Transcriptome analysis of the Tap efflux pump mutant in Mycobacterium bovis BCG.

We used DNA microarrays to define the physiological roles of the Tap efflux pump in M. bovis BCG during the exponential and the stationary phase of in vitro growth. For this purpose we constructed a M. bovis BCG strain in which the tap gene was inactivated by the insertion of a hygromycin resistance cassette (Ω-hyg). When the gene expression patterns of the tap mutant were compared to the wild-type strain, almost no differences were observed during exponential growth; only seven genes slightly increased their expression. In contrast, more that 100 genes showed a variation in their level of expression during stationary growth. More than ten representative genes were chosen from the microarray experiments and their expression was measured by quantitative RT-PCR using sigA as invariant internal control. In support to the gene expression profiling data, the mRNA levels of all selected genes was significantly different in the tap mutant strain relative to control. A functional category analysis (http://tuberculist.epfl.ch/index.html) of the genes differentially expressed revealed a high proportion belonging to the Virulence, Detoxification, Adaptation (VDA), Intermediary Metabolism and Respiration (IMR), Conserved Hypotheticals (CH), and Cell Wall and Cell Processing (CWCP) categories suggesting a major adaptation to a stress generated by inactivation of the tap efflux pump gene. We compared the global gene expression of the tap mutant versus the wild-type strain of M. bovis BCG during the exponential (one week; OD540= 0.2-0.3) and stationary (six weeks; OD540= 0.8-1.0) growth. Hybridizations were performed using RNA extracted from two different biological samples. Each sample was hybridized twice through swap labeling of the respective cDNAs.