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A key aspect of bacterial survival is the ability to evolve while migrating across spatially varying environmental challenges. Laboratory experiments, however, often study evolution in well-mixed systems. Here, we introduce an experimental device, the microbial evolution and growth arena (MEGA)-plate, in which bacteria spread and evolved on a large antibiotic landscape (120×60 centimeters) that allowed visual observation of mutation and selection in a migrating bacterial front. While resistance increased consistently, multiple coexisting lineages diversified both phenotypically and genotypically. Analyzing mutants at and behind the propagating front, we found that evolution is not always led by the most resistant mutants; highly resistant mutants may be trapped behind more sensitive lineages. The MEGA-plate provides a versatile platform for studying microbial adaption and directly visualizing evolutionary dynamics.

Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development. Biocontainment is a key to developing safe genetically-engineered microbes (GEMs). Here the authors demonstrate genetically stable CRISPR-based kill switches that control GEMs’ viability in animal hosts, enabling their safe biomedical applications.

Differences in the presence of even a few genes between otherwise identical bacterial strains may result in critical phenotypic differences. Here we systematically identify microbial genomic structural variants (SVs) and find them to be prevalent in the human gut microbiome across phyla and to replicate in different cohorts. SVs are enriched for CRISPR-associated and antibiotic-producing functions and depleted from housekeeping genes, suggesting that they have a role in microbial adaptation. We find multiple associations between SVs and host disease risk factors, many of which replicate in an independent cohort. Exploring genes that are clustered in the same SV, we uncover several possible mechanistic links between the microbiome and its host, including a region in Anaerostipes hadrus that encodes a composite inositol catabolism-butyrate biosynthesis pathway, the presence of which is associated with lower host metabolic disease risk. Overall, our results uncover a nascent layer of variability in the microbiome that is associated with microbial adaptation and host health. The authors systematically characterize structural variation in the genomes of gut microbiota and show that they are associated with bacterial fitness and with host risk factors, and that examining genes coded in these regions facilitates investigation of mechanisms that may underlie these associations.

Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here, we provide in vitro evidence of Staphylococcus aureus persisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. Intracellular S. aureus persisters remain metabolically active but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell wall stress, SOS and heat shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellular S. aureus persisters may constitute a reservoir for relapsing infection and could contribute to therapeutic failures. Bacterial persister cells exhibit a transient non-growing state and antibiotic tolerance. Here, Peyrusson et al. provide evidence of metabolically active Staphylococcus aureus persisters within infected host cells exposed to antibiotics and analyse transcriptomic alterations associated with persistence.

Construction of a series of genomically recoded organisms whose growth is restricted by the expression of essential genes dependent on exogenously supplied synthetic amino acids introduces novel orthogonal barriers between these engineered organisms and the environment, thereby creating safer genetically modified organisms. Two routes to safer GMOs Two manuscripts published in this issue of Nature describe independent approaches towards generating an organism dependent on unnatural amino acids, a development which could find applications for biocontainment and exploration of previously unsampled fitness landscapes. George Church and colleagues redesigned essential enzymes in an organism ( Escherichia coli ) with an altered genetic code to make it metabolically dependent on non-standard amino acids for survival. The resulting genetically modified organisms (GMOs) cannot metabolically circumvent their biocontainment mechanisms and show unprecedented resistance to evolutionary escape. The few escapees are rapidly outcompeted by unmodified organisms. Using multiplex automated genome engineering, Farren Isaacs and colleagues construct a series of genomically recoded organisms whose growth is restricted by the expression of essential genes that depend on exogenously supplied synthetic amino acids. They constructed synthetic auxotrophs with advanced orthogonal barriers between engineered organisms and the environment, thereby creating safer GMOs. Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals 1 . Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems 2 , 3 , which include bioremediation 4 and probiotics 5 . Although safeguards have been designed to control cell growth by essential gene regulation 6 , inducible toxin switches 7 and engineered auxotrophies 8 , these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations 9 , 10 . Here we describe the construction of a series of genomically recoded organisms (GROs) 11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering 12 , we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residues 13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼10 11 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches 2 , 3 , 6 , 7 , 8 , 9 , 10 . We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer 11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

Eukaryotic genomes are generally organized in multiple chromosomes. Here we have created a functional single-chromosome yeast from a Saccharomyces cerevisiae haploid cell containing sixteen linear chromosomes, by successive end-to-end chromosome fusions and centromere deletions. The fusion of sixteen native linear chromosomes into a single chromosome results in marked changes to the global three-dimensional structure of the chromosome due to the loss of all centromere-associated inter-chromosomal interactions, most telomere-associated inter-chromosomal interactions and 67.4% of intra-chromosomal interactions. However, the single-chromosome and wild-type yeast cells have nearly identical transcriptome and similar phenome profiles. The giant single chromosome can support cell life, although this strain shows reduced growth across environments, competitiveness, gamete production and viability. This synthetic biology study demonstrates an approach to exploration of eukaryote evolution with respect to chromosome structure and function. Successive fusion of yeast chromosomes is used to produce a single-chromosome strain that is viable, albeit with slightly reduced fitness.

Introns are ubiquitous features of all eukaryotic cells. Introns need to be removed from nascent messenger RNA through the process of splicing to produce functional proteins. Here we show that the physical presence of introns in the genome promotes cell survival under starvation conditions. A systematic deletion set of all known introns in budding yeast genes indicates that, in most cases, cells with an intron deletion are impaired when nutrients are depleted. This effect of introns on growth is not linked to the expression of the host gene, and was reproduced even when translation of the host mRNA was blocked. Transcriptomic and genetic analyses indicate that introns promote resistance to starvation by enhancing the repression of ribosomal protein genes that are downstream of the nutrient-sensing TORC1 and PKA pathways. Our results reveal functions of introns that may help to explain their evolutionary preservation in genes, and uncover regulatory mechanisms of cell adaptations to starvation. Transcriptomic and genetic analyses of a deletion set of all known introns in genes of the budding yeast Saccharomyces cerevisiae indicate that introns promote resistance to starvation.

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic ‘dual RNA-seq’ approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK–STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes. Using dual RNA-seq technology to profile RNA expression simultaneously in the bacterial pathogen Salmonella and its host during infection reveals molecular phenotypes of small noncoding RNAs in the infection process. RNA expression during bacterial infection This study applies 'dual RNA-seq' to simultaneously profile RNA expression in the pathogen Salmonella enterica serovar Typhimurium and its eukaryotic host cell during infection, to reveal the role of bacterial riboregulators. Among several bacterial small RNAs (sRNAs) discovered to function in the infection process, the authors identify an sRNA (termed PinT) which temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival of the pathogen, and which also alters the expression of both coding and non-coding transcripts of the host. These findings are a proof-of-principle for the utility of this high-throughput screen to uncover potentially novel pathogenic strategies that are important during infection.

Persister cells, rare phenotypic variants that survive normally lethal levels of antibiotics, present a major barrier to clearing bacterial infections 1 . However, understanding the precise physiological state and genetic basis of persister formation has been a longstanding challenge. Here we generated a high-resolution single-cell 2 RNA atlas of Escherichia coli growth transitions, which revealed that persisters from diverse genetic and physiological models converge to transcriptional states that are distinct from standard growth phases and instead exhibit a dominant signature of translational deficiency. We then used ultra-dense CRISPR interference 3 to determine how every E. coli gene contributes to persister formation across genetic models. Among critical genes with large effects, we found lon , which encodes a highly conserved protease 4 , and yqgE , a poorly characterized gene whose product strongly modulates the duration of post-starvation dormancy and persistence. Our work reveals key physiologic and genetic factors that underlie starvation-triggered persistence, a critical step towards targeting persisters in recalcitrant bacterial infections. Single-cell transcriptome analyses of growth phases in Escherichia coli identify a distinct transcriptional state occupied by antibiotic-tolerant persisters in which lon and yqgE have major roles.

The authors develop a mouse model of Enterococcus faecalis colonization to show that enterococci harbouring the bacteriocin-expressing plasmid pPD1 replace indigenous enterococci and have the ability to transfer the plasmid to other enterococci, which enhances the stability of the bacteriocin-expressing bacteria in the gut; this result suggests a therapeutic approach that leverages niche-specificity to eliminate antibiotic-resistant bacteria from infected individuals. Competition defeats multidrug-resistant bacteria Enterococcus faecalis is a normal gut bacterium and is usually harmless, but it can cause a variety of hospital-acquired infections in which its acquisition of antibiotic resistance makes it hard to treat. Nita Salzman and colleagues develop a mouse model of E. faecalis infection and use it to show that enterococci harbouring the bacteriocin-expressing plasmid pPD1 replace indigenous enterococci and have the ability to transfer the plasmid to other enterococci, thereby enhancing the stability of the bacteriocin-expressing bacteria in the gut. However, colonization by a strain in which the plasmid was not passed on resulted in clearance of other enterococci strains from the gut — including those resistant to the antibiotic vancomycin. This result suggests a way of using bacteriocin-producing bacteria as targeted therapeutics designed to clear competing multidrug-resistant strains from infected individuals. Enterococcus faecalis is both a common commensal of the human gastrointestinal tract and a leading cause of hospital-acquired infections 1 . Systemic infections with multidrug-resistant enterococci occur subsequent to gastrointestinal colonization 2 . Preventing colonization by multidrug-resistant E. faecalis could therefore be a valuable approach towards limiting infection. However, little is known about the mechanisms E. faecalis uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive conjugative plasmids encoding bacteriocins are common among enterococcal strains 3 and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of colonization of the mouse gut with E. faecalis , without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 21 (ref. 4 ) in enterococcal colonization. Here we show that E. faecalis harbouring pPD1 replaces indigenous enterococci and outcompetes E. faecalis lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other E. faecalis strains by conjugation, enhancing their survival. Colonization with an E. faecalis strain carrying a conjugation-defective pPD1 mutant subsequently resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore, bacteriocin expression by commensal bacteria can influence niche competition in the gastrointestinal tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multidrug-resistant bacteria, without profound disruption of the indigenous microbiota.