Phylogenetic analysis reveals diversity in glycan biosynthesis in "Candidatus Accumulibacter".

Biological wastewater treatment relies on microorganisms that grow as flocs, biofilms, or granules for efficient separation of biomass from cleaned water. This biofilm structure emerges from the interactions between microbes that produce, and are embedded in, extracellular polymeric substances (EPS). The true composition and structure of the EPS responsible for dense biofilm formation are still obscure. We conducted a bottom-up approach utilizing advanced glycomic techniques to explore the glycan diversity in the EPS from a highly enriched "Candidatus Accumulibacter" granular sludge. Rare novel sugar monomers such as N-Acetylquinovosamine (QuiNAc) and 2-O-Methylrhamnose (2-OMe-Rha) were identified to be present in the EPS of both enrichments. Further, a high diversity in the glycoprotein structures of said EPS was identified by means of lectin based microarrays. We explored the genetic potential of "Ca. Accumulibacter" high quality metagenome assembled genomes (MAGs) to showcase the shortcoming of top-down bioinformatics based approaches at predicting EPS composition and structure, especially when dealing with glycans and glycoconjugates. This work suggests that more bottom-up research is necessary to understand the composition and complex structure of EPS in biofilms since genome based inference cannot directly predict glycan structures and glycoconjugate diversity.

Pseudaminic and legionaminic acids are a subgroup of nonulosonic acids (NulOs) unique to bacterial species. There is a lack of advances in the study of these NulOs due to their complex synthesis and production. Recently, it was seen that "Candidatus Accumulibacter" can produce Pse or Leg analogues as part of its extracellular polymeric substances (EPS). In order to employ a "Ca. Accumulibacter" enrichment as production platform for bacterial sialic acids, it is necessary to determine which fractions of the EPS of "Ca. Accumulibacter" contain NulOs and how to enrich and/or isolate them. We extracted the EPS from granules enriched with "Ca. Accumulibcater" and used size-exclusion chromatography (SEC) to separate them into different molecular weight (MW) fractions. This separation resulted in two high molecular weight (> 5500kDa) fractions dominated by polysaccharides, with a NulO content up to 4 times higher than the extracted EPS. This suggests that NulOs in "Ca. Accumulibacter" are likely located in high molecular weight polysaccharides. Additionally, it was seen that the extracted EPS and the NulO-rich fractions can bind and neutralize histones. This opens the possibility of EPS and NulO-rich fractions as potential source for sepsis treatment drugs. KEY POINTS: • NulOs in "Ca. Accumulibacter" are likely located in high MW polysaccharides • SEC allows to obtain high MW polysaccharide-rich fractions enriched with NulOs • EPS and the NulOs-rich fractions are a potential source for sepsis treatment drugs.

Identifying structural proteins within the extracellular polymeric substances (EPS) will provide a better understanding of the stability of aerobic granular sludge (AGS) and biofilms in general. In this work, an abundant surface protein was identified and localized in the extracellular matrix of seawater-adapted AGS. Granules with good phosphate removal were cultivated in a sequencing batch bubble column reactor with acetate as a carbon source dissolved in seawater. "Candidatus Accumulibacter" was observed as the most dominant community member through fluorescent in-situ hybridization. A surface protein of 74.5 kDa was identified in the EPS extract of the seawater-adapted AGS by SDS-PAGE and mass spectrometry. The surface protein was produced by an Accumulibacter species and showed homology to S-layer proteins. A type 1 secretion system was found adjacent to the gene encoding for the surface protein, suggesting a possible export system. Antibodies generated from a unique peptide of the surface protein confirmed the extracellular location of the surface protein. Microscopy observations with antibody staining showed the surface protein forms dense structures within the Accumulibacter microcolonies and larger fiber structures around the microcolonies. These observations highlight the importance of the protein for the structural properties of the granule. To detect more structural proteins in the EPS, optimization of the EPS extraction and in situ imaging validation methods are essential.

Nanoscale zero-valent iron alleviated horizontal transfer of antibiotic resistance genes in soil: The important role of extracellular polymeric substances

Antibiotic resistance genes (ARGs) in bacteria are emerging contaminants as their proliferation in the environment poses significant threats to human health. It is well recognized that extracellular polymeric substances (EPS) can protect microorganisms against stress or damage from exogenous contaminants. However, it is not clear whether EPS could affect the lateral transfer of ARGs into bacteria, which is one of the major processes for the dissemination of ARGs. This study investigated the lateral transfer of ARGs carried by plasmids (pUC19, pHSG298, and pHSG396) into competent Escherichia coli cells with and without EPS. Transformant numbers and transformation efficiency for E. coli without EPS were up to 29 times of those with EPS at pH 7.0 in an aqueous system. The EPS removal further increased cell permeability in addition to the enhanced cell permeability by Ca2+, which could be responsible for the enhanced lateral transfer of ARGs. The fluorescence quenching experiments showed that EPS could strongly bind to plasmid DNA in the presence of Ca2+ and the binding strength (LogKA = 10.65–15.80 L mol-1) between EPS and plasmids was positively correlated with the enhancement percentage of transformation efficiency resulting from the EPS removal. X-ray photoelectron spectroscopy (XPS) analyses and model computation further showed that Ca2+ could electrostatically bind with EPS mainly through the carboxyl group, hydroxyl group, and RC-O-CR in glucoside, thus bridging the plasmid and EPS. As a result, the binding of plasmids with EPS hindered the lateral transfer of plasmid-borne ARGs. This study improved our understanding on the function of EPS in controlling the fate and transport of ARGs on the molecular and cellular scales.

The spread of antibiotic resistance genes (ARGs) via horizontal gene transfer (HGT) in wastewater treatment processes presents a critical One-Health challenge. While extracellular polymeric substances (EPS) are known to envelop microbial cells and mediate intercellular interactions, their role in conjugation, the predominant HGT mode, remains unclear. Herein, we developed an in vivo framework to investigate the impacts of EPS on conjugation. Simulating the generation of antibiotic-resistant Pseudomonas aeruginosa, a critical ESKAPE pathogen, we found that EPS significantly shaped conjugative behaviors with their depletion consistently reducing conjugation occurrences. Mechanistic investigations revealed that while EPS removal increased the cell membrane permeability, community-level reactive oxygen species (ROS), and virulence gene expression, it also led to decreased intracellular energy production and diminished transcription of key conjugation components. Furthermore, EPS depletion compromised the physical integrity of microbial community structures such as biofilms, weakened cell-to-cell contact, and reduced biomass of microbes involved in conjugation. These factors collectively determine the fate of conjugation events. To further validate the regulatory role of EPS, we engineered a CRISPR-ddCas12a system to repress EPS biosynthesis, significantly suppressing the conjugation of ARGs. This work provides critical insights into conjugation mechanisms and underscores the potential of targeting EPS to limit conjugation in wastewater treatment.

The long-term effects of environmental conditions, such as seawater salinity, on the extracellular investigated EPS changes during a stepwise increase in salinity (0-4%), renewing over 90% of biomass at each condition. Stable granulation, complete anaerobic acetate uptake, and phosphate removal were maintained throughout. FT-IR of granules showed significant changes in glycans (1025 cm⁻¹) and sialic acid (1730 cm⁻¹), which were reflected in the EPS. Lectin microarray revealed that increasing salinity reduced glycan diversity in EPS glycoproteins, while increasing negatively charged groups, including sialic acids and sulfated groups. At 4% salinity, EPS negative charge increased by 19.8% compared to 0%. Microbial community composition shifted from a diverse mix (Dechloromonas; 23%, "Candidatus Competibacter"; 13%, "Candidatus Accumulibacter"; 28%) at 0% to a dominant (69% - 75%) unclassified Accumulibacter clade I species at 1 - 4% salinity. Metaproteomic analysis showed strong upregulation of genes of "Ca. Accumulibacter" involved in monosaccharide, lipopolysaccharide, and peptidoglycan biosynthesis from 3% - 4% salinity, indicating its adaptation to salinity stress. Dechloromonas and "Ca. Competibacter" represented a minor or a non-significant fraction of those proteins related to glycan synthesis across the salinities. Despite that no glycoprotein biosynthesis pathways were identified in the metaproteomic data, three putative glycoproteins produced by "Ca. Accumulibacter" were detected across all conditions. They were downregulated as the salinity increased. These findings highlight how "Ca.Accumulibacter" dynamically adapts its EPS, particularly glycoprotein glycans, in response to increasing salinity, offering new insights into EPS adaptation under environmental stress.

Evolutionary Genomics: Transdomain Gene Transfers

Extracellular polymeric substances (EPSs) produced by actinomycetes of the genus Rhodococcus play crucial roles in their ecological success, metabolic versatility, and biotechnological value. This review summarizes existing studies of Rhodococcus EPSs, emphasizing the biochemical composition, functional attributes, and practical significance of EPSs, as well as their importance in biomedicine, bioremediation, and other applications (food industry, biomineralization) with respect to the EPS chemical composition and biological roles. Rhodococcus species synthesize complex EPSs composed primarily of polysaccharides, proteins and lipids that, like in other bacteria, support cell adhesion, aggregation, biofilm formation, and horizontal gene transfer (and can prevent exogenous DNA binding) and are highly important for resistance against toxicants and dissolution/assimilation of hydrophobic compounds. EPSs produced by different species of Rhodococcus exhibit diverse structures (soluble EPSs, loosely bound and tightly bound fractions, capsules, linear and branched chains, amorphous coils, rigid helices, mushroom-like structures, extracellular matrix, and a fibrillar structure with a sheet-like texture), leading to variations in their properties (rheological features, viscosity, flocculation, sorption abilities, compression, DNA binding, and interaction with hydrophobic substrates). Notably, the EPSs exhibit marked emulsifying and flocculating properties, contributing to their recognized role in bioremediation. Furthermore, EPSs possess antiviral, antibiofilm, anti-inflammatory, and anti-proliferating activities and high viscosity, which are valuable in terms of biomedical and food applications. Despite extensive industrial and environmental interest, the molecular regulation, biosynthetic pathways, and structural diversity of Rhodococcus EPSs remain insufficiently characterized. Advancing our understanding of these biopolymers could expand new applications in biomedicine, bioremediation, and biotechnology.

Biomineralization and AHLs-guided quorum sensing enhanced phosphorus recovery in the alternating aerobic/anaerobic biofilm system under metal ion stress

Enhanced biological phosphorus removal (EBPR) is widely used for removal of phosphorus from wastewater. In this study, a metagenome (18.2 Gb) was generated using Illumina sequencing from a full-scale EBPR plant to study the community structure and genetic potential. Quantitative fluorescence in situ hybridization (qFISH) was applied as an independent method to evaluate the community structure. The results were in qualitative agreement, but a DNA extraction bias against gram positive bacteria using standard extraction protocols was identified, which would not have been identified without the use of qFISH. The genetic potential for community function showed enrichment of genes involved in phosphate metabolism and biofilm formation, reflecting the selective pressure of the EBPR process. Most contigs in the assembled metagenome had low similarity to genes from currently sequenced genomes, underlining the need for more reference genomes of key EBPR species. Only the genome of 'Candidatus Accumulibacter', a genus of phosphorus-removing organisms, was closely enough related to the species present in the metagenome to allow for detailed investigations. Accumulibacter accounted for only 4.8% of all bacteria by qFISH, but the depth of sequencing enabled detailed insight into their microdiversity in the full-scale plant. Only 15% of the reads matching Accumulibacter had a high similarity (>95%) to the sequenced Accumulibacter clade IIA strain UW-1 genome, indicating the presence of some microdiversity. The differences in gene complement between the Accumulibacter clades were limited to genes for extracellular polymeric substances and phage-related genes, suggesting a selective pressure from phages on the Accumulibacter diversity.

Microbially produced extracellular polymeric substances (EPS) have been linked with many important ecological functions in natural sediments; yet, most information has been derived from marine systems. The present paper is the first comprehensive study on EPS (i.e., carbohydrates and proteins) dynamics in riverine sediments addressing spatial (six reservoirs and four groyne fields across three European rivers), temporal (all seasons in 2003-2005), and vertical (over a 50-cm sediment depth transect) pattern. The variation in hydrodynamic regime found in the reservoirs and groyne fields was reflected in the biomass and composition of the benthic microorganisms that produce EPS. The microphytobenthic communities consisted mainly of diatoms and a higher algal biomass (up to 248 microg g(-1) dry weight, DW) seemed to be indicative for higher amounts of secreted colloidal carbohydrates. Consequently, the model proposed by Underwood and Smith (1998) for the relation chlorophyll-colloidal carbohydrates was also applicable for upper riverine sediment layers. The close relation between algal biomass and bacterial cell counts (10(8)-10(9) cells g(-1) DW) supports the idea of bacterial use of the secreted EPS. However, the data also suggest a contribution to the EPS pool through bacterial secretion of proteins/extracellular enzymes and possibly carbohydrates. Over depth, the relationships between microorganisms and EPS became increasingly decoupled along with increasing ratios of bound (refractory) to colloidal (labile) EPS. These data suggest fresh production of polymeric substances in upper sediment layers and mainly accumulation of refractory, biodegraded material in deeper layers. The high contents of EPS colloidal and bound carbohydrates (0.1-1.8 and 1.3-6.7 mg g(-1) DW, respectively) and EPS proteins (0.4-12.9 mg g(-1) DW) at the freshwater study sites might indicate an important role in sediment ecology.

Twenty years ago the concept of horizontal or lateral gene transfer (the nongenealogical transmission of genetic material between organisms) was introduced as an explanation to incongruence in phylogenetic reconstructions using different genes. Today, horizontal gene transfer is accepted as an important force modulating evolution of prokaryotes and unicellular eukaryotes, whereas the extent to which it impacts on the evolution of metazoan eukaryotes remains unknown. Evidence for the role of horizontal gene transfer in evolution put in question the metaphor of the tree of life and the traditional view of evolution as a slow process. A more pluralistic approach to evolution is emerging that encompasses different evolutionary mechanisms operating at different levels of complexity. Key Concepts: Horizontal gene transfer is the nongenealogical transmission of genetic material between different organisms. Horizontal gene transfer plays an important role in prokaryotic evolution. Horizontal gene transfer also modulates evolution of unicellular eukaryotes. The importance of horizontal gene transfer in metazoan evolution is uncertain. Horizontal gene transfer challenges the tree of life metaphor. Horizontal gene transfer challenges the traditional view of evolution as a slow process. A pluralistic approach to evolution is needed.

Response of methanotroph-heterotroph consortia to different environmental factors

Most bacteria live in microbial assemblages like biofilms and granules, and each layer of these assemblages provides a niche for certain bacteria with specific metabolic functions. In this study, a gentle (non-destructive) extraction approach based on a cation exchange resin and defined shear was employed to gradually disintegrate biomass and collect single layers of aerobic granules from a full-scale municipal wastewater treatment plant. The microbial community composition of granule layers was characterized using next-generation sequencing (NGS) targeting the 16S rRNA gene, and protein composition was investigated using metaproteomics. The chemical composition of eroded layers was explored using Fourier Transformed Infrared Spectroscopy. On the surface of the granules, the microbial structure (flocculation-supporting Nannocystis sp.) as well as composition of extracellular polymers (extracellular DNA) and proteome (chaperonins and binding proteins) favored microbial aggregation. Extracellular polymeric substances in the granules were composed of mostly proteins and EPS-producers, such as Tetrasphaera sp. and Zoogloea sp., were evenly distributed throughout the granule structure. The interior of the granules harbored several denitrifiers (e.g., Thauera sp.), phosphate-accumulating denitrifiers (Candidatus Accumulibacter, Dechloromonas sp.) and nitrifiers (Candidatus Nitrotoga). Proteins associated with glycolytic activity were identified in the outer and middle granule layers, and proteins associated with phosphorus conversions, in the deeper layers. In conclusion, the use of an existing cation-exchange resin for gradual biomass disintegration, combined with NGS and metaproteomic analysis was demonstrated as a promising approach for simultaneously investigating the identity and functions of microbes in multilayered biofilm structures.