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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 8 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 nanoscale secondary ion mass spectrometry (NanoSIMS). By coupling FISH with bioorthogonal noncanonical 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. Multicellular magnetotactic bacteria (MMB) are unique in having no unicellular stage in their life cycle. This study characterizes the genomics and physiology of MMB consortia at single cell resolution, revealing that individual cells are not genetically identical and exhibit significant metabolic differentiation, highlighting their level of complexity. [ABSTRACT FROM AUTHOR]

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic (ANME) archaea 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 used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it. A unique syntrophic partnership between anaerobic methanotrophic archaea and sulfate-reducing bacteria exists in deep-sea ecosystems, where they live in multicellular consortia. This study reveals a possible evolutionary trajectory of the bacterial partner towards adaptation to this syntrophic lifestyle. [ABSTRACT FROM AUTHOR]

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 that 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. A comparative genomics study of anaerobic methanotrophic (ANME) archaea reveals the genetic "parts list" associated with the repeated evolutionary transition between methanogenic and methanotrophic metabolism in the archaeal domain of life. [ABSTRACT FROM AUTHOR]

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called “marine” group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as “Candidatus Nitrosotenuis uzonensis”, is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, “Candidatus N. uzonensis” also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in “Ca. N. uzonensis”, which potentially include genetic determinants of ecological niche differentiation. [ABSTRACT FROM AUTHOR]

The discovery of ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota and the high abundance of archaeal ammonia monooxygenase subunit A encoding gene sequences in many environments have extended our perception of nitrifying microbial communities. Moreover, AOA are the only aerobic ammonia oxidizers known to be active in geothermal environments. Molecular data indicate that in many globally distributed terrestrial high-temperature habits a thaumarchaeotal lineage within the Nitrosopumilus cluster (also called "marine" group I.1a) thrives, but these microbes have neither been isolated from these systems nor functionally characterized in situ yet. In this study, we report on the enrichment and genomic characterization of a representative of this lineage from a thermal spring in Kamchatka. This thaumarchaeote, provisionally classified as "Candidatus Nitrosotenuis uzonensis", is a moderately thermophilic, non-halophilic, chemolithoautotrophic ammonia oxidizer. The nearly complete genome sequence (assembled into a single scaffold) of this AOA confirmed the presence of the typical thaumarchaeotal pathways for ammonia oxidation and carbon fixation, and indicated its ability to produce coenzyme F420 and to chemotactically react to its environment. Interestingly, like members of the genus Nitrosoarchaeum, "Candidatus N. uzonensis" also possesses a putative artubulin-encoding gene. Genome comparisons to related AOA with available genome sequences confirmed that the newly cultured AOA has an average nucleotide identity far below the species threshold and revealed a substantial degree of genomic plasticity with unique genomic regions in "Ca. N. uzonensis", which potentially include genetic determinants of ecological niche differentiation.