Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes

Abstract

Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.

Coordinated Regulation of the EII Man and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways

“…Mutagenesis of a putative F-6-P kinase in UA159 (fruP; SMU.113) showed no impact on growth on any sugars tested (data not shown). In contrast, repeated attempts to delete a third, apparent 6-phosphofructokinase (SMU.1191) in UA159 have failed, suggesting that the SMU.1191 gene encodes the dominant F-6-P kinase that provides substrates for the EMP in S. mutans, as has been reported for related bacteria (37,38). Therefore, the significant growth defects observed with the fruK mutants ( Fig.…”

PIMMS (Pragmatic Insertional Mutation Mapping System) Laboratory Methodology a Readily Accessible Tool for Identification of Essential Genes in Streptococcus

“…The movement toward next generation high-throughput transposon insertion-site sequencing in Streptococci has been shown in studies on Streptococcus pneumoniae (van Opijnen et al, 2009), Streptococcus pyogenes (Le Breton et al, 2015), and Streptococcus agalactiae (Hooven et al, 2016) and these provide a benchmark for the PIMMS protocol. In all cases the Tn-seq protocol was used for detection of the insertion junction sequences.…”

“…These data further support the assertion that pGh9:ISS1 has neither a transposition bias to a specific insertion motif (Green et al, 2012) nor holds an insertional preference to specific structural features of DNA (Lampe et al, 1998); either of which can lead to pseudo-random insertions, limiting the range and variability of mutations that can be created within a population. In the studies in S. pyogenes (Le Breton et al, 2015) and S. agalactiae (Hooven et al, 2016) the mutation/mutant ratios were not reported.…”

“…S. pyogenes , also contains multiple sequences encoding both activities capable of interconversion of glycreate-2P and glycerate-3P (Pancholi and Caparon, 2016). An investigation of two strains of S. pyogenes (Le Breton et al, 2015) identified three orthologs of gpm. In one strain of S. pyogenes, gpmA was essential whilst in another strain none of the sequences was clearly identified as essential.…”

Essential Genes Predicted in the Genome of Rubrivivax gelatinosus

“…Organisms with more genomic content tend to have lower essential gene content by percentage. For example, Salmonella typhi has 356 essential ORFs of its total 4,537 (7.8%) (13), Agrobacterium tumefaciens has 372 of 5,460 (6.8%) (15), and Pseudomonas aeruginosa has 352 of 5,678 (6.2%) (16); conversely, Caulobacter crescentus has 480 of 3,876 (12.4%) (14), Brevundimonas subvibrioides has 447 of 3,393 (13.2%) (15), and Streptococcus pyogenes has 241 of 1,785 (13.5%) (20). Another contributing factor to lower essential gene content is the general lack of metabolic genes.…”