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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

Genes that influence fitness at high temperature. (A) Genes contributing to fitness at 43°C were classified by cellular function. Genes associated with the cell envelope constituted the largest fraction of the hits. (B) Six genes known to be associated with the heat-shock response were found to be temperature-sensitive. The number of reads in each gene normalized to 5 million sample reads is shown at the various temperatures tested. Loss of lyrA and mprF was found to have opposite phenotypes at 43°C, with an increase (C) and decrease (D) in fitness being observed, respectively. (E) To validate these phenotypes,  mutants in S. aureus HG003 were grown to mid-log phase, diluted, and grown at 43°C. While ΔmprF did not grow, confirming temperature sensitivity, the ΔlyrA strain grew at nearly twice the rate of WT.
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Fig6: Genes that influence fitness at high temperature. (A) Genes contributing to fitness at 43°C were classified by cellular function. Genes associated with the cell envelope constituted the largest fraction of the hits. (B) Six genes known to be associated with the heat-shock response were found to be temperature-sensitive. The number of reads in each gene normalized to 5 million sample reads is shown at the various temperatures tested. Loss of lyrA and mprF was found to have opposite phenotypes at 43°C, with an increase (C) and decrease (D) in fitness being observed, respectively. (E) To validate these phenotypes, mutants in S. aureus HG003 were grown to mid-log phase, diluted, and grown at 43°C. While ΔmprF did not grow, confirming temperature sensitivity, the ΔlyrA strain grew at nearly twice the rate of WT.

Mentions: A significant number of genes were found to be affected by transposon insertion when grown at 43°C, with 42 being enriched and 77 being depleted. Because this method for creating transposon libraries does not involve a high-temperature plasmid curing step, we expected to retain many temperature-sensitive mutants. We used two methods for identifying temperature-sensitive mutants represented in the library. The first was the Mann-Whitney U analysis used for every temperature. For the second analysis, we used the 43°C data and the essential gene analysis described in the previous section to identify genes essential at 43°C that were not identified as essential at 30°C. We confirmed that the number of reads mapping to these genes had decreased at 43°C from 30°C. We compared these genes to other essential genes lists [29,31-34], and identified 19 genes that had been annotated as essential in at least two other transposon library analyses, but were only temperature-sensitive with our method (Table 2). To further analyze the temperature sensitive gene subset, we classified them according to cellular function (Figure 6A). Because protection from temperature stress relies on the coordinated response of many different pathways regulated by signaling systems and alternative transcription factors, we were not surprised to find that 11 regulatory genes were significantly depleted at 43°C, including yycH (SAOUHSC_00022) and yycI (SAOUHSC_00023), which were identified as essential in another transposon library [29]. The yycH and yycI genes negatively regulate the two component system, walKR, which is a major regulator of peptidoglycan metabolism that controls autolytic activity [54,55]. Deletions of yycH and yycI result in upregulation of walKR, which induces cell wall defects [54,56]. As walKR positively regulates autolytic activity [57], increased autolysis of peptidoglycan could explain how derepression of walKR produces a temperature-sensitive phenotype. We also found, as expected, that many genes implicated in the heat shock response were required for survival at 43°C. These included the chaperones dnaK (SAOUHSC_01683) and dnaJ (SAOUHSC_01682) [58], the transcriptional regulator hrcA (SAOUHSC_01685) [59], the protease clpC (SAOUHSC_00505) [60], and mcsB (SAOUHSC_00504; also known as yakI), an arginine phosphotransferase that negatively regulates the stress response repressor ctsR [61,62]. GrpE (SAOUHSC_01684), which acts in a complex with DnaK and DnaJ, narrowly missed our cutoffs (Figure 6B) [63].Table 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)

Genes that influence fitness at high temperature. (A) Genes contributing to fitness at 43°C were classified by cellular function. Genes associated with the cell envelope constituted the largest fraction of the hits. (B) Six genes known to be associated with the heat-shock response were found to be temperature-sensitive. The number of reads in each gene normalized to 5 million sample reads is shown at the various temperatures tested. Loss of lyrA and mprF was found to have opposite phenotypes at 43°C, with an increase (C) and decrease (D) in fitness being observed, respectively. (E) To validate these phenotypes,  mutants in S. aureus HG003 were grown to mid-log phase, diluted, and grown at 43°C. While ΔmprF did not grow, confirming temperature sensitivity, the ΔlyrA strain grew at nearly twice the rate of WT.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Genes that influence fitness at high temperature. (A) Genes contributing to fitness at 43°C were classified by cellular function. Genes associated with the cell envelope constituted the largest fraction of the hits. (B) Six genes known to be associated with the heat-shock response were found to be temperature-sensitive. The number of reads in each gene normalized to 5 million sample reads is shown at the various temperatures tested. Loss of lyrA and mprF was found to have opposite phenotypes at 43°C, with an increase (C) and decrease (D) in fitness being observed, respectively. (E) To validate these phenotypes, mutants in S. aureus HG003 were grown to mid-log phase, diluted, and grown at 43°C. While ΔmprF did not grow, confirming temperature sensitivity, the ΔlyrA strain grew at nearly twice the rate of WT.
Mentions: A significant number of genes were found to be affected by transposon insertion when grown at 43°C, with 42 being enriched and 77 being depleted. Because this method for creating transposon libraries does not involve a high-temperature plasmid curing step, we expected to retain many temperature-sensitive mutants. We used two methods for identifying temperature-sensitive mutants represented in the library. The first was the Mann-Whitney U analysis used for every temperature. For the second analysis, we used the 43°C data and the essential gene analysis described in the previous section to identify genes essential at 43°C that were not identified as essential at 30°C. We confirmed that the number of reads mapping to these genes had decreased at 43°C from 30°C. We compared these genes to other essential genes lists [29,31-34], and identified 19 genes that had been annotated as essential in at least two other transposon library analyses, but were only temperature-sensitive with our method (Table 2). To further analyze the temperature sensitive gene subset, we classified them according to cellular function (Figure 6A). Because protection from temperature stress relies on the coordinated response of many different pathways regulated by signaling systems and alternative transcription factors, we were not surprised to find that 11 regulatory genes were significantly depleted at 43°C, including yycH (SAOUHSC_00022) and yycI (SAOUHSC_00023), which were identified as essential in another transposon library [29]. The yycH and yycI genes negatively regulate the two component system, walKR, which is a major regulator of peptidoglycan metabolism that controls autolytic activity [54,55]. Deletions of yycH and yycI result in upregulation of walKR, which induces cell wall defects [54,56]. As walKR positively regulates autolytic activity [57], increased autolysis of peptidoglycan could explain how derepression of walKR produces a temperature-sensitive phenotype. We also found, as expected, that many genes implicated in the heat shock response were required for survival at 43°C. These included the chaperones dnaK (SAOUHSC_01683) and dnaJ (SAOUHSC_01682) [58], the transcriptional regulator hrcA (SAOUHSC_01685) [59], the protease clpC (SAOUHSC_00505) [60], and mcsB (SAOUHSC_00504; also known as yakI), an arginine phosphotransferase that negatively regulates the stress response repressor ctsR [61,62]. GrpE (SAOUHSC_01684), which acts in a complex with DnaK and DnaJ, narrowly missed our cutoffs (Figure 6B) [63].Table 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