High‐throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae. Issue 5 (10th May 2017)

• Record Type: Journal Article
• Authors: Liu, Xue; Gallay, Clement; Kjos, Morten; Domenech, Arnau; Slager, Jelle; van Kessel, Sebastiaan P; Knoops, Kèvin; Sorg, Robin A; Zhang, Jing‐Ren; Veening, Jan‐Willem
• Is Part Of: Molecular systems biology. Volume 13:Issue 5(2017:May)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2017-05-10
• Subjects: bacterial cell wall -- competence -- DNA replication -- gene essentiality -- teichoic acid biosynthesis; Molecular biology -- Periodicals; Systems biology -- Periodicals; 572.8
• DOI: 10.15252/msb.20167449
• Languages: English
• ISSNs: 1744-4292

Abstract

Abstract: Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae . However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets. Synopsis: A CRISPRi knockdown library targeting 348 potentially essential genes in Streptococcus pneumoniae strain D39, in combination with high‐throughput phenotyping identifies new essential genes involved in cell wall synthesis and in competence regulation. A CRISPRi knockdown library was constructed targeting 348 potentially essential genes in Streptococcus pneumoniae strain D39, as determined by Tn‐seq. 254 out of 348 targeted genes showed growth phenotypes, providing a useful platform for the functional identification of hypothetical genes. High‐content microscopy allowed linking genotypes with phenotypes and identified TarP and TarQ as being involved in polymerization of teichoic acid precursors. The essential ATPase ClpX, together with ClpP was shown to regulate competence development. Abstract : A CRISPRi knockdown library targeting 348 potentially essential genes in Streptococcus pneumoniae strain D39, in combination with high‐throughput phenotyping identifies new essential genes involved in cell wall synthesis and in competence regulation.