Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Microbial Primer: Transposon directed insertion site sequencing (TraDIS): A high throughput method for linking genotype to phenotype.

Genetic screens are a key tool for linking phenotype and genotype. Transposon mutagenesis was one of the first genetic methodologies to associate genetic loci with phenotypes. The advent of next-generation sequencing transformed the use of this technique allowing rapid interrogation of whole genomes for genes that correlate with phenotype. One method is transposon directed insertion-site sequencing (TraDIS). Here we describe the method, recent developments in technology, and the advantages and disadvantages of this method compared to other genetic screening tools.

Recognize and Alleviate a Resource Management Conundrum Facing Science Diaspora Networks.

Increasingly, science diaspora networks are managed by formal organizations such as embassies or non-profit organizations. Researchers have studied these networks to understand how they influence international collaborations and science diplomacy, and to determine which network activities foster those outcomes and which do not. In this perspective, we suggest that many of these network organizations confront an underappreciated conundrum for managing resources: organizations with few resources must learn how to obtain more resources despite lacking means to do so. To substantiate our suggestion, we do the following. We review exploratory results from a study of network organizations that indicate that these organizations generally lack resources, learn too little from each other, and struggle to overcome the resource conundrum. We also show that this conundrum is expected from organizational theory based on bounded rationality. To help organizations confront the issue, we do the following. First we provide a new database of operating science diaspora networks. We encourage managers of network organizations to use it as a resource to identify peers with whom to regularly exchange knowledge about securing resources. We also suggest that other scientific organizations should infuse network organizations with fresh resources. Ultimately, we urge all relevant stakeholders to recognize that the conundrum results not from the shortcomings of individual managers, but rather is a legitimate organizational phenomena that must be addressed by organizational design.

LI-Detector: a Method for Curating Ordered Gene-Replacement Libraries.

In recent years the availability of genome sequence information has grown logarithmically resulting in the identification of a plethora of uncharacterized genes. To address this gap in functional annotation, many high-throughput screens have been devised to uncover novel gene functions. Gene-replacement libraries are one such tool that can be screened in a high-throughput way to link genotype and phenotype and are key community resources. However, for a phenotype to be attributed to a specific gene, there needs to be confidence in the genotype. Construction of large libraries can be laborious and occasionally errors will arise. Here, we present a rapid and accurate method for the validation of any ordered library where a gene has been replaced or disrupted by a uniform linear insertion (LI). We applied our method (LI-detector) to the well-known Keio library of Escherichia coli gene-deletion mutants. Our method identified 3,718 constructed mutants out of a total of 3,728 confirmed isolates, with a success rate of 99.7% for identifying the correct kanamycin cassette position. This data set provides a benchmark for the purity of the Keio mutants and a screening method for mapping the position of any linear insertion, such as an antibiotic resistance cassette in any ordered library. IMPORTANCE The construction of ordered gene replacement libraries requires significant investment of time and resources to create a valuable community resource. During construction, technical errors may result in a limited number of incorrect mutants being made. Such mutants may confound the output of subsequent experiments. Here, using the remarkable E. coli Keio knockout library, we describe a method to rapidly validate the construction of every mutant.