Chemical genomics informs antibiotic and essential gene function in .

The Gram-negative pathogen, , poses a serious threat to human health due to its role in nosocomial infections that are resistant to treatment with current antibiotics. Despite this, our understanding of fundamental biology remains limited, as many essential genes have not been experimentally characterized. These essential genes are critical for bacterial survival and, thus, represent promising targets for drug discovery.

Modular, inducible, and titratable expression systems for and .

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. In this study, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium K-12 as well as strains of the antibiotic-resistant pathogen, .

Modular, inducible, and titratable expression systems for and .

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. Here, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium K-12 as well as strains of the antibiotic-resistant pathogen, We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design.

Essential gene knockdowns reveal genetic vulnerabilities and antibiotic sensitivities in .

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, , is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities.

Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in .

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, , is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities.

Programmable Gene Knockdown in Diverse Bacteria Using Mobile-CRISPRi.

Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene-knockdown tool that uses an RNA-protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes.

Rapid de novo evolution of lysis genes in single-stranded RNA phages.

Leviviruses are bacteriophages with small single-stranded RNA genomes consisting of 3-4 genes, one of which (sgl) encodes a protein that induces the host to undergo autolysis and liberate progeny virions. Recent meta-transcriptomic studies have uncovered thousands of leviviral genomes, but most of these lack an annotated sgl, mainly due to the small size, lack of sequence similarity, and embedded nature of these genes. Here, we identify sgl genes in 244 leviviral genomes and functionally characterize them in Escherichia coli.

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ.

The L protein of the single-stranded RNA phage MS2 causes lysis of without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (nsensitivity to ysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L.