Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae

Abstract

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

“…Finally, transformation efficiencies were quantified by flow-cytometry ( Figure 4A). In line with studies using classical plating methods to assess transformation efficiencies (Keller et al, 2019;Lee et al, 1998), higher transformation frequencies were observed at higher donor DNA concentrations and with longer homology regions ( Figure 4B). Interestingly, the frequency of transformation plateaued at ~50% regardless of the concentration of donor DNA and sequence homology length ( Figure 4B).…”

“…In order to overcome these concerns and analyze successful recombination events during transformation at the single-cell level, we developed a fluorescence-based reporter system inspired by a system previously used to observe natural transformation in S. pneumoniae ( Bergé et al, 2013 ) and other bacterial species ( Boonstra et al, 2018 ; Corbinais et al, 2016 ; Godeux et al, 2018 ). To do so, we utilized a fluorescent donor strain in which the gene encoding the abundant histone-like protein HlpA (aka HU) was fused in frame with the gene encoding the red fluorescent protein mScarlet-I integrated at the native hlpA locus at 169° on the circular chromosome ( Keller et al, 2019 ) (strain VL1780) ( Figure 3A ). A recipient, non-fluorescent strain was constructed (strain VL1784) in which hlpA was separated from mScarlet-I by a stop codon mutation (G > T) ( Figure 3A–C and Figure 3—figure supplement 2A , hlpA-stop-mScarlet-I ).…”

“…To construct transcriptional fusion of ssbB and mScarlet-I, mScarlet-I was amplified by PCR with OVL1415/OVL1418 from genomic DNA of VL1787 (cep:spv_1159-mScarlet-I, spcR) (Keller et al, 2019). Upper and downer fragments were amplified with OVL166/OVL1168 and OVL1199/OVL167 using genomic DNA of VL599 (ssbB::ssbB_luc, kanR) as template, respectively.…”

Phosphorylcholine esterase is critical for Dolichos biflorus and Helix pomatia agglutinin binding to pneumococcal teichoic acid

“…Pneumococci are emerging as a preferred model organism for these surface structure studies because of its acknowledged clinical importance as a human pathogen and because of its inherent genetic malleability. For instance, the expression of pce can be regulated by inducible promoters [24,36] and DBA can be used to identify the subcellular location where Pce functions. Thus, these lectins will help us study the synthesis and attachment of WTA/LTA to PG.…”

Exploration of bacterial bottlenecks andStreptococcus pneumoniaepathogenesis by CRISPRi-seq

“…The E. coli library could also be used as a reservoir of sgRNAs for construction of reproducible CRISPRi 190 libraries among S. pneumoniae D39V strains with different genetic backgrounds (e.g. mutant strains) or to transform 191 other pneumococcal strains that contain the pPEPZ integration region, which is harbored by 64.21% of all sequenced 192 pneumococcal strains (Keller et al, 2019). The plasmid pool was then transformed into S. pneumoniae D39V with the 193 above described tet-inducible dcas9 ( Figure 2E).…”