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.

In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages The Gram-negative proteobacterium Burkholderia pseudomallei can survive and multiply within a variety of eukaryotic cells, including macrophages. This property is believed to be important for its ability to cause the disease melioidosis in a wide range of animal species, including humans. To identify determinants that are important for the ability of B. pseudomallei to survive within macrophages, in vivo expression technology (IVET) was employed. Several putative macrophage-inducible genes were identified that are likely to contribute to the virulence of B. pseudomallei, including three genes (tssH-5, tssI-5 and tssM-5) located within the same type VI secretion system cluster (tss-5), mntH, encoding a natural resistance-associated macrophage protein (NRAMP)-like manganese ion transporter, and a haem acquisition gene, bhuT. The macrophage-inducibility of the tss-5 gene cluster was confirmed by reporter gene analysis. Construction of tssH-5 and bhuT null mutants indicated that expression of the tss-5 unit and the bhu operon were not required for intramacrophage survival. A further five tss units were identified within the B. pseudomallei genome that, together with tss-5, account for approximately 2.3 % of the total genome size. The presence of six type VI secretion systems in this organism is likely to be an important factor in making this bacterium such a versatile pathogen. INTRODUCTIONMelioidosis is the name given to any infection caused by Burkholderia pseudomallei, a saprophytic, Gram-negative bacillus found in wet soil and pooled water, particularly in south-east Asia and northern Australia (White, 2003). It is principally acquired following inoculation of lesions in the skin by contaminated soil and water, with the highest incidence of the disease occurring during the rainy and monsoon seasons (Dance, 1991;White, 2003). Another important route of infection is inhalation of contaminated particles. The clinical spectrum of melioidosis ranges from an acute fulminant septicaemia to chronic localized infections, often affecting the lung, and is usually characterized by abscess formation. No vaccines are available, and antibiotic therapy is problematic due to the intrinsic high resistance of B. pseudomallei to many antibiotics. The overall mortality of melioidosis patients is 50 % (White, 2003).Certain features of melioidosis suggest that B. pseudomallei is a facultative intracellular pathogen. These include the occurrence of long periods of latency (a recent case report suggests this can be as long as 62 years), relapses due to recrudescence of a persistent primary infection, and the activation of a cellular immune response during melioidosis (Chaowagul et al., 1993;Ngauy et al., 2005). Consistent with this, B. pseudomallei has been shown to survive and multiply within non-phagocytic cells, macrophages and free-living amoebae (Pruksachartvuthi et al., 1990;Jones et a...

Genes required for infection of mice by Salmonella Typhimurium can be identified by the interrogation of random transposon mutant libraries for mutants that cannot survive in vivo. Inactivation of such genes produces attenuated S. Typhimurium strains that have potential for use as live attenuated vaccines. A quantitative screen, Transposon Mediated Differential Hybridisation (TMDH), has been developed that identifies those members of a large library of transposon mutants that are attenuated. TMDH employs custom transposons with outward-facing T7 and SP6 promoters. Fluorescently-labelled transcripts from the promoters are hybridised to whole-genome tiling microarrays, to allow the position of the transposon insertions to be determined. Comparison of microarray data from the mutant library grown in vitro (input) with equivalent data produced after passage of the library through mice (output) enables an attenuation score to be determined for each transposon mutant. These scores are significantly correlated with bacterial counts obtained during infection of mice using mutants with individual defined deletions of the same genes. Defined deletion mutants of several novel targets identified in the TMDH screen are effective live vaccines.

The ferric uptake regulator (Fur) functions as a transcription repressor of many genes in bacteria in response to iron, but the presence of a functional equivalent of this protein has not been demonstrated in Burkholderia cepacia. A segment of the Burkholderia pseudomallei fur gene was amplified using degenerate primers and used to identify chromosomal restriction fragments containing the entire fur genes of B. cepacia and B. pseudomallei. These fragments were cloned and sequenced, revealing the Fur protein of both species to be a polypeptide of 142 amino acids possessing a high degree of amino acid sequence identity to Fur of other members of the β subclass of the Proteobacteria. Primer extension analysis demonstrated that transcription of B. cepacia fur originated from a single promoter located 36 bp upstream from the fur translation initiation codon. The Fur polypeptide of B. cepacia was shown to functionally substitute for Fur in an Escherichia coli fur mutant. Single copy fur-lacZ fusions were constructed and used to examine the regulation of B. cepacia fur. The B. cepacia fur promoter was not responsive to iron availability, the presence of hydrogen peroxide or the superoxide generator methyl viologen. In addition, fur expression was not significantly influenced by carbon source. Interestingly, the presence of the divergently transcribed omlA/smpA gene upstream of fur in some members of the γ subclass of the Proteobacteria is retained in several genera within the β taxon, including Burkholderia.

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