Benthic Biofilms in Glacier-Fed Streams from Scandinavia to the Himalayas Host Distinct Bacterial Communities Compared with the Streamwater.

Microbial life in glacier-fed streams (GFSs) is dominated by benthic biofilms which fulfill critical ecosystem processes. However, it remains unclear how the bacterial communities of these biofilms assemble in stream ecosystems characterized by rapid turnover of benthic habitats and high suspended sediment loads. Using16S rRNA gene amplicon sequence data collected from 54 GFSs across the Himalayas, European Alps, and Scandinavian Mountains, we found that benthic biofilms harbor bacterial communities that are distinct from the bacterial assemblages suspended in the streamwater. Our data showed a decrease in species richness in the benthic biofilms compared to the bacterial cells putatively free-living in the water. The benthic biofilms also differed from the suspended water fractions in terms of community composition. Differential abundance analyses highlighted bacterial families that were specific to the benthic biofilms and the suspended assemblages. Notably, source-sink models suggested that the benthic biofilm communities are not simply a subset of the suspended assemblages. Rather, we found evidence that deterministic processes (e.g., species sorting) shape the benthic biofilm communities. This is unexpected given the high vertical mixing of water and contained bacterial cells in GFSs and further highlights the benthic biofilm mode of life as one that is determined through niche-related processes. Our findings therefore reveal a "native" benthic biofilm community in an ecosystem that is currently threatened by climate-induced glacier shrinkage. IMPORTANCE Benthic biofilms represent the dominant form of life in glacier-fed streams. However, it remains unclear how bacterial communities within these biofilms assemble. Our findings from glacier-fed streams from three major mountain ranges across the Himalayas, the European Alps and the Scandinavian Mountains reveal a bacterial community associated with benthic biofilms that is distinct from the assemblage in the overlying streamwater. Our analyses suggest that selection is the underlying process to this differentiation. This is unexpected given that bacterial cells that are freely living or attached to the abundant sediment particles suspended in the water continuously mix with the benthic biofilms. The latter colonize loose sediments that are subject to high turnover owing to the forces of the water flow. Our research unravels the existence of a microbiome specific to benthic biofilms in glacier-fed streams, now under major threats due to global warming.

Glacier shrinkage will accelerate downstream decomposition of organic matter and alters microbiome structure and function.

The shrinking of glaciers is among the most iconic consequences of climate change. Despite this, the downstream consequences for ecosystem processes and related microbiome structure and function remain poorly understood. Here, using a space-for-time substitution approach across 101 glacier-fed streams (GFSs) from six major regions worldwide, we investigated how glacier shrinkage is likely to impact the organic matter (OM) decomposition rates of benthic biofilms. To do this, we measured the activities of five common extracellular enzymes and estimated decomposition rates by using enzyme allocation equations based on stoichiometry. We found decomposition rates to average 0.0129 (% d-1 ), and that decreases in glacier influence (estimated by percent glacier catchment coverage, turbidity, and a glacier index) accelerates decomposition rates. To explore mechanisms behind these relationships, we further compared decomposition rates with biofilm and stream water characteristics. We found that chlorophyll-a, temperature, and stream water N:P together explained 61% of the variability in decomposition. Algal biomass, which is also increasing with glacier shrinkage, showed a particularly strong relationship with decomposition, likely indicating their importance in contributing labile organic compounds to these carbon-poor habitats. We also found high relative abundances of chytrid fungi in GFS sediments, which putatively parasitize these algae, promoting decomposition through a fungal shunt. Exploring the biofilm microbiome, we then sought to identify bacterial phylogenetic clades significantly associated with decomposition, and found numerous positively (e.g., Saprospiraceae) and negatively (e.g., Nitrospira) related clades. Lastly, using metagenomics, we found evidence of different bacterial classes possessing different proportions of EEA-encoding genes, potentially informing some of the microbial associations with decomposition rates. Our results, therefore, present new mechanistic insights into OM decomposition in GFSs by demonstrating that an algal-based "green food web" is likely to increase in importance in the future and will promote important biogeochemical shifts in these streams as glaciers vanish.

The dark side of the moon: first insights into the microbiome structure and function of one of the last glacier-fed streams in Africa.

The glaciers on Africa's 'Mountains of the Moon' (Rwenzori National Park, Uganda) are predicted to disappear within the next decades owing to climate change. Consequently, the glacier-fed streams (GFSs) that drain them will vanish, along with their resident microbial communities. Despite the relevance of microbial communities for performing ecosystem processes in equatorial GFSs, their ecology remains understudied. Here, we show that the benthic microbiome from the Mt. Stanley GFS is distinct at several levels from other GFSs. Specifically, several novel taxa were present, and usually common groups such as Chrysophytes and Polaromonas exhibited lower relative abundances compared to higher-latitude GFSs, while cyanobacteria and diatoms were more abundant. The rich primary producer community in this GFS likely results from the greater environmental stability of the Afrotropics, and accordingly, heterotrophic processes dominated in the bacterial community. Metagenomics revealed that almost all prokaryotes in the Mt. Stanley GFS are capable of organic carbon oxidation, while greater than 80% have the potential for fermentation and acetate oxidation. Our findings suggest a close coupling between photoautotrophs and other microbes in this GFS, and provide a glimpse into the future for high-latitude GFSs globally where primary production is projected to increase with ongoing glacier shrinkage.

Glacier-Fed Stream Biofilms Harbor Diverse Resistomes and Biosynthetic Gene Clusters.

Antimicrobial resistance (AMR) is a universal phenomenon the origins of which lay in natural ecological interactions such as competition within niches, within and between micro- to higher-order organisms. To study these phenomena, it is crucial to examine the origins of AMR in pristine environments, i.e., limited anthropogenic influences. In this context, epilithic biofilms residing in glacier-fed streams (GFSs) are an excellent model system to study diverse, intra- and inter-domain, ecological crosstalk. We assessed the resistomes of epilithic biofilms from GFSs across the Southern Alps (New Zealand) and the Caucasus (Russia) and observed that both bacteria and eukaryotes encoded twenty-nine distinct AMR categories. Of these, beta-lactam, aminoglycoside, and multidrug resistance were both abundant and taxonomically distributed in most of the bacterial and eukaryotic phyla. AMR-encoding phyla included Bacteroidota and Proteobacteria among the bacteria, alongside Ochrophyta (algae) among the eukaryotes. Additionally, biosynthetic gene clusters (BGCs) involved in the production of antibacterial compounds were identified across all phyla in the epilithic biofilms. Furthermore, we found that several bacterial genera (Flavobacterium, Polaromonas, Superphylum Patescibacteria) encode both atimicrobial resistance genes (ARGs) and BGCs within close proximity of each other, demonstrating their capacity to simultaneously influence and compete within the microbial community. Our findings help unravel how naturally occurring BGCs and AMR contribute to the epilithic biofilms mode of life in GFSs. Additionally, we report that eukaryotes may serve as AMR reservoirs owing to their potential for encoding ARGs. Importantly, these observations may be generalizable and potentially extended to other environments that may be more or less impacted by human activity. IMPORTANCE Antimicrobial resistance is an omnipresent phenomenon in the anthropogenically influenced ecosystems. However, its role in shaping microbial community dynamics in pristine environments is relatively unknown. Using metagenomics, we report the presence of antimicrobial resistance genes and their associated pathways in epilithic biofilms within glacier-fed streams. Importantly, we observe biosynthetic gene clusters associated with antimicrobial resistance in both pro- and eukaryotes in these biofilms. Understanding the role of resistance in the context of this pristine environment and complex biodiversity may shed light on previously uncharacterized mechanisms of cross-domain interactions.

Viral diversity is linked to bacterial community composition in alpine stream biofilms.

Biofilms play pivotal roles in fluvial ecosystems, yet virtually nothing is known about viruses in these communities. Leveraging an optimized sample-to-sequence pipeline, we studied the spatiotemporal turnover of dsDNA viruses associated with stream biofilms and found an astounding diversity to be structured by seasons and along the longitudinal gradient in the stream. While some vOTUs were region- or season-specific, we also identified a large group of permanent biofilm phages, taxonomically dominated by Myoviridae. Comparison of the observed viral distribution with predictions based on neutral community assembly indicated that chance and dispersal may be important for structuring stream biofilm viral communities. Deviation from neutral model predictions suggests that certain phages distribute efficiently across distant locations within the stream network. This dispersal capacity appears to be linked to EPS depolymerases that enable phages to efficiently overcome the biofilm barrier. Other phages, particularly vOTUs classified as Siphoviridae, appear locally overrepresented and to rely on a lysogenic life cycle, potentially to exploit the spatial distribution of bacterial populations in stream biofilms. Overall, biofilm viral and bacterial community turnover were significantly coupled. Yet, viral communities were linked to the presence of the most abundant bacterial community members. With this work, we provide a foundational ecological perspective on factors that structure viral diversity in stream biofilms and identify potentially important viral traits related to the biofilm mode of life.

Microdiversity characterizes prevalent phylogenetic clades in the glacier-fed stream microbiome.

Glacier-fed streams (GFSs) are extreme and rapidly vanishing ecosystems, and yet they harbor diverse microbial communities. Although our understanding of the GFS microbiome has recently increased, we do not know which microbial clades are ecologically successful in these ecosystems, nor do we understand potentially underlying mechanisms. Ecologically successful clades should be more prevalent across GFSs compared to other clades, which should be reflected as clade-wise distinctly low phylogenetic turnover. However, methods to assess such patterns are currently missing. Here we developed and applied a novel analytical framework, "phyloscore analysis", to identify clades with lower spatial phylogenetic turnover than other clades in the sediment microbiome across twenty GFSs in New Zealand. These clades constituted up to 44% and 64% of community α-diversity and abundance, respectively. Furthermore, both their α-diversity and abundance increased as sediment chlorophyll a decreased, corroborating their ecological success in GFS habitats largely devoid of primary production. These clades also contained elevated levels of putative microdiversity than others, which could potentially explain their high prevalence in GFSs. This hitherto unknown microdiversity may be threatened as glaciers shrink, urging towards further genomic and functional exploration of the GFS microbiome.

Spatial patterns of benthic biofilm diversity among streams draining proglacial floodplains.

Glacier shrinkage opens new proglacial terrain with pronounced environmental gradients along longitudinal and lateral chronosequences. Despite the environmental harshness of the streams that drain glacier forelands, their benthic biofilms can harbor astonishing biodiversity spanning all domains of life. Here, we studied the spatial dynamics of prokaryotic and eukaryotic photoautotroph diversity within braided glacier-fed streams and tributaries draining lateral terraces predominantly fed by groundwater and snowmelt across three proglacial floodplains in the Swiss Alps. Along the lateral chronosequence, we found that benthic biofilms in tributaries develop higher biomass than those in glacier-fed streams, and that their respective diversity and community composition differed markedly. We also found spatial turnover of bacterial communities in the glacier-fed streams along the longitudinal chronosequence. These patterns along the two chronosequences seem unexpected given the close spatial proximity and connectivity of the various streams, suggesting environmental filtering as an underlying mechanism. Furthermore, our results suggest that photoautotrophic communities shape bacterial communities across the various streams, which is understandable given that algae are the major source of organic matter in proglacial streams. Overall, our findings shed new light on benthic biofilms in proglacial streams now changing at rapid pace owing to climate-induced glacier shrinkage.

Genomic and metabolic adaptations of biofilms to ecological windows of opportunity in glacier-fed streams.

In glacier-fed streams, ecological windows of opportunity allow complex microbial biofilms to develop and transiently form the basis of the food web, thereby controlling key ecosystem processes. Using metagenome-assembled genomes, we unravel strategies that allow biofilms to seize this opportunity in an ecosystem otherwise characterized by harsh environmental conditions. We observe a diverse microbiome spanning the entire tree of life including a rich virome. Various co-existing energy acquisition pathways point to diverse niches and the exploitation of available resources, likely fostering the establishment of complex biofilms during windows of opportunity. The wide occurrence of rhodopsins, besides chlorophyll, highlights the role of solar energy capture in these biofilms while internal carbon and nutrient cycling between photoautotrophs and heterotrophs may help overcome constraints imposed by oligotrophy in these habitats. Mechanisms potentially protecting bacteria against low temperatures and high UV-radiation are also revealed and the selective pressure of this environment is further highlighted by a phylogenomic analysis differentiating important components of the glacier-fed stream microbiome from other ecosystems. Our findings reveal key genomic underpinnings of adaptive traits contributing to the success of complex biofilms to exploit environmental opportunities in glacier-fed streams, which are now rapidly changing owing to global warming.

Microbial Ecology of Methanotrophy in Streams Along a Gradient of CH4 Availability.

Despite the recognition of streams and rivers as sources of methane (CH4) to the atmosphere, the role of CH4 oxidation (MOX) in these ecosystems remains poorly understood to date. Here, we measured the kinetics of MOX in stream sediments of 14 sites to resolve the ecophysiology of CH4 oxidizing bacteria (MOB) communities. The streams cover a gradient of land cover and associated physicochemical parameter and differed in stream- and porewater CH4 concentrations. Michealis-Menten kinetic parameter of MOX, maximum reaction velocity (V max ), and CH4 concentration at half V max (K S ) increased with CH4 supply. K S values in the micromolar range matched the CH4 concentrations measured in shallow stream sediments and indicate that MOX is mostly driven by low-affinity MOB. 16S rRNA gene sequencing identified MOB classified as Methylococcaceae and particularly Crenothrix. Their relative abundance correlated with pmoA gene counts and MOX rates, underscoring their pivotal role as CH4 oxidizers in stream sediments. Building on the concept of enterotypes, we identify two distinct groups of co-occurring MOB. While there was no taxonomic difference among the members of each cluster, one cluster contained abundant and common MOB, whereas the other cluster contained rare operational taxonomic units (OTUs) specific to a subset of streams. These integrated analyses of changes in MOB community structure, gene abundance, and the corresponding ecosystem process contribute to a better understanding of the distal controls on MOX in streams.

Optimised biomolecular extraction for metagenomic analysis of microbial biofilms from high-mountain streams.

Glacier-fed streams (GFS) are harsh ecosystems dominated by microbial life organized in benthic biofilms, yet the biodiversity and ecosystem functions provided by these communities remain under-appreciated. To better understand the microbial processes and communities contributing to GFS ecosystems, it is necessary to leverage high throughput sequencing. Low biomass and high inorganic particle load in GFS sediment samples may affect nucleic acid extraction efficiency using extraction methods tailored to other extreme environments such as deep-sea sediments. Here, we benchmarked the utility and efficacy of four extraction protocols, including an up-scaled phenol-chloroform protocol. We found that established protocols for comparable sample types consistently failed to yield sufficient high-quality DNA, delineating the extreme character of GFS. The methods differed in the success of downstream applications such as library preparation and sequencing. An adapted phenol-chloroform-based extraction method resulted in higher yields and better recovered the expected taxonomic profile and abundance of reconstructed genomes when compared to commercially-available methods. Affordable and straight-forward, this method consistently recapitulated the abundance and genomes of a mock community, including eukaryotes. Moreover, by increasing the amount of input sediment, the protocol is readily adjustable to the microbial load of the processed samples without compromising protocol efficiency. Our study provides a first systematic and extensive analysis of the different options for extraction of nucleic acids from glacier-fed streams for high-throughput sequencing applications, which may be applied to other extreme environments.

Patterns and Drivers of Extracellular Enzyme Activity in New Zealand Glacier-Fed Streams.

Glacier-fed streams (GFSs) exhibit near-freezing temperatures, variable flows, and often high turbidities. Currently, the rapid shrinkage of mountain glaciers is altering the delivery of meltwater, solutes, and particulate matter to GFSs, with unknown consequences for their ecology. Benthic biofilms dominate microbial life in GFSs, and play a major role in their biogeochemical cycling. Mineralization is likely an important process for microbes to meet elemental budgets in these systems due to commonly oligotrophic conditions, and extracellular enzymes retained within the biofilm enable the degradation of organic matter and acquisition of carbon (C), nitrogen (N), and phosphorus (P). The measurement and comparison of these extracellular enzyme activities (EEA) can in turn provide insight into microbial elemental acquisition effort relative to environmental availability. To better understand how benthic biofilm communities meet resource demands, and how this might shift as glaciers vanish under climate change, we investigated biofilm EEA in 20 GFSs varying in glacier influence from New Zealand's Southern Alps. Using turbidity and distance to the glacier snout normalized for glacier size as proxies for glacier influence, we found that bacterial abundance (BA), chlorophyll a (Chl a), extracellular polymeric substances (EPS), and total EEA per gram of sediment increased with decreasing glacier influence. Yet, when normalized by BA, EPS decreased with decreasing glacier influence, Chl a still increased, and there was no relationship with total EEA. Based on EEA ratios, we found that the majority of GFS microbial communities were N-limited, with a few streams of different underlying bedrock geology exhibiting P-limitation. Cell-specific C-acquiring EEA was positively related to the ratio of Chl a to BA, presumably reflecting the utilization of algal exudates. Meanwhile, cell-specific N-acquiring EEA were positively correlated with the concentration of dissolved inorganic nitrogen (DIN), and both N- and P-acquiring EEA increased with greater cell-specific EPS. Overall, our results reveal greater glacier influence to be negatively related to GFS biofilm biomass parameters, and generally associated with greater microbial N demand. These results help to illuminate the ecology of GFS biofilms, along with their biogeochemical response to a shifting habitat template with ongoing climate change.

Unraveling the biophysical underpinnings to the success of multispecies biofilms in porous environments.

Biofilms regulate critical processes in porous ecosystems. However, the biophysical underpinnings of the ecological success of these biofilms are poorly understood. Combining experiments with fluidic devices, sequencing and modeling, we reveal that architectural plasticity enhances space exploitation by multispecies biofilms in porous environments. Biofilms consistently differentiated into an annular base biofilm coating the grains and into streamers protruding from the grains into the pore space. Although different flow-related processes governed the differentiation of these architectures, both BB and streamers were composed of similar bacterial assemblages. This is evidence for architectural plasticity. Architectural plasticity allowed for complementary use of the space provided by the grain-pore complexes, which increased biofilm carrying capacity at the larger scale of the porous system. This increase comes potentially at the cost of a tradeoff. Contrasting time scales of oxygen replenishment and consumption, we show that streamers locally inhibit the growth of the BB downstream from the grains. Our study provides first insights into the biophysical underpinnings to the success of multispecies biofilms in porous environments.

Screening fecal enterococci from Greek healthy infants for susceptibility to antimicrobial agents.

Enterococci are among the first lactic acid bacteria to colonize the neonatal gastrointestinal tract, but they are also characterized as significant nosocomial pathogens. The aim of this study was to investigate the incidence of antibiotic resistance in enterococci isolated from neonates' gut microbiota as well as the presence of genetic determinants encoding for certain antibiotic resistance traits. A total of 263 fecal samples derived from 97 infants were collected on day 4, 30, and 90 after delivery. Enterococcus faecalis was the most frequently identified species (54.6%) followed by E. faecium, while E. casseliflavus/E. flavescens and E. gallinarum were also traced. The isolates were examined for their resistance to 12 antibiotics. Rifampicin resistance was the highest observed (53.2%), followed by resistance to tetracycline (42.0%), erythromycin (35.7%), and vancomycin (11.2%). Multiresistant strains were highly prevalent. Only intrinsic vancomycin resistance (vanC1 and vanC2/C3) was traced. The ermB gene was detected in 49 out of 96 erythromycin-resistant isolates, while tet genes were detected in 51 out of 113 tetracycline-resistant strains, with tet(L) being the most frequently observed. In conclusion, antibiotic-resistant enterococci are already established in the fecal microbiota of healthy neonates, from the first days of an infant's life.

Complete mitochondrial genome sequence of the wine yeast Candida zemplinina: intraspecies distribution of a novel group-IIB1 intron with eubacterial affiliations.

The mtDNA of the ascomycetous wine yeast Candida zemplinina is a circularly mapping genome of 23,114 bp. It contains 35 genes coding for the seven basic subunits of oxidative phosporylation found in yeasts (the genes encoding for NADH oxidoreductase subunits are absent), the ribosomal protein Var1, two rRNAs and 25 tRNA genes. Although protein phylogenetic analysis showed a divergent mitochondrial genome, several traits appeared preserved. The conserved gene blocks between the mtDNAs of C. zemplinina and Candida glabrata were maintained and changes in gene order and putative promoters were due to restricted genome reshuffling. New heterogeneous hairpin elements were identified scattered throughout cox1 introns. The large subunit rRNA gene harboured the first group-IIB1 intron containing a putative active reverse transcriptase (RT) in mitochondrial genomes of fungi. Phylogenetic analysis of the RT protein confirmed its closer relationship to eubacterial intronic RTs, while being only distantly related to all other fungal mitochondrial group-II introns and RTs. The findings point towards an early migration event of a eubacterial group-II intron to the mitochondrial genome of C. zemplinina.

The mitochondrial genome of the wine yeast Hanseniaspora uvarum: a unique genome organization among yeast/fungal counterparts.

The complete sequence of the apiculate wine yeast Hanseniaspora uvarum mtDNA has been determined and analysed. It is an extremely compact linear molecule containing the shortest functional region ever found in fungi (11 094 bp long), flanked by Type 2 telomeric inverted repeats. The latter contained a 2704-bp-long subterminal region and tandem repeats of 839-bp units. In consequence, a population of mtDNA molecules that differed at the number of their telomeric reiterations was detected. The functional region of the mitochondrial genome coded for 32 genes, which included seven subunits of respiratory complexes and ATP synthase (the genes encoding for NADH oxidoreductase subunits were absent), two rRNAs and 23 tRNA genes which recognized codons for all amino acids. A single intron interrupted the cytochrome oxidase subunit 1 gene. A number of reasons contributed towards its strikingly small size, namely: (1) the remarkable size reduction (by >40%) of the rns and rnl genes; (2) that most tRNA genes and five of the seven protein-coding genes were the shortest among known yeast homologs; and (3) that the noncoding regions were restricted to 5.1% of the genome. In addition, the genome showed multiple changes in the orientation of transcription and the gene order differed drastically from other yeasts. When all protein coding gene sequences were considered as one unit and were compared with the corresponding molecules from all other complete mtDNAs of yeasts, the phylogenetic trees constructed robustly supported its placement basal to the yeast species of the 'Saccharomyces complex', demonstrating the advantage of this approach over single-gene or multigene approaches of unlinked genes.