Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild-type rats.

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as αSN, which is central in the development of Parkinson's disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order ß-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.

Characterizing the growing microorganisms at species level in 46 anaerobic digesters at Danish wastewater treatment plants: A six-year survey on microbial community structure and key drivers.

Anaerobic digestion (AD) is a key technology at many wastewater treatment plants (WWTPs) for converting primary and surplus activated sludge to methane-rich biogas. However, the limited number of surveys and the lack of comprehensive datasets have hindered a deeper understanding of the characteristics and associations between key variables and the microbial community composition. Here, we present a six-year survey of 46 anaerobic digesters, located at 22 WWTPs in Denmark, which is the first and largest known study of the microbial ecology of AD at WWTPs at a regional scale. For three types of AD (mesophilic, mesophilic with thermal hydrolysis pretreatment, and thermophilic), we present the typical value range of 12 key parameters including operational variables and performance parameters. High-resolution bacterial and archaeal community analyses were carried out at species level using amplicon sequencing of >1,000 samples and the new ecosystem-specific MiDAS 3 reference database. We detected 42 phyla, 1,600 genera, and 3,584 species in the bacterial community, where 70% of the genera and 93% of the species represented environmental taxa that were only classified based on MiDAS 3 de novo placeholder taxonomy. More than 40% of the bacterial species were found not to grow in the mesophilic and thermophilic digesters and were only present due to immigration with the feed sludge. Ammonium concentration was the main driver shaping the bacterial community while temperature and pH were main drivers for the archaea in the three types of ADs. Sub-setting for the growing microbes improved significantly the correlation analyses and revealed the main drivers for the presence of specific species. Within mesophilic digesters, feed sludge composition and other key parameters (organic loading rate, biogas yield, and ammonium concentration) correlated with specific growing species. This survey provides a comprehensive insight into community structure at species level, providing a foundation for future studies of the ecological significance/characteristics and function of the many novel or poorly described taxa.

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax).

High-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here, we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) resolved reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. Reference databases processed with AutoTax greatly improves the classification of short-read 16S rRNA ASVs at the genus- and species-level, compared with the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

MiDAS 3: An ecosystem-specific reference database, taxonomy and knowledge platform for activated sludge and anaerobic digesters reveals species-level microbiome composition of activated sludge.

The function of the microbiomes in wastewater treatment systems and anaerobic digesters is dictated by the physiological activity of their members and complex interactions between them. Since functional traits are often conserved at low taxonomic ranks (genus, species, strain), high resolution taxonomic classification is crucial to understand the role of microbes in any ecosystem. Here we present MiDAS 3, a comprehensive 16S rRNA gene reference database based on full-length 16S rRNA gene amplicon sequence variants (FL-ASVs) derived from activated sludge and anaerobic digester systems in Denmark. The new database proposes unique provisional names for all unclassified microorganisms down to species level, providing a new and much-needed tool for microbiome research. The MiDAS 3 database was used to analyze the microbiome in 20 Danish wastewater treatment plants with nutrient removal, sampled over 13 years. The 50 most abundant species belonged to 42 genera, including 14 genera with provisional 'midas' name. Of those, 20 have no known function in the system, which highlights the need for more efforts towards elucidating the role of important members of wastewater treatment ecosystems. The new MiDAS 3 database also forms the backbone of the MiDAS Field Guide - an online resource linking the identity of microorganisms in wastewater treatment systems to available data related to their functional importance. The new field guide contains a complete list of genera (>1800) and species (>4200) found in activated sludge and anaerobic digesters in Denmark, but is also relevant to wastewater systems across the world. The identity of the microbes is linked to functional information, where available, and the website provides the possibility to BLAST new sequences against the MiDAS 3 database. The MiDAS Field Guide is a collaborative platform acting as an online knowledge repository, facilitating understanding of wastewater treatment ecosystem function.

Novel syntrophic bacteria in full-scale anaerobic digesters revealed by genome-centric metatranscriptomics.

Short-chain fatty acid (SCFA) degradation is an important process in methanogenic ecosystems, and is usually catalyzed by SCFA-oxidizing bacteria in syntrophy with methanogens. Current knowledge of this functional guild is mainly based on isolates or enrichment cultures, but these may not reflect the true diversity and in situ activities of the syntrophs predominating in full-scale systems. Here we obtained 182 medium to high quality metagenome-assembled genomes (MAGs) from the microbiome of two full-scale anaerobic digesters. The transcriptomic response of individual MAG was studied after stimulation with low concentrations of acetate, propionate, or butyrate, separately. The most pronounced response to butyrate was observed for two MAGs of the recently described genus Candidatus Phosphitivorax (phylum Desulfobacterota), expressing a butyrate beta-oxidation pathway. For propionate, the largest response was observed for an MAG of a novel genus in the family Pelotomaculaceae, transcribing a methylmalonyl-CoA pathway. All three species were common in anaerobic digesters at Danish wastewater treatment plants as shown by amplicon analysis, and this is the first time their syntrophic features involved in SCFA oxidation were revealed with transcriptomic evidence. Further, they also possessed unique genomic features undescribed in well-characterized syntrophs, including the metabolic pathways for phosphite oxidation, nitrite and sulfate reduction.

Extracellular polymeric substances of biofilms: Suffering from an identity crisis.

Microbial biofilms can be both cause and cure to a range of emerging societal problems including antimicrobial tolerance, water sanitation, water scarcity and pollution. The identities of extracellular polymeric substances (EPS) responsible for the establishment and function of biofilms are poorly understood. The lack of information on the chemical and physical identities of EPS limits the potential to rationally engineer biofilm processes, and impedes progress within the water and wastewater sector towards a circular economy and resource recovery. Here, a multidisciplinary roadmap for addressing this EPS identity crisis is proposed. This involves improved EPS extraction and characterization methodologies, cross-referencing between model biofilms and full-scale biofilm systems, and functional description of isolated EPS with in situ techniques (e.g. microscopy) coupled with genomics, proteomics and glycomics. The current extraction and spectrophotometric characterization methods, often based on the principle not to compromise the integrity of the microbial cells, should be critically assessed, and more comprehensive methods for recovery and characterization of EPS need to be developed.

The Sheaths of Methanospirillum Are Made of a New Type of Amyloid Protein.

The genera Methanospirillum and Methanosaeta contain species of anaerobic archaea that grow and divide within proteinaceous tubular sheaths that protect them from environmental stressors. The sheaths of Methanosaeta thermophila PT are composed of the 60.9 kDa major sheath protein MspA. In this study we show that sheaths purified from Methanospirillum hungatei JF-1 are regularly striated tubular structures with amyloid-like properties similar to those of M. thermophila PT. Depolymerizing the sheaths from M. hungatei JF-1 allowed us to identify a 40.6 kDa protein (WP_011449234.1) that shares 23% sequence similarity to MspA from M. thermophila PT (ABK14853.1), indicating that they might be distant homologs. The genome of M. hungatei JF-1 encodes six homologs of the identified MspA protein. Several homologs also exist in the related strains Methanospirillum stamsii Pt1 (7 homologs, 28-66% sequence identity), M. lacunae Ki8-1 C (15 homologs, 29-60% sequence identity) and Methanolinea tarda NOBI-1 (2 homologs, 31% sequence identity). The MspA protein discovered here could accordingly represent a more widely found sheath protein than the MspA from M. thermophila PT, which currently has no homologs in the NCBI Reference Sequence database (RefSeq).

Novel prosthecate bacteria from the candidate phylum Acetothermia.

Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an operational taxonomic unit belonging to Acetothermia was detected at high abundance in four full-scale anaerobic digesters by 16S rRNA gene amplicon sequencing. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with long nanopore reads. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtains energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy, and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

High-Quality Draft Genome Sequence of Sphaerisporangium cinnabarinum ATCC 31213.

A high-quality draft genome sequence of Sphaerisporangium cinnabarinum ATCC 31213 is presented here. This bacterium produces several important bioactive compounds and may also produce functional amyloids. This is the first sequenced genome from the genus Sphaerisporangium, and it will be essential in determining the nature of the potential amyloid protein.

Genomic insights into members of the candidate phylum Hyd24-12 common in mesophilic anaerobic digesters.

Members of the candidate phylum Hyd24-12 are globally distributed, but no genomic information or knowledge about their morphology, physiology or ecology is available. In this study, members of the Hyd24-12 lineage were shown to be present and abundant in full-scale mesophilic anaerobic digesters at Danish wastewater treatment facilities. In some samples, a member of the Hyd24-12 lineage was one of the most abundant genus-level bacterial taxa, accounting for up to 8% of the bacterial biomass. Three closely related and near-complete genomes were retrieved using metagenome sequencing of full-scale anaerobic digesters. Genome annotation and metabolic reconstruction showed that they are Gram-negative bacteria likely involved in acidogenesis, producing acetate and hydrogen from fermentation of sugars, and may play a role in the cycling of sulphur in the digesters. Fluorescence in situ hybridization revealed single rod-shaped cells dispersed within the flocs. The genomic information forms a foundation for a more detailed understanding of their role in anaerobic digestion and provides the first insight into a hitherto undescribed branch in the tree of life.

Genomic and in situ investigations of the novel uncultured Chloroflexi associated with 0092 morphotype filamentous bulking in activated sludge.

Overgrowth of filamentous bacteria in activated sludge wastewater treatment plants (WWTPs) leads to impaired sludge settleability, a condition known as bulking, which is a common operational problem worldwide. Filaments with the Eikelboom 0092 morphotype are commonly associated with such bulking episodes. Members of the uncultured B45 phylotype, which is embraced within the phylum Chloroflexi, were recently shown to exhibit this morphology. Although these organisms are among the most abundant populations recorded in activated sludge processes, nothing is known about their metabolic characteristics. In this study, a genome sequence, representing the B45 phylotype, was retrieved from a metagenome generated from an activated sludge WWTP. The genome consisted of two chromosomes and one plasmid, which were 4.0, 1.0 and 0.04 Mbps in size, respectively. A metabolic model was constructed for this organism, based on annotation of its genome, showing its ability to generate energy by respiration, utilizing oxygen, nitrite or nitrous oxide as electron acceptors, or by fermentation of sugars. The ability of B45 members to ferment sugars under anaerobic conditions was validated in situ with microautoradiography-fluorescence in situ hybridization. The provisional name of 'Candidatus Promineofilum breve' is proposed for this species. This study represents the first detailed information on an uncultured genus of filamentous organisms from activated sludge.

The fungal community changes over time in developing wheat heads.

Under normal conditions, wheat is colonized by a multitude of fungi that can have beneficial or adverse effects on plant growth and yield. To study the effect of spraying wheat heads with fungicides on the fungal community from emergence to harvest we applied an amplicon sequencing approach on single wheat heads. The climatic data showed that the spring of 2014 was very dry and without precipitation in the two weeks around flowering. An initial quantitative PCR showed that the total amount of fungal DNA increased during the entire period, without significant difference between sprayed and control wheat heads. Amplicon sequencing of the internal transcribed spacer 2 (ITS2) region showed that operational taxonomic units (OTUs) identified as Sporobolomyces roseus dominated in the first weeks, whereas Alternaria infectoria OTUs dominated in the last weeks before harvest. The only observed significant difference was that the control wheat heads contained more of the powdery mildew causing Blumeria graminis f. sp. tritici OTUs compared with the sprayed wheat heads. The dry conditions around flowering most likely also had an effect on Fusarium head blight infection as Fusarium OTUs were only sporadically encountered. Analyses of secondary metabolites produced by Fusarium and Alternaria in the wheat heads confirmed the observations from the amplicon sequencing. Enniatin B was the most frequent contaminant present in four sprayed (49-538 ng/g) and three control (56-355 ng/g) wheat heads. The A. infectoria secondary metabolites infectopyrone and 4Z-infectopyrone were however consistently observed in all samples collected the last five weeks before harvest.

Detection of Pathogenic Biofilms with Bacterial Amyloid Targeting Fluorescent Probe, CDy11.

Bacterial biofilms are responsible for a wide range of persistent infections. In the clinic, diagnosis of biofilm-associated infections relies heavily on culturing methods, which fail to detect nonculturable bacteria. Identification of novel fluorescent probes for biofilm imaging will greatly facilitate diagnosis of pathogenic bacterial infection. Herein, we report a novel fluorescent probe, CDy11 (compound of designation yellow 11), which targets amyloid in the Pseudomonas aeruginosa biofilm matrix through a diversity oriented fluorescent library approach (DOFLA). CDy11 was further demonstrated for in vivo imaging of P. aeruginosa in implant and corneal infection mice models.

Label-free quantification reveals major proteomic changes in Pseudomonas putida F1 during the exponential growth phase.

The physiological adaptation to stationary growth by Pseudomonas putida F1, a model organism for the degradation of aromatic compounds, was investigated by proteome-wide label-free quantification.The data unveiled that entrance to the stationary phase did not involve an abrupt switch within the P. putida F1 proteome, but rather an ongoing adaptation that started already during the mid-exponential growth phase. The proteomic adaptations involved a clear increase in amino acid degradation capabilities and a loss of transcriptional as well as translational capacity. The final entrance to the stationary phase was accompanied by increased oxidative stress protection, although the stress and stationary sigma factor RpoS increased in abundance already during mid-exponential growth. The results show that it is important to consider significant sample variations when exponentially growing cultures are studied alone or compared across proteomic or transcriptomic literature. All MS data have been deposited in the ProteomeXchange with identifier PXD001219 (http://proteomecentral.proteomexchange.org/dataset/PXD001219).

Complete Genome Sequence of the Bacterium Aalborg_AAW-1, Representing a Novel Family within the Candidate Phylum SR1.

Here, we present the complete genome sequence of the candidate phylum SR1 bacterium Aalborg_AAW-1. Its 16S rRNA gene is only 85.5% similar to that of the closest relative, RAAC1_SR1, and the genome of Aalborg_AAW-1 consequently represents the first of a novel family within the candidate phylum SR1.

Complete Genome Sequence of Pseudomonas sp. UK4, a Model Organism for Studies of Functional Amyloids in Pseudomonas.

Here, we present the complete genome of Pseudomonas sp. UK4. This bacterium was the first Pseudomonas strain shown to produce functional amyloids, and it represents a model organism for studies of functional amyloids in Pseudomonas (Fap).

Widespread abundance of functional bacterial amyloid in mycolata and other gram-positive bacteria.

Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, including Escherichia coli, Salmonella, and the gram-positive organism Streptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six other distantly related species examined belonging to the phyla Actinobacteria and Firmicutes. Most of the bacteria produced extracellular fimbriae, sometimes copious amounts of them, and in two cases large extracellular fibrils were also produced. In three cases, FuBA was revealed only after extensive removal of extracellular material by saponification, indicating that there is integrated attachment within the cellular envelope. Spores of species in the genera Streptomyces, Bacillus, and Nocardia were all coated with amyloids. FuBA was purified from Gordonia amarae (from the cell envelope) and Geodermatophilus obscurus, and they had the morphology, tinctorial properties, and beta-rich structure typical of amyloid. The presence of approximately 9-nm-wide amyloids in the cell envelope of G. amarae was visualized by transmission electron microscopy analysis. We conclude that amyloid is widespread among gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spore and the cellular envelope.

Amyloid adhesins are abundant in natural biofilms.

Surface-associated amyloid fibrils have been described by bacteria in the family Enterbacteriaceae, but it is unknown to what extent amyloid adhesins are present in natural biofilms. In this study, amyloid adhesins were specifically stained with Thioflavin T and two conformationally specific antibodies targeting amyloid fibrils. These three independent detection methods were each combined with fluorescence in situ hybridization using fluorescently labelled oligonucleotide probes in order to link phenotype with identity. Escherichia coli mutants with and without amyloid adhesins (curli) served as controls. In biofilms from four different natural habitats, bacteria producing extracellular amyloid adhesins were identified within several phyla: Proteobacteria (Alpha-, Beta-, Gamma- and Deltaproteobacteria), Bacteriodetes, Chloroflexi and Actinobacteria, and most likely also in other phyla. Quantification of the microorganisms producing amyloid adhesins showed that they constituted at least 5-40% of all prokaryotes present in the biofilms, depending on the habitat. Particularly in drinking water biofilms, a high number of amyloid-positive bacteria were identified. Production of amyloids was confirmed by environmental isolates belonging to the Gammaproteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. The new approach is a very useful tool for further culture-independent studies in mixed microbial communities, where the abundance and diversity of bacteria expressing amyloid adhesins seems much greater than hitherto anticipated.