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The effects of electroporation temperature, biochemical pretreatment of cells and stage of culture on electroporation efficiency for slow-growing mycobacteria were investigated. The efficiency of transformation into Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium intracellulare increased markedly with temperature. In contrast, the efficiency of transformation into Mycobacterium smegmatis, a fast-growing species, was higher at 0 degree C and decreased with temperature. While stage of culture had little effect, a further increase in efficiency of 2-4-fold was obtained following glycine or ethionamide pretreatment. Electroporation at 37 degrees C has been chosen as a standard condition for slow-growing species as it usually resulted in a transformation efficiency several orders of magnitude higher than that obtained at 0 degree C.

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Tuberculosis, caused by closely related members of the Mycobacterium tuberculosis complex, continues to have a major impact on human and animal health worldwide and is responsible for the death of approximately two million people each year, primarily in developing nations (14). Mycobacterium bovis, the pathogen responsible for bovine tuberculosis, is a broad-host-range member of the M. tuberculosis complex, and its transmission to humans is probably responsible for some 5% of human tuberculosis deaths (15). The current tuberculosis vaccine, M. bovis bacillus Calmette-Guérin (BCG), has shown highly variable efficacy, and a significantly better vaccine is urgently required.In New Zealand, traditional test and slaughter approaches to eradication of bovine tuberculosis from domestic livestock have been frustrated by the presence of introduced wildlife, particularly the Australian brushtail possum (Trichosurus vulpecula), which maintains a reservoir of infection (23). Extensive wildlife culling operations over many years have failed to eliminate infected possums from many parts of the country, and vaccination of wildlife against tuberculosis is being investigated. Any vaccine developed for tuberculosis control in the New Zealand environment must be compatible with largescale vaccination of animals, and this requirement has directed research towards the development of rationally attenuated strains of M. bovis with vaccine efficacy (9,11,12). Moredetailed investigation of the attenuation of some of these strains (10, 38) is also contributing to our understanding of the molecular determinants required for tuberculosis pathogenesis.Among the known determinants required for virulence in pathogenic mycobacteria are complex lipid components of the mycobacterial cell wall that act as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids produced by members of the M. tuberculosis complex and closely related species (17). PDIMs are built upon polyketide scaffolds and comprise multimethyl-branched long-chain mycocerosic acids diesterified with long-chain phthiocerol or phthiodiolone diols (28) (Fig. 1). Additional PDIM variants include the phenol-and glycosylphenol-PDIMs (Fig. 1). Recent work in several laboratories has established that proteins encoded by genes at the M. tuberculosis fadD26-mmpL7 locus (fa...