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.

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.

Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE: Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

From its earliest beginnings, life’s expansion into new habitats has been profoundly shaped by its reciprocal interactions with a changing Earth. To understand the evolution of life’s metabolic engines, we must therefore uncover the ecological and evolutionary processes that shaped their underlying genes and networks. Here, we focus on nitrogen (N2) fixation, one of life’s most critical metabolisms, and investigate the ecological and evolutionary drivers of complexity within its associated gene machinery. We used a large-scale comparative genomics framework to construct a comprehensive catalog of extant N2 fixation-associated genes and to assess their distribution across diverse microbial genomes and environments. Genomes enriched in N2 fixation genes are generally larger, encode broader metabolic capabilities, span wider habitat ranges, and are predominantly associated with mesophilic and aerobic lifestyles. Evolutionary reconstructions reveal early gene gains in ancestral diazotrophs followed by lineage-specific gene losses in later-diverging taxa, consistent with evolutionary trade-offs driven by changing environments. Together, these findings show that the evolution of N2 fixation is tightly intertwined with the composition and organization of its supporting gene machinery, reflecting feedbacks between genome evolution and shifting environmental and ecological contexts.

From its earliest beginnings, life’s expansion into new habitats has been profoundly shaped by its reciprocal interactions with Earth’s changing environments. Understanding how ancient metabolisms co-evolved with their environments requires uncovering the ecological and evolutionary processes that structured the functionally linked genes and networks underlying these metabolisms. Here, we focus on nitrogen (N ₂ ) fixation, one of life’s most critical metabolisms, and investigate the drivers of complexity in its associated gene machinery today. We used a large-scale comparative genomics framework to construct a comprehensive catalog of extant N ₂ fixation-associated genes and assessed their distribution across diverse microbial genomes and environmental backgrounds. Genomes enriched in N ₂ fixation genes generally have larger genome sizes, broader metabolic capabilities, wider habitat ranges, and are predominantly associated with mesophilic and aerobic lifestyles. Evolutionary reconstructions reveal a pattern of early gene gains in ancestral diazotrophs followed by lineage-specific gene losses in later diverging taxa, suggesting evolutionary trade-offs shaped by changing environments. These findings demonstrate that the evolution of N ₂ fixation has been intertwined with the composition and organization of the genes supporting the overarching N ₂ metabolism, driven by feedback between genome evolution and shifting environmental and ecological conditions.

Molybdenum (Mo) is an essential nutrient for most living organisms, serving as a cofactor in a diverse array of molybdoenzymes that catalyze key reactions in several elemental cycles. However, geochemical data suggest that dissolved Mo concentrations in the Archean ocean (before 2.5 billion years ago) were 1-2 orders of magnitude lower than today, raising questions about its bioavailability to early life. Here, we apply a phylogenomic approach to chart the modern biological and environmental distribution of Mo-related enzymes and use phylogenetic reconciliations to reconstruct the evolutionary history of biological Mo usage. Our results reveal the ubiquity of molybdoenzymes across contemporary organisms inhabiting diverse environments. Furthermore, phylogenetic evidence indicates that the earliest molybdoenzymes stem back to the Paleo/Mesoarchean (~3.5-3.0 Gya), facilitating critical energy-harnessing reactions in some of Earth’s most ancient life forms. Taken together, our findings challenge the prevailing view of limited Mo bioavailability on the anoxic early Earth.