330 Antisense technology has been widely used to regulate gene expression. A tetracycline (tet)-regulated antisense-RNA- expressing system has been developed and used to downregulate chromosomally derived genes expressed in Staphylococcus aureus. This downregulation subsequently provides an evaluation of the virulence factor and drug targets. The regulated antisense RNA library allows for genome-wide analyses of the functions of staphylococcal gene products for growth in culture and survival during infection. Moreover, this antisense RNA technology may provide a key tool to identify mechanisms of novel antibacterial compound action. Addresses Department of Microbiology, GlaxoSmithKline Pharmaceuticals Research and Development, 1250 South Collegeville Road, Collegeville, Pennsylvania 19462, USA *Author for correspondence: Yinduo Ji; e-mail: yinduo_ji-1@gsk.com Current Opinion in Microbiology 2002, 5:330–333 1369-5274/02/$ —see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 7 May 2002 Abbreviations ATc anhydrotetracycline hla α-toxin ORF open reading frame PNA peptide nucleic acid tet tetracycline xyl xylose Introduction The occurrence of antibiotic resistance results in an urgent need to develop new antibiotics. The availability of genomic sequence data, bioinformatic analysis and new molecular technologies make it possible to quickly scan genes essential for bacterial growth and survival during infection. Gene inactivation or disruption technology is still playing an important role in the identification of essential genes, which may be suitable as potential molecular targets for drug discovery. Various techniques have been used in bacterial systems to achieve inactivation of gene products. Most of these involve gene knockout methods that use insertion, deletion (for example, allelic replacement) and point mutation techniques [1–4]. One setback to the afore- mentioned techniques, however, is that strains carrying null mutations in genes essential for growth that are generated by these genetic methods are typically not recoverable for further analysis. Conditional mutagenesis approaches may overcome this problem and have been used successfully to identify essential genes for viability [5,6]. Regulated gene expression systems, which place genes downstream of the regulatable promoters and then delete the normal gene, provide an alternative strategy. This strategy allows selective genes to be turned on or off and to be expressed at certain levels, have the potential to provide more quantitative data on the gene product. These systems have been used widely in Escherichia coli [7,8] and Bacillus subtilis [9,10], and are now being developed for bacterial pathogens [11–13]. In this review, we focus on genomic analysis using regulated antisense RNA and antisense oligonucleotide technology. The antisense mechanism Antisense technology is an effective approach to down- regulate expression of specific genes for the validation of drug targets as well as the rational design of gene therapeutic drugs. It has been widely used to interfere with eukaryotic gene expression through injection of synthetic oligo- nucleotides complementary to mRNA [14,15] and by the synthesis of antisense RNA from DNA cloned in an antisense orientation [16]. The mechanism of function through which expressed antisense RNA can decrease the production of the protein encoded by the endogenous gene probably involves duplex RNA formation by binding of complementary regions of the normal sense mRNA and the antisense RNA strand. Therefore, duplex RNA can inhibit translation of the mRNA to protein or cause mRNA degradation [16]. There have been three mechanisms of action proposed [17] for the ability of antisense oligo- nucleotides to downregulate gene expression. Firstly, antisense oligonucleotides might hybridize to their target mRNA in a strictly base-pair-specific manner and, thus, block translation; secondly, antisense oligonucleotides might bind to the genomic DNA and then block transcription; thirdly, the oligonucleotide might bind to a target protein. Construction of a tetracycline transcription regulatory system for antisense studies in Staphylococcus aureus Until recently, the antisense method has not been routinely used to inhibit gene expression in bacteria, although antisense-RNA-mediated control of gene expression occurs naturally in bacteria during plasmid, phage and chromosomal replication [18,19]. Kernodle et al. [16] constructed an α-toxin (hla) antisense isogenic staphylococcal strain by cloning a 600 bp fragment of hla into an E. coli–S. aureus shuttle vector in an antisense orientation. Utilizing this antisense vector, they demonstrated that the isogenic strain expressing hla antisense RNA generated 16-fold less α-toxin than either its parent or a control strain containing vector DNA and significantly attenuated lethal activity in a murine model. This suggested that the antisense RNA technology might be useful in creating novel live-attenuated strains of bacteria for use as vaccine candidates. Genomic analysis using conditional phenotypes generated by antisense RNA Dezhong Yin and Yinduo Ji*