Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Eukaryotic Acquisition of a Bacterial Operon.

Operons are a hallmark of bacterial genomes, where they allow concerted expression of functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here, we report the horizontal operon transfer of a siderophore biosynthesis pathway from relatives of Escherichia coli into a group of budding yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are expressed, exhibit eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer, several genetic changes occurred during subsequent evolution, including the gain of new transcription start sites that were sometimes within protein-coding sequences, acquisition of polyadenylation sites, structural rearrangements, and integration of eukaryotic genes into the cluster. We conclude that the genes were likely acquired as a unit, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly competitive, iron-limited environment.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria.

Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.

Functional and evolutionary significance of unknown genes from uncultivated taxa.

Many of the Earth's microbes remain uncultured and understudied, limiting our understanding of the functional and evolutionary aspects of their genetic material, which remain largely overlooked in most metagenomic studies1. Here we analysed 149,842 environmental genomes from multiple habitats2-6 and compiled a curated catalogue of 404,085 functionally and evolutionarily significant novel (FESNov) gene families exclusive to uncultivated prokaryotic taxa. All FESNov families span multiple species, exhibit strong signals of purifying selection and qualify as new orthologous groups, thus nearly tripling the number of bacterial and archaeal gene families described to date. The FESNov catalogue is enriched in clade-specific traits, including 1,034 novel families that can distinguish entire uncultivated phyla, classes and orders, probably representing synapomorphies that facilitated their evolutionary divergence. Using genomic context analysis and structural alignments we predicted functional associations for 32.4% of FESNov families, including 4,349 high-confidence associations with important biological processes. These predictions provide a valuable hypothesis-driven framework that we used for experimental validatation of a new gene family involved in cell motility and a novel set of antimicrobial peptides. We also demonstrate that the relative abundance profiles of novel families can discriminate between environments and clinical conditions, leading to the discovery of potentially new biomarkers associated with colorectal cancer. We expect this work to enhance future metagenomics studies and expand our knowledge of the genetic repertory of uncultivated organisms.

A Pseudomonas aeruginosa small RNA regulates chronic and acute infection.

The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.

A naturally inspired antibiotic to target multidrug-resistant pathogens.

Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections1,2. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin3. Bacteria-derived antibiotics often appear in nature as collections of similar structures that are encoded by evolutionarily related biosynthetic gene clusters. This structural diversity is, at least in part, expected to be a response to the development of natural resistance, which often mechanistically mimics clinical resistance. Here we propose that a solution to mcr-1-mediated resistance might have evolved among naturally occurring colistin congeners. Bioinformatic analysis of sequenced bacterial genomes identified a biosynthetic gene cluster that was predicted to encode a structurally divergent colistin congener. Chemical synthesis of this structure produced macolacin, which is active against Gram-negative pathogens expressing mcr-1 and intrinsically resistant pathogens with chromosomally encoded phosphoethanolamine transferase genes. These Gram-negative bacteria include extensively drug-resistant Acinetobacter baumannii and intrinsically colistin-resistant Neisseria gonorrhoeae, which, owing to a lack of effective treatment options, are considered among the highest level threat pathogens4. In a mouse neutropenic infection model, a biphenyl analogue of macolacin proved to be effective against extensively drug-resistant A. baumannii with colistin-resistance, thus providing a naturally inspired and easily produced therapeutic lead for overcoming colistin-resistant pathogens.

Promoter recruitment drives the emergence of proto-genes in a long-term evolution experiment with Escherichia coli.

The phenomenon of de novo gene birth-the emergence of genes from non-genic sequences-has received considerable attention due to the widespread occurrence of genes that are unique to particular species or genomes. Most instances of de novo gene birth have been recognized through comparative analyses of genome sequences in eukaryotes, despite the abundance of novel, lineage-specific genes in bacteria and the relative ease with which bacteria can be studied in an experimental context. Here, we explore the genetic record of the Escherichia coli long-term evolution experiment (LTEE) for changes indicative of "proto-genic" phases of new gene birth in which non-genic sequences evolve stable transcription and/or translation. Over the time span of the LTEE, non-genic regions are frequently transcribed, translated and differentially expressed, with levels of transcription across low-expressed regions increasing in later generations of the experiment. Proto-genes formed downstream of new mutations result either from insertion element activity or chromosomal translocations that fused preexisting regulatory sequences to regions that were not expressed in the LTEE ancestor. Additionally, we identified instances of proto-gene emergence in which a previously unexpressed sequence was transcribed after formation of an upstream promoter, although such cases were rare compared to those caused by recruitment of preexisting promoters. Tracing the origin of the causative mutations, we discovered that most occurred early in the history of the LTEE, often within the first 20,000 generations, and became fixed soon after emergence. Our findings show that proto-genes emerge frequently within evolving populations, can persist stably, and can serve as potential substrates for new gene formation.

A metabolomics pipeline for the mechanistic interrogation of the gut microbiome.

Gut microorganisms modulate host phenotypes and are associated with numerous health effects in humans, ranging from host responses to cancer immunotherapy to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microorganisms has hindered efforts to define mechanistic connections between individual microbial strains and host phenotypes. One key way in which the gut microbiome influences host physiology is through the production of small molecules1-3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect the products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites in diverse sample types. We report the metabolic profiles of 178 gut microorganism strains using our library of 833 metabolites. Using this metabolomics resource, we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover a previously undescribed type of metabolism in Bacteroides, and reveal candidate biochemical pathways using comparative genomics. Microbiota-dependent metabolites can be detected in diverse biological fluids from gnotobiotic and conventionally colonized mice and traced back to the corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microorganisms and interactions between microorganisms and their host.

Transcriptional Pausing as a Mediator of Bacterial Gene Regulation.

Cellular life depends on transcription of DNA by RNA polymerase to express genetic information. RNA polymerase has evolved not just to read information from DNA and write it to RNA but also to sense and process information from the cellular and extracellular environments. Much of this information processing occurs during transcript elongation, when transcriptional pausing enables regulatory decisions. Transcriptional pauses halt RNA polymerase in response to DNA and RNA sequences and structures at locations and times that help coordinate interactions with small molecules and transcription factors important for regulation. Four classes of transcriptional pause signals are now evident after decades of study: elemental pauses, backtrack pauses, hairpin-stabilized pauses, and regulator-stabilized pauses. In this review, I describe current understanding of the molecular mechanisms of these four classes of pause signals, remaining questions about how RNA polymerase responds to pause signals, and the many exciting directions now open to understand pausing and the regulation of transcript elongation on a genome-wide scale.

Action of a minimal contractile bactericidal nanomachine.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1-4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

Large-scale duplication events underpin population-level flexibility in tRNA gene copy number in Pseudomonas fluorescens SBW25.

The complement of tRNA genes within a genome is typically considered to be a (relatively) stable characteristic of an organism. Here, we demonstrate that bacterial tRNA gene set composition can be more flexible than previously appreciated, particularly regarding tRNA gene copy number. We report the high-rate occurrence of spontaneous, large-scale, tandem duplication events in laboratory populations of the bacterium Pseudomonas fluorescens SBW25. The identified duplications are up to ∼1 Mb in size (∼15% of the wildtype genome) and are predicted to change the copy number of up to 917 genes, including several tRNA genes. The observed duplications are inherently unstable: they occur, and are subsequently lost, at extremely high rates. We propose that this unusually plastic type of mutation provides a mechanism by which tRNA gene set diversity can be rapidly generated, while simultaneously preserving the underlying tRNA gene set in the absence of continued selection. That is, if a tRNA set variant provides no fitness advantage, then high-rate segregation of the duplication ensures the maintenance of the original tRNA gene set. However, if a tRNA gene set variant is beneficial, the underlying duplication fragment(s) may persist for longer and provide raw material for further, more stable, evolutionary change.

CRISPR-Cas immunity is repressed by the LysR-type transcriptional regulator PigU.

Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.

Genome-wide fitness profiling reveals molecular mechanisms that bacteria use to interact with Trichoderma atroviride exometabolites.

Trichoderma spp. are ubiquitous rhizosphere fungi capable of producing several classes of secondary metabolites that can modify the dynamics of the plant-associated microbiome. However, the bacterial-fungal mechanisms that mediate these interactions have not been fully characterized. Here, a random barcode transposon-site sequencing (RB-TnSeq) approach was employed to identify bacterial genes important for fitness in the presence of Trichoderma atroviride exudates. We selected three rhizosphere bacteria with RB-TnSeq mutant libraries that can promote plant growth: the nitrogen fixers Klebsiella michiganensis M5aI and Herbaspirillum seropedicae SmR1, and Pseudomonas simiae WCS417. As a non-rhizosphere species, Pseudomonas putida KT2440 was also included. From the RB-TnSeq data, nitrogen-fixing bacteria competed mainly for iron and required the siderophore transport system TonB/ExbB for optimal fitness in the presence of T. atroviride exudates. In contrast, P. simiae and P. putida were highly dependent on mechanisms associated with membrane lipid modification that are required for resistance to cationic antimicrobial peptides (CAMPs). A mutant in the Hog1-MAP kinase (Δtmk3) gene of T. atroviride showed altered expression patterns of many nonribosomal peptide synthetase (NRPS) biosynthetic gene clusters with potential antibiotic activity. In contrast to exudates from wild-type T. atroviride, bacterial mutants containing lesions in genes associated with resistance to antibiotics did not show fitness defects when RB-TnSeq libraries were exposed to exudates from the Δtmk3 mutant. Unexpectedly, exudates from wild-type T. atroviride and the Δtmk3 mutant rescued purine auxotrophic mutants of H. seropedicae, K. michiganensis and P. simiae. Metabolomic analysis on exudates from wild-type T. atroviride and the Δtmk3 mutant showed that both strains excrete purines and complex metabolites; functional Tmk3 is required to produce some of these metabolites. This study highlights the complex interplay between Trichoderma-metabolites and soil bacteria, revealing both beneficial and antagonistic effects, and underscoring the intricate and multifaceted nature of this relationship.

Single cobalt atoms anchored on Ti3C2Tx with dual reaction sites for efficient adsorption-degradation of antibiotic resistance genes.

The assimilation of antibiotic resistance genes (ARGs) by pathogenic bacteria poses a severe threat to public health. Here, we reported a dual-reaction-site-modified CoSA/Ti3C2Tx (single cobalt atoms immobilized on Ti3C2Tx MXene) for effectively deactivating extracellular ARGs via peroxymonosulfate (PMS) activation. The enhanced removal of ARGs was attributed to the synergistic effect of adsorption (Ti sites) and degradation (Co-O3 sites). The Ti sites on CoSA/Ti3C2Tx nanosheets bound with PO43- on the phosphate skeletons of ARGs via Ti-O-P coordination interactions, achieving excellent adsorption capacity (10.21 × 1010 copies mg-1) for tetA, and the Co-O3 sites activated PMS into surface-bond hydroxyl radicals (•OHsurface), which can quickly attack the backbones and bases of the adsorbed ARGs, resulting in the efficient in situ degradation of ARGs into inactive small molecular organics and NO3. This dual-reaction-site Fenton-like system exhibited ultrahigh extracellular ARG degradation rate (k > 0.9 min-1) and showed the potential for practical wastewater treatment in a membrane filtration process, which provided insights for extracellular ARG removal via catalysts design.

Bacterial origin of a key innovation in the evolution of the vertebrate eye.

The vertebrate eye was described by Charles Darwin as one of the greatest potential challenges to a theory of natural selection by stepwise evolutionary processes. While numerous evolutionary transitions that led to the vertebrate eye have been explained, some aspects appear to be vertebrate specific with no obvious metazoan precursor. One critical difference between vertebrate and invertebrate vision hinges on interphotoreceptor retinoid-binding protein (IRBP, also known as retinol-binding protein, RBP3), which enables the physical separation and specialization of cells in the vertebrate visual cycle by promoting retinoid shuttling between cell types. While IRBP has been functionally described, its evolutionary origin has remained elusive. Here, we show that IRBP arose via acquisition of novel genetic material from bacteria by interdomain horizontal gene transfer (iHGT). We demonstrate that a gene encoding a bacterial peptidase was acquired prior to the radiation of extant vertebrates >500 Mya and underwent subsequent domain duplication and neofunctionalization to give rise to vertebrate IRBP. Our phylogenomic analyses on >900 high-quality genomes across the tree of life provided the resolution to distinguish contamination in genome assemblies from true instances of horizontal acquisition of IRBP and led us to discover additional independent transfers of the same bacterial peptidase gene family into distinct eukaryotic lineages. Importantly, this work illustrates the evolutionary basis of a key transition that led to the vertebrate visual cycle and highlights the striking impact that acquisition of bacterial genes has had on vertebrate evolution.

A neglected and emerging antimicrobial resistance gene encodes for a serine-dependent macrolide esterase.

The discovery of unreported antimicrobial resistance genes (ARGs) remains essential. Here, we report the identification and preliminary characterization of an α/ß-hydrolase that inactivates macrolides. This serine-dependent macrolide esterase co-occurs with emerging ARGs in the environment, animal microbiomes, and pathogens.

The bacterial genetic determinants of Escherichia coli capacity to cause bloodstream infections in humans.

Escherichia coli is both a highly prevalent commensal and a major opportunistic pathogen causing bloodstream infections (BSI). A systematic analysis characterizing the genomic determinants of extra-intestinal pathogenic vs. commensal isolates in human populations, which could inform mechanisms of pathogenesis, diagnostic, prevention and treatment is still lacking. We used a collection of 912 BSI and 370 commensal E. coli isolates collected in France over a 17-year period (2000-2017). We compared their pangenomes, genetic backgrounds (phylogroups, STs, O groups), presence of virulence-associated genes (VAGs) and antimicrobial resistance genes, finding significant differences in all comparisons between commensal and BSI isolates. A machine learning linear model trained on all the genetic variants derived from the pangenome and controlling for population structure reveals similar differences in VAGs, discovers new variants associated with pathogenicity (capacity to cause BSI), and accurately classifies BSI vs. commensal strains. Pathogenicity is a highly heritable trait, with up to 69% of the variance explained by bacterial genetic variants. Lastly, complementing our commensal collection with an older collection from 1980, we predict that pathogenicity continuously increased through 1980, 2000, to 2010. Together our findings imply that E. coli exhibit substantial genetic variation contributing to the transition between commensalism and pathogenicity and that this species evolved towards higher pathogenicity.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

The F pilus serves as a conduit for the DNA during conjugation between physically distant bacteria.

Horizontal transfer of F-like plasmids by bacterial conjugation is responsible for disseminating antibiotic resistance and virulence determinants among pathogenic Enterobacteriaceae species, a growing health concern worldwide. Central to this process is the conjugative F pilus, a long extracellular filamentous polymer that extends from the surface of plasmid donor cells, allowing it to probe the environment and make contact with the recipient cell. It is well established that the F pilus can retract to bring mating pair cells in tight contact before DNA transfer. However, whether DNA transfer can occur through the extended pilus has been a subject of active debate. In this study, we use live-cell microscopy to show that while most transfer events occur between cells in direct contact, the F pilus can indeed serve as a conduit for the DNA during transfer between physically distant cells. Our findings enable us to propose a unique model for conjugation that revises our understanding of the DNA transfer mechanism and the dissemination of drug resistance and virulence genes within complex bacterial communities.