Spatial organization plays a critical role in shaping microbial community structure and function, influencing ecological stability, resource utilization, and evolutionary dynamics. Microbial interactions such as competition and cooperation are key drivers of spatial patterning, yet the environmental factors modulating these interactions remain incompletely understood. Here, we investigated how toxic substrates influence the spatial organization of synthetic microbial communities engaged in metabolic cross-feeding. Using a synthetic Pseudomonas stutzeri consortium consisting of the detoxifier and consumer that cooperatively degrade the toxic compound salicylate, we found that increasing the substrate concentration leads to a distinct shift in spatial organization: the detoxifier increasingly dominates the outer periphery of the expanding colony, forming a “detoxifier-first” succession pattern. Mathematical modeling further revealed that this spatial arrangement emerges from substrate toxicity, which selectively favors the detoxifier. Substrate toxicity inhibits consumer proliferation. However, the detoxifier, capable of degrading the substrate, locally reduces toxicity and creates a protective microenvironment that enables nearby consumer cells to survive and grow. In return, the consumer provides essential final products that support the growth and expansion of the detoxifier. This reciprocal interaction establishes a directional dynamic in which the detoxifier, favored by its detoxification capability, colonizes first, paving the way for subsequent consumer proliferation. Our findings demonstrate that substrate toxicity is a crucial environmental factor shaping spatial organization and diversity in microbial communities. This study highlights the importance of considering both metabolic interactions and substrate properties in understanding microbial ecology.

Bacterial persister cells that exhibit a transient state of antibiotic tolerance play a key role in chronic and recurring infections. Despite advances in our understanding of persisters, many aspects of their phenotypic plasticity, particularly their metabolism, are poorly characterized. In this Perspective, we examine the static and dynamic characteristics of persister metabolism that are shaped by genetics, environmental cues, and the intrinsic variability in cellular processes. These factors underlie much of the diversity observed among persister cells and largely explain the inconsistency in expression of classic persister hallmarks such as biphasic killing curves or metabolic dormancy. Further, the literature suggests that persisters are not uniformly metabolically dormant but represent a range of metabolic states. We will focus on the unique rewiring of the metabolic mechanisms in persisters, which depends on both internal and external factors. Bacterial persister cells exhibit a transient state of antibiotic tolerance and are commonly assumed to be metabolically ‘dormant’. In this Perspective, Orman et al. re-examine common assumptions and emphasize that persisters represent a range of metabolic states, driven by internal and external factors, which may explain the inconsistent expression of classic persister hallmarks.

Plants face constant environmental stresses that induce conflicts between growth and defense. The rhizosphere microbiome helps resolve this conflict by enhancing nutrient-uptake efficiency and activating plant immunity simultaneously. In this review, we first outline the mechanisms and limitations of plant-intrinsic growth-defense trade-offs; we then describe the integrated support that rhizosphere microbial communities provide for plant nutrition and defense. Finally, we propose the “microbial-damper” framework and further elucidate the interactions and feedback mechanisms that constitute this system. The microbial damper is a conceptual framework describing the capacity of the rhizosphere microbiome to stabilize a plant’s internal growth-defense resource allocation, which is otherwise perturbed by stresses such as nutrient imbalance and other environmental stresses. This framework highlights how the rhizosphere microbiome can reduce stress-induced plant growth-defense resource-allocation conflicts, thereby providing actionable strategies for designing sustainable agricultural systems.

Optimizing nitrogen (N) utilization in ruminant production systems holds both economic and environmental significance. However, traditional paradigms of N metabolism, derived primarily from well-studied model rumen bacteria, do not fully reflect the diverse and complex N metabolism in the rumen ecosystem. To address this gap, we utilized comparative genomics and genome-resolved multi-omics analyses using a curated set of microbial genomes to investigate N assimilation and regulation in rumen microbes. We discovered that well-established mechanisms of ammonia assimilation and regulation, such as the glutamine synthetase (GS)/glutamate synthase (GOGAT) pathways and their regulatory proteins, are absent in many of the predominant rumen microbes, which likely utilize alternative pathways for ammonia assimilation. These findings challenge the applicability of E. coli-based N regulation models to rumen bacteria in response to ammonia availability. We further linked polysaccharide utilization and ammonia assimilation across hundreds of rumen microbial species. Furthermore, we identified specific microbial species involved in ureolysis and denitrification, as well as phages carrying auxiliary metabolic genes involved in N assimilation. Using an animal trial involving 11 pairs of lamb twins in a crossover design, we demonstrated that dietary crude protein (CP) at 10% and 13% had minimal impact on rumen microbiome composition and expression of N assimilation genes. Instead, changes in concentrate levels altered N assimilation, notably increasing expression of amino acid biosynthesis pathways. These findings indicate a nuanced, species-specific microbial response to dietary interventions, highlighting the limitations of traditional N metabolism models applied to rumen microbes and the need for more granular studies of rumen microbial ecosystems.

Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Although PAM-relaxed Cas9 variants have been developed, expanded targeting is often accompanied by compromised target specificity. Here, we report the rational engineering of eSaCas9-NNG, a SaCas9 variant that recognizes relaxed NNG PAMs while maintaining high target fidelity, thereby overcoming a fundamental trade-off in Cas9-based genome editing. eSaCas9-NNG efficiently induces indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice, with editing efficiencies comparable to those of other PAM-relaxed nucleases, including SpRY, SpG, and iGeoCas9, but with reduced off-target activity. We further determine the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for its relaxed PAM recognition, improved target specificity, and nuclease activation. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases. CRISPR-Cas9-based genome editing is powerful but limited by target range, specificity, and delivery constraints. Here, authors engineer a compact SaCas9 that recognizes NNG PAMs for efficient genome and base editing in cells and mice, and reveal its activation mechanism via cryo-EM structural analysis.