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Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.

Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.

Although microbiome signatures have been identified in various contexts (ie, pathogenesis of non-communicable diseases and treatment response), qualified microbiome-based biomarkers are currently not in use in clinical practice. The Human Microbiome Action consortium initiated a Delphi survey to establish a consensus on the needs, challenges, and limitations in developing qualified microbiome-based biomarkers. The questionnaire was developed by a scientific committee via literature review and expert interviews. To ensure broad applicability of the results, 307 experts were invited to participate; 114 of them responded to the first round of the survey, 93 of whom completed the second and final round as well. The survey highlighted the experts’ confidence in the potential of microbiome-based biomarkers for several indications or pathologies. The paucity of validated analytical methods appears to be the principal factor hindering the qualification of these biomarkers. The survey also showed that clinical implementation of these biomarkers would only be possible if kitted and validated molecular assays with simple interpretation are developed. This initiative serves as a foundation for designing and implementing public-private collaborative projects to overcome the challenges and promote clinical application of microbiome-based biomarkers.

Randomly barcoded transposon mutant libraries are powerful tools for studying gene function and organization, assessing gene essentiality and pathways, discovering potential therapeutic targets, and understanding the physiology of gut bacteria and their interactions with the host. However, construction of high-quality libraries with uniform representation can be challenging. In this review, we survey various strategies for barcoded library construction, including transposition systems, methods of transposon delivery, optimal library size, and transconjugant selection schemes. We discuss the advantages and limitations of each approach, as well as factors to consider when selecting a strategy. In addition, we highlight experimental and computational advances in arraying condensed libraries from mutant pools. We focus on examples of successful library construction in gut bacteria and their application to gene function studies and drug discovery. Given the need for understanding gene function and organization in gut bacteria, we provide a comprehensive guide for researchers to construct randomly barcoded transposon mutant libraries.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. The in situ ribosome structures upon tetracycline treatment show a virtually identical drug binding-site in both strains, yet the distribution of ribosomal complexes clearly differs. While K-12 retains ribosomes in a translation competent state, tRNAs are lost in the vast majority of ED1a ribosomes. A differential response is also reflected in proteome-wide abundance and thermal stability assessment. Our study underlines the need to include molecular analyses and to consider gut bacteria when addressing antibiotic mode of action.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

To explore favourable niches while avoiding threats, many bacteria use a chemotaxis navigation system. Despite decades of studies on chemotaxis, most signals and sensory proteins are still unknown. Many bacterial species release d-amino acids to the environment; however, their function remains largely unrecognized. Here we reveal that d-arginine and d-lysine are chemotactic repellent signals for the cholera pathogen Vibrio cholerae. These d-amino acids are sensed by a single chemoreceptor MCPDRK co-transcribed with the racemase enzyme that synthesizes them under the control of the stress-response sigma factor RpoS. Structural characterization of this chemoreceptor bound to either d-arginine or d-lysine allowed us to pinpoint the residues defining its specificity. Interestingly, the specificity for these d-amino acids appears to be restricted to those MCPDRK orthologues transcriptionally linked to the racemase. Our results suggest that d-amino acids can shape the biodiversity and structure of complex microbial communities under adverse conditions.

Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities., Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities.

Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities., Multi-omics analyses are used in microbiome studies to understand molecular changes in microbial communities exposed to different conditions. However, it is not always clear how much each omics data type contributes to our understanding and whether they are concordant with each other. Here, we map the molecular response of a synthetic community of 32 human gut bacteria to three non-antibiotic drugs by using five omics layers (16S rRNA gene profiling, metagenomics, metatranscriptomics, metaproteomics and metabolomics). We find that all the omics methods with species resolution are highly consistent in estimating relative species abundances. Furthermore, different omics methods complement each other for capturing functional changes. For example, while nearly all the omics data types captured that the antipsychotic drug chlorpromazine selectively inhibits Bacteroidota representatives in the community, the metatranscriptome and metaproteome suggested that the drug induces stress responses related to protein quality control. Metabolomics revealed a decrease in oligosaccharide uptake, likely caused by Bacteroidota depletion. Our study highlights how multi-omics datasets can be utilized to reveal complex molecular responses to external perturbations in microbial communities.

Fluctuating conditions and diverse stresses are typical in natural environments. In response, cells mount complex responses across multiple scales, including adjusting their shape to withstand stress. In enterobacteria, the Rcs phosphorelay is activated by cell envelope damage and by changes to periplasmic dimensions and cell width. Here, we investigated the physiological and morphological consequences of Rcs activation in Escherichia coli in the absence of stresses, using an inducible version of RcsF that mislocalizes to the inner membrane, RcsFIM. Expression of RcsFIM immediately reduced cellular growth rate and the added length per cell cycle in a manner that was directly dependent on induction levels, but independent of Rcs-induced capsule production. At the same time, cells increased intracellular concentration of the cell division protein FtsZ, and decreased the distance between division rings in filamentous cells. Depletion of the Rcs negative regulator IgaA phenocopied RcsFIM induction, indicating that IgaA is essential due to growth inhibition in its absence. However, A22 treatment did not affect growth rate or FtsZ intracellular concentration, despite activating the Rcs system. These findings suggest that the effect of Rcs activation on FtsZ levels is mediated indirectly through growth-rate changes, and highlight feedbacks among the Rcs stress response, growth dynamics, and cell-size control.

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we fou

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we fou

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we fou

Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly-ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell-based ubiquitin replacement strategy to enable selective and conditional abrogation of K27-linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27-linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97-proteasome pathway model substrate (Ub(G76V)-GFP) is directly modified by K27-linked ubiquitylation, and that disabling the formation of K27-linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage-specific binder UCHL3 impedes Ub(G76V)-GFP turnover at the level of p97 function. Our findings suggest a critical role of K27-linked ubiquitylation in supporting cell fitness by facilitating p97-dependent processing of ubiquitylated nuclear proteins.

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we fou

The ubiquitous presence of toxic arsenate (AsV) in the environment has raised mechanisms of resistance in all living organisms. Generally, bacterial detoxification of AsV relies on its reduction to arsenite (AsIII) by ArsC, followed by the export of AsIII by ArsB. However, how pathogenic species resist this metalloid remains largely unknown. Here, we found that Vibrio cholerae, the etiologic agent of the diarrheal disease cholera, outcompetes other enteropathogens when grown on millimolar concentrations of AsV. To do so, V. cholerae uses, instead of ArsCB, the AsV-inducible vc1068-1071 operon (renamed var for vibrio arsenate resistance), which encodes the arsenate repressor ArsR, an alternative glyceraldehyde-3-phosphate dehydrogenase, a putative phosphatase, and the AsV transporter ArsJ. In addition to Var, V. cholerae induces oxidative stress-related systems to counter reactive oxygen species (ROS) production caused by intracellular AsV. Characterization of the var mutants suggested that these proteins function independently from one another and play critical roles in preventing deleterious effects on the cell membrane potential and growth derived from the accumulation AsV. Mechanistically, we demonstrate that V. cholerae complexes AsV with the glycolytic intermediate 3-phosphoglycerate into 1-arseno-3-phosphoglycerate (1As3PG). We further show that 1As3PG is not transported outside the cell; instead, it is subsequently dissociated to enable extrusion of free AsV through ArsJ. Collectively, we propose the formation of 1As3PG as a transient metabolic storage of AsV to curb the noxious effect of free AsV. This study advances our understanding of AsV resistance in bacteria and underscores new points of vulnerability that might be an attractive target for antimicrobial interventions. IMPORTANCE Even though resistance to arsenate has been extensively investigated in environmental bacteria, how enteric pathogens tolerate this toxic compound remains unknown. Here, we fou

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping ofthe interactions between drugs and bacteria has only started recently' and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth ofthe bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine bindsto several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegansto duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. However, the systematic mapping ofthe interactions between drugs and bacteria has only started recently' and the main underlying mechanism proposed is the chemical transformation of drugs by microorganisms (biotransformation). Here we investigated the depletion of 15 structurally diverse drugs by 25 representative strains of gut bacteria. This revealed 70 bacteria-drug interactions, 29 of which had not to our knowledge been reported before. Over half of the new interactions can be ascribed to bioaccumulation; that is, bacteria storing the drug intracellularly without chemically modifying it, and in most cases without the growth ofthe bacteria being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using click chemistry, thermal proteome profiling and metabolomics. We find that duloxetine bindsto several metabolic enzymes and changes the metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the composition of the community through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioural response of Caenorhabditis elegansto duloxetine. Together, our results show that bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, probably in an individual manner.

Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome.

Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome.

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.

Treatment options for gonorrhoea are scarce. Drug repurposing of bioactive molecules approved for other conditions might therefore be of value. We developed a method for wide-scale, systematic drug repurposing screen to identify molecules with activity against Neisseria gonorrhoeae and screened the Prestwick Chemical Library (1200 FDA-approved drugs). As a proof-of-concept, we further examined one promising and interesting screening hit (auranofin; antirheumatic agent). Three WHO gonococcal reference strains (WHO F, O, P) were used for the Library screening. The strains were grown in presence of a fixed concentration of the library drugs in 384-well plates for 12 h, and the remaining bacterial respiration, to reflect growth, was then quantitatively measured using optical density (OD) 450 nm and a resazurin assay. The activity of auranofin was further examined using in vitro susceptibility testing (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)) against genetically diverse antimicrobial-resistant N. gonorrhoeae strains and time-kill assays. Sixty-eight molecules significantly inhibited bacterial growth of WHO F, O and P. Auranofin showed potent in vitro bactericidal activity (in MIC-, MBC- and time-kill assays) against four WHO reference strains. No cross-resistance between auranofin and any antimicrobial currently or previously used for gonorrhoea treatment was found when examining 51 selected antimicrobial-resistant gonococcal strains. In conclusion, this is the first wide-scale systematic screening effort for repurposing drugs for future treatment of gonorrhoea. Additional studies examining mechanism(s) of action, resistance development, in vivo anti-gonococcal activity and pharmacokinetics/pharmacodynamics for gonococcal infections of auranofin and several other significant screening hits would be valuable.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Treatment options for gonorrhoea are scarce. Drug repurposing of bioactive molecules approved for other conditions might therefore be of value. We developed a method for wide-scale, systematic drug repurposing screen to identify molecules with activity against Neisseria gonorrhoeae and screened the Prestwick Chemical Library (1200 FDA-approved drugs). As a proof-of-concept, we further examined one promising and interesting screening hit (auranofin; antirheumatic agent). Three WHO gonococcal reference strains (WHO F, O, P) were used for the Library screening. The strains were grown in presence of a fixed concentration of the library drugs in 384-well plates for 12 hours and the remaining bacterial respiration, to reflect growth, was then quantitatively measured using optical density (OD) 450 nm and a resazurin assay. The activity of auranofin was further examined using in vitro susceptibility testing (minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)) against genetically diverse antimicrobial-resistant N. gonorrhoeae strains and time-kill assays. Sixty-eight molecules significantly inhibited bacterial growth of WHO F, O and, P. Auranofin showed potent in vitro bactericidal activity (in MIC-, MBC-, and time-kill assays) against four WHO reference strains. No cross resistance between auranofin and any antimicrobial currently or previously used for gonorrhoea treatment was found when examining 51 selected antimicrobial-resistant gonococcal strains. In conclusion, this is the first wide-scale systematic screening effort for repurposing drugs for future treatment of gonorrhoea. Additional studies examining mechanism(s) of action, resistance development, in vivo anti-gonococcal activity, and pharmacokinetics/pharmacodynamics for gonococcal infections of auranofin and several other significant screening hits would be valuable., Funding Agencies:SystemsX.ch (The Swiss Initiative for Systems Biology) Örebro County Council Research Committee Foundation for Medical Research at Örebro University Hospital, Sweden County Council of Norrbotten NLL-393301 NLL-393021Umeå University County Council of Västerbotten (ALF Universitets-ST)

Complex regulatory circuits determine whether DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng et al [1] report that a novel p53-suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR-mediated DSB repair and proliferation of p53-deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.

Complex regulatory circuits determine whether DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng et al [1] report that a novel p53-suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR-mediated DSB repair and proliferation of p53-deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term "magic pools." Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes. IMPORTANCE Molecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a "magic pool." The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA "parts," we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.

In this paper, we consider the problem of learning the genetic interaction map, i.e., the topology of a directed acyclic graph (DAG) of genetic interactions from noisy double-knockout (DK) data. Based on a set of well-established biological interaction models, we detect and classify the interactions between genes. We propose a novel linear integer optimization program called the Genetic-Interactions-Detector (GENIE) to identify the complex biological dependencies among genes and to compute the DAG topology that matches the DK measurements best. Furthermore, we extend the GENIE program by incorporating genetic interaction profile (GI-profile) data to further enhance the detection performance. In addition, we propose a sequential scalability technique for large sets of genes under study, in order to provide statistically significant results for real measurement data. Finally, we show via numeric simulations that the GENIE program and the GI-profile data extended GENIE (GI-GENIE) program clearly outperform the conventional techniques and present real data results for our proposed sequential scalability technique.

In this paper, we consider the problem of learning the genetic interaction map, i.e., the topology of a directed acyclic graph (DAG) of genetic interactions from noisy double-knockout (DK) data. Based on a set of well-established biological interaction models, we detect and classify the interactions between genes. We propose a novel linear integer optimization program called the Genetic-Interactions-Detector (GENIE) to identify the complex biological dependencies among genes and to compute the DAG topology that matches the DK measurements best. Furthermore, we extend the GENIE program by incorporating genetic interaction profile (GI-profile) data to further enhance the detection performance. In addition, we propose a sequential scalability technique for large sets of genes under study, in order to provide statistically significant results for real measurement data. Finally, we show via numeric simulations that the GENIE program and the GI-profile data extended GENIE (GI-GENIE) program clearly outperform the conventional techniques and present real data results for our proposed sequential scalability technique.

In this paper, we consider the problem of learning the genetic interaction map, i.e., the topology of a directed acyclic graph (DAG) of genetic interactions from noisy double-knockout (DK) data. Based on a set of well-established biological interaction models, we detect and classify the interactions between genes. We propose a novel linear integer optimization program called the Genetic-Interactions-Detector (GENIE) to identify the complex biological dependencies among genes and to compute the DAG topology that matches the DK measurements best. Furthermore, we extend the GENIE program by incorporating genetic interaction profile (GI-profile) data to further enhance the detection performance. In addition, we propose a sequential scalability technique for large sets of genes under study, in order to provide statistically significant results for real measurement data. Finally, we show via numeric simulations that the GENIE program and the GI-profile data extended GENIE (GI-GENIE) program clearly outperform the conventional techniques and present real data results for our proposed sequential scalability technique.

In this paper, we consider the problem of learning the genetic interaction map, i.e., the topology of a directed acyclic graph (DAG) of genetic interactions from noisy double-knockout (DK) data. Based on a set of well-established biological interaction models, we detect and classify the interactions between genes. We propose a novel linear integer optimization program called the Genetic-Interactions-Detector (GENIE) to identify the complex biological dependencies among genes and to compute the DAG topology that matches the DK measurements best. Furthermore, we extend the GENIE program by incorporating genetic interaction profile (GI-profile) data to further enhance the detection performance. In addition, we propose a sequential scalability technique for large sets of genes under study, in order to provide statistically significant results for real measurement data. Finally, we show via numeric simulations that the GENIE program and the GI-profile data extended GENIE (GI-GENIE) program clearly outperform the conventional techniques and present real data results for our proposed sequential scalability technique.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division.