RESUMO
SUMMARYFunctional genomics is the use of systematic gene perturbation approaches to determine the contributions of genes under conditions of interest. Although functional genomic strategies have been used in bacteria for decades, recent studies have taken advantage of CRISPR (clustered regularly interspaced short palindromic repeats) technologies, such as CRISPRi (CRISPR interference), that are capable of precisely modulating expression of all genes in the genome. Here, we discuss and review the use of CRISPRi and related technologies for bacterial functional genomics. We discuss the strengths and weaknesses of CRISPRi as well as design considerations for CRISPRi genetic screens. We also review examples of how CRISPRi screens have defined relevant genetic targets for medical and industrial applications. Finally, we outline a few of the many possible directions that could be pursued using CRISPR-based functional genomics in bacteria. Our view is that the most exciting screens and discoveries are yet to come.
RESUMO
Small Multidrug Resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the Proton Motive Force (PMF), reducing viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from P. aeruginosa, transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens.
