Because the majority of microbial organisms still evade cultivation attempts, genomic insights into many taxa are limited to cultivation-independent approaches. However, current methods of metagenomics and single-cell genome sequencing have individual drawbacks, which can limit the quality and completeness of the reconstructed genomes. Current attempts to combine both approaches still use whole-genome amplification techniques, which are prone to bias. Here, we propose a novel approach for the purpose of genome reconstructions that utilizes the potential of cell sorting for targeted enrichment and depletion of different cell types to create distinct cell fractions with sufficient DNA amounts, circumventing amplification. By distributing sequencing efforts over these fractions as well as the original sample, coassemblies become highly optimized for coabundance variation-based binning approaches. "Midi-metagenomics" enables accurate metagenome-assembled genome (MAG) reconstruction from individual sorted samples with higher quality than coassembly and binning of multiple distinct samples and therefore improves analyses of uncultivated microorganisms.

Abstract Microbial responses to xenobiotic compounds are difficult to resolve due to environmental complexity and limited functional annotation. Here, we establish a materials-based cultivation framework using macroporous elastomeric silicone foams (MESIF) to capture microbial adaptation across environmental contexts and timescales. Using glyphosate as a model compound and per- and polyfluoroalkyl substances (PFAS) as a poorly understood class, we show that responses differ depending on the availability of established metabolic pathways. Glyphosate exposure induced rapid, pathway-specific functional enrichment with minimal taxonomic change. In contrast, PFAS exposure did not yield consistent taxonomic or annotation-based signals but instead produced responses that emerged over time and were primarily detectable at protein and genome-resolved levels. LC-MS analyses revealed transformation dynamics, including formation of shorter-chain products. These responses were not explained by known degrader taxa but involved uncharacterized proteins and microbial populations, highlighting the extent of functional “dark matter” in microbial responses to persistent contaminants.

Flagellar motility is crucial for the swim-and-stick lifestyle and plays an important role in bacterial-algal interactions of Rhodobacterales. This alphaproteobacterial order contains three distinct types of flagellar gene clusters (FGCs) for the formation of a functional flagellum. Our phylogenetically broad taxon sampling of more than 300 genomes revealed that the most common FGC, the fla1-type, was probably already present in the common ancestor of Rhodobacterales and was strictly vertically inherited, while the other two FGC types, fla2 and fla3, were spread via horizontal operon transfers. Swimming of the marine model organism Phaeobacter inhibens DSM 17395 (Roseobacteraceae) is mediated by the archetypal fla1-type flagellum. Screening of 13,000 transposon mutants of P. inhibens on soft agar plates revealed that 40 genes, including four genes encoding conserved but not yet characterized proteins (CP1-4) within the FGC, are essential for motility. Exoproteome analyses indicated that CP1-4 are required at different stages of flagellar assembly. Only eight genes outside the FGC were identified as essential for swimming motility, including all three genes of the CtrA phosphorelay. Using comparative transcriptomics of ΔcckA, ΔchpT, and ΔctrA mutants of the distantly related model organisms P. inhibens and Dinoroseobacter shibae DSM 16493, we identified genes for the flagellum and cyclic di-GMP turnover as core targets of the CtrA phosphorelay and a conserved connection with quorum sensing across members of the Rhodobacterales. IMPORTANCE:The bacterial flagellum is a sophisticated nanomachine for swimming motility and rapid chemotactic response to gradients of attractants or repellents in the environment. It is structurally highly conserved and has been intensively studied in gammaproteobacterial model bacteria such as Escherichia coli and Salmonella enterica. However, the flagellar gene clusters of different alphaproteobacterial orders have distinct structures and compositions, as demonstrated by the three flagellar systems of Rhodobacterales investigated in the current study. The archetypal fla1-type flagellum originated in its common ancestor and evolved synchronously with the host. The universal presence of four as yet uncharacterized essential genes in fla1-type FGCs (CP1-4) reflects the order-specific adaptation of the flagellar system during bacterial evolution. Comparative transcriptome analyses of ΔcckA, ΔchpT, and ΔctrA mutants showed that the core function of the CtrA phosphorelay in Rhodobacterales is the transcriptional control of flagellar genes.

Since the majority of microbial organisms still evade cultivation attempts, genomic insights into many taxa are limited to cultivation-independent approaches. However, current methods of metagenomics and single cell genome sequencing have individual drawbacks, which can limit the quality as well as completeness of the reconstructed genomes. Current attempts to combine both approaches still use amplification techniques which are prone to bias. Here, we propose a novel approach for the purpose of genome reconstructions, that utilizes the potential of cell sorting for targeted enrichment and depletion of different cell types to create distinct cell fractions of sufficient size, circumventing amplification. By distributing sequencing efforts over these fractions as well as the original sample, co-assemblies become highly optimized for co-abundance variation based binning approaches. “Midi-metagenomics” enables accurate metagenome assembled genome (MAG) reconstruction from individual sorted samples with higher quality than co-assembly of multiple distinct samples and has potential for the targeted enrichment and sequencing of microbial dark matter.

The phylum Chloroflexota (formerly Chloroflexi) encompasses metabolically diverse bacteria that often have high prevalence in terrestrial and aquatic habitats, some even with biotechnological application. However, there is substantial disagreement in public databases which lineage should be considered a member of the phylum and at what taxonomic level. Here, we addressed these issues through extensive phylogenomic analyses. The analyses were based on a collection of >5000 Chloroflexota genomes and metagenome-assembled genomes (MAGs) from public databases, novel environmental sites, as well as newly generated MAGs from publicly available sequence reads via an improved binning approach incorporating covariance information. Based on calculated relative evolutionary divergence, we propose that Candidatus Dormibacterota should be listed as a class (i.e., Ca. Dormibacteria) within Chloroflexota together with the classes Anaerolineae, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Ca. Limnocylindria, Thermomicrobia, and two other classes containing only uncultured members. All other Chloroflexota lineages previously listed at the class rank appear to be rather orders or families in the Anaerolineae and Dehalococcoidia, which contain the vast majority of genomes and exhibited the strongest phylogenetic radiation within the phylum. Furthermore, the study suggests that a common ecophysiological capability of members of the phylum is to successfully cope with low energy fluxes.

The marine deep subsurface is home to a vast microbial ecosystem, affecting biogeochemical cycles on a global scale. One of the better-studied deep biospheres is the Juan de Fuca (JdF) Ridge, where hydrothermal fluid introduces oxidants into the sediment from below, resulting in two sulfate methane transition zones (SMTZs). In this study, we present the first shotgun metagenomics study of unamplified DNA from sediment samples from different depths in this stratified environment. Bioinformatic analyses showed a shift from a heterotrophic, Chloroflexota-dominated community above the upper SMTZ to a chemolithoautotrophic Proteobacteria-dominated community below the secondary SMTZ. The reintroduction of sulfate likely enables respiration and boosts active cells that oxidize acetate, iron, and complex carbohydrates to degrade dead biomass in this low-abundance, low-diversity environment. In addition, analyses showed many proteins of unknown function as well as novel metagenome-assembled genomes (MAGs). The study provides new insights into microbial communities in this habitat, enabled by an improved DNA extraction protocol that allows a less biased view of taxonomic composition and metabolic activities, as well as uncovering novel taxa. Our approach presents the first successful attempt at unamplified shotgun sequencing samples from beyond 50 meters below the seafloor and opens new ways for capturing the true diversity and functional potential of deep-sea sediments.

The roseobacter group of marine bacteria is characterized by a mosaic distribution of ecologically important phenotypes. These are often encoded on mobile extrachromosomal replicons. So far, conjugation had only been experimentally proven between the two model organisms Phaeobacter inhibens and Dinoroseobacter shibae . Here, we show that two large natural RepABC-type plasmids from D. shibae can be transferred into representatives of all known major Rhodobacterales lineages. Complete genome sequencing of the newly established Phaeobacter inhibens transconjugants confirmed their genomic integrity. The conjugated plasmids were stably maintained as single copy number replicons in the genuine as well as the new host. Co-cultivation of Phaeobacter inhibens and the transconjugants with the dinoflagellate Prorocentrum minimum demonstrated that Phaeobacter inhibens is a probiotic strain that improves the yield and stability of the dinoflagellate culture. The transconjugant carrying the 191 kb plasmid, but not the 126 kb sister plasmid, killed the dinoflagellate in co-culture.

The environmental prevalence of antimicrobial resistance (AMR) has come into focus under the One Health concept. Wastewater treatment systems are among the significant sources of AMR in the environment. In such systems, it is uncertain to which extent antimicrobials present at sub-inhibitory concentrations constitute a selective pressure for bacterial maintenance and acquisition of antibiotic resistance (AR) genes. Here, we mapped AMR to inhibitors of folate biosynthesis in an aerated and a non-aerated horizontal subsurface flow treatment wetland receiving the same pre-treated municipal wastewater. General water characteristics and the concentrations of folate inhibitors were determined to define the ambient conditions over the longitudinal axis of the two treatment wetlands. Profiling of AMR as well as class 1 integrons, a carrier of AR genes against folate inhibitors and other antimicrobials, was conducted by cultivation-dependent and -independent methods. The wetlands achieved mean reductions of AR gene copy numbers in the effluents of at least 2 log, with the aerated system performing better. The folate inhibitors had no noticeable effect on the prevalence of respective AR genes. However, there was a transient increase of AR gene copy numbers and AR gene cassette composition in class 1 integrons in the aerated wetland. The comparison of all data from both wetlands suggests that higher levels of cellular stress in the aerated system promoted the mobility of AR genes via enhancing the activity of the DNA recombinase of the class 1 integron. The findings highlight that environmental conditions that modulate the activity of this genetic element can be more important for the fate of associated AR genes in treatment wetlands than the ambient concentration of the respective antimicrobial agents. By extrapolation, the results suggest that cellular stress also contributes to the mobility of AR gene in other wastewater treatment systems.

As of today, the majority of environmental microorganisms remain uncultured and is therefore referred to as 'microbial dark matter' (MDM). Hence, genomic insights into these organisms are limited to cultivation-independent approaches such as single-cell- and metagenomics. However, without access to cultured representatives for verifying correct taxon-assignments, MDM genomes may cause potentially misleading conclusions based on misclassified or contaminant contigs, thereby obfuscating our view on the uncultured microbial majority. Moreover, gradual database contaminations by past genome submissions can cause error propagations which affect present as well as future comparative genome analyses. Consequently, strict contamination detection and filtering need to be applied, especially in the case of uncultured MDM genomes. Current genome reporting standards, however, emphasize completeness over purity and the de facto gold standard genome assessment tool, checkM, discriminates against uncultured taxa and fragmented genomes. To tackle these issues, we present a novel contig classification, screening, and filtering workflow and corresponding open-source python implementation called MDMcleaner, which was tested and compared to other tools on mock and real datasets. MDMcleaner revealed substantial contaminations overlooked by current screening approaches and sensitively detects misattributed contigs in both novel genomes and the underlying reference databases, thereby greatly improving our view on 'microbial dark matter'.

Actinobacteria, one of the largest bacterial phyla, are ubiquitous in many of Earth's ecosystems and often act as defensive symbionts with animal hosts. Members of the phylum have repeatedly been isolated from basidiomycete-cultivating fungus-farming termites that maintain a monoculture fungus crop on macerated dead plant substrate. The proclivity for antimicrobial and enzyme production of Actinobacteria make them likely contributors to plant decomposition and defense in the symbiosis. To test this, we analyzed the prophylactic (biosynthetic gene cluster [BGC]) and metabolic (carbohydrate-active enzyme [CAZy]) potential in 16 (10 existing and six new genomes) termite-associated Actinobacteria and compared these to the soil-dwelling close relatives. Using antiSMASH, we identified 435 BGCs, of which 329 (65 unique) were similar to known compound gene clusters, while 106 were putatively novel, suggesting ample prospects for novel compound discovery. BGCs were identified among all major compound categories, including 26 encoding the production of known antimicrobial compounds, which ranged in activity (antibacterial being most prevalent) and modes of action that might suggest broad defensive potential. Peptide pattern recognition analysis revealed 823 (43 unique) CAZymes coding for enzymes that target key plant and fungal cell wall components (predominantly chitin, cellulose, and hemicellulose), confirming a substantial degradative potential of these bacteria. Comparison of termite-associated and soil-dwelling bacteria indicated no significant difference in either BGC or CAZy potential, suggesting that the farming termite hosts may have coopted these soil-dwelling bacteria due to their metabolic potential but that they have not been subject to genome change associated with symbiosis.IMPORTANCEActinobacteria have repeatedly been isolated in fungus-farming termites, and our genome analyses provide insights into the potential roles they may serve in defense and for plant biomass breakdown. These insights, combined with their relatively higher abundances in fungus combs than in termite gut, suggest that they are more likely to play roles in fungus combs than in termite guts. Up to 25% of the BGCs we identify have no similarity to known clusters, indicating a large potential for novel chemistry to be discovered. Similarities in metabolic potential of soil-dwelling and termite-associated bacteria suggest that they have environmental origins, but their consistent presence with the termite system suggests their importance for the symbiosis.

Gene abundances measured by qPCR:Antimicrobial resistance: (intI1, sul1, sul2, dfrA1),E.coli (uidA),and total bacterial: 16S rRNA genefrom pilot scale constructed wetlands treating municipal wastewater.

The present study reports the isolation of antibacterial exhibiting Bacillus pumilus (B. pumilus) SF-4 from soil field. The genome of this strain SF-4 was sequenced and analyzed to acquire in-depth genomic level insight related to functional diversity, evolutionary history, and biosynthetic potential. The genome of the strain SF-4 harbor 12 Biosynthetic Gene Clusters (BGCs) including four Non-ribosomal peptide synthetases (NRPSs), two terpenes, and one each of Type III polyketide synthases (PKSs), hybrid (NRPS/PKS), lipopeptide, β-lactone, and bacteriocin clusters. Plant growth-promoting genes associated with de-nitrification, iron acquisition, phosphate solubilization, and nitrogen metabolism were also observed in the genome. Furthermore, all the available complete genomes of B. pumilus strains were used to highlight species boundaries and diverse niche adaptation strategies. Phylogenetic analyses revealed local diversification and indicate that strain SF-4 is a sister group to SAFR-032 and 150a. Pan-genome analyses of 12 targeted strains showed regions of genome plasticity which regulate function of these strains and proposed direct strain adaptations to specific habitats. The unique genome pool carries genes mostly associated with “biosynthesis of secondary metabolites, transport, and catabolism” (Q), “replication, recombination and repair” (L), and “unknown function” (S) clusters of orthologous groups (COG) categories. Moreover, a total of 952 unique genes and 168 exclusively absent genes were prioritized across the 12 genomes. While newly sequenced B. pumilus SF-4 genome consists of 520 accessory, 59 unique, and seven exclusively absent genes. The current study demonstrates genomic differences among 12 B. pumilus strains and offers comprehensive knowledge of the respective genome architecture which may assist in the agronomic application of this strain in future.

As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as "microbial dark matter." In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique-out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages-in whole or in part-have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.

In many countries, emission of insufficiently treated wastewater into water bodies appears to be an important factor in spreading clinically relevant antimicrobial resistant bacteria. In this study, we looked for the presence of Enterobacteriaceae strains with resistance to 3rd generation cephalosporin antibiotics in four urban wetlands in southwestern Nigeria by isolation, whole genome sequencing and qPCR enumeration of marker genes. Genome analysis of multi-drug resistant and potentially pathogenic Escherichia coli isolates (members of the widely distributed ST10 complex) revealed the presence of the extended spectrum beta-lactamase gene blaCTX-M-15 on self-transmissible IncF plasmids. The gene was also present together with a blaTEM-1B gene on self-transmissible IncH plasmids in multi-drug resistant Enterobacter cloacae isolates. A Citrobacter freundii isolate carried blaTEM-1B on an IncR-type plasmid without discernable conjugation apparatus. All strains were isolated from a wetland for which previous qPCR enumeration of marker genes, in particular the ratio of intI1 to 16S rRNA gene copy numbers, had indicated a strong anthropogenic impact. Consistent with the isolation origin, qPCR analysis in this study showed that the blaCTX-M gene was present at an abundance of 1x10-4 relative to bacterial 16S rRNA gene copy numbers. The results indicate that contamination of these urban aquatic ecosystems with clinically relevant antibiotic resistant bacteria is substantial in some areas. Measures should therefore be put in place to mitigate the propagation of clinically relevant antimicrobial resistance within the Nigerian aquatic ecosystems.

Seagrass meadows are ubiquitous, fragile and endangered marine habitats, which serve as fish breeding grounds, stabilize ocean floor substrates, retain nutrients and serve as important carbon sinks, counteracting climate change. In the Mediterranean Sea, seagrass meadows are mostly formed by the slow-growing endemic plant Posidonia oceanica (Neptune grass), which is endangered by global warming and recreational motorboating. Despite its importance, surprisingly little is known about the leaf surface microbiome of P. oceanica. Using amplicon sequencing, we here show that species belonging to the phylum Planctomycetes can dominate the biofilms of young and aged P. oceanica leaves. Application of selective cultivation techniques allowed for the isolation of two novel planctomycetal strains belonging to two yet uncharacterized genera.

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20T and RB56T, were determined using a polyphasic approach. Both were isolated from the fungus-farming termite Macrotermes natalensis. Results of 16S rRNA gene sequence analysis revealed that both strains are members of the genus Nocardia with the closest phylogenetic neighbours Nocardia miyunensis JCM12860T (98.9 %) and Nocardia nova DSM44481T (98.5 %) for RB20T and Nocardia takedensis DSM 44801T (98.3 %), Nocardia pseudobrasiliensis DSM 44290T (98.3 %) and Nocardia rayongensis JCM 19832T (98.2 %) for RB56T. Digital DNA–DNA hybridization (DDH) between RB20T and N. miyunensis JCM12860T and N. nova DSM 44481T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56T and N. takedensis DSM44801T and N. pseudobrasiliensis DSM44290T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56T and N. rayongensis JCM19832T resulted in 10.2 % (14.5 %) similarity. Both strains showed morphological and chemotaxonomic features typical for the genus Nocardia , such as the presence of meso-diaminopimelic acid (A2pm) within the cell wall, arabinose and galactose as major sugar components within whole cell-wall hydrolysates, the presence of mycolic acids and major phospholipids (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol), and the predominant menaquinone MK-8 (H4, ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C16 : 0), 10-methyloctadecanoic acid (10-methyl C18 : 0) and cis-9-octadecenoic acid (C18 : 1 ω9c). We propose two novel species within the genus Nocardia : Nocardia macrotermitis sp. nov. with the type strain RB20T (=VKM Ac-2841T=NRRL B65541T) and Nocardia aurantia sp. nov. with the type strain RB56T (=VKM Ac-2842T=NRRL B65542T).

Seagrass meadows are ubiquitous, fragile and endangered marine habitats, which serve as fish breeding grounds, stabilize ocean floor substrates, retain nutrients and serve as important carbon sinks, counteracting climate change. In the Mediterranean Sea, seagrass meadows are mostly formed by the slow-growing endemic plantPosidonia oceanica(Neptune grass), which is endangered by global warming and recreational motorboating. Despite its importance, surprisingly little is known about the leaf surface microbiome ofP. oceanica. Using amplicon sequencing, we here show that species belonging to the phylumPlanctomycetescan dominate the biofilms of young and agedP. oceanicaleaves. Application of selective cultivation techniques allowed for the isolation of two novel planctomycetal strains belonging to two yet uncharacterized genera.

The taxonomic positions of two novel aerobic, Gram-positive actinobacteria, designated strains RB29 T and RB68 T , were determined using a polyphasic approach. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of RB29 T were identified as Actinomadura rayongensis DSM 102126 T (99.2 % similarity) and Actinomadura atramentaria DSM 43919 T (98.7 %), and for strain RB68 T was Actinomadura hibisca DSM 44148 T (98.3 %). Digital DNA–DNA hybridization (dDDH) between RB29 T and its closest phylogenetic neighbours, A. rayongensis DSM 102126 T and A. atramentaria DSM 43919 T , resulted in similarity values of 53.2 % (50.6–55.9 %) and 26.4 % (24.1–28.9 %), respectively. Additionally, the average nucleotide identity (ANI) was 93.2 % (94.0 %) for A. rayongensis DSM 102126 T and 82.3 % (78.9 %) for A. atramentaria DSM 43919 T . dDDH analysis between strain RB68 T and A. hibisca DSM 44148 T gave a similarity value of 24.5 % (22.2–27.0 %). Both strains, RB29 T and RB68 T , revealed morphological characteristics and chemotaxonomic features typical for the genus Actinomadura , such as the presence of meso -diaminopimelic acid in the cell wall, galactose and glucose as major sugar components within whole-cell hydrolysates and the absence of mycolic acids. The major phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside. Predominant menaquinones were MK-9(H 6 ) and MK-9(H 8 ) for RB29 T and MK-9(H 4 ) and MK-9(H 6 ) for RB68 T . The main fatty acids were identified as 10-methyloctadecanoic acid (10-methyl C 18:0 ), 14-methylpentadecanoic acid (iso-C 16:0 ), hexadecanoic acid (C 16:0 ) and cis -9-octadecanoic acid (C 18 : 1 ω9 c ). Here, we propose two novel species of the genus Actinomadura : Actinomadura rubteroloni sp. nov. with the type strain RB29 T (=CCUG 72668 T =NRRL B-65537 T ) and Actinomadura macrotermitis sp. nov. with the type strain RB68 T (=CCUG 72669 T =NRRL B-65538 T ).

Bei Plasmiden handelt es sich um ringförmige, doppelsträngige DNA‐Moleküle, die unabhängig vom Bakterienchromosom in der Bakterienzelle vorkommen. Bakterien nutzen Plasmide unter anderem für den horizontalen Gentransfer. Forscherinnen und Forscher um Professorin Anne‐Kristin Kaster, Direktorin des Institute for Biological Interfaces (IBG 5) des Karlsruher Instituts für Technologie (KIT), vom Leibniz‐Institut DSMZ und der University of East Anglia (UEA) haben nun erstmals ein hundertprozentig identisches Plasmid in verschiedenen Mikroorganismen an geographisch weit entfernten Standorten nachgewiesen. Ihre Ergebnisse publizierten die Forscher in dem renommierten Fachmagazin Proceedings of the National Academy of Sciences (PNAS).