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Abstract
BackgroundStaphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. High throughput, parallel-sequencing methods for analyzing transposon mutant libraries have the potential to revolutionize studies of S. aureus, but the genetic tools to take advantage of the power of next generation sequencing have not been fully developed.ResultsHere we report a phage-based transposition system to make ultra-high density transposon libraries for genome-wide analysis of mutant fitness in any Φ11-transducible S. aureus strain. The high efficiency of the delivery system has made it possible to multiplex transposon cassettes containing different regulatory elements in order to make libraries in which genes are over- or under-expressed as well as deleted. By incorporating transposon-specific barcodes into the cassettes, we can evaluate how null mutations and changes in gene expression levels affect fitness in a single sequencing data set. Demonstrating the power of the system, we have prepared a library containing more than 690,000 unique insertions. Because one unique feature of the phage-based approach is that temperature-sensitive mutants are retained, we have carried out a genome-wide study of S. aureus genes involved in withstanding temperature stress. We find that many genes previously identified as essential are temperature sensitive and also identify a number of genes that, when disrupted, confer a growth advantage at elevated temperatures.ConclusionsThe platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1361-3) contains supplementary material, which is available to authorized users.
Highlights
Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology
694,755 found to be significantly enriched at high temperature. Mutations in this pathway have previously been found in small colony variant (SCV) strains isolated from in vivo infections that are resistant to antibiotics, and our results suggest that heat stress is one condition that may select for these mutants
Adapting the transposon system for compatibility with any Staphylococcus aureus strain Recently, we reported a phage-based approach for S. aureus that enables high efficiency delivery of transposons [35], but it was not compatible with generation sequencing
Summary
Staphylococcus aureus readily develops resistance to antibiotics and achieving effective therapies to overcome resistance requires in-depth understanding of S. aureus biology. Several variations of high throughput transposon insertion site sequencing have been reported, including transposon directed insertionsite sequencing (TraDIS), insertion sequencing (INSeq), high-throughput insertion tracking by deep sequencing (HITS), and transposon sequencing (Tn-Seq) [22,23,24,25] These approaches vary in the method of transposon delivery, being tailored for a particular species, and in the preparation of library DNA to map transposon insertion sites. Most high coverage transposon libraries in S. aureus typically utilize a temperature sensitive plasmid containing the transposon and require high-temperature plasmid-curing steps to remove the plasmid delivery vehicle after transposition has occurred [29,30,31,32,33,34] During this curing step, temperature-sensitive transposon mutants may be culled, making it challenging to differentiate essential genes from those that are required for growth at elevated temperatures
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