Abstract
ABSTRACT For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria.
Highlights
Identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion
Sequencing the 14 libraries yielded an average of 2.5 million unique transposonchromsosome junctions, which could be mapped to 42% to 64% of the TA dinucleotide sites in the chromosome in each individual library
This work describes the first use of a Himar1 library that has reached the practical limit of saturation for the definition of essential genomic regions
Summary
Identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features. These analyses rely on the characterization of a single transposon library to identify regions that are devoid of transposon insertions and likely to encode functions that are necessary for growth [6, 13] Many of these procedures rely on the Himar transposon, which is used because it is thought to lack sequence specificity except for the required TA dinucleotide insertion site [14]. ~13% of the ORFs of M. tuberculosis (538 of 3,990 genes) were effectively excluded from TnSeq profiling in those prior studies
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