Biology subjects

Malmstrom, R.

Publications and source records attributed to Malmstrom, R..

9 recordsLinked to original sources

bioRxiv2024

scMicrobe PTA: Near Complete Genomes From Single Bacterial Cells

Microbial genomes produced by single-cell amplification are largely incomplete. Here, we show that primary template amplification (PTA), a novel single-cell amplification technique, generated nearly complete genomes from three bacterial isolate species. Furthermore, taxonomically diverse genomes recovered from aquatic and soil microbiomes using PTA had a median completeness of 81%, whereas genomes from standard amplification approaches were usually <30% complete. PTA-derived genomes also included more associated viruses and biosynthetic gene clusters.

genomics↗

bioRxiv2023

Multicellular magnetotactic bacterial consortia are metabolically differentiated and not clonal

Consortia of multicellular magnetotactic bacteria (MMB) are currently the only known example of bacteria without a unicellular stage in their life cycle. Because of their recalcitrance to cultivation, most previous studies of MMB have been limited to microscopic observations. To study the biology of these unique organisms in more detail, we use multiple culture-independent approaches to analyze the genomics and physiology of MMB consortia at single cell resolution. We separately sequenced the metagenomes of 22 individual MMB consortia, representing eight new species, and quantified the genetic diversity within each MMB consortium. This revealed that, counter to conventional views, cells within MMB consortia are not clonal. Single consortia metagenomes were then used to reconstruct the species-specific metabolic potential and infer the physiological capabilities of MMB. To validate genomic predictions, we performed stable isotope probing (SIP) experiments and interrogated MMB consortia using fluorescence in situ hybridization (FISH) combined with nano-scale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal non-canonical amino acid tagging (BONCAT) we explored their in situ activity as well as variation of protein synthesis within cells. We demonstrate that MMB consortia are mixotrophic sulfate reducers and that they exhibit metabolic differentiation between individual cells, suggesting that MMB consortia are more complex than previously thought. These findings expand our understanding of MMB diversity, ecology, genomics, and physiology, as well as offer insights into the mechanisms underpinning the multicellular nature of their unique lifestyle. Significance statementThe emergence of multicellular lifeforms represents a pivotal milestone in Earths history, ushering in a new era of biological complexity. Because of the relative scarcity of multicellularity in the domains Bacteria and Archaea, research on the evolution of multicellularity has predominantly focused on eukaryotic model organisms. In this study, we explored the complexity of the only known bacteria without a unicellular stage in their life cycle, consortia of multicellular magnetotactic bacteria (MMB). Genomic and physiological analyses revealed that cells within individual MMB consortia are not clonal and exhibit metabolic differentiation. This implies a higher level of complexity than previously assumed for MMB consortia, prompting a reevaluation of the evolutionary factors that have led to the emergence of multicellularity. Because of their unique biology MMB consortia are ideally suited to become a model system to explore the underpinnings of bacterial multicellularity.

microbiology↗

bioRxiv2023

Hybridization breaks species barriers in long-term coevolution of a cyanobacterial population

Bacterial species often undergo rampant recombination yet maintain cohesive genomic identity. Ecological differences can generate recombination barriers between species and sustain genomic clusters in the short term. But can these forces prevent genomic mixing during long-term coevolution? Cyanobacteria in Yellowstone hot springs comprise several diverse species that have coevolved for hundreds of thousands of years, providing a rare natural experiment. By analyzing more than 300 single-cell genomes, we show that despite each species forming a distinct genomic cluster, much of the diversity within species is the result of hybridization driven by selection, which has mixed their ancestral genotypes. This widespread mixing is contrary to the prevailing view that ecological barriers can maintain cohesive bacterial species and highlights the importance of hybridization as a source of genomic diversity.

microbiology↗

bioRxiv2022

Physiological adaptation of sulfate reducing bacteria in syntrophic partnership with anaerobic methanotrophic archaea

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we explore the metabolic adaptation of four syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a and Seep-SRB1g) from a phylogenomics perspective, tracing the evolution of conserved proteins in the syntrophic SRB clades, and comparing the genomes of syntrophic SRB to their nearest evolutionary neighbors in the phylum Desulfobacterota. We note several examples of gain, loss or biochemical adaptation of proteins within pathways involved in extracellular electron transfer, electron transport chain, nutrient sharing, biofilm formation and cell adhesion. We demonstrate that the metabolic adaptations in each of these syntrophic clades are unique, suggesting that they have independently evolved, converging to a syntrophic partnership with ANME. Within the clades we also investigated the specialization of different syntrophic SRB species to partnerships with different ANME clades, using metagenomic sequences obtained from ANME and SRB partners in individual consortia after fluorescent-sorting of cell aggregates from anaerobic sediments. In one instance of metabolic adaptation to different partnerships, we show that Seep-SRB1a partners of ANME-2c appear to lack nutritional auxotrophies, while the related Seep-SRB1a partners of a different methanotrophic archaeal lineage, ANME-2a, are missing the cobalamin synthesis pathway, suggesting that the Seep-SRB1a partners of ANME-2a may have a nutritional dependence on its partner. Together, our paired genomic analysis of AOM consortia highlights the specific adaptation and diversification of syntrophic SRB clades linked to their associated ANME lineages.

microbiology↗

bioRxiv2022

HT-SIP: A semi-automated Stable Isotope Probing pipeline identifies interactions in the hyphosphere of arbuscular mycorrhizal fungi

BackgroundLinking the identity of wild microbes with their ecophysiological traits and environmental functions is a key ambition for microbial ecologists. Of many techniques that strive to meet this goal, Stable Isotope Probing--SIP--remains the most comprehensive for studying whole microbial communities in situ. In DNA-SIP, active microorganisms that take up an isotopically heavy substrate build heavier DNA, which can be partitioned by density into multiple fractions and sequenced. However, SIP is relatively low throughput and requires significant hands-on labor. We designed and tested a semi-automated DNA-SIP pipeline to support well-replicated, temporally-resolved amplicon or metagenomics experiments that enable studies of dynamic microbial communities over space and time. To test this pipeline, we assembled SIP-metagenome assembled genomes (MAGs) from the hyphosphere zone surrounding arbuscular mycorrhizal fungi (AMF), in combination with a 13CO2 plant labelling study. ResultsOur semi-automated pipeline for DNA fractionation, cleanup, and nucleic acid quantification of SIP density gradients requires six times less hands-on labor compared to manual SIP and allows 16 samples to be processed simultaneously. Automated density fractionation increased the reproducibility of SIP gradients and reduced variation compared to manual fractionation, and we show adding a non-ionic detergent to the gradient buffer improved SIP DNA recovery. We then tested this pipeline on samples from a highly-constrained soil microhabitat with significant ecological importance, the AMF fungal hyphosphere. Processing via our quantitative SIP pipeline confirmed the AMF Rhizophagus intraradices and its associated microbiome were highly 13C enriched, even though the soils overall enrichment was only 1.8 atom% 13C. We assembled 212 13C-enriched hyphosphere MAGs, and the hyphosphere taxa that assimilated the most AMF-derived 13C (range 10-33 atom%) were from the phlya Myxococcota, Fibrobacterota, Verrucomicrobiota, and the ammonia oxidizing archaeon genus Nitrososphaeara. ConclusionsOur semi-automated SIP approach decreases operator time and errors and improves reproducibility by targeting the most labor-intensive steps of SIP--fraction collection and cleanup. Here, we illustrate this approach in a unique and understudied soil microhabitat--generating MAGs of active microbes living in the AMF hyphosphere (without plant roots). Their phylogenetic composition and gene content suggest predation, decomposition, and ammonia oxidation may be key processes in hyphosphere nutrient cycling.

microbiology↗

bioRxiv2022

A centimeter-long bacterium with DNA compartmentalized in membrane-bound organelles

Cells of most bacterial species are around 2 {micro}m in length, with some of the largest specimens reaching 750 {micro}m. Using fluorescence, x-ray, and electron microscopy in conjunction with genome sequencing, we characterized Ca. Thiomargarita magnifica, a bacterium with an average cell length greater than 9,000 {micro}m that is visible to the naked eye. We found that these cells grow orders of magnitude over theoretical limits for bacterial cell size through unique biology, display unprecedented polyploidy of more than half a million copies of a very large genome, and undergo a dimorphic life cycle with asymmetric segregation of chromosomes in daughter cells. These features, along with compartmentalization of genomic material and protein synthesis in membrane-bound organelles, indicate gain of complexity in the Thiomargarita lineage, and challenge traditional concepts of bacterial cells. One Sentence SummaryCa. T. magnifica are compartmentalized centimeter-long bacteria

cell biology↗

bioRxiv2021

Comparative genomics reveals electron transfer and syntrophic mechanisms differentiating methanotrophic and methanogenic archaea

The anaerobic oxidation of methane coupled to sulfate reduction is a microbially mediated process requiring a syntrophic partnership between anaerobic methanotrophic (ANME) archaea and sulfate reducing bacteria (SRB). Based on genome taxonomy, ANME lineages are polyphyletic within the phylum Halobacterota, none of which have been isolated in pure culture. Here we reconstruct 28 ANME genomes from environmental metagenomes and flow sorted syntrophic consortia. Together with a reanalysis of previously published datasets, these genomes enable a comparative analysis of all marine ANME clades. We review the genomic features which separate ANME from their methanogenic relatives and identify what differentiates ANME clades. Large multiheme cytochromes and bioenergetic complexes predicted to be involved in novel electron bifurcation reactions are well-distributed and conserved in the ANME archaea, while significant variations in the anabolic C1 pathways exists between clades. Our analysis raises the possibility that methylotrophic methanogenesis may have evolved from a methanotrophic ancestor.

microbiology↗

bioRxiv2021

Subsurface Hydrocarbon Degradation Strategies in Low- and High-Sulfate Coal Seam Communities Identified with Activity-Based Metagenomics

Environmentally relevant metagenomes and BONCAT-FACS derived translationally active metagenomes from Powder River Basin coal seams were investigated to elucidate potential genes and functional groups involved in hydrocarbon degradation to methane in coal seams with high- and low-sulfate levels. An advanced subsurface environmental sampler allowed the establishment of coal-associated microbial communities under in situ conditions for metagenomic analyses from environmental and translationally active populations. Metagenomic sequencing demonstrated that biosurfactants, aerobic dioxygenases, and anaerobic phenol degradation pathways were present in active populations across the sampled coal seams. In particular, results suggested the importance of anaerobic degradation pathways under high-sulfate conditions with an emphasis on fumarate addition. Under low-sulfate conditions, a mixture of both aerobic and anaerobic pathways were observed but with a predominance of aerobic dioxygenases. The putative low-molecular weight biosurfactant, lichysein, appeared to play a more important role compared to rhamnolipids. The methods used in this study--subsurface environmental samplers in combination with metagenomic sequencing of both total and translationally active metagenomes--offer a deeper and environmentally relevant perspective on community genetic potential from coal seams poised at different redox conditions broadening the understanding of degradation strategies for subsurface carbon. One Sentence SummaryIdentifying hydrocarbon degradation strategies across different redox conditions via metagenomic analysis of environmental and translationally active (BONCAT-FACS) samples from subsurface coal beds.

microbiology↗

bioRxiv2018

Connections between freshwater carbon and nutrient cycles revealed through reconstructed population genomes

Metabolic processes at the microbial scale influence ecosystem functions because microbes are responsible for much of the carbon and nutrient cycling in freshwater. One approach to predict the metabolic capabilities of microbial communities is to search for functional marker genes in metagenomes. However, this approach does not provide context about co-occurrence with other metabolic traits within an organism or detailed taxonomy about those organisms. Here, we combine a functional marker gene analysis with metabolic pathway prediction of microbial population genomes (MAGs) assembled from metagenomic time series in eutrophic Lake Mendota and humic Trout Bog to identify how carbon and nutrient cycles are connected in freshwater. We found that phototrophy, carbon fixation, and nitrogen fixation pathways co-occurred in Cyanobacteria MAGs in Lake Mendota and in Chlorobiales MAGs in Trout Bog. Cyanobacteria MAGs also had strong temporal correlations to functional marker genes for nitrogen fixation in several years. Genes encoding steps in the nitrogen and sulfur cycles varied in abundance and taxonomy by lake, potentially reflecting the availability and composition of inorganic nutrients in these systems. We were also able to identify which populations contained the greatest density and diversity of genes encoding glycoside hydrolases. Populations with many glycoside hydrolases also encoded pathways for sugar degradation. By using both MAGs and marker genes, we were better able to link functions to specific taxonomic groups in our metagenomic time series, enabling a more detailed understanding of freshwater microbial carbon and nutrient cycling.

microbiology↗