Search

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. [ABSTRACT FROM AUTHOR]

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 β‐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. [ABSTRACT FROM AUTHOR]

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. Microbes play key roles in wastewater treatment. Here, Singleton et al. use long-read and short-read sequencing to recover 1083 high-quality metagenome-assembled genomes from 23 wastewater treatment plants, and combine this information with amplicon data, Raman microspectroscopy and FISH to reveal functionally important lineages. [ABSTRACT FROM AUTHOR]

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." [ABSTRACT FROM AUTHOR]

Cable bacteria of the family Desulfobulbaceae form centimeterlong 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Escherichia coli 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article reflects on the study related to the role of functional bacterial amyloid in increasing Pseudomonas biofilm hydrophobicity and stiffness and deletion of any one of the particular members of in the fap operon destroys this ability to increase biofilm stiffness.

Escherichia coli and a few other members of the Enterobacteriales can produce functional amyloids known as curli. These extracellular fibrils are involved in biofilm formation and studies have shown that they may act as virulence factors during infections. It is not known whether curli fibrils are restricted to the Enterobacteriales or if they are phylogenetically widespread. The growing number of genome-sequenced bacteria spanning many phylogenetic groups allows a reliable bioinformatic investigation of the phylogenetic diversity of the curli system. Here we show that the curli system is phylogenetically much more widespread than initially assumed, spanning at least four phyla. Curli fibrils may consequently be encountered frequently in environmental as well as pathogenic biofilms, which was supported by identification of curli genes in public metagenomes from a diverse range of habitats. Identification and comparison of curli subunit (CsgA/B) homologs show that these proteins allow a high degree of freedom in their primary protein structure, although a modular structure of tightly spaced repeat regions containing conserved glutamine, asparagine and glycine residues has to be preserved. In addition, a high degree of variability within the operon structure of curli subunits between bacterial taxa suggests that the curli fibrils might have evolved to fulfill specific functions. Variations in the genetic organization of curli genes are also seen among different bacterial genera. This suggests that some genera may utilize alternative regulatory pathways for curli expression. Comparison of phylogenetic trees of Csg proteins and the 16S rRNA genes of the corresponding bacteria showed remarkably similar overall topography, suggesting that horizontal gene transfer is a minor player in the spreading of the curli system. [ABSTRACT FROM AUTHOR]

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA–F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation. [ABSTRACT FROM AUTHOR]

Functional bacterial amyloids (FuBA) are important components in many environmental biofilms where they provide structural integrity to the biofilm, mediate bacterial aggregation and may function as virulence factor by binding specifically to host cell molecules. A novel FuBA system, the Fap system, was previously characterized in the genus Pseudomonas, however, very little is known about the phylogenetic diversity of bacteria with the genetic capacity to apply this system. Studies of genomes and public metagenomes from a diverse range of habitats showed that the Fap system is restricted to only three classes in the phylum Proteobacteria, the Beta-, Gamma- and Deltaproteobacteria. The structural organization of the fap genes into a single fapABCDEF operon is well conserved with minor variations such as a frequent deletion of fapA. A high degree of variation was seen within the primary structure of the major Fap fibril monomers, FapC, whereas the minor monomers, FapB, showed less sequence variation. Comparison of phylogenetic trees based on Fap proteins and the 16S rRNA gene of the corresponding bacteria showed remarkably similar overall topology. This indicates, that horizontal gene transfer is an infrequent event in the evolution of the Fap system. [ABSTRACT FROM AUTHOR]