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.
Highlights
Utilising generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality
The probability of either an A or a T occuring at any position between the insertion site and 20 bp downstream, was between 54% to 70% per bp highlighting a modest preference of ISS1 for AT rich regions, which is in agreement with the overall AT content of the S. equi genome (58.7%) [2]
Our analysis revealed that the essential/critical/ambiguous genes of S. equi, S. pyogenes and S. agalactiae were attributed to 45, 41 and 41 Kyoto encyclopaedia of genes and genomes (KEGG) categories, respectively, 39 of which were shared between the three species (Fig. 4a) (Additional file 6)
Summary
Utilising generation sequencing to interrogate saturated bacterial mutant libraries provides unprecedented information for the assignment of genome-wide gene essentiality. We present a barcoded transposon directed insertion-site sequencing (TraDIS) system to define an essential gene list for Streptococcus equi subsp. The gene essentiality data for this group C Streptococcus was compared to that of group A and B streptococci. The increased accessibility of next-generation sequencing (NGS) technologies has facilitated the development of a variety of transposon-genome junction sequencing techniques, which combine dense mutant libraries and sequencing to identify essential bacterial genomes and assign gene function. NGS of transposon-genome junctions in saturated transposon mutant libraries permits the simultaneous identification of potentially hundreds of thousands of unique insertion sites, providing data pertaining to gene essentiality at the most basic level.
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