Abstract
BackgroundUnderstanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited. Here we report use of a diverse collection of 616 carriage isolates of Streptococcus pneumoniae and their genome sequences to help identify a novel determinant of pneumococcal colonization.ResultsA spontaneously arising laboratory variant (SpnYL101) of a capsule-switched TIGR4 strain (TIGR4:19F) showed reduced ability to establish mouse nasal colonization and lower resistance to non-opsonic neutrophil-mediated killing in vitro, a phenotype correlated with in vivo success. Whole genome sequencing revealed 5 single nucleotide polymorphisms (SNPs) affecting 4 genes in SpnYL101 relative to its ancestor. To evaluate the effect of variation in each gene, we performed an in silico screen of 616 previously published genome sequences to identify pairs of closely-related, serotype-matched isolates that differ at the gene of interest, and compared their resistance to neutrophil-killing. This method allowed rapid examination of multiple candidate genes and found phenotypic differences apparently associated with variation in SP_1645, a RelA/ SpoT homolog (RSH) involved in the stringent response. To establish causality, the alleles corresponding to SP_1645 were switched between the TIGR4:19F and SpnYL101. The wild-type SP_1645 conferred higher resistance to neutrophil-killing and competitiveness in mouse colonization. Using a similar strategy, variation in another RSH gene (TIGR4 locus tag SP_1097) was found to alter resistance to neutrophil-killing.ConclusionsThese results indicate that analysis of naturally existing genomic diversity complements traditional genetics approaches to accelerate genotype-phenotype analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1573-6) contains supplementary material, which is available to authorized users.
Highlights
Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited
Genomic diversity of existing microbial populations can be used to infer the consequences of polymorphisms by evaluating phenotypic differences between naturally occurring variants, if 1) the naturally occurring variants harbor the genetic variation of interest and 2) other known genetic determinants of the phenotypic difference can be accounted for
A variant of pneumococcus with reduced fitness We observed a variant of the TIGR4:19F strain (SpnYL101) showing reduced ability to compete against other strains during nasal colonization, compared to the originally constructed TIGR4:19F (Figure 2A)
Summary
Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited. Genomic diversity of existing microbial populations can be used to infer the consequences of polymorphisms by evaluating phenotypic differences between naturally occurring variants, if 1) the naturally occurring variants harbor the genetic variation of interest and 2) other known genetic determinants of the phenotypic difference can be accounted for. For genetic variations outside the capsular polysaccharide synthesis (cps) region, much less is known about their contribution to resistance to surface killing or ability to compete during nasopharyngeal colonization
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