SUMMARY A systems level understanding of Gram-positive bacteria is important from both an environmental and health perspective, and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3968 and 3970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.

The cell envelope of Gram-negative bacteria is a formidable barrier that is difficult for antimicrobial drugs to penetrate. Thus, the list of treatments effective against these organisms is small and with the rise of new resistance mechanisms is shrinking rapidly. New therapies to treat Gram-negative bacterial infections are therefore sorely needed. This goal will be greatly aided by a detailed mechanistic understanding of envelope assembly. Although excellent progress in the identification of essential envelope biogenesis systems has been made in recent years, many aspects of the process remain to be elucidated. We therefore developed a simple, quantitative, and high-throughput assay for mutants with envelope biogenesis defects and used it to screen an ordered single-gene deletion library of Escherichia coli. The screen was robust and correctly identified numerous mutants known to be involved in envelope assembly. Importantly, the screen also implicated 102 genes of unknown function as encoding factors that likely impact envelope biogenesis. As a proof of principle, one of these factors, ElyC (YcbC), was characterized further and shown to play a critical role in the metabolism of the essential lipid carrier used for the biogenesis of cell wall and other bacterial surface polysaccharides. Further analysis of the function of ElyC and other hits identified in our screen is likely to uncover a wealth of new information about the biogenesis of the Gram-negative envelope and the vulnerabilities in the system suitable for drug targeting. Moreover, the screening assay described here should be readily adaptable to other organisms to study the biogenesis of different envelope architectures.

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.

Advances in our ability to systematically introduce and track controlled genetic variance in microbes have fueled high-throughput reverse genetics approaches in the past decade. When coupled to quantitative readouts, such approaches are extremely powerful at elucidating gene function and providing insights into the underlying pathways and the overall cellular network organization. Yet, until now all efforts for quantifying microbial macroscopic phenotypes have been restricted to monitoring growth in a small number of model microbes. We developed an image analysis software named Iris, which allows for systematic exploration of a number of orthogonal-to-growth processes, including biofilm formation, colony morphogenesis, envelope biogenesis, sporulation and reporter activity. In addition, Iris provides more sensitive growth measurements than current available software, and is compatible with a variety of different microbes, as well as with endpoint or kinetic data. We used Iris to reanalyze existing chemical genomics data in Escherichia coli and to perform proof-of-principle screens on colony biofilm formation and morphogenesis of different bacterial species and the pathogenic fungus, Candida albicans. Thereby we recapitulated existing knowledge but also identified a plethora of additional genes and pathways involved in both processes.

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Retrons are genetic retroelements, commonly found in bacterial genomes and recently repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has remained elusive. We establish that the retron-Sen2 in Salmonella Typhimurium encodes a toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, and the msDNA enabling the antitoxin activity. Using another E. coli retron, we establish that this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate.Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems..

Chemical-genetic approaches are based on measuring the cellular outcome of combining genetic and chemical perturbations in large-numbers in tandem. In these approaches the contribution of every gene to the fitness of an organism is measured upon exposure to different chemicals. Current technological advances enable the application of chemical genetics to almost any organism and at an unprecedented throughput. Here we review the underlying concepts behind chemical genetics, present its different vignettes and illustrate how such approaches can propel drug discovery.

Fighting antibiotic resistance requires a deeper understanding of the genetic factors that determine the antibiotic susceptibility of bacteria. Here we describe a chemical-genomic screen in Escherichia coli K-12 that was designed to discover new aspects of antibiotic resistance by focusing on a set of 26 antibiotics and other stresses with poorly characterized mode-of-action and determinants of resistance. We show that the screen identifies new resistance determinants for these antibiotics including a common signature from two antimicrobials, kasugamycin and blasticidin S, used to treat crop diseases like rice blast and fire blight. Following this signature, we further investigated the mechanistic basis for susceptibility to kasugamycin and blasticidin S in E. coli using both genetic and biochemical approaches. We provide evidence that these compounds hijack an overlapping set of peptide ABC-importers to enter the bacterial cell. Loss of uptake may be an underappreciated mechanism for the development of kasugamycin resistance in bacterial plant pathogens.