In situ Sub-Cellular Identification of Functional Amyloids in Bacteria and Archaea by Infrared Nanospectroscopy.

Formation of amyloid structures is originally linked to human disease. However, amyloid materials are found extensively in the animal and bacterial world where they stabilize intra- and extra-cellular environments like biofilms or cell envelopes. To date, functional amyloids have largely been studied using optical microscopy techniques in vivo, or after removal from their biological context for higher-resolution studies in vitro. Furthermore, conventional microscopies only indirectly identify amyloids based on morphology or unspecific amyloid dyes. Here, the high chemical and spatial (≈20 nm) resolution of Infrared Nanospectroscopy (AFM-IR) to investigate functional amyloid from Escherichia coli (curli), Pseudomonas (Fap), and the Archaea Methanosaeta (MspA) in situ is exploited. It is demonstrated that AFM-IR identifies amyloid protein within single intact cells through their cross ß-sheet secondary structure, which has a unique spectroscopic signature in the amide I band of protein. Using this approach, nanoscale-resolved chemical images and spectra of purified curli and Methanosaeta cell wall sheaths are provided. The results highlight significant differences in secondary structure between E. coli cells with and without curli. Taken together, these results suggest that AFM-IR is a new and powerful label-free tool for in situ investigations of the biophysical state of functional amyloid and biomolecules in general.

Low Global Diversity of Candidatus Microthrix, a Troublesome Filamentous Organism in Full-Scale WWTPs.

Candidatus Microthrix is one of the most common bulking filamentous microorganisms found in activated sludge wastewater treatment plants (WWTPs) across the globe. One species, Ca. M. parvicella, is frequently observed, but global genus diversity, as well as important aspects of its ecology and physiology, are still unknown. Here, we use the MiDAS ecosystem-specific 16S rRNA gene database in combination with amplicon sequencing of Danish and global WWTPs to investigate Ca. Microthrix spp. diversity, distribution, and factors affecting their global presence. Only two species were abundant across the world confirming low diversity of the genus: the dominant Ca. M. parvicella and an unknown species typically present along with Ca. M. parvicella, although usually in lower abundances. Both species were mostly found in Europe at low-to-moderate temperatures and their growth was favored in municipal WWTPs with advanced process designs. As no isolate is available for the novel species, we propose the name "Candidatus Microthrix subdominans." Ten high-quality metagenome-assembled genomes recovered from Danish WWTPs, including 6 representing the novel Ca. M. subdominans, demonstrated high genetic similarity between the two species with a likely preference for lipids, a putative capability to reduce nitrate and nitrite, and the potential to store lipids and poly-P. Ca. M. subdominans had a potentially more versatile metabolism including additional sugar transporters, higher oxygen tolerance, and the potential to use carbon monoxide as energy source. Newly designed fluorescence in situ hybridization probes revealed similar filamentous morphology for both species. Raman microspectroscopy was used to quantify the in situ levels of intracellular poly-P. Despite the observed similarities in their physiology (both by genomes and in situ), the two species showed different seasonal dynamics in Danish WWTPs through a 13-years survey, possibly indicating occupation of slightly different niches. The genomic information provides the basis for future research into in situ gene expression and regulation, while the new FISH probes provide a useful tool for further characterization in situ. This study is an important step toward understanding the ecology of Ca. Microthrix in WWTPs, which may eventually lead to optimization of control strategies for its growth in this ecosystem.

Connecting structure to function with the recovery of over 1000 high-quality metagenome-assembled genomes from activated sludge using long-read sequencing.

Microorganisms play crucial roles in water recycling, pollution removal and resource recovery in the wastewater industry. The structure of these microbial communities is increasingly understood based on 16S rRNA amplicon sequencing data. However, such data cannot be linked to functional potential in the absence of high-quality metagenome-assembled genomes (MAGs) for nearly all species. Here, we use long-read and short-read sequencing to recover 1083 high-quality MAGs, including 57 closed circular genomes, from 23 Danish full-scale wastewater treatment plants. The MAGs account for ~30% of the community based on relative abundance, and meet the stringent MIMAG high-quality draft requirements including full-length rRNA genes. We use the information provided by these MAGs in combination with >13 years of 16S rRNA amplicon sequencing data, as well as Raman microspectroscopy and fluorescence in situ hybridisation, to uncover abundant undescribed lineages belonging to important functional groups.

"Candidatus Galacturonibacter soehngenii" Shows Acetogenic Catabolism of Galacturonic Acid but Lacks a Canonical Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase Complex.

Acetogens have the ability to fixate carbon during fermentation by employing the Wood-Ljungdahl pathway (WLP), which is highly conserved across Bacteria and Archaea. In a previous study, product stoichometries in galacturonate-limited, anaerobic enrichment cultures of "Candidatus Galacturonibacter soehngenii," from a novel genus within the Lachnospiraceae, suggested the simultaneous operation of a modified Entner-Doudoroff pathway for galacturonate fermentation and a WLP for acetogenesis. However, a draft metagenome-assembled genome (MAG) based on short reads did not reveal homologs of genes encoding a canonical WLP carbon-monoxide-dehydrogenase/acetyl-Coenzyme A synthase (CODH/ACS) complex. In this study, NaH13CO3 fed to chemostat-grown, galacturonate-limited enrichment cultures of "Ca. G. soehngenii" was shown to be incorporated into acetate. Preferential labeling of the carboxyl group of acetate was consistent with acetogenesis via a WLP in which the methyl group of acetate was predominately derived from formate. This interpretation was further supported by high transcript levels of a putative pyruvate-formate lyase gene and very low transcript levels of a candidate gene for formate dehydrogenase. Reassembly of the "Ca. G. soehngenii" MAG with support from long-read nanopore sequencing data produced a single-scaffold MAG, which confirmed the absence of canonical CODH/ACS-complex genes homologs. However, high CO-dehydrogenase activities were measured in cell extracts of "Ca. G. soehngenii" enrichment cultures, contradicting the absence of corresponding homologs in the MAG. Based on the highly conserved amino-acid motif associated with anaerobic Ni-CO dehydrogenase proteins, a novel candidate was identified which could be responsible for the observed activities. These results demonstrate operation of an acetogenic pathway, most probably as a yet unresolved variant of the Wood-Ljungdahl pathway, in anaerobic, galacturonate-limited cultures of "Ca. G. soehngenii."

On the evolution and physiology of cable bacteria.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO2 using the Wood-Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N2, and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

The Proteome of Tetrasphaera elongata is adapted to Changing Conditions in Wastewater Treatment Plants.

The activated sludge in wastewater treatment plants (WWTP) designed for enhanced biological phosphorus removal (EBPR) experiences periodically changing nutrient and oxygen availability. Tetrasphaera is the most abundant genus in Danish WWTP and represents up to 20-30% of the activated sludge community based on 16S rRNA amplicon sequencing and quantitative fluorescence in situ hybridization analyses, although the genus is in low abundance in the influent wastewater. Here we investigated how Tetrasphaera can successfully out-compete most other microorganisms in such highly dynamic ecosystems. To achieve this, we analyzed the physiological adaptations of the WWTP isolate T. elongata str. LP2 during an aerobic to anoxic shift by label-free quantitative proteomics and NMR-metabolomics. Escherichia coli was used as reference organism as it shares several metabolic capabilities and is regularly introduced to wastewater treatment plants without succeeding there. When compared to E. coli, only minor changes in the proteome of T. elongata were observed after the switch to anoxic conditions. This indicates that metabolic pathways for anaerobic energy harvest were already expressed during the aerobic growth. This allows continuous growth of Tetrasphaera immediately after the switch to anoxic conditions. Metabolomics furthermore revealed that the substrates provided were exploited far more efficiently by Tetrasphaera than by E. coli. These results suggest that T. elongata prospers in the dynamic WWTP environment due to adaptation to the changing environmental conditions.

Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal - as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the "Candidatus Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

Ecology and Biogenesis of Functional Amyloids in Pseudomonas.

Functional amyloids can be found in the extracellular matrix produced by many bacteria during biofilm growth. They mediate the initial attachment of bacteria to surfaces and provide stability and functionality to mature biofilms. Efficient amyloid biogenesis requires a highly coordinated system of amyloid subunits, molecular chaperones and transport systems. The functional amyloid of Pseudomonas (Fap) represents such a system. Here, we review the phylogenetic diversification of the Fap system, its potential ecological role and the dedicated machinery required for Fap biogenesis, with a particular focus on the amyloid exporter FapF, the structure of which has been recently resolved. We also present a sequence covariance-based in silico model of the FapC fiber-forming subunit. Finally, we highlight key questions that remain unanswered and we believe deserve further attention by the scientific community.

Retrieval of a million high-quality, full-length microbial 16S and 18S rRNA gene sequences without primer bias.

Small subunit ribosomal RNA (SSU rRNA) genes, 16S in bacteria and 18S in eukaryotes, have been the standard phylogenetic markers used to characterize microbial diversity and evolution for decades. However, the reference databases of full-length SSU rRNA gene sequences are skewed to well-studied ecosystems and subject to primer bias and chimerism, which results in an incomplete view of the diversity present in a sample. We combine poly(A)-tailing and reverse transcription of SSU rRNA molecules with synthetic long-read sequencing to generate high-quality, full-length SSU rRNA sequences, without primer bias, at high throughput. We apply our approach to samples from seven different ecosystems and obtain more than a million SSU rRNA sequences from all domains of life, with an estimated raw error rate of 0.17%. We observe a large proportion of novel diversity, including several deeply branching phylum-level lineages putatively related to the Asgard Archaea. Our approach will enable expansion of the SSU rRNA reference databases by orders of magnitude, and contribute to a comprehensive census of the tree of life.

The impact of immigration on microbial community composition in full-scale anaerobic digesters.

Anaerobic digestion is widely applied to treat organic waste at wastewater treatment plants. Characterisation of the underlying microbiology represents a source of information to develop strategies for improved operation. Hence, we investigated microbial communities of thirty-two full-scale anaerobic digesters over a six-year period using 16S rRNA gene amplicon sequencing. Sampling of the sludge fed into these systems revealed that several of the most abundant populations were likely inactive and immigrating with the influent. This observation indicates that a failure to consider immigration will interfere with correlation analysis and give an inaccurate picture of the growing microbial community. Furthermore, several abundant OTUs could not be classified to genus level with commonly applied taxonomies, making inference of their function unreliable and comparison to other studies problematic. As such, the existing MiDAS taxonomy was updated to include these abundant phylotypes. The communities of individual digesters surveyed were remarkably similar - with only 300 OTUs representing 80% of the total reads across all plants, and 15% of these identified as non-growing and possibly inactive immigrating microbes. By identifying abundant and growing taxa in anaerobic digestion, this study paves the way for targeted characterisation of the process-important organisms towards an in-depth understanding of the microbiology.

Direct Identification of Functional Amyloid Proteins by Label-Free Quantitative Mass Spectrometry.

Functional amyloids are important structural and functional components of many biofilms, yet our knowledge of these fascinating polymers is limited to a few examples for which the native amyloids have been isolated in pure form. Isolation of the functional amyloids from other cell components represents a major bottleneck in the search for new functional amyloid systems. Here we present a label-free quantitative mass spectrometry method that allows identification of amyloid proteins directly in cell lysates. The method takes advantage of the extreme structural stability and polymeric nature of functional amyloids and the ability of concentrated formic acid to depolymerize the amyloids. An automated data processing pipeline that provides a short list of amyloid protein candidates was developed based on an amyloid-specific sigmoidal abundance signature in samples treated with increasing concentrations of formic acid. The method was evaluated using the Escherichiacoli curli and the Pseudomonas Fap system. It confidently identified the major amyloid subunit for both systems, as well as the minor subunit for the curli system. A few non-amyloid proteins also displayed the sigmoidal abundance signature. However, only one of these contained a sec-dependent signal peptide, which characterizes most of all secreted proteins, including all currently known functional bacterial amyloids.

Culture-Independent Analyses Reveal Novel Anaerolineaceae as Abundant Primary Fermenters in Anaerobic Digesters Treating Waste Activated Sludge.

Anaerobic digestion for biogas production is reliant on the tightly coupled synergistic activities of complex microbial consortia. Members of the uncultured A6 phylotype, within the phylum Chloroflexi, are among the most abundant genus-level-taxa of mesophilic anaerobic digester systems treating primary and surplus sludge from wastewater treatment plants, yet are known only by their 16S rRNA gene sequence. This study applied metagenomics to obtain a complete circular genome (2.57 Mbp) from a representative of the A6 taxon. Preliminary annotation of the genome indicates these organisms to be anaerobic chemoorganoheterotrophs with a fermentative metabolism. Given their observed abundance, they are likely important primary fermenters in digester systems. Application of fluorescence in situ hybridisation probes designed in this study revealed their morphology to be short filaments present within the flocs. The A6 were sometimes co-located with the filamentous Archaea Methanosaeta spp. suggesting potential undetermined synergistic relationships. Based on its genome sequence and morphology we propose the species name Brevefilum fermentans gen. nov. sp. nov.

Epigallocatechin Gallate Remodels Overexpressed Functional Amyloids in Pseudomonas aeruginosa and Increases Biofilm Susceptibility to Antibiotic Treatment.

Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea. It has antimicrobial properties and disrupts the ordered structure of amyloid fibrils involved in human disease. The antimicrobial effect of EGCG against the opportunistic pathogen Pseudomonas aeruginosa has been shown to involve disruption of quorum sensing (QS). Functional amyloid fibrils in P. aeruginosa (Fap) are able to bind and retain quorum-sensing molecules, suggesting that EGCG interferes with QS through structural remodeling of amyloid fibrils. Here we show that EGCG inhibits the ability of Fap to form fibrils; instead, EGCG stabilizes protein oligomers. Existing fibrils are remodeled by EGCG into non-amyloid aggregates. This fibril remodeling increases the binding of pyocyanin, demonstrating a mechanism by which EGCG can affect the QS function of functional amyloid. EGCG reduced the amyloid-specific fluorescent thioflavin T signal in P. aeruginosa biofilms at concentrations known to exert an antimicrobial effect. Nanoindentation studies showed that EGCG reduced the stiffness of biofilm containing Fap fibrils but not in biofilm with little Fap. In a combination treatment with EGCG and tobramycin, EGCG had a moderate effect on the minimum bactericidal eradication concentration against wild-type P. aeruginosa biofilms, whereas EGCG had a more pronounced effect when Fap was overexpressed. Our results provide a direct molecular explanation for the ability of EGCG to disrupt P. aeruginosa QS and modify its biofilm and strengthens the case for EGCG as a candidate in multidrug treatment of persistent biofilm infections.

Functional bacterial amyloid increases Pseudomonas biofilm hydrophobicity and stiffness.

The success of Pseudomonas species as opportunistic pathogens derives in great part from their ability to form stable biofilms that offer protection against chemical and mechanical attack. The extracellular matrix of biofilms contains numerous biomolecules, and it has recently been discovered that in Pseudomonas one of the components includes ß-sheet rich amyloid fibrils (functional amyloid) produced by the fap operon. However, the role of the functional amyloid within the biofilm has not yet been investigated in detail. Here we investigate how the fap-based amyloid produced by Pseudomonas affects biofilm hydrophobicity and mechanical properties. Using atomic force microscopy imaging and force spectroscopy, we show that the amyloid renders individual cells more resistant to drying and alters their interactions with hydrophobic probes. Importantly, amyloid makes Pseudomonas more hydrophobic and increases biofilm stiffness 20-fold. Deletion of any one of the individual members of in the fap operon (except the putative chaperone FapA) abolishes this ability to increase biofilm stiffness and correlates with the loss of amyloid. We conclude that amyloid makes major contributions to biofilm mechanical robustness.

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids.

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.

Survival and activity of individual bioaugmentation strains.

Successful application of bioaugmentation for enhanced degradation of environmental pollutants is often limited by the lack of methods to monitor the survival and activity of individual bioaugmentation strains. However, recent advancements in sequencing technologies and molecular techniques now allow us to address these limitations. Here a complementing set of general applicable molecular methods are presented that provides detailed information on the performance of individual bioaugmentation strains under in situ conditions. The approach involves genome sequencing to establish highly specific qPCR and RT-qPCR tools for cell enumerations and expression of involved genes, stable isotope probing to follow growth on the target compounds and GFP-tagging to visualize the bioaugmentation strains directly in samples, all in combination with removal studies of the target compounds. The concept of the approach is demonstrated through a case study involving degradation of aromatic hydrocarbons in activated sludge augmented with the bioaugmentation strain Pseudomonas monteilii SB3078.

Functional amyloids keep quorum-sensing molecules in check.

The mechanism by which extracellular metabolites, including redox mediators and quorum-sensing signaling molecules, traffic through the extracellular matrix of biofilms is poorly explored. We hypothesize that functional amyloids, abundant in natural biofilms and possessing hydrophobic domains, retain these metabolites. Using surface plasmon resonance, we demonstrate that the quorum-sensing (QS) molecules, 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone, and the redox mediator pyocyanin bind with transient affinity to functional amyloids from Pseudomonas (Fap). Their high hydrophobicity predisposes them to signal-amyloid interactions, but specific interactions also play a role. Transient interactions allow for rapid association and dissociation kinetics, which make the QS molecules bioavailable and at the same time secure within the extracellular matrix as a consequence of serial bindings. Retention of the QS molecules was confirmed using Pseudomonas aeruginosa PAO1-based 2-heptyl-3-hydroxy-4(1H)-quinolone and N-(3-oxododecanoyl)-l-homoserine lactone reporter assays, showing that Fap fibrils pretreated with the QS molecules activate the reporters even after sequential washes. Pyocyanin retention was validated by electrochemical analysis of pyocyanin-pretreated Fap fibrils subjected to the same washing process. Results suggest that QS molecule-amyloid interactions are probably important in the turbulent environments commonly encountered in natural habitats.

Major proteomic changes associated with amyloid-induced biofilm formation in Pseudomonas aeruginosa PAO1.

The newly identified functional amyloids in Pseudomonas (Fap) are associated with increased aggregation and biofilm formation in the opportunistic pathogen P. aeruginosa; however, whether this phenomenon can be simply ascribed to the mechanical properties of the amyloid fibrils remains undetermined. To gain a deeper understanding of the Fap-mediated biofilm formation, the physiological consequences of Fap expression were investigated using label-free protein quantification. The functional amyloids were found to not solely act as inert structural biofilm components. Their presence induced major changes in the global proteome of the bacterium. These included the lowered abundance of classical virulence factors such as elastase B and the secretion system of alkaline protease A. Amyloid-mediated biofilm formation furthermore increased abundance of the alginate and pyoverdine synthesis machinery, which turned P. aeruginosa PAO1 into an unexpected mucoid phenotype. The results imply a significant impact of functional amyloids on the physiology of P. aeruginosa with subsequent implications for biofilm formation and chronic infections.

Complete Genome Sequence of Actinobaculum schaalii Strain CCUG 27420.

Complete genome sequencing of the emerging uropathogen Actinobaculum schaalii indicates that an important mechanism of its virulence is attachment pili, which allow the organism to adhere to the surface of animal cells, greatly enhancing the ability of this organism to colonize the urinary tract.

Complete Genome Sequences of Pseudomonas monteilii SB3078 and SB3101, Two Benzene-, Toluene-, and Ethylbenzene-Degrading Bacteria Used for Bioaugmentation.

Pseudomonas monteilii SB3078 and SB3101 are benzene-, toluene-, and ethylbenzene-degrading strains used for bioaugmentation in relation to treatment of wastewater contaminated with petrochemical hydrocarbons. Complete genome sequencing of the bioaugmentation strains confirms that they are very closely related (100.0% average nucleotide identity). Both strains contain extensive integration of phage elements, with the main difference being insertion of additional phage elements in the SB3078 genome.