High-throughput CRISPRi phenotyping in Streptococcus pneumoniae identifies new essential genes involved in cell wall synthesis and competence development

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 SPD1416 and SPD1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD1198 and SPD1197 (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 Clpproteolytic 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.


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While several previous studies have identified many pneumococcal genes that are likely to 111 be essential, the precise contribution to pneumococcal biology has remained to be defined 112 for most of these genes. Here, we aim to characterize the functions of these proteins in the 113 commonly used S. pneumoniae serotype 2 strain D39 by the CRISPRi approach. Therefore,

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To develop the CRISPR interference system, we first engineered the commonly used LacI-123 based isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible system for S. pneumoniae 124 (see Methods). dcas9 was placed under control of this new IPTG-inducible promoter, named 125 PL, and was integrated into the chromosome via double crossover (Fig. 1A-B). To confirm 126 the reliability of the CRISPRi system, we tested it in a reporter strain expressing firefly 127 luciferase (luc), in which a sgRNA targeting luc was placed under the constitutive P3 6 promoter (Sorg et al, 2015) and integrated at a non-essential locus (Fig. 1B). To obtain high 129 efficiency of transcriptional repression, we used the optimized sgRNA sequence as reported 130 previously (Chen et al, 2013) (Fig. S1A).

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Induction of dCas9 with 1 mM IPTG resulted in quick reduction of luciferase activity; 132 approximately 30-fold repression of luciferase expression was obtained within 2 hr without 133 substantial impact on bacterial growth (Fig. 1C). Furthermore, the level of repression was 134 tunable by using different concentrations of IPTG (Fig. 1C). By comparing strains with or 135 without sgRNAluc, we found that repression in our CRISPRi system is stringently dependent 136 on expression of both dCas9 and the sgRNA (Fig. S1B)

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~600 fold upon addition of 1 mM IPTG after 2.5 hr. Upon dCas9 induction, the luc gene was 142 significantly repressed (~ 84 fold) (Fig. 1D). Our RNA-seq data showed that the genes 143 (spd0424, spd0425, lacE-1, lacG-1, lacF-1) that are downstream of luc, which was driven by 144 a strong constitutive promoter without terminator, were significantly repressed as well, 145 confirming the reported polar effect of CRISPRi . Interestingly, by comparing 146 the transcriptome of strain with (XL28) or without (XL29) sgRNAluc cultivated in medium with 147 1 mM IPTG, we found that galT-2, galK and galR were specifically induced by IPTG, and 148 fluctuation of the competence system was also observed which is known to be highly noisy  Table 2). Taken together, the 150 IPTG-inducible CRISPRi system is highly specific.

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Construction and growth analysis of the CRISPRi library

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We next used the CRISPRi system to construct an expression knock-down library of 154 pneumococcal essential genes. A sgRNA to each of the 391 potentially essential genes was 155 designed as described previously   (Supplementary Table 3). Note that 7 CRISPRi also inhibits expression of co-transcribed downstream genes (Fig. 1D) (Qi et al, 157 2013). Thus, some of the genes may be targeted multiple times (in case of more than one 158 essential gene within the operon). We used an infusion cloning technique to construct the 159 library as this was more efficient than the previously reported PCR-based sgRNA 160 construction method (see SI text) (Figs 2A and 2B). All sgRNA strains were sequence 161 verified, and we considered them genetically functional when the sgRNA did not contain 162 more than 1 mismatch to the designed sgRNA and no mismatches in the first 14-nt prior to 163 the PAM. Using this approach, after a single round of cloning and sequencing, we

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Using the same approach, we could also functionally verify several genes that were 197 studied before but had not been annotated in the D39 genome. These included pcsB 198 (spd2043), vicR (spd1085), divIC (spd0008) and rafX (spd1672). Transcriptional repression 199 led to significant growth defects and cellular changes in shape and size (SI text and       12 seemed to match the molecular weight of the RU, suggesting that SPD1197 and SPD1198 297 play a role in TA precursor polymerization. Although repression of the genes associated with 298 peptidoglycan synthesis (murT, gatD and pbp2x) made the 4 main TA bands weaker, the 299 pattern of the TA bands was not changed. Likely, the reduction of the TA of these three 300 strains is due to the reduction of peptidoglycan, which constitutes the anchor for wall TA.

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The TA chains of S. pneumoniae are thought to be polymerized before they are was enhanced, while depleting any of the other ATPase subunits (ClpC, ClpE and ClpL) had 329 no effect on competence (Fig. S15). This shows that ClpX is the main ATPase subunit 330 responsible for ClpP-dependent repression of competence.

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Here, we developed an IPTG-inducible CRISPRi system to study essential genes in S. 334 pneumoniae (Fig. 1). In addition, we adopted a simple and efficient one-step sgRNA cloning 335 strategy using infusion cloning, which results in ~89% positive sgRNA clones (

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This work and other studies highlight that high-throughput phenotyping by CRISPRi is 353 a powerful approach for hypothesis-forming and functional characterization of essential 354 genes (Peters et al, 2016). We also show that CRISPRi can be used to unravel gene-355 regulatory networks in which essential genes play a part (Fig. 6). While we shed light on the 356 function of just several previously uncharacterized essential genes, the here-described 357 library contains richer information that needs to be further explored. In addition, CRISPRi                    Tables 876 Table 1. Cellular pathways selected for CRISPRi phenotyping 1 .

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1 The genes highlighted in bold were included in Fig. 3.