DOI: 10.4121/21680384.v2
title: Transcriptomic analysis of Streptococcus suis Small Colony variants (SCVs) immune to Cas9-mediated genome editing

description: <strong>Description:</strong>The formation of small colony variants is a phenomenon that has been described in numerous diverse bacterial species. In many cases SCVs are formed by pathogenic bacteria and are often recovered from clinical specimens. Defining characteristics of SCVs are the slow growth and low metabolic rate, resulting in the smaller colony phenotype. Depending on the species, the SCV phenotype can be transient (phase-variable) or non-reversible. Previous studies have found a link between the SCV phenotype and increased resistance to various classes of antibiotics and other external stress factors such as oxidative stress and low pH as well as an increased mutation rate. It has been suggested that SCVs could lead to “persister” cell formation, resulting in recurrent infections. Here we show that S. suis can form SCVs upon transformation with pSStarget, a plasmid expressing a CRISPR/Cas9 system that targets the chromosome. The SCV phenotype was reversible, as growth under non-selective conditions resulted in a normal growth phenotype. We compared the transcriptome of the SCVs and a strain transformed with a non-targeting version of the pSStarget plasmid to unravel the molecular mechanisms underlying SCV formation. In line with previous reports we found characteristic alterations of the transcriptome in the SCVs, such as reduced expression of genes involved in fatty acid biosynthesis and TCA as well as upregulation of chromosomal toxin-antitoxin systems. Furthermore, all plasmid-encoded genes were strongly downregulated in the SCVs, suggesting a possible reduction of plasmid copy number.<strong>Explanation of variables:</strong>All files contain Illumina reads in the FASTQ (.fq) format in zipped (.gz) format.SG1, SG2 and SG3 refer to the three biological replicates of SCVs recovered after transformation with pSStarget-sg6 targeting the capsular polysaccharide (<em>cps</em>) locus.SG4, SG5 and SG6 refer to the three biological replicates of normally sized colonies that were transformed with pSStarget-NT, a non-targeting version of the CRISPR plasmid which does not contain a sgRNA matching any sequence in the S. suis genome.The second number of the sample name, '_1' and '_2' refers to each member of a paired-end read pair.<br><strong>Materials and methods:</strong>Bacterial strains, plasmids and culture conditions Liquid cultures of <em>S. suis</em> were grown in Todd-Hewitt broth (Oxoid) supplemented with 0.2% Bacto™ yeast extract (BD Biosciences) (THY) at 37 °C with 5% CO2 without agitation. When required, 5 µg/ml chloramphenicol (Cml) (Sigma-Aldrich) was added to the culture media. <em>S. suis</em> strain P1/7 was transformed with plasmid pSStarget-sg6 (targeting the <em>cps</em> locus) or pSStarget-NT (control plasmid lacking a sgRNA with matching genomic target) as previously described, utilizing the natural competence-inducing peptide<sup>1</sup>. Briefly, <em>S. suis </em>overnight cultures were diluted 40-fold and grown to an OD600 of approximately 0.04-0.05. Aliquots of 100µl bacterial culture were transferred to sterile microcentrifuge tube and 1-10µg of transforming DNA were added to each tube. The competence-inducing peptide (Genscript) was added to a final concentration of 250µM and the cultures were incubated for 2h at 37°C with 5% CO2 before plating 100µl transformation mix on selective agar plates. Appropriate dilutions were plated to allow selecting single coloniesRNA extraction and quality controlBacterial colonies were collected from the transformation plate using a sterile loop and resuspended in 1ml of THY containing Cml to a density (OD600) of 1. In the case of the SCV condition, special care was taken to ensure that only the small colony variants were collected without disturbing the normal-sized colonies. The bacterial suspensions were used to inoculate 6ml THY + Cml in triplicate at a starting OD600 of 0.06. The cultures were grown for 2.5 hours at 37°C with 5% CO2 and the bacteria were collected by centrifugation. The pellet was resuspended in QIAzol lysis reagent (Qiagen), transferred to a tube containing 0.1mm silica beads (Lysing Matrix B, MP Biomedicals) and lysed by bead beating for 40 seconds at 4.0 m/s using a FastPrep-24 5G (MP Biomedicals). The lysates were cleared by centrifugation at 16.000x for 10 minutes. RNA isolation from the supernatants has been performed using the miRNeasy mini kit (Qiagen) according to manufacturer’s instructions. The quantity and quality of the purified RNA was determined using the Qubit 4 Fluorometer (ThermoFisher) and DS-11 UV-Vis spectrophotometer (DeNovix). The RNA integrity was quantified using the 2200 TapeStation electrophoresis system (Agilent). The Illumina library preparation and sequencing using the Hiseq Illumina PE150 platform has been performed by Novogene Bioinformatics Technology Co., Ltd., in Hong Kong. Library preparation and sequencingStrand-specific libraries were prepared by Novogene as follows: After total RNA sample QC, Ribo-zero kits were used to remove rRNA, then fragmentation buffer was added to fragment the mRNAs. Using random hexamers as primers, RNA fragments were reverse-transcribed to single stranded cDNAs. After synthesis of single stranded cDNAs, a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. The double stranded cDNAs were purified using AMPure XP beads, end-repaired, polyadenylated, ligated with adapter sequences and size-selected using AMPure XP beads. Then the uracil containing strands were degraded by USEREnzyme, and the remaining strands were amplified using PCR and purified using AMPure XP beads. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/μl before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR.Sequencing was carried out by Novogene using the Hiseq Illumina PE150 platform to generate short paired-end reads. The original image data from Illumina are transformed to raw data (sequenced reads) by CASAVA base calling analysis. The raw data was delivered in FASTQ format, containing sequence information and corresponding sequencing quality information. Error rates were 0.03%, and sequencing resulted in 1.4-1.7 Gb of clean bases per sample; detailed QC data per sample are available upon request.<strong>Reference:</strong>(1) Zaccaria, E.; van Baarlen, P.; de Greeff, A.; Morrison, D. A.; Smith, H.; Wells, J. M. Control of Competence for DNA Transformation in Streptococcus Suis by Genetically Transferable Pherotypes. PLoS One 2014, 9 (6), e99394. https://doi.org/10.1371/journal.pone.0099394.<strong>License</strong>This dataset is published under the CC BY-SA (Attribution ShareAlike) license.