% of the variation. Based on these results, bacterial communities could be included in the Habitats Directive to improve the monitoring, as microorganisms are more sensitive to changes in the environment compared to vegetation, which the current monitoring is based on.

Genetic potential for exopolysaccharide synthesis in activated sludge bacteria uncovered by genome-resolved metagenomics.

A good floc formation of activated sludge (AS) is crucial for solid-liquid separation and production of clean effluent during wastewater treatment. Floc formation is partly controlled by self-produced extracellular polymeric substances (EPS) such as exopolysaccharides, proteins, and nucleic acids. Little is known about the composition, structure, and function of EPS in AS and which bacteria produce them. To address this knowledge gap for the exopolysaccharides, we took advantage of 1083 high-quality metagenome-assembled genomes (MAGs) obtained from 23 Danish wastewater treatment plants. We investigated the genomic potential for exopolysaccharide biosynthesis in bacterial species typical in AS systems based on genome mining and gene synteny analyses. Putative gene clusters associated with the biosynthesis of alginate, cellulose, curdlan, diutan, hyaluronic acids, Pel, poly-ß-1,6-N-acetyl-d-glucosamine (PNAG), Psl, S88 capsular polysaccharide, salecan, succinoglycan, and xanthan were identified and linked to individual MAGs, providing a comprehensive overview of the genome-resolved potential for these exopolysaccharides in AS bacteria. The approach and results provide a starting point for a more comprehensive understanding of EPS composition in wastewater treatment systems, which may facilitate a more refined regulation of the activated sludge process for improved stability.

Functional Amyloids in Pseudomonas aeruginosa Are Essential for the Proteome Modulation That Leads to Pathoadaptation in Pulmonary Niches.

Persistence and survival of Pseudomonas aeruginosa in chronic lung infections is closely linked to the biofilm lifestyle. One biofilm component, functional amyloid of P. aeruginosa (Fap), imparts structural adaptations for biofilms; however, the role of Fap in pathogenesis is still unclear. Conservation of the fap operon encoding Fap and P. aeruginosa being an opportunistic pathogen of lung infections prompted us to explore its role in lung infection. We found that Fap is essential for establishment of lung infection in rats, as its genetic exclusion led to mild focal infection with quick resolution. Moreover, without an underlying cystic fibrosis (CF) genetic disorder, overexpression of Fap reproduced the CF pathotype. The molecular basis of Fap-mediated pulmonary adaptation was explored through surface-associated proteomics in vitro. Differential proteomics positively associated Fap expression with activation of known proteins related to pulmonary pathoadaptation, attachment, and biofilm fitness. The aggregative bacterial phenotype in the pulmonary niche correlated with Fap-influenced activation of biofilm sustainability regulators and stress response regulators that favored persistence-mediated establishment of pulmonary infection. Fap overexpression upregulated proteins that are abundant in the proteome of P. aeruginosa in colonizing CF lungs. Planktonic lifestyle, defects in anaerobic pathway, and neutrophilic evasion were key factors in the absence of Fap that impaired establishment of infection. We concluded that Fap is essential for cellular equilibration to establish pulmonary infection. Amyloid-induced bacterial aggregation subverted the immune response, leading to chronic infection by collaterally damaging tissue and reinforcing bacterial persistence. IMPORTANCE Pseudomonas aeruginosa is inextricably linked with chronic lung infections. In this study, the well-conserved Fap operon was found to be essential for pathoadaptation in pulmonary infection in a rat lung model. Moreover, the presence of Fap increased pathogenesis and biofilm sustainability by modulating bacterial physiology. Hence, a pathoadaptive role of Fap in pulmonary infections can be exploited for clinical application by targeting amyloids. Furthermore, genetic conservation and extracellular exposure of Fap make it a commendable target for such interventions.

Application of ecosystem-specific reference databases for increased taxonomic resolution in soil microbial profiling.

Intensive agriculture systems have paved the way for a growing human population. However, the abundant use of mineral fertilizers and pesticides may negatively impact nutrient cycles and biodiversity. One potential alternative is to harness beneficial relationships between plants and plant-associated rhizobacteria to increase nutrient-use efficiency and provide pathogen resistance. Plant-associated microbiota profiling can be achieved using high-throughput 16S rRNA gene amplicon sequencing. However, interrogation of these data is limited by confident taxonomic classifications at high taxonomic resolution (genus- or species level) with the commonly applied universal reference databases. High-throughput full-length 16S rRNA gene sequencing combined with automated taxonomy assignment (AutoTax) can be used to create amplicon sequence variant resolved ecosystems-specific reference databases that are superior to the traditional universal reference databases. This approach was used here to create a custom reference database for bacteria and archaea based on 987,353 full-length 16S rRNA genes from Askov and Cologne soils. We evaluated the performance of the database using short-read amplicon data and found that it resulted in the increased genus- and species-level classification compared to commonly use universal reference databases. The custom database was utilized to evaluate the ecosystem-specific primer bias and taxonomic resolution of amplicon primers targeting the V5-V7 region of the 16S rRNA gene commonly used within the plant microbiome field. Finally, we demonstrate the benefits of custom ecosystem-specific databases through the analysis of V5-V7 amplicon data to identify new plant-associated microbes for two legumes and two cereal species.

MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants.

Microbial communities are responsible for biological wastewater treatment, but our knowledge of their diversity and function is still poor. Here, we sequence more than 5 million high-quality, full-length 16S rRNA gene sequences from 740 wastewater treatment plants (WWTPs) across the world and use the sequences to construct the 'MiDAS 4' database. MiDAS 4 is an amplicon sequence variant resolved, full-length 16S rRNA gene reference database with a comprehensive taxonomy from domain to species level for all sequences. We use an independent dataset (269 WWTPs) to show that MiDAS 4, compared to commonly used universal reference databases, provides a better coverage for WWTP bacteria and an improved rate of genus and species level classification. Taking advantage of MiDAS 4, we carry out an amplicon-based, global-scale microbial community profiling of activated sludge plants using two common sets of primers targeting regions of the 16S rRNA gene, revealing how environmental conditions and biogeography shape the activated sludge microbiota. We also identify core and conditionally rare or abundant taxa, encompassing 966 genera and 1530 species that represent approximately 80% and 50% of the accumulated read abundance, respectively. Finally, we show that for well-studied functional guilds, such as nitrifiers or polyphosphate-accumulating organisms, the same genera are prevalent worldwide, with only a few abundant species in each genus.

Microbial communities across activated sludge plants show recurring species-level seasonal patterns.

Microbial communities in activated sludge (AS) are the core of sanitation in wastewater treatment plants (WWTPs). Microbial communities in AS have shown seasonal changes, however, long-term experiments (>2 years) are rarely conducted, limiting our understanding of the true seasonal dynamics in WWTPs. In this study, we resolved the microbial seasonal dynamics at the species level in four municipal full-scale WWTPs, sampled every 7-10 days, during 3-5 consecutive years. By applying a new time-series analysis approach, we revealed that the seasonal pattern was species-specific, where species belonging to the same functional guild or genus may show different seasonal dynamics. Species could be grouped into cohorts according to their seasonal patterns, where seasonal cohorts showed repeatable annual dynamics across years and plants. Species were also grouped according to their net growth rate in the AS (i.e., growing species and disappearing species). Growing species were more prevailing in spring and autumn cohorts, while disappearing species, which were only present due to the continuous immigration from influent wastewater, were mostly associated with winter and spring cohorts. Most known process-critical species, such as nitrifiers, polyphosphate accumulating organisms and filamentous organisms, showed distinct species-specific patterns. Overall, our study showed that overarching seasonal patterns affected microbial species in full-scale AS plants, with similar seasonal patterns across plants for many dominant species. These recurrent seasonal variations should be taken into account in the operation, understanding and management of the WWTPs.