High-throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae

Mol Syst Biol. 2017 May 10;13(5):931. doi: 10.15252/msb.20167449.


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.

Keywords: DNA replication; bacterial cell wall; competence; gene essentiality; teichoic acid biosynthesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / genetics
  • Cell Cycle Proteins / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endopeptidase Clp / genetics
  • Gene Expression Regulation, Bacterial*
  • Gene Library
  • Gene Regulatory Networks
  • Genes, Bacterial
  • Genes, Essential*
  • High-Throughput Nucleotide Sequencing / methods*
  • Peptidoglycan / biosynthesis
  • Peptidoglycan / genetics
  • Streptococcus pneumoniae / genetics*
  • Teichoic Acids / biosynthesis
  • Teichoic Acids / genetics


  • Bacterial Proteins
  • Cell Cycle Proteins
  • PcsB protein, Streptococcus pneumoniae
  • Peptidoglycan
  • Teichoic Acids
  • YycF protein, Bacteria
  • ClpP protein, Streptococcus pneumoniae
  • Endopeptidase Clp