Burkholderia pseudomallei gene expression in low iron conditions and identification of a novel virulence factor

B. pseudomallei is sensitive to free iron concentrations in the growth media which provide a signal for regulation of gene expression. As low iron conditions are thought to mimic the host environment, genes that are upregulated in these conditions may play a role in bacterial virulence. Screening of a B. pseudomallei 1026b random promoter library led to the identification of 39 genes. These 39 promoters consistently showed greater than 2-fold higher expression in low iron compared to high iron conditions. Only 2 of the 39 genes were also identified from a microarray analysis of iron-responsive genes. Mutants of genes encoding an uncharacterized virulence factor (mviN) and an uncharacterized Fe-S protein (BPSS0707) showed the highest increase in LD50 compared to wildtype. In addition to iron, the mviN promoter also appears to respond to bacterial growth or density. The fully functional mviN gene is required for B. pseudomallei virulence and ability to invade eukaryotic cells. Deletion of 82% of the mviN structural gene is lethal in B. pseudomallei demonstrating that the gene is essential. The mviN gene encodes for an integral membrane protein with 12 predicted transmembrane domains that is conserved among bacteria and classified as a member of the multidrug/oligosaccharide-lipid/polysaccharide (MOP) exporter superfamily. As with mviN the BPSS0707 gene appears important for full B. pseudomallei virulence and ability to invade eukaryotic cells. We conclude that a random promoter library screen in low and high iron conditions identifies previously unknown iron-responsive genes and provides an alternative tool for the identification of novel virulence factors. Further characterization of B. pseudomallei mviN will advance the knowledge of B. pseudomallei pathogenesis and the development of treatment and preventive strategies for melioidosis.