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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

Reduction of transposon-plasmid junction NGS reads with flankingNotI restriction sites. (A) Inverse PCR was used to amplify the ITR2 transposon junctions for twelve colonies as has been described [39]. Three out of twelve of these colonies also contained transposon-plasmid junctions (~160 bp DNA band). This ratio increased to seven out of twelve when the canonical ITR sequence was altered to incorporate a MmeI recognition site (Additional file 1: Figure S2). Results are representative of multiple independent experimental replicates. (B) The putative mechanism for transposase catalyzed integration of transposon-plasmid junctions may involve engagement of non-contiguous ITR repeats (dashed lines), resulting in chromosomally integrated transposon multimers. In contrast, when both ITR sequences are optimal, contiguous ITRs are most frequently mobilized (solid lines). (C) Colors are used to identify the positions of the sequences in this drawing. To selectively remove transposon-plasmid junctions, we introduced two NotI sites into the transposon construct that flanked the MmeI modified ITR2. In addition, we included a P7 Illumina sequencing primer site with a unique 3-bp DNA barcode to identify the Pout promoter that faces outward from ITR1 during NGS sequencing. (D) After first digesting gDNA with NotI, the transposon-plasmid junction content was substantially reduced in comparison to Figure 3A.
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Fig3: Reduction of transposon-plasmid junction NGS reads with flankingNotI restriction sites. (A) Inverse PCR was used to amplify the ITR2 transposon junctions for twelve colonies as has been described [39]. Three out of twelve of these colonies also contained transposon-plasmid junctions (~160 bp DNA band). This ratio increased to seven out of twelve when the canonical ITR sequence was altered to incorporate a MmeI recognition site (Additional file 1: Figure S2). Results are representative of multiple independent experimental replicates. (B) The putative mechanism for transposase catalyzed integration of transposon-plasmid junctions may involve engagement of non-contiguous ITR repeats (dashed lines), resulting in chromosomally integrated transposon multimers. In contrast, when both ITR sequences are optimal, contiguous ITRs are most frequently mobilized (solid lines). (C) Colors are used to identify the positions of the sequences in this drawing. To selectively remove transposon-plasmid junctions, we introduced two NotI sites into the transposon construct that flanked the MmeI modified ITR2. In addition, we included a P7 Illumina sequencing primer site with a unique 3-bp DNA barcode to identify the Pout promoter that faces outward from ITR1 during NGS sequencing. (D) After first digesting gDNA with NotI, the transposon-plasmid junction content was substantially reduced in comparison to Figure 3A.

Mentions: To adapt the transposon system for NGS, modifications to the donor plasmids were required. The initial design included the incorporation of: 1) the P7 Illumina adapter sequence within the transposon cassette to enable Illumina based NGS, 2) unique three base pair barcodes specific to each outward facing promoter element for de-multiplexing after sequencing, and 3) a MmeI site to capture the transposon-genome junction. The MmeI restriction site was embedded within one ITR of the transposon by a single base pair change, and facilitates processing of transposon insertion sites by cutting non-specifically 20-base pairs downstream of its recognition site (16-base pairs downstream of the Himar1 TA dinucleotide insertion site) [22]. Early efforts to prepare and sequence our transposon libraries using the reengineered constructs were plagued by high plasmid-transposon junction read counts, despite the fact that the vast majority of ermR colonies arose via bona fide transposition events (Figure 2A). When we used PCR to probe the transposon junctions in isolated transposon mutant colonies, we observed a small population harboring both plasmid- and genomic-transposon junctions (Figure 3A), as previously reported [35]. However, when a single base pair was changed in the canonical ITR DNA sequence to create the MmeI site, the population of transposon insertion mutants containing plasmid-transposon junctions increased to over 50% (Additional file 1: Figure S2). We hypothesized that the MmeI modified base in the ITR was important for recognition by the Himar1 transposase in vivo, resulting in a transposase-DNA complex that often failed to engage the initially encountered ITR and instead read through to a downstream non, contiguous ITR within the concatemer (Figure 3B) [38]. The ensuing transposition event would thus capture and introduce the intervening plasmid region into the recipient genome. Therefore, we added two NotI sites to the plasmid, one immediately after the MmeI-modified ITR within the plasmid backbone and the second immediately upstream of the P7 adapter sequence (Figure 3C). Following an added NotI digestion step, the plasmid-transposon junctions could now be selectively removed as described below and in the Additional file 1 (Figure 3D).Figure 3


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)

Reduction of transposon-plasmid junction NGS reads with flankingNotI restriction sites. (A) Inverse PCR was used to amplify the ITR2 transposon junctions for twelve colonies as has been described [39]. Three out of twelve of these colonies also contained transposon-plasmid junctions (~160 bp DNA band). This ratio increased to seven out of twelve when the canonical ITR sequence was altered to incorporate a MmeI recognition site (Additional file 1: Figure S2). Results are representative of multiple independent experimental replicates. (B) The putative mechanism for transposase catalyzed integration of transposon-plasmid junctions may involve engagement of non-contiguous ITR repeats (dashed lines), resulting in chromosomally integrated transposon multimers. In contrast, when both ITR sequences are optimal, contiguous ITRs are most frequently mobilized (solid lines). (C) Colors are used to identify the positions of the sequences in this drawing. To selectively remove transposon-plasmid junctions, we introduced two NotI sites into the transposon construct that flanked the MmeI modified ITR2. In addition, we included a P7 Illumina sequencing primer site with a unique 3-bp DNA barcode to identify the Pout promoter that faces outward from ITR1 during NGS sequencing. (D) After first digesting gDNA with NotI, the transposon-plasmid junction content was substantially reduced in comparison to Figure 3A.
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Related In: Results  -  Collection

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Fig3: Reduction of transposon-plasmid junction NGS reads with flankingNotI restriction sites. (A) Inverse PCR was used to amplify the ITR2 transposon junctions for twelve colonies as has been described [39]. Three out of twelve of these colonies also contained transposon-plasmid junctions (~160 bp DNA band). This ratio increased to seven out of twelve when the canonical ITR sequence was altered to incorporate a MmeI recognition site (Additional file 1: Figure S2). Results are representative of multiple independent experimental replicates. (B) The putative mechanism for transposase catalyzed integration of transposon-plasmid junctions may involve engagement of non-contiguous ITR repeats (dashed lines), resulting in chromosomally integrated transposon multimers. In contrast, when both ITR sequences are optimal, contiguous ITRs are most frequently mobilized (solid lines). (C) Colors are used to identify the positions of the sequences in this drawing. To selectively remove transposon-plasmid junctions, we introduced two NotI sites into the transposon construct that flanked the MmeI modified ITR2. In addition, we included a P7 Illumina sequencing primer site with a unique 3-bp DNA barcode to identify the Pout promoter that faces outward from ITR1 during NGS sequencing. (D) After first digesting gDNA with NotI, the transposon-plasmid junction content was substantially reduced in comparison to Figure 3A.
Mentions: To adapt the transposon system for NGS, modifications to the donor plasmids were required. The initial design included the incorporation of: 1) the P7 Illumina adapter sequence within the transposon cassette to enable Illumina based NGS, 2) unique three base pair barcodes specific to each outward facing promoter element for de-multiplexing after sequencing, and 3) a MmeI site to capture the transposon-genome junction. The MmeI restriction site was embedded within one ITR of the transposon by a single base pair change, and facilitates processing of transposon insertion sites by cutting non-specifically 20-base pairs downstream of its recognition site (16-base pairs downstream of the Himar1 TA dinucleotide insertion site) [22]. Early efforts to prepare and sequence our transposon libraries using the reengineered constructs were plagued by high plasmid-transposon junction read counts, despite the fact that the vast majority of ermR colonies arose via bona fide transposition events (Figure 2A). When we used PCR to probe the transposon junctions in isolated transposon mutant colonies, we observed a small population harboring both plasmid- and genomic-transposon junctions (Figure 3A), as previously reported [35]. However, when a single base pair was changed in the canonical ITR DNA sequence to create the MmeI site, the population of transposon insertion mutants containing plasmid-transposon junctions increased to over 50% (Additional file 1: Figure S2). We hypothesized that the MmeI modified base in the ITR was important for recognition by the Himar1 transposase in vivo, resulting in a transposase-DNA complex that often failed to engage the initially encountered ITR and instead read through to a downstream non, contiguous ITR within the concatemer (Figure 3B) [38]. The ensuing transposition event would thus capture and introduce the intervening plasmid region into the recipient genome. Therefore, we added two NotI sites to the plasmid, one immediately after the MmeI-modified ITR within the plasmid backbone and the second immediately upstream of the P7 adapter sequence (Figure 3C). Following an added NotI digestion step, the plasmid-transposon junctions could now be selectively removed as described below and in the Additional file 1 (Figure 3D).Figure 3

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