Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival.

BACKGROUND: Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage. RESULTS: Although previously identified groups or classes of genes required for virulence and survival were represented in the screen, additional novel identifications were revealed and may be attributable to the difference in insertion sequence biases of the two transposons. Mutants identified using a fluorescence-based macrophage screen were further evaluated using gentamicin-based protection assay in macrophages, survival in the mouse splenic clearance model and growth in vitro to identify mutants with reduced growth rates. CONCLUSION: The identification of novel genes within previously described groups was expected, and nearly two-thirds of the 95 genes had not been previously reported as contributing to survival and virulence using random Tn5-based mutagenesis. The results of this work provide added insight with regard to the regulatory elements, nutritional demands and mechanisms required for efficient intracellular growth and survival of the organism.