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Cover cropping is an effective method to protect agricultural soils from erosion, promote nutrient and moisture retention, encourage beneficial microbial activity, and maintain soil structure. Re-utilization of winter cover crop root channels by maize roots during summer allows the cash crop to extract resources from distal regions in the soil horizon. In this study, we investigated how cover cropping during winter followed by maize ( Zea mays L.) during summer affects the spatiotemporal composition and function of the bacterial communities in the maize rhizosphere and surrounding soil samples using quantitative polymerase chain reaction (PCR), 16S ribosomal ribonucleic acid (rRNA) gene amplicon sequencing, and metaproteomics. We found that the bacterial community differed significantly among cover crop species, soil depths, and maize growth stages. Bacterial abundance increased in reused root channels, and it continued to increase as cover crop diversity changed from monocultures to mixtures. Mixing Fabaceae with Brassicaceae or Poaceae enhanced the overall contributions of several steps of the bacterial carbon and nitrogen cycles, especially glycolysis and the pentose phosphate pathway. The deeper root channels of Fabaceae and Brassicaceae as compared to Poaceae corresponded to higher bacterial 16S rRNA gene copy numbers and improved community presence in the subsoil regimes, likely due to the increased availability of root exudates secreted by maize roots. In conclusion, root channel reuse improved the expression of metabolic pathways of the carbon and nitrogen cycles and the bacterial communities, which is beneficial to the soil and to the growing crops., Competing Interests: The authors declare that they have no conflict of interests that could have influenced the work being reported in this manuscript., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Drug-induced differential gene expression analysis (DGEA) is essential for uncovering the molecular basis of cell phenotypic changes and understanding individual tumor responses to anticancer drugs. Performing high throughput DGEA is challenging due to the high cost and labor-intensive multi-step sample preparation protocols. In particular, performing drug-induced DGEA on cancer cells derived from patient biopsies is even more challenging due to the scarcity of available cells. A novel, miniaturized, nanoliter-scale method for drug-induced DGEA is introduced, enabling high-throughput and parallel analysis of patient-derived cell drug responses, overcoming the limitations and laborious nature of traditional protocols. The method is based on the Droplet Microarray (DMA), a microscope glass slide with hydrophilic spots on a superhydrophobic background, facilitating droplet formation for cell testing. DMA allows microscopy-based phenotypic analysis, cDNA extraction, and DGEA. The procedure includes cell lysis for mRNA isolation and cDNA conversion followed by droplet pooling for qPCR analysis. In this study, the drug-induced DGEA protocol on the DMA platform is demonstrated using patient-derived chronic lymphocytic leukemia (CLL) cells. This methodology is critical for DGEA with limited cell numbers and promise for applications in functional precision oncology. This method enables molecular profiling of patient-derived samples after drug treatment, crucial for understanding individual tumor responses to anticancer drugs., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Microorganisms serve as linchpins in agricultural systems. Classic examples include microbial composting for nutrient recovery, using microorganisms in biogas technology for agricultural waste utilization, and employing biofilters to reduce emissions from stables or improve water quality in aquaculture. This mini-review highlights the importance of microbiome analysis in understanding microbial diversity, dynamics, and functions, fostering innovations for a more sustainable agriculture. In this regard, customized microorganisms for soil improvement, replacements for harmful agrochemicals or antibiotics in animal husbandry, and (probiotic) additives in animal nutrition are already in or even beyond the testing phase for a large-scale conventional agriculture. Additionally, as climate change reduces arable land, new strategies based on closed-loop systems and controlled environment agriculture, emphasizing microbial techniques, are being developed for regional food production. These strategies aim to secure the future food supply and pave the way for a sustainable, resilient, and circular agricultural economy. KEY POINTS: • Microbial strategies facilitate the integration of multiple trophic levels, essential for cycling carbon, nitrogen, phosphorus, and micronutrients. • Exploring microorganisms in integrated biological systems is essential for developing practical agricultural solutions. • Technological progress makes sustainable closed-entity re-circulation systems possible, securing resilient future food production., (© 2024. The Author(s).)

Pseudomonas stutzeri KC can rapidly degrade carbon tetrachloride (CCl4) to CO2 by a fortuitous reaction with pyridine-2,6-bis(thiocarboxylic acid), a metal chelator encoded by pdt genes. These genes were first identified after a spontaneous mutant, strain CTN1, lost the ability to degrade CCl4. Here we report the complete genome of strain KC and show that these pdt genes are located on an integrative and conjugative element (ICE), designated ICEPsstKC. Comparative genome analyses revealed homologues of pdt genes in genomes of members of other gammaproteobacterial orders. Discrepancies between the tree topologies of the deduced pdt gene products and the host phylogeny based on 16S rRNA provided evidence for horizontal gene transfer (HGT) in several sequenced strains of these orders. In addition to ICEPsstKC, HGT may be have been facilitated by other mobile genetic elements, as indicated by the location of the pdt gene cluster adjacent to fragments of other ICEs and prophages in several genome assemblies. We could here show that the majority of cells from the culture collection DSMZ had lost the ICE. The presence of the pdt gene cluster on mobile genetic elements has important implications for the bioremediation of CCl4 for bioremediation of CCl4 and needs consideration when selecting suitable strains., (The Author(s). Published by S. Karger AG, Basel.)

Glyphosate is used worldwide as a compound of pesticides and is detectable in many environmental compartments. It enters water bodies primarily through drift from agricultural areas so that aquatic organisms are exposed to this chemical, especially after rain events. Glyphosate is advertised and sold as a highly specific herbicide, which interacts with the EPSP synthase, an enzyme of the shikimate metabolism, resulting in inhibition of the synthesis of vital aromatic amino acids. However, not only plants but also bacteria can possess this enzyme so that influences of glyphosate on the microbiomes of exposed organisms cannot be excluded. Those influences may result in subtle and long-term effects, e.g., disturbance of the symbiotic interactions of bionts with microorganisms of their microbiomes. Mechanisms how the transformation product aminomethylphosphonic acid (AMPA) of glyphosate might interfere in this context have not understood so far. In the present study, molecular biological fingerprinting methods showed concentration-dependent effects of glyphosate and AMPA on fish microbiomes. In addition, age-dependent differences in the composition of the microbiomes regarding abundance and diversity were detected. Furthermore, the effect of exposure to glyphosate and AMPA was investigated for several fish pathogens of gut microbiomes in terms of their gene expression of virulence factors associated with pathogenicity. In vitro transcriptome analysis with the fish pathogen Yersinia ruckeri revealed that it is questionable whether the observed effect on the microbiome is caused by the intended mode of action of glyphosate, such as the inhibition of EPSP synthase activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hembach, Drechsel, Sobol, Kaster, Köhler, Triebskorn and Schwartz.)

Sustainable approaches to circular economy in animal agriculture are still poorly developed. Here, we report an approach to reduce gaseous emissions of CO 2 and NH 3 from animal housing while simultaneously using them to produce value-added biomass. To this end, a cone-shaped, helical photobioreactor was developed that can be integrated into animal housing by being freely suspended, thereby combining a small footprint with a physically robust design. The photobioreactor was coupled with the exhaust air of a chicken house to allow continuous cultivation of a mixed culture of Arthrospira spec. (Spirulina). Continuous quantification of CO 2 and NH 3 concentration showed that the coupled algae reactor effectively purifies the exhaust air from the chicken house while producing algal biomass. Typical production rates of greater than 0.3 g/l*day dry mass were obtained, and continuous operation was possible for several weeks. Morphological, biochemical, and genomic characterization of Spirulina cultures yielded insights into the dynamics and metabolic processes of the microbial community. We anticipate that further optimization of this approach will provide new opportunities for the generation of value-added products from gaseous CO 2 and NH 3 waste emissions, linking resource-efficient production of microalgae with simultaneous sequestration of animal emissions. KEY POINTS: • Coupling a bioreactor with exhaust gases of chicken coop for production of biomass. • Spirulina mixed culture removes CO 2 and NH 3 from chicken house emissions. • High growth rates and biodiversity adaptation for nitrogen metabolism. Towards a sustainable circular economy in livestock farming. The functional coupling of a helical tube photobioreactor with exhaust air from a chicken house enabled the efficient cultivation of Spirulina microalgae while simultaneously sequestering the animals' CO 2 and NH 3 emissions., (© 2023. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Metze, Vollmers, Lenk and Kaster.)

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 plethora of stress factors that can damage microbial cells has evolved sophisticated stress response mechanisms. While existing bioreporters can monitor individual responses, sensors for detecting multimodal stress responses in living microorganisms are still lacking. Orthogonally detectable red, green, and blue fluorescent proteins combined in a single plasmid, dubbed RGB-S reporter, enable simultaneous, independent, and real-time analysis of the transcriptional response of Escherichia coli using three promoters which report physiological stress (PosmY for RpoS), genotoxicity (PsulA for SOS), and cytotoxicity (PgrpE for RpoH). The bioreporter is compatible with standard analysis and Fluorescent Activated Cell Sorting (FACS) combined with subsequent transcriptome analysis. Various stressors, including the biotechnologically relevant 2-propanol, activate one, two, or all three stress responses, which can significantly impact non-stress-related metabolic pathways. Implemented in microfluidic cultivation with confocal fluorescence microscopy imaging, the RGB-S reporter enabled spatiotemporal analysis of live biofilms revealing stratified subpopulations of bacteria with heterogeneous stress responses., (© 2023. Springer Nature Limited.)

Microbial single-cell genomics (SCG) provides access to the genomes of rare and uncultured microorganisms and is a complementary method to metagenomics. Due to the femtogram-levels of DNA in a single microbial cell, sequencing the genome requires whole genome amplification (WGA) as a preliminary step. However, the most common WGA method, multiple displacement amplification (MDA), is known to be costly and biased against specific genomic regions, preventing high-throughput applications and resulting in uneven genome coverage. Thus, obtaining high-quality genomes from many taxa, especially minority members of microbial communities, becomes difficult. Here, we present a volume reduction approach that significantly reduces costs while improving genome coverage and uniformity of DNA amplification products in standard 384-well plates. Our results demonstrate that further volume reduction in specialized and complex setups (e.g., microfluidic chips) is likely unnecessary to obtain higher-quality microbial genomes. This volume reduction method makes SCG more feasible for future studies, thus helping to broaden our knowledge on the diversity and function of understudied and uncharacterized microorganisms in the environment.

Annotating protein sequences according to their biological functions is one of the key steps in understanding microbial diversity, metabolic potentials, and evolutionary histories. However, even in the best-studied prokaryotic genomes, not all proteins can be characterized by classical in vivo, in vitro, and/or in silico methods-a challenge rapidly growing alongside the advent of next-generation sequencing technologies and their enormous extension of 'omics' data in public databases. These so-called hypothetical proteins (HPs) represent a huge knowledge gap and hidden potential for biotechnological applications. Opportunities for leveraging the available 'Big Data' have recently proliferated with the use of artificial intelligence (AI). Here, we review the aims and methods of protein annotation and explain the different principles behind machine and deep learning algorithms including recent research examples, in order to assist both biologists wishing to apply AI tools in developing comprehensive genome annotations and computer scientists who want to contribute to this leading edge of biological research., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

Transcriptomics, or more specifically mRNA sequencing, is a powerful tool to study gene expression at the single-cell level (scRNA-seq) which enables new insights into a plethora of biological processes. While methods for single-cell RNA-seq in eukaryotes are well established, application to prokaryotes is still challenging. Reasons for that are rigid and diverse cell wall structures hampering lysis, the lack of polyadenylated transcripts impeding mRNA enrichment, and minute amounts of RNA requiring amplification steps before sequencing. Despite those obstacles, several promising scRNA-seq approaches for bacteria have been published recently, albeit difficulties in the experimental workflow and data processing and analysis remain. In particular, bias is often introduced by amplification which makes it difficult to distinguish between technical noise and biological variation. Future optimization of experimental procedures and data analysis algorithms are needed for the improvement of scRNA-seq but also to aid in the emergence of prokaryotic single-cell multi-omics. to help address 21st century challenges in the biotechnology and health sector., (Copyright © 2023. Published by Elsevier Inc.)

To date, the vast majority of prokaryotic organisms escapes detailed characterization because they cannot be isolated in axenic cultures. These organisms are referred to as microbial dark matter. Targeted labelling and sorting of these microorganisms pave the way for single-cell, enrichment, or cultivation approaches. In this review, we describe an array of different methods ranging from labeling-free to specific labelling techniques. In addition, different cell sorting methods and their combinations with targeting strategies are summarized and downstream applications like sequencing and cultivation are reviewed. Recent advances, challenges, and limitations of the particular methods are discussed with respect to cell viability, genome integrity as well as throughput, in order to help researchers select the most suitable methods for their specific research questions., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Self-transferable plasmids of the incompatibility group P-1 (IncP-1) are considered important carriers of genes for antibiotic resistance and other adaptive functions. In the laboratory, these plasmids have a broad host range; however, little is known about their in situ host profile. In this study, we discovered that Thauera aromatica K172 T , a facultative denitrifying microorganism capable of degrading various aromatic compounds, contains a plasmid highly similar to the IncP-1 ε archetype pKJK5. The plasmid harbours multiple antibiotic resistance genes and is maintained in strain K172 T for at least 1000 generations without selection pressure from antibiotics. In a subsequent search, we found additional nine IncP-type plasmids in a total of 40 sequenced genomes of the closely related genera Aromatoleum and Thauera. Six of these plasmids form a novel IncP-1 subgroup designated θ, four of which carry genes for anaerobic or aerobic degradation of aromatic compounds. Pentanucleotide sequence analyses (k-mer profiling) indicated that Aromatoleum spp. and Thauera spp. are among the most suitable hosts for the θ plasmids. Our results highlight the importance of IncP-1 plasmids for the genetic adaptation of these common facultative denitrifying bacteria and provide novel insights into the in situ host profile of these plasmids., (© 2022 The Authors. Environmental Microbiology published by Applied Microbiology International and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Natural evolution has produced an almost infinite variety of microorganisms that can colonize almost any conceivable habitat. Since the vast majority of these microbial consortia are still unknown, there is a great need to elucidate this "microbial dark matter" (MDM) to enable exploitation in biotechnology. We report the fabrication and application of a novel device that integrates a matrix of macroporous elastomeric silicone foam (MESIF) into an easily fabricated and scalable chip design that can be used for decoding MDM in environmental microbiomes. Technical validation, performed with the model organism Escherichia coli expressing a fluorescent protein, showed that this low-cost, bioinert, and widely modifiable chip is rapidly colonized by microorganisms. The biological potential of the chip was then illustrated through targeted sampling and enrichment of microbiomes in a variety of habitats ranging from wet, turbulent moving bed biofilters and wastewater treatment plants to dry air-based environments. Sequencing analyses consistently showed that MESIF chips are not only suitable for sampling with high robustness but also that the material can be used to detect a broad cross section of microorganisms present in the habitat in a short time span of a few days. For example, results from the biofilter habitat showed efficient enrichment of microorganisms belonging to the enigmatic Candidate Phyla Radiation , which comprise ∼70% of the MDM. From dry air, the MESIF chip was able to enrich a variety of members of Actinobacteriota , which is known to produce specific secondary metabolites. Targeted sampling from a wastewater treatment plant where the herbicide glyphosate was added to the chip's reservoir resulted in enrichment of Cyanobacteria and Desulfobacteria , previously associated with glyphosate degradation. These initial case studies suggest that this chip is very well suited for the systematic study of MDM and opens opportunities for the cultivation of previously unculturable microorganisms.

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'., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Planctomycetes are bacteria that were long thought to be unculturable, of low abundance, and therefore neglectable in the environment. This view changed in recent years, after it was shown that members of the phylum Planctomycetes can be abundant in many aquatic environments, e.g., in the epiphytic communities on macroalgae surfaces. Here, we analyzed three different macroalgae from the North Sea and show that Planctomycetes is the most abundant bacterial phylum on the alga Fucus sp., while it represents a minor fraction of the surface-associated bacterial community of Ulva sp. and Laminaria sp. Especially dominant within the phylum Planctomycetes were Blastopirellula sp., followed by Rhodopirellula sp., Rubripirellula sp., as well as other Pirellulaceae and Lacipirellulaceae , but also members of the OM190 lineage. Motivated by the observed abundance, we isolated four novel planctomycetal strains to expand the collection of species available as axenic cultures since access to different strains is a prerequisite to investigate the success of planctomycetes in marine environments. The isolated strains constitute four novel species belonging to one novel and three previously described genera in the order Pirellulales , class Planctomycetia , phylum Planctomycetes .

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., Competing Interests: JR is an employee of cytena GmbH, a company that is developing single-cell analysis and handling solutions. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wiegand, Dam, Riba, Vollmers and Kaster.)

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. IMPORTANCE Actinobacteria 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., (Copyright © 2021 Murphy et al.)

Thirteen novel planctomycetal strains were isolated from five different aquatic sampling locations. These comprise the hydrothermal vent system close to Panarea Island (Italy), a biofilm on the surface of kelp at Monterey Bay (CA, USA), sediment and algae on Mallorca Island (Spain) and Helgoland Island (Germany), as well as a seawater aquarium in Braunschweig, Germany. All strains were shown to belong to the genus Gimesia. Their genomes cover a size range from 7.22 to 8.29 Mb and have a G+C content between 45.1 and 53.7%. All strains are mesophilic (T opt 26-33 °C) with generation times between 12 and 32 h. Analysis of fatty acids yielded palmitic acid (16:0) and a fatty acid with the equivalent chain length of 15.817 as major compounds. While five of the novel strains belong to the already described species Gimesia maris and Gimesia chilikensis, the other strains belong to novel species, for which we propose the names Gimesia alba (type strain Pan241w T  = DSM 100744 T  = LMG 31345 T  = CECT 9841 T  = VKM B-3430 T ), Gimesia algae (type strain Pan161 T  = CECT 30192 T = STH00943 T  = LMG 29130 T ), Gimesia aquarii (type strain V144 T  = DSM 101710 T  = VKM B-3433 T ), Gimesia fumaroli (type strain Enr17 T  = DSM 100710 T  = VKM B-3429 T ) and Gimesia panareensis (type strain Enr10 T  = DSM 100416 T  = LMG 29082 T ). STH numbers refer to the Jena Microbial Resource Collection (JMRC).

Eight novel strains of the phylum Planctomycetes were isolated from different aquatic habitats. Among these habitats were the hydrothermal vent system close to Panarea Island, a public beach at Mallorca Island, the shore of Costa Brava (Spain), and three sites with brackish water in the Baltic Sea. The genome sizes of the novel strains range from 4.33 to 6.29 Mb with DNA G+C contents between 52.8 and 66.7%. All strains are mesophilic (T opt 24-30 °C) and display generation times between 17 and 94 h. All eight isolates constitute novel species of either already described or novel genera within the family Lacipirellulaceae. Two of the novel species, Posidoniimonas polymericola (type strain Pla123a T  = DSM 103020 T  = LMG 29466 T ) and Bythopirellula polymerisocia (type strain Pla144 T  = DSM 104841 T  = VKM B-3442 T ), belong to established genera, while the other strains represent the novel genera Aeoliella gen. nov., Botrimarina gen. nov., Pirellulimonas gen. nov. and Pseudobythopirellula gen. nov. Based on our polyphasic analysis, we propose the species Aeoliella mucimassa sp. nov. (type strain Pan181 T  = DSM 29370 T  = LMG 31346 T  = CECT 9840 T  = VKM B-3426 T ), Botrimarina colliarenosi sp. nov. (type strain Pla108 T  = DSM 103355 T  = LMG 29803 T ), Botrimarina hoheduenensis sp. nov. (type strain Pla111 T  = DSM 103485 T  = STH00945 T , Jena Microbial Resource Collection JMRC), Botrimarina mediterranea sp. nov. (type strain Spa11 T  = DSM 100745 T  = LMG 31350 T  = CECT 9852 T  = VKM B-3431 T ), Pirellulimonas nuda sp. nov. (type strain Pla175 T  = DSM 109594 T  = CECT 9871 T  = VKM B-3448 T ) and Pseudobythopirellula maris sp. nov. (type strain Mal64 T  = DSM 100832 T  = LMG 29020 T ).

The taxonomic position of a novel aerobic, Gram-positive actinobacteria, designated strain RB5 T , was determined using a polyphasic approach. The strain, isolated from the gut of the fungus-farming termite Macrotermes natalensis , showed morphological, physiological and chemotaxonomic properties typical of the genus Streptomyces . Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of RB5 T was Streptomyces polyrhachis DSM 42102 T (98.87 %). DNA-DNA hybridization experiments between strain RB5 T and S. polyrhachis DSM 42102 T resulted in a value of 27.4 % (26.8 %). The cell wall of strain RB5 T contained ll-diaminopimelic acid as the diagnostic amino acid. Mycolic acids and diagnostic sugars in whole-cell hydrolysates were not detected. The strain produced the following major phospholipids: diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol-mannoside and phosphatidylserine. The menaquinone profile showed hexa- and octahydrogenated menaquinones containing nine isoprene units [MK-9(H 6 ) and MK-9(H 8 )]. The strain exhibited a fatty acid profile containing the following major fatty acids: 12-methyltridecanoic acid (iso-C 14 : 0 ) 12-methyltetradecanoic acid (anteiso-C 15 : 0 ), 13-methyltetradecanoic acid (iso-C 15 : 0 ) and 14-methylpentadecanoic acid (iso-C 16 : 0 ). Here, we propose a novel species of the genus Streptomyces - Streptomyces smaragdinus with the type strain RB5 T (=VKM Ac-2839 T =NRRL B65539 T ).

Based on high-resolution tandem mass spectrometry (HR-MS 2 ) and global natural products social molecular networking (GNPS), we found that plant-derived daidzein and genistein derivatives are polyhalogenated by termite-associated Actinomadura species RB99. MS-guided purification from extracts of bacteria grown under optimized conditions led to the isolation of eight polychlorinated isoflavones, including six unreported derivatives, and seven novel polybrominated derivatives, two of which showed antimicrobial activity.

Single-cell genomics and transcriptomics can provide reliable context for assembled genome fragments and gene expression activity on the level of individual prokaryotic genomes. These methods are rapidly emerging as an essential complement to cultivation-based, metagenomics, metatranscriptomics, and microbial community-focused research approaches by allowing direct access to information from individual microorganisms, even from deep-branching phylogenetic groups that currently lack cultured representatives. Their integration and binning with environmental 'omics data already provides unprecedented insights into microbial diversity and metabolic potential, enabling us to provide information on individual organisms and the structure and dynamics of natural microbial populations in complex environments. This review highlights the pitfalls and recent advances in the field of single-cell omics and its importance in microbiological and biotechnological studies. KEY POINTS: • Single-cell omics expands the tree of life through the discovery of novel organisms, genes, and metabolic pathways. • Disadvantages of metagenome-assembled genomes are overcome by single-cell omics. • Functional analysis of single cells explores the heterogeneity of gene expression. • Technical challenges still limit this field, thus prompting new method developments.

The taxonomic positions of two novel aerobic, Gram-stain-positive Actinobacteria, designated RB20 T and RB56 T , 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 JCM12860 T (98.9 %) and Nocardia nova DSM44481 T (98.5 %) for RB20 T and Nocardia takedensis DSM 44801 T (98.3 %), Nocardia pseudobrasiliensis DSM 44290 T (98.3 %) and Nocardia rayongensis JCM 19832 T (98.2 %) for RB56 T . Digital DNA-DNA hybridization (DDH) between RB20 T and N. miyunensis JCM12860 T and N. nova DSM 44481 T resulted in similarity values of 33.9 and 22.0 %, respectively. DDH between RB56 T and N. takedensis DSM44801 T and N. pseudobrasiliensis DSM44290 T showed similarity values of 20.7 and 22.3 %, respectively. In addition, wet-lab DDH between RB56 T and N. rayongensis JCM19832 T 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 (A 2 pm) 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 (H 4 , ω-cyclo). The main fatty acids for both strains were hexadecanoic acid (C 16 : 0 ), 10-methyloctadecanoic acid (10-methyl C 18 : 0 ) and cis -9-octadecenoic acid (C 18 : 1 ω9 c ). We propose two novel species within the genus Nocardia : Nocardia macrotermitis sp. nov. with the type strain RB20 T (=VKM Ac-2841 T =NRRL B65541 T ) and Nocardia aurantia sp. nov. with the type strain RB56 T (=VKM Ac-2842 T =NRRL B65542 T ).

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 ).

Rare members of environmental microbial communities are often overlooked and unexplored, primarily due to the lack of techniques capable of acquiring their genomes. Chloroflexi belong to one of the most understudied phyla, even though many of its members are ubiquitous in the environment and some play important roles in biochemical cycles or biotechnological applications. We here used a targeted cell-sorting approach, which enables the selection of specific taxa by fluorescent labeling and is compatible with subsequent single-cell genomics, to enrich for rare Chloroflexi species from a wastewater-treatment plant and obtain their genomes. The combined workflow was able to retrieve a substantially higher number of novel Chloroflexi draft genomes with much greater phylogenetical diversity when compared to a metagenomics approach from the same sample. The method offers an opportunity to access genetic information from rare biosphere members which would have otherwise stayed hidden as microbial dark matter and can therefore serve as an essential complement to cultivation-based, metagenomics, and microbial community-focused research approaches., (Copyright © 2020 Dam, Vollmers, Sobol, Cabezas and Kaster.)

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., (Copyright © 2020 Kohn, Rast, Kallscheuer, Wiegand, Boedeker, Jetten, Jeske, Vollmers, Kaster, Rohde, Jogler and Jogler.)

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., Competing Interests: The authors have declared that no competing interests exist.

Subsurface ecosystems like groundwater harbour diverse microbial communities, including small-sized, putatively symbiotic organisms of the Candidate Phyla Radiation, yet little is known about their ecological preferences and potential microbial partners. Here, we investigated a member of the superphylum Microgenomates (Cand. Roizmanbacterium ADI133) from oligotrophic groundwater using mini-metagenomics and monitored its spatio-temporal distribution using 16S rRNA gene analyses. A Roizmanbacteria-specific quantitative PCR assay allowed us to track its abundance over the course of 1 year within eight groundwater wells along a 5.4 km hillslope transect, where Roizmanbacteria reached maximum relative abundances of 2.3%. In-depth genomic analyses suggested that Cand. Roizmanbacterium ADI133 is a lactic acid fermenter, potentially able to utilize a range of complex carbon substrates, including cellulose. We hypothesize that it attaches to host cells using a trimeric autotransporter adhesin and inhibits their cell wall biosynthesis using a toxin-antitoxin system. Network analyses based on correlating Cand. Roizmanbacterium ADI133 abundances with amplicon sequencing-derived microbial community profiles suggested one potential host organism, classified as a member of the class Thermodesulfovibrionia (Nitrospirae). By providing lactate as an electron donor Cand. Roizmanbacterium ADI133 potentially mediates the transfer of carbon to other microorganisms and thereby is an important connector in the microbial community., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that 'everything is everywhere'. With the discovery of lateral budding in 'Kolteria novifilia' and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how 'microbial dark matter' can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection.

Horizontal gene transfer (HGT) plays an important role in bacterial evolution and serves as a driving force for bacterial diversity and versatility. HGT events often involve mobile genetic elements like plasmids, which can promote their own dissemination by associating with adaptive traits in the gene pool of the so-called mobilome. Novel traits that evolve through HGT can therefore lead to the exploitation of new ecological niches, prompting an adaptive radiation of bacterial species. In this study, we present phylogenetic, biogeographic, and functional analyses of a previously unrecognized RepL-type plasmid found in diverse members of the marine Roseobacter group across the globe. Noteworthy, 100% identical plasmids were detected in phylogenetically and geographically distant bacteria, revealing a so-far overlooked, but environmentally highly relevant vector for HGT. The genomic and functional characterization of this plasmid showed a completely conserved backbone dedicated to replication, stability, and mobilization as well as an interchangeable gene cassette with highly diverse, but recurring motifs. The majority of the latter appear to be involved in mechanisms coping with toxins and/or pollutants in the marine environment. Furthermore, we provide experimental evidence that the plasmid has the potential to be transmitted across bacterial orders, thereby increasing our understanding of evolution and microbial niche adaptation in the environment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The release of abiotic methane from marine seeps into the atmosphere is a major source of this potent greenhouse gas. Methanotrophic microorganisms in methane seeps use methane as carbon and energy source, thus significantly mitigating global methane emissions. Here, we investigated microbial methane oxidation at the sediment-water interface of a shallow marine methane seep. Metagenomics and metaproteomics, combined with 13 C-methane stable isotope probing, demonstrated that various members of the gammaproteobacterial family Methylococcaceae were the key players for methane oxidation, catalysing the first reaction step to methanol. We observed a transfer of carbon to methanol-oxidizing methylotrophs of the betaproteobacterial family Methylophilaceae, suggesting an interaction between methanotrophic and methylotrophic microorganisms that allowed for rapid methane oxidation. From our microcosms, we estimated methane oxidation rates of up to 871 nmol of methane per gram sediment per day. This implies that more than 50% of methane at the seep is removed by microbial oxidation at the sediment-water interface, based on previously reported in situ methane fluxes. The organic carbon produced was further assimilated by different heterotrophic microbes, demonstrating that the methane-oxidizing community supported a complex trophic network. Our results provide valuable eco-physiological insights into this specialized microbial community performing an ecosystem function of global relevance., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Coastal saltmarsh sediments represent an important source of natural methane emissions, much of which originates from quaternary and methylated amines, such as choline and trimethylamine. In this study, we combine DNA stable isotope probing with high throughput sequencing of 16S rRNA genes and 13 C 2 -choline enriched metagenomes, followed by metagenome data assembly, to identify the key microbes responsible for methanogenesis from choline. Microcosm incubation with 13 C 2 -choline leads to the formation of trimethylamine and subsequent methane production, suggesting that choline-dependent methanogenesis is a two-step process involving trimethylamine as the key intermediate. Amplicon sequencing analysis identifies Deltaproteobacteria of the genera Pelobacter as the major choline utilizers. Methanogenic Archaea of the genera Methanococcoides become enriched in choline-amended microcosms, indicating their role in methane formation from trimethylamine. The binning of metagenomic DNA results in the identification of bins classified as Pelobacter and Methanococcoides. Analyses of these bins reveal that Pelobacter have the genetic potential to degrade choline to trimethylamine using the choline-trimethylamine lyase pathway, whereas Methanococcoides are capable of methanogenesis using the pyrrolysine-containing trimethylamine methyltransferase pathway. Together, our data provide a new insight on the diversity of choline utilizing organisms in coastal sediments and support a syntrophic relationship between Bacteria and Archaea as the dominant route for methanogenesis from choline in this environment.

There are increasing concerns about possible dissemination of clinically relevant antibiotic resistance genes, including genes encoding for carbapenemases in the environment. However, little is known about environmental distribution of antibiotic resistance in Africa. In this study, four polluted urban wetlands in Nigeria were investigated as potential reservoirs of carbapenem-resistant bacteria (CRB). CRB were isolated from the wetlands, characterized by Blue-Carba test, MIC determinations and whole genome sequencing (WGS). Nine of 65 bacterial isolates identified as members of the Pseudomonas putida group (P. plecoglossicida and P. guariconensis, respectively) harboured the metallo-beta-lactamase gene bla VIM-5 . WGS revealed the bla VIM-5 in three novel Tn402-like class 1 integron structures containing the cassette arrays aadB|bla VIM-5 |bla PSE-1 , aadB|bla VIM-5 |aadB|bla PSE-1 , and bla VIM-5 |aadB|tnpA|bla PSE-1 |smr2|tnpA, respectively. Strains carrying the aadB|bla VIM-5 |bla PSE-1 cassette also carried an identical integron without bla VIM-5 . In addition , the strains harboured another Tn402-like class 1 integron carrying bcr2, several multidrug resistance efflux pumps, and at least one of ampC, aph(3")-lb, aph(6)-ld, tetB, tetC, tetG, floR, and macAB. This is the first report of a carbapenemase gene in bacteria from environmental sources in Nigeria and the first report of bla VIM-5 in environmental bacteria isolates. This result underscores the role of the Nigerian environment as reservoir of bacteria carrying clinically relevant antibiotic resistance genes.

The Roseobacter group comprises a significant group of marine bacteria which are involved in global carbon and sulfur cycles. Some members are methylotrophs, using one-carbon compounds as a carbon and energy source. It has recently been shown that methylotrophs generally require a rare earth element when using the methanol dehydrogenase enzyme XoxF for growth on methanol. Addition of lanthanum to methanol enrichments of coastal seawater facilitated the isolation of a novel methylotroph in the Roseobacter group: Marinibacterium anthonyi strain La 6. Mutation of xoxF5 revealed the essential nature of this gene during growth on methanol and ethanol. Physiological characterization demonstrated the metabolic versatility of this strain. Genome sequencing revealed that strain La 6 has the largest genome of all Roseobacter group members sequenced to date, at 7.18 Mbp. Multilocus sequence analysis (MLSA) showed that whilst it displays the highest core gene sequence similarity with subgroup 1 of the Roseobacter group, it shares very little of its pangenome, suggesting unique genetic adaptations. This research revealed that the addition of lanthanides to isolation procedures was key to cultivating novel XoxF-utilizing methylotrophs from the marine environment, whilst genome sequencing and MLSA provided insights into their potential genetic adaptations and relationship to the wider community.

Understanding of global methane sources and sinks is a prerequisite for the design of strategies to counteract global warming. Microbial methane oxidation in soils represents the largest biological sink for atmospheric methane. However, still very little is known about the identity, metabolic properties and distribution of the microbial group proposed to be responsible for most of this uptake, the uncultivated upland soil cluster α (USCα). Here, we reconstructed a draft genome of USCα from a combination of targeted cell sorting and metagenomes from forest soil, providing the first insights into its metabolic potential and environmental adaptation strategies. The 16S rRNA gene sequence recovered was distinctive and suggests this crucial group as a new genus within the Beijerinckiaceae, close to Methylocapsa. Application of a fluorescently labelled suicide substrate for the particulate methane monooxygenase enzyme (pMMO) coupled to 16S rRNA fluorescence in situ hybridisation (FISH) allowed for the first time a direct link of the high-affinity activity of methane oxidation to USCα cells in situ. Analysis of the global biogeography of this group further revealed its presence in previously unrecognized habitats, such as subterranean and volcanic biofilm environments, indicating a potential role of these environments in the biological sink for atmospheric methane., (© 2018 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive dehalogenation, as well as the biology of abundant deep-sea Chloroflexi IMPORTANCE The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marine Chloroflexi Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenating Dehalococcoides ., (Copyright © 2017 Sewell et al.)

Polychlorinated dibenzo-p-dioxin (PCDD)-contaminated sites are widespread and associated with a variety of anthropogenic sources. PCDDs and other organohalide pollutants can serve as terminal electron acceptors for anaerobic respiration by specialized bacteria containing reductive dehalogenases (RdhA). These microorganisms, therefore, play an important role in the bioremediation of PCDD-contaminated sites. Two anaerobic enrichment cultures established using sediments collected from the PCDD-polluted Hackensack (USA) and Kymijoki (Finland) rivers showed robust reductive dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD). Here, we report on the draft genome reconstructions of the two predominant Dehalococcoides strains from the metagenomes of these dehalogenating enrichment cultures. Furthermore, we gathered a complete list of RdhA in the two predominant Dehalococcoides strains, and determined which are likely to be responsible for the reductive dechlorination of PCDDs. The divergent rdhA gene profiles of the Dehalococcoides strains likely reflect their exposure to different organohalide compounds in their original habitats. Both draft genomes contained a full length rdhA gene with high sequence similarity to a rdhA gene, i.e. cbrA, found in Dehalococcoides mccartyi CBDB1 known to reductively dechlorinate 1,2,3,4-tetrachlorobenzene. This gene homologue might also be responsible for reductive dechlorination of 1,2,3,4-TeCDD in the enrichments and could be used as a biomarker to determine the potential for the bioremediation of PCDD-contaminated sediments., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The discovery of six new, highly substituted tropolone alkaloids, rubterolones A-F, from Actinomadura sp. 5-2, isolated from the gut of the fungus-growing termite Macrotermes natalensis is reported. Rubterolones were identified by using fungus-bacteria challenge assays and a HRMS-based dereplication strategy, and characterised by NMR and HRMS analyses and by X-ray crystallography. Feeding experiments and subsequent chemical derivatisation led to a first library of rubterolone derivatives (A-L). Genome sequencing and comparative analyses revealed their putative biosynthetic pathway, which was supported by feeding experiments. This study highlights how gut microbes can present a prolific source of secondary metabolites., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The kelp forest of the Pacific temperate rocky marine coastline of Monterey Bay in California is a dominant habitat for large brown macro-algae in the order of Laminariales . It is probably one of the most species-rich, structurally complex and productive ecosystems in temperate waters and well-studied in terms of trophic ecology. However, still little is known about the microorganisms thriving in this habitat. A growing body of evidence suggests that bacteria associated with macro-algae represent a huge and largely untapped resource of natural products with chemical structures that have been optimized by evolution for biological and ecological purposes. Those microorganisms are most likely attracted by algae through secretion of specific carbohydrates and proteins that trigger them to attach to the algal surface and to form biofilms. The algae might then employ those bacteria as biofouling control, using their antimicrobial secondary metabolites to defeat other bacteria or eukaryotes. We here analyzed biofilm samples from the brown macro-algae Macrocystis pyrifera sampled in November 2014 in the kelp forest of Monterey Bay by a metagenomic shotgun and amplicon sequencing approach, focusing on Planctomycetes and Verrucomicrobia from the PVC superphylum. Although not very abundant, we were able to find novel Planctomycetal and Verrucomicrobial species by an innovative binning approach. All identified species harbor secondary metabolite related gene clusters, contributing to our hypothesis that through inter-species interaction, microorganisms might have a substantial effect on kelp forest wellbeing and/or disease-development.

With the constant improvement in cost-efficiency and quality of Next Generation Sequencing technologies, shotgun-sequencing approaches -such as metagenomics- have nowadays become the methods of choice for studying and classifying microorganisms from various habitats. The production of data has dramatically increased over the past years and processing and analysis steps are becoming more and more of a bottleneck. Limiting factors are partly the availability of computational resources, but mainly the bioinformatics expertise in establishing and applying appropriate processing and analysis pipelines. Fortunately, a large diversity of specialized software tools is nowadays available. Nevertheless, choosing the most appropriate methods for answering specific biological questions can be rather challenging, especially for non-bioinformaticians. In order to provide a comprehensive overview and guide for the microbiological scientific community, we assessed the most common and freely available metagenome assembly tools with respect to their output statistics, their sensitivity for low abundant community members and variability in resulting community profiles as well as their ease-of-use. In contrast to the highly anticipated "Critical Assessment of Metagenomic Interpretation" (CAMI) challenge, which uses general mock community-based assembler comparison we here tested assemblers on real Illumina metagenome sequencing data from natural communities of varying complexity sampled from forest soil and algal biofilms. Our observations clearly demonstrate that different assembly tools can prove optimal, depending on the sample type, available computational resources and, most importantly, the specific research goal. In addition, we present detailed descriptions of the underlying principles and pitfalls of publically available assembly tools from a microbiologist's perspective, and provide guidance regarding the user-friendliness, sensitivity and reliability of the resulting phylogenetic profiles., Competing Interests: The authors have declared that no competing interests exist.

Cell sorting coupled with single-cell genomics is a powerful tool to circumvent cultivation of microorganisms and reveal microbial 'dark matter'. Single-cell Raman spectra (SCRSs) are label-free biochemical 'fingerprints' of individual cells, which can link the sorted cells to their phenotypic information and ecological functions. We employed a novel Raman-activated cell ejection (RACE) approach to sort single bacterial cells from a water sample in the Red Sea based on SCRS. Carotenoids are highly diverse pigments and play an important role in phototrophic bacteria, giving strong and distinctive Raman spectra. Here, we showed that individual carotenoid-containing cells from a Red Sea sample were isolated based on the characteristic SCRS. RACE-based single-cell genomics revealed putative novel functional genes related to carotenoid and isoprenoid biosynthesis, as well as previously unknown phototrophic microorganisms including an unculturable Cyanobacteria spp. The potential of Raman sorting coupled to single-cell genomics has been demonstrated., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Members of the phylum Planctomycetes are ubiquitous bacteria that dwell in aquatic and terrestrial habitats. While planctomycetal species are important players in the global carbon and nitrogen cycle, this phylum is still undersampled and only few genome sequences are available. Here we describe strain NH11 T , a novel planctomycete obtained from a crustacean shell (Wadden Sea, Germany). The phylogenetically closest related cultivated species is Gimesia maris , sharing only 87% 16S rRNA sequence identity. Previous isolation attempts have mostly yielded members of the genus Rhodopirellula from water of the German North Sea. On the other hand, only one axenic culture of the genus Pirellula was obtained from a crustacean thus far. However, the 16S rRNA gene sequence of strain NH11 T shares only 80% sequence identity with the closest relative of both genera, Rhodopirellula and Pirellula . Thus, strain NH11 T is unique in terms of origin and phylogeny. While the pear to ovoid shaped cells of strain NH11 T are typical planctomycetal, light-, and electron microscopic observations point toward an unusual variation of cell division through budding: during the division process daughter- and mother cells are connected by an unseen thin tubular-like structure. Furthermore, the periplasmic space of strain NH11 T was unusually enlarged and differed from previously known planctomycetes. The complete genome of strain NH11 T , with almost 9 Mb in size, is among the largest planctomycetal genomes sequenced thus far, but harbors only 6645 protein-coding genes. The acquisition of genomic components by horizontal gene transfer is indicated by the presence of numerous putative genomic islands. Strikingly, 45 "giant genes" were found within the genome of NH11 T . Subsequent analysis of all available planctomycetal genomes revealed that Planctomycetes as such are especially rich in "giant genes". Furthermore, Multilocus Sequence Analysis (MLSA) tree reconstruction support the phylogenetic distance of strain NH11 T from other cultivated Planctomycetes of the same phylogenetic cluster. Thus, based on our findings, we propose to classify strain NH11 T as Fuerstia marisgermanicae gen. nov., sp. nov., with the type strain NH11 T , within the phylum Planctomycetes.

The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita's core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.

The phylum Chloroflexi is one of the most frequently detected phyla in the subseafloor of the Pacific Ocean margins. Dehalogenating Chloroflexi (Dehalococcoidetes) was originally discovered as the key microorganisms mediating reductive dehalogenation via their key enzymes reductive dehalogenases (Rdh) as sole mode of energy conservation in terrestrial environments. The frequent detection of Dehalococcoidetes-related 16S rRNA and rdh genes in the marine subsurface implies a role for dissimilatory dehalorespiration in this environment; however, the two genes have never been linked to each other. To provide fundamental insights into the metabolism, genomic population structure and evolution of marine subsurface Dehalococcoidetes sp., we analyzed a non-contaminated deep-sea sediment core sample from the Peruvian Margin Ocean Drilling Program (ODP) site 1230, collected 7.3 m below the seafloor by a single cell genomic approach. We present for the first time single cell genomic data on three deep-sea Chloroflexi (Dsc) single cells from a marine subsurface environment. Two of the single cells were considered to be part of a local Dehalococcoidetes population and assembled together into a 1.38-Mb genome, which appears to be at least 85% complete. Despite a high degree of sequence-level similarity between the shared proteins in the Dsc and terrestrial Dehalococcoidetes, no evidence for catabolic reductive dehalogenation was found in Dsc. The genome content is however consistent with a strictly anaerobic organotrophic or lithotrophic lifestyle.