Limits...
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.

Show MeSH

Related in: MedlinePlus

The essential gene sets at 30°C. (A) The Circos program was used to map transposon insertion sites across the genome, with a histogram depicting the number of reads per TA site in purple. The innermost blue rings depict the locations of non-essential genes for which a fitness cost was not observed, while the outermost red ring depicts those genes identified as essential. The middle green ring represents those genes that were found to be domain essential. (B) Venn diagram comparing our essential gene list to Chaudhuri et al. and Valentino et al [29,33]. There is extensive overlap between the three studies. 211 genes were classified as essential in all three works. These represent a core set of genes required for cell growth regardless of experimental and analytical variations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: The essential gene sets at 30°C. (A) The Circos program was used to map transposon insertion sites across the genome, with a histogram depicting the number of reads per TA site in purple. The innermost blue rings depict the locations of non-essential genes for which a fitness cost was not observed, while the outermost red ring depicts those genes identified as essential. The middle green ring represents those genes that were found to be domain essential. (B) Venn diagram comparing our essential gene list to Chaudhuri et al. and Valentino et al [29,33]. There is extensive overlap between the three studies. 211 genes were classified as essential in all three works. These represent a core set of genes required for cell growth regardless of experimental and analytical variations.

Mentions: The Blunt construct, which is not fitted with gene expression modulating elements, does not include promoters that affect proximal gene expression and is most similar to previously investigated transposons [29-34]. Therefore, we used it for the rest of the analyses so that we could compare our results to previous studies. In two biological replicates, there were ~126,000 unique insertion sites due to the Blunt construct. Using the Circos program to display the number of reads per TA site [48], we visually confirmed randomly distributed insertions throughout the length of the genome (Figure 5A). Of the 2723 coding regions of the genome that are not part of the Φ11 family lysogen, EL-ARTIST called 2212 non-essential, 190 domain essential, and 321 essential (Additional file 2).Figure 5


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 essential gene sets at 30°C. (A) The Circos program was used to map transposon insertion sites across the genome, with a histogram depicting the number of reads per TA site in purple. The innermost blue rings depict the locations of non-essential genes for which a fitness cost was not observed, while the outermost red ring depicts those genes identified as essential. The middle green ring represents those genes that were found to be domain essential. (B) Venn diagram comparing our essential gene list to Chaudhuri et al. and Valentino et al [29,33]. There is extensive overlap between the three studies. 211 genes were classified as essential in all three works. These represent a core set of genes required for cell growth regardless of experimental and analytical variations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: The essential gene sets at 30°C. (A) The Circos program was used to map transposon insertion sites across the genome, with a histogram depicting the number of reads per TA site in purple. The innermost blue rings depict the locations of non-essential genes for which a fitness cost was not observed, while the outermost red ring depicts those genes identified as essential. The middle green ring represents those genes that were found to be domain essential. (B) Venn diagram comparing our essential gene list to Chaudhuri et al. and Valentino et al [29,33]. There is extensive overlap between the three studies. 211 genes were classified as essential in all three works. These represent a core set of genes required for cell growth regardless of experimental and analytical variations.
Mentions: The Blunt construct, which is not fitted with gene expression modulating elements, does not include promoters that affect proximal gene expression and is most similar to previously investigated transposons [29-34]. Therefore, we used it for the rest of the analyses so that we could compare our results to previous studies. In two biological replicates, there were ~126,000 unique insertion sites due to the Blunt construct. Using the Circos program to display the number of reads per TA site [48], we visually confirmed randomly distributed insertions throughout the length of the genome (Figure 5A). Of the 2723 coding regions of the genome that are not part of the Φ11 family lysogen, EL-ARTIST called 2212 non-essential, 190 domain essential, and 321 essential (Additional file 2).Figure 5

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