SUMMARY Human gut microbiome research still relies heavily on fecal sampling because stool is noninvasive, scalable, repeatable, and essential for population-level and longitudinal studies. These strengths have made stool central to human microbiome science, but fecal output is not a neutral representation of every intestinal compartment. It reflects material that has persisted through digestion, transport, microbial turnover, host absorption, and distal colonic processing. This distinction becomes important when studies move from detecting microbial associations to explaining localized activity, host-interface interaction, inducible viral states, metabolic flux, or clinical consequences. This review proposes a framework for aligning sampling context with inferential scope across five evidence domains. These include distal output detection, regional luminal measurement, host-interface measurement, linked functional response, and patient-level consequence. The framework is not intended to rank specimen types or require every study to escalate toward invasive sampling. Instead, it clarifies which claims are supported by stool, luminal sampling, mucosal or host-proximal measurements, experimental models, and clinical metadata when used alone or in combination. Prophage induction and fecal short-chain fatty acid measurements are used as examples of a broader methodological issue. Detection in feces can be highly informative, but it does not by itself localize activity, establish mechanism, or demonstrate host consequence. As gut microbiome research increasingly seeks mechanistic explanations, sampling strategy should be treated as part of inference itself.
SUMMARY For single-celled organisms, the cell envelope is the thin barrier between life and death, responding dynamically to both the ever-expanding self and the ever-changing environment. Failure to balance these responses can lead to disastrous consequences, such as lysis. In gram-negative bacteria, the cell envelope is tripartite, consisting of two lipid bilayers with the periplasm, an aqueous space containing the thin peptidoglycan sacculus, between them. The outer of these lipid bilayers—aptly called the outer membrane—is asymmetric, bearing phospholipids on the inner leaflet and lipopolysaccharide (LPS) on the outer leaflet. Surface-exposed, highly immunoreactive, and essential to nearly all tested gram-negative bacteria, the overall architecture of LPS is largely conserved, but its modifications are subject to a myriad of variations. LPS has been studied extensively for its roles in pathogenesis and immunobiology and as a target of potential novel antibiotics. However, LPS exists in both free-living and pathogenic bacteria, suggesting it plays a critical role in bacterial physiology independent of the host. This aspect of LPS biology and its contribution to overall gram-negative cell physiology remains an area of active investigation. Addressing this gap, here we describe recent work detailing the essential contributions of LPS to bacterial physiology: particularly its role in cell envelope rigidity, its contributions to outer membrane capacity and fluidity, and the mechanisms integrating LPS synthesis into diverse aspects of cell envelope biogenesis during growth and division. We end with a discussion of the subset of gram-negative organisms that survive—either naturally or through genetic manipulation—in the absence of LPS and the adaptations that make this possible.
SUMMARYRibosomes produce the staggering array of proteins that perform the structural and enzymatic feats of the cell. Therefore, most of the cell's energy goes toward producing ribosomes and the work performed by them. The work of the ribosome is relatively simple-decode mRNA codons and catalyze the formation of peptide bonds. By marching iteratively along the length of an open reading frame, a complete peptide is produced. Ribosomes catalyze the formation of peptide bonds at a rate of approximately 15 amino acids per second. However, bonds between amino acids do not form with equal efficiency, and ribosomes can become stalled at difficult-to-translate sequences. Proline is unique among the amino acids in that its side chain is covalently bonded to the peptide backbone to form a rigid ring. The rigidity of proline, and especially tracts of proline, makes it a difficult substrate for peptide bond formation, but it is also an essential motif in many protein structures. Elongation factor P (EF-P) is the star player for facilitating translation of polyproline tracts. However, recently identified factors play an important supporting role, and loss of these factors incurs a severe fitness defect in the absence of EF-P. These factors include an EF-P paralog, EfpL, as well as the ABCF ATPase YfmR/Uup and YebC2. The abundance and partial redundancy of factors that prevent ribosome stalling at polyprolines highlights the structural importance of polyproline tracts and the need to facilitate their translation. Here, we review recently identified translation factors that prevent ribosome stalling at polyprolines in bacteria.
SUMMARYLiquid-liquid phase separation (LLPS) drives the formation of biomolecular condensates, a conserved phenomenon across eukaryotes. This process governs diverse cellular programs, from stress response and morphogenesis to disease pathology. Over the past two decades, the regulatory impact of biomolecular condensates in fungal biology has become increasingly recognized. In this review, we examine the fundamental molecular mechanisms driving LLPS, evaluate the current evidence for LLPS in macromolecular organization and cellular regulation in fungi, and outline the tools employed to study this phenomenon. Lastly, we highlight the challenges of bridging the gap between the in vitro behavior of biomolecular condensates and their complex regulatory functions in vivo within fungal biology.
SUMMARYHow temperate bacteriophages choose between lysis and lysogeny (dormancy) was one of the very first-and remains among the best studied-models of gene regulation. As over 75% of bacteria have a dormant phage within their genomes, the impact of this decision is far-reaching. It is also underappreciated, with much of microbiology studied independently of this decision; for example, the ubiquitous lab E. coli strain K-12 was cured of phage lambda to make it a "better" model. Here, we review why phages "choose" to lyse or not to lyse, the wide variety of signals they can respond to, how these signals are tapped into by the phage, how the timing of the decision impacts its outcome, and how phages interact around this decision. This decision underpins so much of bacteriology that an understanding of it, and the resulting ability to bias it, will help explain phenomena ranging from failures in culturing bacteria to successes of antibiotic treatments.
SUMMARYFungi in the genera Fusarium, Metarhizium, and Trichoderma (FMT) are traditionally defined by their roles as plant pathogens, insect pathogens, and mycoparasites, respectively. However, these classifications obscure both their shared hypocrealean ancestry and the remarkable ecological plasticity that characterizes all three genera. Across these lineages, plant endophytism appears to represent the predominant ecological state, with frequent transitions among saprotrophy, symbiosis, pathogenicity, and antagonism. Comparative genomics reveals that FMT fungi possess two-speed genomes comprising conserved core chromosomes and dynamic accessory regions enriched in host-interaction and secondary metabolism genes. These architectures support a shared hypocrealean genomic toolkit that has been differentially modified across lineages. In Fusarium, transitions along the mutualism-to-pathogenicity continuum appear to be driven primarily by regulatory plasticity rather than by major changes in gene content. By contrast, Metarhizium and Trichoderma expanded from ancestral pathogenic states toward broader plant associations through horizontal gene transfer, gene duplication, and diversification of host-recognition, signaling, and metabolite-production pathways. Reproductive strategies similarly reflect ecological divergence. Generalist lineages are predominantly clonal, whereas specialists more frequently retain sexual reproduction, facilitating adaptation to predictable hosts and environments. Ecologically, FMT fungi occupy overlapping but distinct niches: Trichoderma dominates stable environments through mycoparasitism and competitive exclusion; Fusarium thrives in disturbed habitats through rapid colonization of stressed plants; and Metarhizium bridges soil, plant, and insect environments through combined root association and insect pathogenicity. Collectively, FMT fungi illustrate how divergent ecological strategies can emerge through differential modification and regulatory deployment of a shared hypocrealean genomic toolkit.
SUMMARYMicroorganisms can cope with stress by entering dormancy, a viable state of reduced metabolic activity that enables persistence, dispersal, and long-term survival. However, microbial life in environmental systems is best understood as a spectrum of metabolic activity, spanning from highly active, dividing cells to deeply dormant phenotypes. This spectrum reflects dynamic survival strategies under fluctuating conditions, with critical implications for ecosystem stability, gene dissemination, and resilience to disturbances in natural and human-influenced systems. Yet, microbial activity is often treated as binary (active vs. dormant), oversimplifying a biological continuity that remains technically difficult to quantify. Here, we synthesize advances in microbial dormancy to reconceptualize activity as a spectrum. We review current and emerging methods to quantify environmental activity, linking each to the Central Dogma of molecular biology (DNA to RNA to protein) to guide interpretation along a generalizable continuum. Through a literature synthesis of terrestrial, aquatic, and wastewater treatment ecosystems, we compare how methods estimate active cells and populations. We recommend standardized reporting of total community size, active cell abundance, and proportional activity to enrich the interpretation of microbiome 'omics data, with activity intensity and active-inactive switching providing deeper insights. To achieve this, we advocate for increased accessibility and throughput of precise activity-discriminating technologies, alongside renewed use of reliable methods like direct cell counts and activity stains. Adopting this spectrum-based perspective will improve our ability to tackle key societal challenges, such as understanding microbial contributions to ecosystem function under climate change and gene dispersal at human-environment interfaces.
SUMMARYMendel's Law of Segregation posits an equal probability for each allele to be inherited during sexual reproduction. This process, however, is subverted by killer meiotic drivers (KMDs)-selfish genetic elements that enhance their own transmission in driver+/driver- heterozygotes by selectively eliminating meiotic products lacking the driver allele. Such elements arise recurrently during evolution, and the genomic conflicts they generate are considered potent evolutionary forces shaping genome architecture and sexual reproductive systems. While documented across diverse eukaryotes-including plants, fungi, and animals-most KMDs remain molecularly uncharacterized, and their actual prevalence in nature remains elusive. In fungi, KMDs can act at two distinct life-cycle stages: by directly killing sexual spores (fungal gametes)-hence termed "spore killers"-or by targeting haploid progeny after spore germination. Fungal models have profoundly advanced our understanding of these elements. This review synthesizes current knowledge on characterized fungal KMDs (from Neurospora, Podospora, Fusarium, and Schizosaccharomyces), emphasizing their molecular basis and interplay with the host. Approaches for KMD discovery are also discussed. Growing evidence from fungi suggests that these selfish elements are likely far more prevalent than previously appreciated.
SUMMARYThe mycobacterial cell envelope, one of the most complex membranes found in bacteria, plays a major role in bacterial pathogenesis, virulence, and antimicrobial resistance. Biogenesis and modeling of this cell envelope are heavily influenced by the mycobacterial membrane protein large (MmpL) family of transporters due to their ability to export fatty acids and lipid components. Select MmpL transporters can also function as siderophore exporters to help regulate the acquisition of iron, which is critical for mycobacterial survival. Additionally, certain MmpLs can participate in active efflux of antimycobacterial drugs, directly contributing to antimicrobial resistance. Given the physiological significance of these MmpL membrane proteins and their potential to serve as important antimycobacterial targets, questions regarding their functional roles, cellular assemblies, interactions, and regulation need to be fully addressed. In this review, we summarize our current knowledge on the structures and functions of these MmpL transporters. It is our hope that researchers in the field will continue to build upon these efforts and apply various structural, biophysical, and biochemical methodologies to fully elucidate how MmpL transporters coordinate to participate in cell envelope biogenesis, cell elongation and division, and antimicrobial resistance.
SUMMARYClass I fusion proteins are trimeric viral membrane proteins that mediate fusion between the virion and cellular membranes. In their prefusion state, they comprise three domains: a globular "head" domain that binds the target cell receptor, a helical "stalk" domain, and a transmembrane domain (TMD) at the carboxy-terminal end that anchors the protein in the viral membrane. However, it is now evident that the role of the TMD extends beyond simple membrane anchoring. This review explores the dynamic and regulatory functions of the TMD throughout the viral life cycle. TMDs contribute to intracellular trafficking and modulate membrane fusion activity and receptor binding. During virion assembly, they facilitate the incorporation of fusion proteins into budding particles, and influence virion formation and release. Furthermore, TMDs mediate interactions between viral and host proteins, shaping the structural organization of viral complexes, and impacting cellular responses to infection. Collectively, these findings highlight the TMD as a critical determinant of viral fitness and infectivity, underscoring its potential as a novel therapeutic target.
SUMMARYReptiles are a diverse and speciose class of animals that are broadly threatened by habitat loss, climate change, and other factors. From a microbiological perspective, reptiles have historically been examined as a source of disease, particularly salmonellosis, with most studies being culture-based investigations into causative agents of disease and potential for zoonoses. More recent work has sought to characterize the oral, skin, and gut microbiomes of reptiles more broadly to understand their contribution to reptile health and digestion. Non-avian reptiles are particularly interesting as ectothermic tetrapods, which usually lay eggs and have limited interaction with their young, as their digestion and life history strategies diverge substantially from the more well-studied mammals. Here, we review the reptile skin, oral, gut, eggshell, and nest microbiomes, along with the relationship between the microbiome and temperature stress. We present findings that distinguish the reptile microbiome from those of other studied vertebrate taxa, and place them in the context of their phylogenetic and ecological similarities to other animals. We discuss major disease-causing agents in reptiles, which was historically the main lens through which to view reptile microbiology, along with potential zoonoses. Finally, we examine how temperature and thermoregulation interact with the microbiome in reptiles, and how the microbiome may play a role in reptile conservation.
SUMMARYPersistent Staphylococcus aureus infections pose a major therapeutic challenge due to the formation of metabolically dormant persister cells that survive antibiotic exposure without acquiring genetic resistance. Despite their potent bactericidal activity, aminoglycosides fail against these persisters due to their reliance on energy-dependent uptake driven by the proton motive force (PMF). This review synthesizes emerging strategies designed to overcome this critical bottleneck. Metabolic stimulation using specific carbon sources or PMF-modulating agents reactivates membrane energetics, thereby restoring aminoglycoside uptake in dormant cells. Membrane-targeting adjuvants bypass PMF altogether, enabling antibiotic entry via biophysical remodeling and disruption of the lipid bilayer. Additionally, we discuss rationally engineered aminoglycoside hybrids, such as peptide-conjugated variants, that achieve self-directed, energy-independent penetration while preserving ribosomal targeting. Collectively, these approaches highlight that aminoglycoside failure against S. aureus persisters can be a modifiable physiological limitation rather than an issue of intrinsic resistance. The convergence of metabolic and membrane-based potentiation underscores the therapeutic potential of combinatorial regimens tailored to the unique bioenergetic state of persisters. Their clinical translation, combined with efforts to address toxicity, may transform the treatment landscape of recalcitrant S. aureus infections and mitigate the risk of relapse.
SUMMARYThe transcriptional regulator NrdR is present in most bacteria and in some archaea, but is lacking in eukaryotes. It controls the expression of operons of the universal and essential ribonucleotide reductase (RNR) enzymes, which provide building blocks of DNA by reducing ribonucleotides to their corresponding deoxyribonucleotides. The NrdR protein consists of an N-terminal zinc-ribbon domain that can bind to specific NrdR boxes in DNA, followed by an ATP-cone domain that binds adenosine nucleotides. Discovered 20 years ago, it was not until the recent high-resolution structures of NrdR in its DNA-bound and -unbound forms that its intricate mechanism of action could be described in detail. Contrary to early assumptions, the ATP-cone in NrdR has two nucleotide-binding sites, an inner and an outer site. When cellular dATP is low, NrdR is loaded with ATP in both sites and forms oligomers unable to bind to DNA, allowing transcription of RNR-encoding operons, and DNA replication and repair. When dATP levels increase, ATP in the outer site is substituted for dATP, NrdR will bind to DNA, and the expression of RNR-encoding genes will be inhibited. Interestingly, many RNRs also carry an ATP-cone that binds either one or two adenosine nucleotides, and that acts as an allosteric on/off switch of its enzyme activity. On the basis of current knowledge, this is a unique utilization of the same horizontally transferable domain for controlling both enzyme expression and enzyme activity.
SUMMARYInfluenza viruses remain a serious global health problem, causing annual epidemics and potential pandemics with significant morbidity and mortality. Antiviral therapy, particularly with direct-acting antivirals (DAAs), is a critical component of influenza control. This comprehensive review analyzes the current landscape of DAA drugs, with special attention paid to the complexity of synthesis, pharmacokinetic properties, and the development of antiviral resistance. We evaluated approved treatments, including neuraminidase (NA) inhibitors, viral RNA polymerase inhibitors, hemagglutinin (HA) inhibitors, and M2 ion channel blockers. We identify several critical obstacles to effective treatment: (i) the high genetic variability of influenza viruses, which facilitates the emergence of resistance, particularly in the case of M2 blockers (widespread) and, to a lesser but concerning extent, NA and polymerase inhibitors, both through natural evolution and selective pressure during drug treatment, and (ii) the suboptimal pharmacokinetic profiles of many existing drugs. This review provides a crucial framework for evaluating existing and investigational drugs for influenza, emphasizing the need to develop balanced therapeutic strategies that consider efficacy, resistance management, and global accessibility. The novelty of this review is a comprehensive comparative analysis of not only the drugs officially recommended by the WHO but also a wide range of other anti-influenza drugs approved in individual countries or under development. We have identified key comparative aspects that are discussed in detail here and are not always brought together in other reviews. The purpose of the article is to provide a generalized overview of the current state of knowledge, identify key trends and problems, and discuss future prospects without providing new primary data or experimental results.
SUMMARYMushroom-forming Agaricomycete fungi underpin global nutrient cycling and carbon sequestration, and support large and growing markets across food, medicinal supplements, and biomaterials. Yet most commercial and research uses still rely on wild-type strains, highlighting the opportunity for genetic engineering to expand possibilities for both fundamental research and biotechnological applications. In this review, we highlight progress toward synthetic biology in Agaricomycetes, and outline the main barriers that limit predictable genetic engineering. We emphasize engineering constraints unique to mushroom biology, including complex sexual cycles, heterokaryosis, and strain instability during transformation and outgrowth. We then transition to gene expression bottlenecks: the scarcity of characterized promoters and terminators, the challenges for gene integration posed by the condensed nature of Agaricomycete genomes, and the effects of introns and specific sequence motifs. Finally, drawing inspiration from progress in related fungi and other eukaryotes, we highlight the priorities for the field: systematic cross-species evaluation of genetic parts, development of more sophisticated gene-editing strategies, higher-throughput screening methods, and the establishment of a unifying model system. These advances would enable new possibilities in the study and use of Agaricomycetes, establishing these elusive organisms as programmable platforms for sustainable biomanufacturing, designer biomaterials, climate solutions, and mechanistic studies of fungal biology.
SUMMARYViral myocarditis is an inflammatory disease of the heart muscle caused by diverse viral pathogens and shaped by complex interactions between viruses and host responses, ultimately leading to myocardial inflammation and dysfunction. While environmental factors contribute to disease onset, growing evidence highlights a central role for host genetic determinants in influencing susceptibility, severity, and clinical outcomes. In addition, sex-specific differences have emerged as important modifiers of immune responses and disease progression. This review examines the genetic architecture of viral myocarditis, integrating human genetic studies with mechanistic insights derived largely from coxsackievirus B3 (CVB3) models, which provide the most comprehensive experimental framework for dissecting gene-disease relationships in vivo. By integrating genetic associations with their functional implications, we aim to deepen the understanding of viral myocarditis and inform future research directions and potential therapeutic strategies.
SUMMARYLive-cell study at the molecular level is essential for understanding the complexities of bacterial systems and protein functions in vivo. While ensemble biochemical approaches have been widely used to characterize protein functions, they suffer from heterogeneities arising from variations in individual cells' different physiological states and microenvironments. Single-molecule techniques have enhanced our abilities to resolve these heterogeneities by enabling the observation of individual cells and molecules, one at a time. Particularly, fluorescence single-molecule localization microscopy (SMLM) and single-molecule tracking (SMT) have emerged as powerful tools for real-time, in vivo analysis of biomolecular dynamics. SMT can reveal the heterogeneity and dynamic behaviors of individual molecules within their native cellular contexts, providing insights that are often obscured in ensemble studies. This review highlights recent progress on imaging probes and modalities and focuses on several key scientific discoveries in microbial systems, including metal regulation, electron transfer, cell division, and DNA repair. By showcasing novel insights gained from single-molecule imaging techniques, this review underscores their vital role in advancing our understanding of cellular processes at the single-molecule level and discusses future challenges and opportunities in the field.
SUMMARYThe velvet family of fungal regulatory proteins constitutes a unique and evolutionarily conserved set of transcriptional and epigenetic regulators that coordinate development, secondary metabolism, and pathogenicity in filamentous fungi. Among them, VeA, VelB, VelC, and VosA form dynamic protein complexes that act as molecular hubs, integrating environmental cues such as light and nutrient availability with intrinsic developmental signals. Over the past two decades, extensive genetic, molecular, and biochemical studies-particularly in Aspergillus species-have elucidated the central roles of these regulators in governing asexual and sexual development, spore viability, cell wall integrity, and the biosynthesis of secondary metabolites. Recent advances in genome-wide analyses, proteomics, and chromatin mapping have expanded our understanding of how velvet complexes influence chromatin architecture and coordinate transcriptional programs across fungal genomes. Velvet proteins not only regulate gene clusters involved in specialized metabolism but also control developmental transitions and stress responses, including those related to virulence in plant and human fungal pathogens. The interaction between LaeA-a methyltransferase-and velvet proteins further links transcriptional regulation to epigenetic control. This review synthesizes recent findings on the molecular functions, regulatory networks, and evolutionary conservation of velvet regulators, highlighting their emerging significance as master integrators in fungal biology. We also discuss knowledge gaps and future directions, including the therapeutic and biotechnological potential of targeting velvet-mediated regulation.
SUMMARYAntibiotic resistance is a major global health threat, with an estimated 1.14 million deaths in 2021 linked to antibiotic resistance. Mutations naturally arise as bacteria evolve to defend against and survive various environmental challenges, including those exerted by antibiotics. Both overuse and misuse of antibiotics can accelerate selection for resistant bacteria. Misuse can happen when antibiotic treatment ends prematurely, resulting in sub-lethal antibiotic levels. This provides an ideal environment for the proliferation of resistance-causing mutations, which, in some cases, are enhanced further by triggering the synthesis of error-prone DNA polymerases. Low levels of antibiotics are also found in the environment, creating breeding grounds for the evolution of antibiotic resistance. Mutations diminish the impact of antibiotics by three principal mechanisms: (i) reducing antibiotic influx, (ii) elevating antibiotic efflux, and (iii) altering cellular targets of antibiotics. The first two mechanisms confer modest resistance against a broad range of antibiotics; however, in combination with the third target-specific mechanism, they become the foundation of high-level antibiotic resistance. Ultimately, while the manifestation of mutations cannot be prevented, steps can be taken to lower their frequency by carefully considering the need for antibiotic prescription, exploring combination therapies, integrating adjuvants such as efflux pump inhibitors, and minimizing environmental contamination of antibiotics.
