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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

Elimination of non-transposase catalyzed transposon integration in strain HG003. (A) The ratio of erythromycin resistant colonies arising from non-transposase catalyzed (hatched) to transposase dependent (solid shading) events was determined in the S. aureus RN4220 (green) or HG003 (blue) recipient strain background by comparing the number of colonies resulting from transduction of the transposon into the full transposase or truncated transposase expressing strains (Additional file 1: Figure S1). The presence of the phage attachment site in the bacterial chromosome (attB), the phage attachment site in the donor lysate (attP-int), and a Φ11 prophage in the recipient is indicated for each combination. (B) Putative mechanisms for integration of the ermR cassette of the transposon into the recipient chromosome include transposase catalyzed (top), integrase mediated site specific recombination (middle), and homologous recombination (bottom) of phage-transposon hybrids resolved from concatemeric transposon donor DNA. (C) The integrase pathway was blocked by replacing the integrase (int) gene and the attL sequence with a FRT element by allelic replacement. To cure the resulting prophage, the attR site was also replaced with a second FRT site and a phage donor lacking int-attP was isolated by introducing the FLP recombinase. In the process, a recipient HG003 strain was generated from which the Φ11 prophage was specifically cured and replaced with a single FRT element, preventing homologous recombination.
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Fig2: Elimination of non-transposase catalyzed transposon integration in strain HG003. (A) The ratio of erythromycin resistant colonies arising from non-transposase catalyzed (hatched) to transposase dependent (solid shading) events was determined in the S. aureus RN4220 (green) or HG003 (blue) recipient strain background by comparing the number of colonies resulting from transduction of the transposon into the full transposase or truncated transposase expressing strains (Additional file 1: Figure S1). The presence of the phage attachment site in the bacterial chromosome (attB), the phage attachment site in the donor lysate (attP-int), and a Φ11 prophage in the recipient is indicated for each combination. (B) Putative mechanisms for integration of the ermR cassette of the transposon into the recipient chromosome include transposase catalyzed (top), integrase mediated site specific recombination (middle), and homologous recombination (bottom) of phage-transposon hybrids resolved from concatemeric transposon donor DNA. (C) The integrase pathway was blocked by replacing the integrase (int) gene and the attL sequence with a FRT element by allelic replacement. To cure the resulting prophage, the attR site was also replaced with a second FRT site and a phage donor lacking int-attP was isolated by introducing the FLP recombinase. In the process, a recipient HG003 strain was generated from which the Φ11 prophage was specifically cured and replaced with a single FRT element, preventing homologous recombination.

Mentions: We selected HG003 as our strain background for library preparation because a high quality plasmid-delivered library has been made in the same strain and provided a reference for validating our method [29,30]. However, in contrast to RN4220 and COL, a high number of ermR colonies was observed in HG003 even in the absence of functional transposase (<1% for RN4220 versus >90% for HG003; Figure 2A, bars 1 to 4). We initially speculated that phage-transposon hybrid DNA containing an attP site was being integrated within a recipient HG003 subpopulation where the chromosomal attB site had become available through spontaneous excision of the resident prophage, thus leading to the high background of non-transposase catalyzed events (Figure 2B, middle panel). To confirm this possibility, we introduced the pORF5 transposase plasmid into wild type RN4220, which also contains an available attB site. As with HG003, we now observed a high background of non-transposase catalyzed ermR colonies in RN4220 attB+ (Figure 2A, bars 5 and 6), consistent with a role for phage-mediated att-site specific integration in increasing background (Figure 2B, middle panel). To block this pathway, we constructed a strictly virulent transducing phage (Φ11-FRT) by replacing the attP-int site with a FRT site from the yeast 2-μm plasmid site-specific recombination system [36,37] (Figure 2C), thereby preventing integration. The use of Φ11-FRT decreased the background of non-transposase-catalyzed transposon integration in RN4220 as fewer ermR colonies were produced by the truncated transposase expressing strain (Figure 2A, bars 7 and 8), but the background in HG003 remained unacceptably high (Figure 2A, bars 9 and 10). We therefore considered a second mechanism for the production non-transposase catalyzed ermR colonies. Homologous recombination between phage-transposon hybrids carrying the ermR cassette and the resident Φ11 prophage in HG003 could also yield ermR colonies (Figure 2B, bottom panel). To determine whether this was occurring, we constructed a strain of HG003 where the Φ11 prophage was specifically removed using the same att::FRT exchange strategy employed to create the Φ11-FRT donor phage (Figure 2C). The combination of this recipient strain (HG003 Φ11−) with Φ11-FRT packaged transposon donors reduced non-transposase catalyzed ermR background colonies to less than 1% (Figure 2A, bars 11 and 12). With removal of the Φ11 prophage, this strategy now allows us to create high-density transposon mutant libraries with a very low background of non-transposase catalyzed transposon integration using the phage-based transposition system in any strain of S. aureus that is transducible by Φ11.Figure 2


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)

Elimination of non-transposase catalyzed transposon integration in strain HG003. (A) The ratio of erythromycin resistant colonies arising from non-transposase catalyzed (hatched) to transposase dependent (solid shading) events was determined in the S. aureus RN4220 (green) or HG003 (blue) recipient strain background by comparing the number of colonies resulting from transduction of the transposon into the full transposase or truncated transposase expressing strains (Additional file 1: Figure S1). The presence of the phage attachment site in the bacterial chromosome (attB), the phage attachment site in the donor lysate (attP-int), and a Φ11 prophage in the recipient is indicated for each combination. (B) Putative mechanisms for integration of the ermR cassette of the transposon into the recipient chromosome include transposase catalyzed (top), integrase mediated site specific recombination (middle), and homologous recombination (bottom) of phage-transposon hybrids resolved from concatemeric transposon donor DNA. (C) The integrase pathway was blocked by replacing the integrase (int) gene and the attL sequence with a FRT element by allelic replacement. To cure the resulting prophage, the attR site was also replaced with a second FRT site and a phage donor lacking int-attP was isolated by introducing the FLP recombinase. In the process, a recipient HG003 strain was generated from which the Φ11 prophage was specifically cured and replaced with a single FRT element, preventing homologous recombination.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: Elimination of non-transposase catalyzed transposon integration in strain HG003. (A) The ratio of erythromycin resistant colonies arising from non-transposase catalyzed (hatched) to transposase dependent (solid shading) events was determined in the S. aureus RN4220 (green) or HG003 (blue) recipient strain background by comparing the number of colonies resulting from transduction of the transposon into the full transposase or truncated transposase expressing strains (Additional file 1: Figure S1). The presence of the phage attachment site in the bacterial chromosome (attB), the phage attachment site in the donor lysate (attP-int), and a Φ11 prophage in the recipient is indicated for each combination. (B) Putative mechanisms for integration of the ermR cassette of the transposon into the recipient chromosome include transposase catalyzed (top), integrase mediated site specific recombination (middle), and homologous recombination (bottom) of phage-transposon hybrids resolved from concatemeric transposon donor DNA. (C) The integrase pathway was blocked by replacing the integrase (int) gene and the attL sequence with a FRT element by allelic replacement. To cure the resulting prophage, the attR site was also replaced with a second FRT site and a phage donor lacking int-attP was isolated by introducing the FLP recombinase. In the process, a recipient HG003 strain was generated from which the Φ11 prophage was specifically cured and replaced with a single FRT element, preventing homologous recombination.
Mentions: We selected HG003 as our strain background for library preparation because a high quality plasmid-delivered library has been made in the same strain and provided a reference for validating our method [29,30]. However, in contrast to RN4220 and COL, a high number of ermR colonies was observed in HG003 even in the absence of functional transposase (<1% for RN4220 versus >90% for HG003; Figure 2A, bars 1 to 4). We initially speculated that phage-transposon hybrid DNA containing an attP site was being integrated within a recipient HG003 subpopulation where the chromosomal attB site had become available through spontaneous excision of the resident prophage, thus leading to the high background of non-transposase catalyzed events (Figure 2B, middle panel). To confirm this possibility, we introduced the pORF5 transposase plasmid into wild type RN4220, which also contains an available attB site. As with HG003, we now observed a high background of non-transposase catalyzed ermR colonies in RN4220 attB+ (Figure 2A, bars 5 and 6), consistent with a role for phage-mediated att-site specific integration in increasing background (Figure 2B, middle panel). To block this pathway, we constructed a strictly virulent transducing phage (Φ11-FRT) by replacing the attP-int site with a FRT site from the yeast 2-μm plasmid site-specific recombination system [36,37] (Figure 2C), thereby preventing integration. The use of Φ11-FRT decreased the background of non-transposase-catalyzed transposon integration in RN4220 as fewer ermR colonies were produced by the truncated transposase expressing strain (Figure 2A, bars 7 and 8), but the background in HG003 remained unacceptably high (Figure 2A, bars 9 and 10). We therefore considered a second mechanism for the production non-transposase catalyzed ermR colonies. Homologous recombination between phage-transposon hybrids carrying the ermR cassette and the resident Φ11 prophage in HG003 could also yield ermR colonies (Figure 2B, bottom panel). To determine whether this was occurring, we constructed a strain of HG003 where the Φ11 prophage was specifically removed using the same att::FRT exchange strategy employed to create the Φ11-FRT donor phage (Figure 2C). The combination of this recipient strain (HG003 Φ11−) with Φ11-FRT packaged transposon donors reduced non-transposase catalyzed ermR background colonies to less than 1% (Figure 2A, bars 11 and 12). With removal of the Φ11 prophage, this strategy now allows us to create high-density transposon mutant libraries with a very low background of non-transposase catalyzed transposon integration using the phage-based transposition system in any strain of S. aureus that is transducible by Φ11.Figure 2

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