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A new platform for ultra-high density Staphylococcus aureus transposon libraries.

Santiago M, Matano LM, Moussa SH, Gilmore MS, Walker S, Meredith TC - BMC Genomics (2015)

Bottom Line: 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.The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. marinasantiago@fas.harvard.edu.

ABSTRACT

Background: Staphylococcus 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.

Results: Here 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 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.

Conclusions: The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

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Related in: MedlinePlus

Strategy for using phage-based transposition to make high quality transposon libraries for NGS sequencing. (A) The transposon insertion library is made by creating a high-frequency transducing lysate of the transposon cassette that is able to replicate as a plasmid in the donor strain (repC+). The lysate is mixed with the recipient strain (repC−) carrying a temperature sensitive plasmid from which the Himar1 transposase is expressed, and erythromycin resistant transposon insertion mutants are selected. (B) By fitting the transposon cassette with an outward-facing promoter, genes can be up- or down-regulated, or inactivated if non-essential, in a single library pool. In order to cover a wide range of gene expression levels, different promoter containing transposon constructs can be multiplexed and then identified during NGS sequencing by unique DNA barcodes (purple bar) to collect fitness-gene dose relationships by monitoring read counts.
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Fig1: Strategy for using phage-based transposition to make high quality transposon libraries for NGS sequencing. (A) The transposon insertion library is made by creating a high-frequency transducing lysate of the transposon cassette that is able to replicate as a plasmid in the donor strain (repC+). The lysate is mixed with the recipient strain (repC−) carrying a temperature sensitive plasmid from which the Himar1 transposase is expressed, and erythromycin resistant transposon insertion mutants are selected. (B) By fitting the transposon cassette with an outward-facing promoter, genes can be up- or down-regulated, or inactivated if non-essential, in a single library pool. In order to cover a wide range of gene expression levels, different promoter containing transposon constructs can be multiplexed and then identified during NGS sequencing by unique DNA barcodes (purple bar) to collect fitness-gene dose relationships by monitoring read counts.

Mentions: Recently, we reported a phage-based approach for S. aureus that enables high efficiency delivery of transposons [35], but it was not compatible with next generation sequencing. The phage-based transposon system developed previously uses a conditionally replicative transposon donor plasmid, which is moved by generalized phage transduction to a Himar1 transposase-expressing strain that cannot support plasmid replication (Figure 1A). Following transduction, the transposase inserts the transposon into TA dinucleotide sites randomly across the genome. Whereas other transposon systems generate only genotypes due to gene disruption, the phage-based system was designed to allow over- and under expression of genes as well as inactivation [35]. This was achieved by building a set of transposon donor constructs harboring the mariner inverted terminal repeats (ITR) flanking an erythromycin resistance gene under the control of its own promoter and terminator along with an outward-facing weak, medium or strong promoter. Genes proximal to the insertion site can be upregulated or downregulated to different extents depending on the orientation of insertion and the strength of the promoter (Figure 1B). This phage-based transposition system was used to select for determinants conferring resistance to a panel of antibiotics. It was shown to be useful for nominating antibiotic targets through gene upregulation and for identifying other cellular factors involved in antibiotic resistance through deletion/downregulation [35]. However, in its original format the phage-based system was limited to sequencing single colonies isolated after positive selection on agar plates. Thus, its use as a functional genomics discovery tool was limited and we sought to reengineer it to take full advantage of the power of next generation sequencing.Figure 1


A new platform for ultra-high density Staphylococcus aureus transposon libraries.

Santiago M, Matano LM, Moussa SH, Gilmore MS, Walker S, Meredith TC - BMC Genomics (2015)

Strategy for using phage-based transposition to make high quality transposon libraries for NGS sequencing. (A) The transposon insertion library is made by creating a high-frequency transducing lysate of the transposon cassette that is able to replicate as a plasmid in the donor strain (repC+). The lysate is mixed with the recipient strain (repC−) carrying a temperature sensitive plasmid from which the Himar1 transposase is expressed, and erythromycin resistant transposon insertion mutants are selected. (B) By fitting the transposon cassette with an outward-facing promoter, genes can be up- or down-regulated, or inactivated if non-essential, in a single library pool. In order to cover a wide range of gene expression levels, different promoter containing transposon constructs can be multiplexed and then identified during NGS sequencing by unique DNA barcodes (purple bar) to collect fitness-gene dose relationships by monitoring read counts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389836&req=5

Fig1: Strategy for using phage-based transposition to make high quality transposon libraries for NGS sequencing. (A) The transposon insertion library is made by creating a high-frequency transducing lysate of the transposon cassette that is able to replicate as a plasmid in the donor strain (repC+). The lysate is mixed with the recipient strain (repC−) carrying a temperature sensitive plasmid from which the Himar1 transposase is expressed, and erythromycin resistant transposon insertion mutants are selected. (B) By fitting the transposon cassette with an outward-facing promoter, genes can be up- or down-regulated, or inactivated if non-essential, in a single library pool. In order to cover a wide range of gene expression levels, different promoter containing transposon constructs can be multiplexed and then identified during NGS sequencing by unique DNA barcodes (purple bar) to collect fitness-gene dose relationships by monitoring read counts.
Mentions: Recently, we reported a phage-based approach for S. aureus that enables high efficiency delivery of transposons [35], but it was not compatible with next generation sequencing. The phage-based transposon system developed previously uses a conditionally replicative transposon donor plasmid, which is moved by generalized phage transduction to a Himar1 transposase-expressing strain that cannot support plasmid replication (Figure 1A). Following transduction, the transposase inserts the transposon into TA dinucleotide sites randomly across the genome. Whereas other transposon systems generate only genotypes due to gene disruption, the phage-based system was designed to allow over- and under expression of genes as well as inactivation [35]. This was achieved by building a set of transposon donor constructs harboring the mariner inverted terminal repeats (ITR) flanking an erythromycin resistance gene under the control of its own promoter and terminator along with an outward-facing weak, medium or strong promoter. Genes proximal to the insertion site can be upregulated or downregulated to different extents depending on the orientation of insertion and the strength of the promoter (Figure 1B). This phage-based transposition system was used to select for determinants conferring resistance to a panel of antibiotics. It was shown to be useful for nominating antibiotic targets through gene upregulation and for identifying other cellular factors involved in antibiotic resistance through deletion/downregulation [35]. However, in its original format the phage-based system was limited to sequencing single colonies isolated after positive selection on agar plates. Thus, its use as a functional genomics discovery tool was limited and we sought to reengineer it to take full advantage of the power of next generation sequencing.Figure 1

Bottom Line: 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.The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, 02115, USA. marinasantiago@fas.harvard.edu.

ABSTRACT

Background: Staphylococcus 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.

Results: Here 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 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.

Conclusions: The platform described here reliably provides mutant collections of unparalleled genotypic diversity and will enable a wide range of functional genomic studies in S. aureus.

Show MeSH
Related in: MedlinePlus