Abstract
BackgroundIn 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.ResultsWe 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.ConclusionWe 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.
Highlights
In recent years there has been an increasing problem with Staphylococcus aureus strains that are resistant to treatment with existing antibiotics
In order to overcome the limitations of current tag-array approaches we have developed a simple method, Transposon-Mediated Differential Hybridisation (TMDH) [11,12], that combines the advantages of both sequencing- and array-based approaches to determine the repertoire of genes required for the survival and growth of the target organism
Use of this automated scoring system allows unbiased prediction of essential genes from even large mutant libraries. This method could be applied in isolation to identify potential targets for drug discovery or, as is the case here, used as a preliminary screen to reduce dramatically the workload required for a PCR footprinting and sequencing follow-on strategy to locate unambiguously
Summary
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. The most rigorous method is the systematic construction of defined knockout mutants across the whole genome This has been applied to Bacillus subtilis [4], with essential genes defined as those for which a mutant could not be obtained, in many cases being verified by conditional-lethal constructs.
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