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Recent advancements in single-cell genomic and transcriptomic sequencing, in situ sequencing, and molecular imaging-based technologies have facilitated the examination of heterogeneity within microbial communities at the single-cell level. These cutting-edge methodologies permit the capture of phenotypic and genotypic heterogeneity, as well as the spatial organization within microbial communities. This enables in-depth investigation into microbial dark matter, the evaluation of microbial responses to perturbations, and a comprehensive exploration of spatial functions involved in community assembly and social interactions within microbial communities. These interactions include inter-microbial relationships, bacteria-phage interactions, and host-microbe interactions. Here, we highlight the key technological breakthroughs achieved, elucidating the perspectives from which these technologies enable us to interpret microbial heterogeneity at the single-cell level. Additionally, we critically examine the limitations associated with these technologies. Furthermore, we explore how these methods could be combined and also their applications in future studies. The integration of these approaches holds great promise for increasing our understanding of the organization and function of microbes in complex ecosystems.

Indole-3-acetic acid (IAA) is an important plant hormone that regulates a variety of physiological processes in plants, and it is also produced by some microbes. However, the biosynthesis and roles of IAA in microorganisms, particularly in plant pathogens, remain to be determined. In this study, the plant pathogen Xanthomonas campestris pv. campestris (Xcc) strain XC1 was shown to produce IAA via an  l-tryptophan ( l-Trp)-dependent pathway. The intermediate metabolite indole-3-ethanol and Xcc1569 encoding aromatic amino acid aminotransferase were shown to be partially involved in the uncharacterized sub-pathway in an  l-Trp-dependent IAA biosynthetic pathway. IAA positively regulated the viability of XC1, as indicated by its colony-forming units (CFUs), extracellular polysaccharide production, protease activity, and virulence on cabbage. IAA also negatively regulated reactive oxygen species (ROS) production in XC1. Furthermore, RNA-Seq revealed a gene cluster, ilvCGM-leuA, encoding the products responsible for branched-chain amino acid (BCAA) biosynthesis, which was negatively regulated by IAA. High-performance liquid chromatography (HPLC) analysis showed that IAA negatively regulated valine and leucine production. Deletion of ilvC significantly increased the CFUs and reduced the ROS levels of XC1. Exogenous BCAA addition to mutant strain ΔilvC restored the CFU and ROS levels to those of wild-type strain XC1. These results revealed an IAA signaling cascade in XC1 that involved ilvCGM-leuA, BCAA production, ROS production, and colony formation. These IAA-regulated phenotypes contributed to the virulence of Xcc in host plants. Overall, these results explain IAA-mediated plant-Xcc interactions and underscore the potentially significant role of IAA in microbial physiology.

Ribonuclease E (RNase E) is central to bacterial RNA metabolism. In cyanobacteria, its activity is inhibited by RebA, a key mechanism for controlling cell morphology. Here, we demonstrate that rebA is essential for diazotrophic growth of Anabaena PCC 7120, a filamentous cyanobacterium capable of forming heterocysts-specialized nitrogen-fixing cells-upon nitrogen starvation. The rebA mutant strain (ΔrebA) showed severe growth defects in nitrogen-deprived conditions, despite forming more heterocysts than the wild type. With a GFP fusion strain, we show that RebA is transiently upregulated during heterocyst differentiation. Microscopic and ultrastructural analyses revealed that ΔrebA heterocysts accumulated abnormally large cyanophycin granules, while vegetative cells showed reduced pigment levels and disorganized thylakoid membranes, phenotypes indicative of a severe nitrogen deficiency response. However, esculin tracer diffusion and SepJ-GFP localization in ΔrebA were comparable to the wild type, suggesting that cell-cell communication via septal junctions remains functional. Thus, the growth defect likely results from impaired degradation or mobilization of fixed nitrogen. Notably, the ΔrebA phenotype could be rescued only by wild-type RebA, but not by variants unable to bind RNase E, indicating that RebA's function depends on its modulation of RNase E activity. Together, these findings reveal a key posttranscriptional mechanism linking RNase E regulation to heterocyst development and intercellular nutrient transfer, highlighting the importance of regulated RNA metabolism for diazotrophic growth.

Mycoviruses are common in fungi and can change their host's functions. Here, we identify a novel dsRNA mycovirus GbPmV1 from the fungus Gongronella butleri. The genome of GbPmV1 exceeds 10,000 nucleotides and comprises six dsRNAs, with dsRNA1 encoding the RdRp and dsRNA6 encoding the capsid protein. GbPmV1 belongs to the family Polymycoviridae and shows unusual filamentous, virus-like particles. Infection by GbPmV1 enhances the resistance of its fungal host to stresses and antifungal azoles. This study not only identifies a novel mycovirus in the zygomycete fungus G. butleri but also provides insights into the evolution and biological properties of polymycoviruses.

Photosynthetic electron transfer occurs efficiently in specialized internal membranes known as thylakoid membranes. Thylakoid membranes exhibit diverse structural variations across photoautotrophic organisms. We studied how a key protein, CurT, shapes thylakoid membranes of a model cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942), a rod-shaped cyanobacterium with regular concentric thylakoid layers. By guiding the curves and structure of thylakoid membranes, CurT helps the cells capture light efficiently, especially when conditions change. The detailed characterization of the role of CurT in Syn7942 offers new clues about how nature builds high-performance photosynthetic membrane systems in response to environmental fluctuations. These findings may inspire future ways to redesign photosynthetic membranes for better crop yields or cleaner bioenergy production.

To thrive in nature, bacteria have to rapidly proliferate in favorable conditions while constantly adapt to the fluctuating nutrient environments. However, the molecular players that ensure rapid growth of bacteria in favorable conditions remain poorly understood. Here, we focus on the growth physiology of Bacillus subtilis and find that codY knockout strongly compromises cell growth in rich medium. Global proteome allocation analysis has shown that codY knockout causes a "waste" of cellular resources by stimulating unnecessary expression of many proteins, further reducing the cellular investment on translation machinery. Therefore, CodY-dependent repression is crucial in ensuring rapid growth of B. subtilis in rich medium. On the other hand, the relief of CodY-dependent repression could promote the bacterial adaption during transition from rich medium to minimal medium by shifting resource allocation from ribosome synthesis to amino acid biosynthesis. In addition, the relief of CodY-dependent repression in minimal medium also stimulates pathways of alternative functions such as motility and biosynthesis of secondary metabolites. Our study has thus revealed the pivotal role of CodY in bacterial growth control via governing the condition-dependent resource allocation of B. subtilis, further shedding light on the fundamental molecular strategy of bacteria to achieve fitness maximization.

Electroautotrophy-the use of extracellular electrons as the primary energy source for autotrophic metabolism-remains understudied compared to photoautotrophy and chemoautotrophy. Its occurrence in deep-earth and deep-sea environments suggests profound implications for astrobiology, yet electroautotrophic microorganisms remain poorly explored. This review synthesizes the discovery of electroautotrophs and current knowledge from laboratory and field studies, including insights from the deep biosphere. We evaluate their ecological roles on Earth and discuss their potential significance in possible life-supporting ecosystems elsewhere and in life-detection strategies. Finally, we propose six key research priorities to advance the study of electroautotrophy in astrobiological contexts.

Heterotrophic nitrifiers are bacteria that aerobically oxidize ammonia in the presence of organic carbon sources, which differs from autotrophic nitrifiers that extract energy from ammonia oxidation for cell metabolism and growth. The physiological significance of heterotrophic ammonia oxidation remains unclear, even though this process has been known for decades. Here, we demonstrate that direct ammonia oxidation (Dirammox)-a heterotrophic ammonia oxidation process with dinitrogen (N₂) as the primary product-is associated with both redox balance and the electron transport chain in Alcaligenes faecalis. Genetic and proteomic studies indicated that disruption of Dirammox genes (dnfA/dnfB/dnfC) induces a transient redox imbalance and perturbation in energy metabolism, further resulting in delayed growth. In addition, we found via biochemical and physiological studies that endogenous reactive oxygen species (ROS) enhance redox fluxes to ammonia oxidation, and the genetic disruption of cytochrome c peroxidase results in an increased flux of electrons to ammonia oxidation, producing N₂ and N₂O. These unexpected findings provide a more thorough understanding of both the Dirammox process and the physiology of heterotrophic ammonia oxidation.

It has been proposed by Ettema and colleagues, in the two-domain framework for the tree of life, that Eukarya emerged from Heimdallarchaeia, as sister group to Hodarchaeales. Looking at the individual trees of the protein markers used by these authors, I notice that Eukarya are only sister to Hodarchaeales or other Heimdallarchaeia in a minority of trees, whereas they are located far apart from these Asgard archaea in most other trees. Examination of single trees also reveals massive gene transfers from Crenarchaeota and/or Korachaeota to hyperthermophilic Njordarchaeales, explaining why their belonging to Asgard archaea is sometimes difficult to recover. Finally, I discuss several points raised by Ettema and colleagues, such as the phylogeny of Asgard archaea and the hyperthermophilic nature of their last common ancestor. The patchy localization of Eukarya in individual trees relative to Hodarchaeales and other Heimdallarchaeia, as well as the patchy distribution of eukaryotic signature proteins among Asgard archaea, is best explained by suggesting that multiple gene transfers take place between proto-eukaryotes and Asgard archaea in both directions. This suggests that the co-evolution of proto-eukaryotes and Asgard archaea has played a major role in eukaryogenesis but also in shaping the physiology and diversification of Asgard archaea.