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Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH).

Chaudhuri RR, Allen AG, Owen PJ, Shalom G, Stone K, Harrison M, Burgis TA, Lockyer M, Garcia-Lara J, Foster SJ, Pleasance SJ, Peters SE, Maskell DJ, Charles IG - BMC Genomics (2009)

Bottom Line: We have determined the first comprehensive list of S. aureus essential genes.This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds.The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. roy.chaudhuri@gmail.com

ABSTRACT

Background: In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth.

Results: We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes.

Conclusion: We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

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Related in: MedlinePlus

ROC curves showing sensitivity against false positive rate (1-specificity) for the TMDH microarray screens using individual restriction enzymes, and the combined data from both, for cut-off values from -1 to -10.
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Figure 6: ROC curves showing sensitivity against false positive rate (1-specificity) for the TMDH microarray screens using individual restriction enzymes, and the combined data from both, for cut-off values from -1 to -10.

Mentions: The microarray procedure alone was not expected to comprehensively identify the complete S. aureus essential gene list, but was applied as a screen to reduce the workload for the more robust but laborious PCR-based analysis. However, for some applications an exhaustive essential gene list may not be necessary, so it is useful to evaluate the efficacy of the microarray screen. The robust S. aureus essential gene list obtained using PCR footprinting allows the sensitivity (percentage of essential genes identified as such in the screen) and specificity (percentage of non-essential genes correctly identified by the screen) of the microarray procedure to be evaluated. Figure 6 shows a ROC curve of sensitivity against false positive rate (1-specificity) for cut-off values from -1 to -10. The curve suggests that a cut-off value of -4 would give an optimal balance between false positives and false negatives in future studies. This gives a specificity of 80.3%, with a sensitivity of 99.1%.


Comprehensive identification of essential Staphylococcus aureus genes using Transposon-Mediated Differential Hybridisation (TMDH).

Chaudhuri RR, Allen AG, Owen PJ, Shalom G, Stone K, Harrison M, Burgis TA, Lockyer M, Garcia-Lara J, Foster SJ, Pleasance SJ, Peters SE, Maskell DJ, Charles IG - BMC Genomics (2009)

ROC curves showing sensitivity against false positive rate (1-specificity) for the TMDH microarray screens using individual restriction enzymes, and the combined data from both, for cut-off values from -1 to -10.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2721850&req=5

Figure 6: ROC curves showing sensitivity against false positive rate (1-specificity) for the TMDH microarray screens using individual restriction enzymes, and the combined data from both, for cut-off values from -1 to -10.
Mentions: The microarray procedure alone was not expected to comprehensively identify the complete S. aureus essential gene list, but was applied as a screen to reduce the workload for the more robust but laborious PCR-based analysis. However, for some applications an exhaustive essential gene list may not be necessary, so it is useful to evaluate the efficacy of the microarray screen. The robust S. aureus essential gene list obtained using PCR footprinting allows the sensitivity (percentage of essential genes identified as such in the screen) and specificity (percentage of non-essential genes correctly identified by the screen) of the microarray procedure to be evaluated. Figure 6 shows a ROC curve of sensitivity against false positive rate (1-specificity) for cut-off values from -1 to -10. The curve suggests that a cut-off value of -4 would give an optimal balance between false positives and false negatives in future studies. This gives a specificity of 80.3%, with a sensitivity of 99.1%.

Bottom Line: We have determined the first comprehensive list of S. aureus essential genes.This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds.The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK. roy.chaudhuri@gmail.com

ABSTRACT

Background: In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics. An important starting point for the development of new antimicrobial drugs is the identification of "essential" genes that are important for bacterial survival and growth.

Results: We have developed a robust microarray and PCR-based method, Transposon-Mediated Differential Hybridisation (TMDH), that uses novel bioinformatics to identify transposon inserts in genome-wide libraries. Following a microarray-based screen, genes lacking transposon inserts are re-tested using a PCR and sequencing-based approach. We carried out a TMDH analysis of the S. aureus genome using a large random mariner transposon library of around a million mutants, and identified a total of 351 S. aureus genes important for survival and growth in culture. A comparison with the essential gene list experimentally derived for Bacillus subtilis highlighted interesting differences in both pathways and individual genes.

Conclusion: We have determined the first comprehensive list of S. aureus essential genes. This should act as a useful starting point for the identification of potential targets for novel antimicrobial compounds. The TMDH methodology we have developed is generic and could be applied to identify essential genes in other bacterial pathogens.

Show MeSH
Related in: MedlinePlus