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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

The electron transport system influences sensitivity to high temperatures. (A) Nine genes were identified as growth advantaged at 43°C in comparison to 30°C. The number of reads per gene normalized to 5 million reads is shown for each temperature. (B) Pathway analysis revealed that this subset of genes (highlighted in yellow) is involved in the biosynthesis of components within the electron transport system, including protoheme and menaquinones. (C) Blocking the electron transport system at the F-type ATPase level, however, decreased fitness at 43°C (genes highlighted in yellow). The number of reads per gene normalized to 5 million reads is shown for each temperature.
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Fig7: The electron transport system influences sensitivity to high temperatures. (A) Nine genes were identified as growth advantaged at 43°C in comparison to 30°C. The number of reads per gene normalized to 5 million reads is shown for each temperature. (B) Pathway analysis revealed that this subset of genes (highlighted in yellow) is involved in the biosynthesis of components within the electron transport system, including protoheme and menaquinones. (C) Blocking the electron transport system at the F-type ATPase level, however, decreased fitness at 43°C (genes highlighted in yellow). The number of reads per gene normalized to 5 million reads is shown for each temperature.

Mentions: In addition to the mutants that were depleted at 43°C, we identified a number of processes for which disruption resulted in a significant growth advantage compared to growth at 30°C (Figure 7A). Pathway enrichment analysis using BioCyc [81] showed that the number of reads due to transposon insertion were significantly enriched in seven genes in the aromatic amino acid and menaquinol biosynthetic pathways (SAOUHSC_01481, aroB: SAOUHSC_01482, aroF: SAOUHSC_01483, menF: SAOUHSC_00982, menD: SAOUHSC_00983, aroE: SAOUHSC_01699, and aroA: SAOUHSC_01852) (Figure 7A). In addition to producing phenylalanine and tyrosine, the aromatic amino acid pathway provides precursors (chorismate) for menaquinone biosynthesis (Figure 7B) [82]. Menaquinones are isoprenylated electron transport chain cofactors embedded in the membrane that are necessary for oxidative phosphorylation [83]. Insertions in ispA (SAOUHSC_01618), which encodes a putative geranyltransferase, were also enriched at 43°C. Geranyltransferases initiate the condensation of isoprenoid units into longer chains used in the synthesis of menaquinones, and loss of ispA likely decreases the pool of menaquinones (Figure 7B). We also identified insertions in hemY (SAOUHSC_01460), a gene involved in the production of protoheme [84]. Loss of these factors is thought to shift S. aureus growth towards an anaerobic metabolic regime, which markedly impacts S. aureus membrane physiology and generates small colony variants (SCV) [85,86]. The observed growth advantage could also be related to more efficient anaerobic catabolism at high temperature, and/or decreased oxidative stress. Five subunit genes in the F-type ATPase involved in electron transport were found to be essential at 43°C (Figure 7C): α-subunit (SAOUHSC_02345), β-subunit (SAOUHSC_02343), γ-subunit (SAOUHSC_02341), A subunit (SAOUHSC_02350), and B subunit (SAOUHSC_02347) (all were significantly depleted except the β-subunit), suggesting the electron transport system (ETS) mutations can have opposing effects on fitness at elevated temperatures depending on which step in the ETS is blocked. Among cell envelope-related genes, reads due to transposon insertion were notably enriched in lytD (SAOUHSC_01895), encoding a putative β-N-acetylglucosaminidase [87], and lyrA (SAOUHSC_02611), a polytopic membrane protein. Disruptions in lyrA were previously shown to increase lysostaphin resistance and are lethal when wall teichoic acid biosynthesis is blocked [30,88], and both these phenotypes are consistent with an important role for lyrA in cell envelope biogenesis.Figure 7


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)

The electron transport system influences sensitivity to high temperatures. (A) Nine genes were identified as growth advantaged at 43°C in comparison to 30°C. The number of reads per gene normalized to 5 million reads is shown for each temperature. (B) Pathway analysis revealed that this subset of genes (highlighted in yellow) is involved in the biosynthesis of components within the electron transport system, including protoheme and menaquinones. (C) Blocking the electron transport system at the F-type ATPase level, however, decreased fitness at 43°C (genes highlighted in yellow). The number of reads per gene normalized to 5 million reads is shown for each temperature.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: The electron transport system influences sensitivity to high temperatures. (A) Nine genes were identified as growth advantaged at 43°C in comparison to 30°C. The number of reads per gene normalized to 5 million reads is shown for each temperature. (B) Pathway analysis revealed that this subset of genes (highlighted in yellow) is involved in the biosynthesis of components within the electron transport system, including protoheme and menaquinones. (C) Blocking the electron transport system at the F-type ATPase level, however, decreased fitness at 43°C (genes highlighted in yellow). The number of reads per gene normalized to 5 million reads is shown for each temperature.
Mentions: In addition to the mutants that were depleted at 43°C, we identified a number of processes for which disruption resulted in a significant growth advantage compared to growth at 30°C (Figure 7A). Pathway enrichment analysis using BioCyc [81] showed that the number of reads due to transposon insertion were significantly enriched in seven genes in the aromatic amino acid and menaquinol biosynthetic pathways (SAOUHSC_01481, aroB: SAOUHSC_01482, aroF: SAOUHSC_01483, menF: SAOUHSC_00982, menD: SAOUHSC_00983, aroE: SAOUHSC_01699, and aroA: SAOUHSC_01852) (Figure 7A). In addition to producing phenylalanine and tyrosine, the aromatic amino acid pathway provides precursors (chorismate) for menaquinone biosynthesis (Figure 7B) [82]. Menaquinones are isoprenylated electron transport chain cofactors embedded in the membrane that are necessary for oxidative phosphorylation [83]. Insertions in ispA (SAOUHSC_01618), which encodes a putative geranyltransferase, were also enriched at 43°C. Geranyltransferases initiate the condensation of isoprenoid units into longer chains used in the synthesis of menaquinones, and loss of ispA likely decreases the pool of menaquinones (Figure 7B). We also identified insertions in hemY (SAOUHSC_01460), a gene involved in the production of protoheme [84]. Loss of these factors is thought to shift S. aureus growth towards an anaerobic metabolic regime, which markedly impacts S. aureus membrane physiology and generates small colony variants (SCV) [85,86]. The observed growth advantage could also be related to more efficient anaerobic catabolism at high temperature, and/or decreased oxidative stress. Five subunit genes in the F-type ATPase involved in electron transport were found to be essential at 43°C (Figure 7C): α-subunit (SAOUHSC_02345), β-subunit (SAOUHSC_02343), γ-subunit (SAOUHSC_02341), A subunit (SAOUHSC_02350), and B subunit (SAOUHSC_02347) (all were significantly depleted except the β-subunit), suggesting the electron transport system (ETS) mutations can have opposing effects on fitness at elevated temperatures depending on which step in the ETS is blocked. Among cell envelope-related genes, reads due to transposon insertion were notably enriched in lytD (SAOUHSC_01895), encoding a putative β-N-acetylglucosaminidase [87], and lyrA (SAOUHSC_02611), a polytopic membrane protein. Disruptions in lyrA were previously shown to increase lysostaphin resistance and are lethal when wall teichoic acid biosynthesis is blocked [30,88], and both these phenotypes are consistent with an important role for lyrA in cell envelope biogenesis.Figure 7

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