A group B Streptococcus indexed transposon mutant library to accelerate genetic research on an important perinatal pathogen

Abstract

ABSTRACT Group B Streptococcus (GBS) is a major contributor to sepsis, meningitis, and pneumonia in newborns. Indexed bacterial mutant libraries accelerate pathogenesis research by allowing rapid screening of genes that contribute to disease. In this study, we created and characterized a large-scale GBS indexed library of Himar1 mini-transposon mutant strains grown as monocultures from a mixed transposon insertion library that we had previously used for transposon-genome junction sequencing. We used a high-throughput workflow to identify transposon insertion sites in chromosomal DNA purified from individual mutants. Following quality control steps and the removal of isogenic duplicate strains, we isolated 1,919 monocultures of unique transposon insertion mutants with even dispersion across the GBS genome. Our final library, stored in barcoded, traceable glycerol stocks, contains interruptions of 878 genes and 253 intergenic regions. We also validated select library mutants with confirmatory PCR and, when possible, specific phenotypic testing. While the library contains only sparse interruptions of essential or near-essential genes, the genes represented in the set span a wide range of predicted functional categories, including roles in metabolism, structure, and virulence. In conclusion, we developed and applied a high-throughput molecular analysis and bioinformatic pipeline to generate a GBS indexed library of unprecedented scale that we believe will be a useful genetic tool for fellow GBS researchers. IMPORTANCE Group B Streptococcus (GBS) is a significant global cause of serious infections, most of which affect pregnant women, newborns, and infants. Studying GBS genetic mutant strains is a valuable approach for learning more about how these infections are caused and is a key step toward developing more effective preventative and treatment strategies. In this resource report, we describe a newly created library of defined GBS genetic mutants, containing over 1,900 genetic variants, each with a unique disruption to its chromosome. An indexed library of this scale is unprecedented in the GBS field; it includes strains with mutations in hundreds of genes whose potential functions in human disease remain unknown. We have made this resource freely available to the broader research community through deposition in a publicly funded bacterial maintenance and distribution repository.

Keywords

• Streptococcus agalactiae
• transposon
• mutagenesis
• indexed library