Defining the ABC of gene essentiality in streptococci

Abstract

BackgroundUtilising next generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. Exposure of saturated mutant libraries to specific conditions and subsequent sequencing can be exploited to uncover gene essentiality relevant to the condition. Here we present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. equi, the causative agent of strangles in horses, for the first time. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci.ResultsSix barcoded variants of pGh9:ISS1 were designed and used to generate mutant libraries containing between 33,000-66,000 unique mutants. TraDIS was performed on DNA extracted from each library and data were analysed separately and as a combined master pool. Gene essentiality determined that 19.5% of the S. equi genome was essential. Gene essentialities were compared to those of group A and group B streptococci, identifying concordances of 90.2% and 89.4%, respectively and an overall concordance of 83.7% between the three species.ConclusionsThe use of barcoded pGh9:ISS1 to generate mutant libraries provides a highly useful tool for the assignment of gene function in S. equi and other streptococci. The shared essential gene set of group A, B and C streptococci provides further evidence of the close genetic relationships between these important pathogenic bacteria. Therefore, the ABC of gene essentiality reported here provides a solid foundation towards reporting the functional genome of streptococci.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3794-3) contains supplementary material, which is available to authorized users.

Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

“…Deletion of rmlB and rmlC in S. mutans resulted in a phenotype similar as previously observed for an rmlD deletion mutant (van der Beek et al, 2015), underscoring that inhibition of the dTDP-rhamnose biosynthesis pathway severely impacts bacterial viability. Indeed, the rmlABCD genes are essential for S. mutans in the competitive environment of a mutant transposon library (Shields et al, 2018), similar to GAS and GCS (Le Breton et al, 2013;Charbonneau et al, 2017). We are however able to construct rml deletion mutants in S. mutans in isolation in contrast to similar attempts in GAS (van Sorge et al, 2014).…”

Gene fitness landscape of group A streptococcus during necrotizing myositis

“…These two strains were chosen for transposon mutagenesis because (i) strain MGAS2221 is genetically representative of a pandemic serotype M1 clone that arose in the 1980s, rapidly spread worldwide and currently is the most prevalent cause of severe infections globally (18, 20, 47), (ii) strain MGAS27961 is genetically representative of a virulent serotype M28 clone that is prevalent in the United States and elsewhere (48), (iii) both strains have wild-type alleles of all major transcriptional regulators that are known to affect GAS virulence, such as covR and covS , ropB , mga , and rocA , and (iv) both strains have been used previously in animal infection studies (18, 20, 33). Using transposon plasmid pGh9:IS S1 (49), we generated dense transposon mutant libraries in strains MGAS2221 and MGAS27961 containing 154,666 (an insertion every 12 bp on average) and 330,477 (an insertion every 5.5 bp on average) unique transposon insertions, respectively (Figure 1A). This means that on average, the serotype M1 and M28 libraries had 66 and 139 insertions per open reading frame.…”

“…The mutant library generated in serotype M1 strain MGAS2221 using transposon plasmid pGh9:IS S1 was recently described (31, 49). The serotype M28 strain MGAS27961 transposon mutant library was made by essentially identical methods.…”

“…The mutant library genomic DNA preparation and DNA sequencing were performed according to procedures described previously for TraDIS analysis (31, 49). The PCR-amplified libraries were sequenced with a NextSeq550 instrument (Illumina) using a single-end 75-cycle protocol.…”

Streptococcal dTDP-L-rhamnose biosynthesis enzymes: functional characterization and lead compound identification

“…Deletion of rmlB and rmlC in S. mutans resulted in a phenotype similar as previously observed for an rmlD deletion mutant (van der Beek et al , 2015), underscoring that inhibition of the dTDP-rhamnose biosynthesis pathway severely impacts bacterial viability. Indeed, the rmlABCD genes are essential for S. mutans in the competitive environment of a mutant transposon library (Shields et al , 2018), similar to GAS and GCS (Charbonneau et al , 2017, Le Breton et al , 2017, Le Breton et al , 2015). We are however able to construct rml deletion mutants in S. mutans in isolation in contrast to similar attempts in GAS (van Sorge et al , 2014).…”