Alexander J. Probst

Microbial communities influence key processes in oil reservoirs, including souring, corrosion, and hydrocarbon degradation, so characterizing them is important for reservoir management. Reliable characterization depends on obtaining a sample that represents the community living in the subsurface. As nucleic acids are difficult to recover from oil, produced water, the aqueous phase separated from reservoir fluids, is widely used as a convenient proxy for analyzing the reservoir microbiome. However, whether produced water faithfully represents that community, to our knowledge, has never been tested with paired samples. We addressed this question using 16S rRNA gene amplicon and metagenomic sequencing of reservoir fluids and produced water collected from the same production wells across four North Sea oil fields. The two sample types harbored markedly different microbial communities and functional profiles. This difference persisted after accounting for reservoir identity, and it was larger than the differences between reservoirs. Produced water was less diverse, more uniform across fields, and dominated by aerobic genera associated with seawater and aquifers. Reservoir fluids, in contrast, retained the thermophilic, halophilic, and anaerobic taxa characteristic of the deep subsurface. Only 41% of reservoir-fluid genera and 21% of their amplicon sequence variants were also detected in the paired produced water, indicating overlapping broad taxa but largely distinct populations. Notably, the putatively native reservoir community remained detectable in reservoir fluids even after decades of production, while produced water offered only an incomplete, directionally biased view. Reservoir fluids should therefore be sampled wherever feasible to characterize the in-situ microbiome.

Antimicrobial resistance in Escherichia coli is shaped not only by resistance genes themselves but also by their chromosomal or plasmid localization and co-occurrence with biocide/metal resistance genes (BMRGs), virulence-associated genes and mobile genetic elements. We applied chromosome- and plasmid-resolved genomics to 109 extended-spectrum β -lactamase-producing E. coli isolates from unweaned dairy calves ( n =484) in Germany and compared them with 479 human-associated reference genomes. Calf isolates were polyclonal and dominated by phylogroups A and B1. Resistance was predominantly plasmid-borne: 41% of isolates carried antibiotic resistance genes (ARGs) exclusively on plasmids, whereas only 4.6% carried ARGs exclusively on chromosomes. The chromosomal-versus-plasmid distribution of acquired ARGs differed significantly across phylogroups ( P P P =3.66×10 ⁻⁴² ). ARG and BMRG counts were strongly correlated at the plasmid level (ρ=0.574, P =8.0×10 ⁻⁴¹ ), and class 1 integrons marked enriched multidrug plasmids with increased ARGs ( P =6.22×10 ⁻³⁴ ) and BMRGs ( P =3.00×10 ⁻²⁹ ). At the isolate level, calf isolates carried more acquired ARGs in unadjusted comparisons, but this host-associated difference was largely explained by population structure. At the plasmid level, however, host-associated differences persisted after adjustment: human plasmids carried more ARGs (IRR 1.66, P =0.0017) and showed a strong host×mobility interaction (IRR 4.61, P =4.9×10–8), stronger ARG–BMRG coupling and a higher prevalence of integrons. These findings show that antimicrobial resistance ecology in E. coli is shaped not only by which resistance genes are present, but by where they are located, what they are linked to and how readily their genomic carriers can disseminate.

Cosmopolitan microbial lineages are found in anoxic sediments worldwide, but the details about their ecology and evolution remain underexplored. In this study, we identified persistent populations from these cosmopolitan lineages belonging to Planctomycetes , Chloroflexi Atribacteria , and Candidatus Bathyarchaeia from an ~8,000-year sedimentary sequence. To investigate the genomic variations within these persistent populations, a pangenome of each population was constructed using all non-redundant metagenome-assembled genomes (MAGs) recovered from the sedimentary sequence and was screened for enriched functional genes, single-nucleotide polymorphism (SNP) density, dN / DS ratios, and pseudogene content. Our results show that the majority of persistent populations studied possess large variable genomes enriched for energy conservation and transcriptional regulation functions with increasing depth, whereas Planctomycetes retain a highly conserved, SNP-poor core genome. Analysis of SNPs across depths indicates progressive isolation with burial, while a subset of core genes shows signatures of positive selection. Collectively, the data support depth-associated selection acting alongside drift across Lake Cadagno’s persistent sedimentary lineages. IMPORTANCE: Throughout the subsurface, multiple examples of “evolutionary stasis” have been reported in microbial lineages that exhibit lower rates of metabolic activity and cellular turnover. This study uses an ~8,000-year sedimentary record of Lake Cadagno to evaluate how persistent populations of cosmopolitan bacteria and archaea have changed with burial and identifies signals of progressive genetic isolation along with positive selection of population-specific subsets of core genes with depth. Together, these changes show that Lake Cadagno’s persistent populations are not in stasis but diverge over time and burial.

Borgs are giant linear extrachromosomal elements (ECEs) of methane-oxidizing Methanoperedens archaea whose evolutionary origin and ecosystem distribution remain unknown. Here we detected 240 Borgs in diverse saline, soil, and freshwater ecosystems. 27 encode methyl-coenzyme M reductases central to methane metabolism, eight have full rRNA operons related to those of Methanoperedens, and some contain up to four ribosomal proteins. We also identified 323 mini-Borgs whose proteome content and gene phylogenies classify as a distinct ECE type. Based on 105 complete and near-complete genomes, Borgs and mini-Borgs share core genes in conserved order, indicating common ancestry. Phylogenetic and diversity analyses suggest that Borgs evolved via gene acquisition into a backbone inherited from mini-Borgs. This evolutionary trajectory mirrors those proposed for giant viruses of both eukaryotes and bacteria.

Most prokaryotic genomes in public databases are genomes reconstructed from metagenomes, forming a compendium of multiple contiguous sequences (contigs) assembled from shotgun sequencing data. Binning algorithms for assigning contigs to metagenome assembled genomes (MAGs) are manifold and continuously improving in accuracy. However, binning errors, i.e. the incorrect assignment of a contig and coding sequence to a MAG, often propagate through various databases and confound taxonomic, metabolic and/or evolutionary analyses. Here we present itBins, a fully automated python-based software that enables ultra-fast refinement of metagenomic bins using a rule-based approach harnessing information from %GC content (%GC for brevity), coverage, and taxonomy of individual contigs. When applied to the low, medium, and high complexity data of the Critical Assessment of Metagenome Interpretation (CAMI I) challenge [1], itBins produced higher F ₁ scores (the harmonic mean of precision and recall) for all levels compared to other automated refinement tools, i.e., MDMcleaner and Rosella. Compared to manual refinement via uBin, itBins performed similarly well across all three complexity levels of the CAMI I dataset. With an average speed of 61 ms per bin, itBins is faster than all other refinement tools by at least three orders of magnitude when input data is accordingly available (%GC, coverage, and taxonomy), and was similarly fast when input data preparation was included in the processing time. Application to 64 real-world metagenomes from highly complex river mesocosms resulted in 259 medium-quality and 19 high-quality MAGs refined by itBins, while the other automated refinement tools failed in generating output at all or within 5000 hours of runtime. Finally, itBins also utilizes marker genes to determine the overall binning success for individual metagenomes, providing a crucial benchmark for the user to estimate the ecological relevance of their binned data. The herein introduced software itBins is broadly applicable to any type of metagenome data, integrates well with other software like DASTool, and enables swift and reliable refinement of genomes from metagenomes along with estimation of the overall binning success. itBins is distributed via EUPL 1.2 license and available at Codeberg ( codeberg.org/JMK/itBins ), GitHub ( github.com/ProbstLab/itBins ) and through Bioconda [2]( bioconda.github.io/recipes/itbins/README.html ).

Lakes are highly dynamic freshwater ecosystems where complex interactions between environmental factors and microbial communities regulate biogeochemical cycling and ecosystem stability. Seasonal dynamics of rare and dominant bacterial taxa, as well as the links between dominant taxa and environmental drivers in freshwater ecosystems across Croatia’s contrasting climatic regions, remain insufficiently explored. To address this gap, this study investigated six deep karstic lakes across both regions. Amplicon-based 16 S rRNA gene sequencing coupled with FAPROTAX-based functional predictions revealed that microbial community structure and inferred functional potential were strongly influenced by environmental variability. Dissolved organic carbon (DOC) was identified as the primary driver of community composition. Typical freshwater bacterial genera, including hgcl, Cyanobium PCC-6307, and Luteolibacter, dominated across all lakes. Mediterranean lakes exhibited greater heterogeneity in inferred functional potential across seasons, with variable enrichment in predicted pathways associated with carbon, nitrogen, and sulfur cycling. By contrast, continental lakes showed more stable inferred functional potential with less pronounced seasonal fluctuations. Overall, this study highlights how environmental and climatic variability shape the taxonomic composition and inferred functional potential of microbial communities in freshwater systems, emphasizing the ecological dynamics of dominant bacterial taxa, while also revealing seasonal and regional differences between dominant and rare bacterial taxa across contrasting climatic regions.

The growing research into the archaeal lipidome has uncovered a remarkable structural diversity in isoprenoidal glycerol dialkyl glycerol tetraethers (iGDGTs) and revealed complex membrane adaptations, especially in extreme environments. We performed a comprehensive analysis of the lipidome from the subsurface aquifer of the CO ₂ ‐rich, cold‐water Geyser Andernach (Germany), using ultra‐high‐resolution mass spectrometry. We detected iGDGT‐0, presumably derived from the dominant community member Candidatus Altiarchaeum, providing supporting evidence for its ability to synthesise tetraethers, as previously predicted from metagenomic data. Beyond the typical iGDGT‐0 and acyclic glycerol trialkyl glycerol tetraether (iGTGT‐0), we discovered novel structural derivatives, here referred to as extended iGDGTs and iGTGTs, characterised by the asymmetrical addition of up to two isoprenoid units to only one of their hydrocarbon side chains, analogous to those found in extended archaeols. The apparent absence of GDGT ring synthase A and B genes in the corresponding metagenome‐assembled genome raises the possibility that the producing archaea may utilise extended iGDGTs as a membrane adaptation to cope with the nutrient‐depleted conditions of the geyser environment, highlighting the adaptive flexibility of archaea to extreme physicochemical conditions.

The growing research into the archaeal lipidome has uncovered a remarkable structural diversity in glycerol dialkyl glycerol tetraethers (GDGTs) and revealed complex membrane adaptations, especially in extreme environments. We performed a comprehensive analysis of the lipidome from the subsurface water of a CO ₂ -rich, cold-water Geyser Andernach (Germany), using ultra-high-resolution mass spectrometry methods. We detected GDGT-0, presumably derived from the dominant community member Candidatus Altiarchaeum, providing evidence for its ability to synthesize tetraethers as previously predicted from metagenomic data. Beyond the typical GDGT-0 and acyclic glycerol trialkyl glycerol tetraether (GTGT-0), we discovered novel structural analogues, here referred to as extended GDGTs and GTGTs, characterized by the asymmetrical addition of up to two isoprenoid units to only one of their hydrocarbon side chains, analogous to those found in extended archaeols. The lack of GDGT ring synthase A (GrsA) and GrsB homologs in the corresponding metagenome-assembled genome, suggests that the producing archaeon may utilize extended GDGTs as a membrane adaptation to cope with the energy-depleted conditions of the geyser environment, highlighting the adaptive flexibility of archaea to extreme physicochemical conditions.

Background: Stream hyporheic zones represent a unique ecosystem at the interface of stream water and surrounding sediments, characterized by high heterogeneity and accelerated biogeochemical activity. These zones—represented by the top sediment layer in this study—are increasingly impacted by anthropogenic stressors and environmental changes at a global scale, directly altering their microbiomes. Despite their importance, the current body of literature lacks a systematic understanding of active nitrogen and sulfur cycling across stream sediment and surface water microbiomes, particularly across geographic locations and in response to environmental factors. Results: Based on previously published and unpublished datasets, 363 stream metagenomes were combined to build a comprehensive MAG and gene database from stream sediments and surface water including a full-factorial mesocosm experiment which had been deployed to unravel microbial stress response. Metatranscriptomic data from 23 hyporheic sediment samples collected across North America revealed that microbial activity in sediments was distinct from the activity in surface water, contrasting similarly encoded metabolic potential across the two compartments. The expressed energy metabolism of the hyporheic zone was characterized by increased cycling of sulfur and nitrogen compounds, governed by Nitrospirota and Desulfobacterota lineages. While core metabolic functions like energy conservation were conserved across sediments, temperature and stream order change resulted in differential expression of stress response genes previously observed in mesocosm studies. Conclusions: The hyporheic zone is a microbial hotspot in stream ecosystems, surpassing the activity of overlaying riverine surface waters. Metabolic activity in the form of sulfur and nitrogen cycling in hyporheic sediments is governed by multiple taxa interacting through metabolic handoffs. Despite the spatial heterogeneity of streams, the hyporheic sediment microbiome encodes and expresses conserved stress responses to anthropogenic stressors, e.g. , temperature, in streams of separate continents. The high number of uncharacterized differentially expressed genes as a response to tested stressors is a call-to-action to deepen the study of stream systems.