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

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.

Figure 1

Diagrammatic representation of the TMDH process. a) Illustration of the mariner transposon b) A saturated genome-wide transposon library is produced. Cells with a transposon disrupting an essential gene will not survive. Genomic DNA from the library is restriction digested, and Cy5-labelled RNA run-offs are produced from the T7 promoters by in vitro transcription. c) The labelled RNA is hybridised to a tiling oligonucleotide microarray. Probes that are downstream of a transposon give an "on" signal, other probes give an "off" signal. Note that some probes within an essential gene can give "on" signals, if they can be influenced by transposons outside the gene. Such probes are "non-informative" (see Results and Figure 4). Similarly, probes within a non-essential gene may give an "off" signal if they are upstream of the first transposon.

Figure 2

Cumulative distribution of transposons identified in preliminary experiment. Transposons were located using either the PCR/sequencing method or an initial microarray-based screen. From these experiments it was concluded that the mariner transposon inserted throughout the S. aureus chromosome and hence was suitable for use in TMDH.

Figure 3

Illustration of the TMDH microarray scoring system (see text). a) Histogram of log₂ of the raw microarray signals. b) As a), but overlaid with histogram of log₂ of the microarray signals for the probes which do not hybridise to the S. aureus NCTC 8325 genome sequence, and hence indicate the background signal level in the absence of labelled transcript. c) A normal curve is fitted empirically to the data to model the distribution of background signals. d) Cut-offs are defined to classify the probe signals as reflecting the presence (+1) or absence (-1) of transposons. Probes with an intermediate signal are scored as 0, and effectively omitted from the analysis.

Figure 4

Procedure for identifying "informative probes" for the automated scoring system. a) Probes are only informative for a gene if they are downstream of an intragenic restriction site. Other probes may be influenced by transposons located outside the gene, and give an "on" microarray signal (red lines) even if the gene is essential. b) The exception to this rule is if there are no "on" signals anywhere within a restriction fragment. In that case all the probes within the fragment that overlap the gene are informative, since there are no interfering signals.

Figure 5

PCR footprinting strategy used to confirm or reject putative essential genes. a) Diagrammatic representation of the strategy. PCR is performed using a gene specific primer 50–300 bases upstream of the start codon, and a primer corresponding to the transposon sequence. b) Agarose gel showing sample results. M – size markers, lane 1 – SAOUHSC_00862 (non-essential), lane 2 – SAOUHSC_01672 (essential). Each band represents a PCR product of a different size, corresponding to a transposon insertion in a different position. The white boxes represent the product size range expected for transposon insertions within the gene. For essential genes this region does not contain bands, since cells with insertions in this region are not viable.

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.