Figure 2.

Transcriptional pattern of the M. bovis BCG orf60K-parB region. (A): Schematic representation of the M. bovis BCG orf60K-parB region showing the position of the transcriptional start sites (TSSs). The parS sequences are represented by solid grey rectangles. Cotranscripts identified by RT-qPCR are shown as horizontal bold arrows. TSSs are showed as bent arrows. The position of the TSSs mapped are in parenthesis and it localization is related to the start of the gene immediately downstream. (B): Transcriptional fusions to gfp and measurement of the fluorescence emission. Recombinant plasmids were obtained by cloning of PCR fragments (white rectangles) upstream of the gfp. The coordinates (5' and 3' ends with respect to the start codon of the gene being evaluated), of the cloned fragments are shown in parenthesis together with the plasmid name. The length (in bp) of the cloned fragments is indicated within the white rectangles and the grey arrows represent the cloning direction and the gfp gene. Promoter activity was measured by fluorimetry as Relative Fluorescent Units (RFU) in M. smegmatis corrected by subtracting pFPV27 mediated background fluorescence.The bars on the graphic represent RFU (means ± SE of at least three independently experiments) during stationary phase of growth. (C): Mapping of the mRNA 5' termini on the jag-gidB-parA-parB region of M. bovis BCG by primer extension. The mRNA 5'-ends or TSSs using specific oligos are indicated (T1jag, transcription start site for the promoter 1 of gene jag, etc.). Sequencing reaction with the same primers is shown alongside. The ParA1B primer was annealed to total RNA at 48°C. The highlighted boxed region defines the -35 and -10 promoter sequences identified upstream of each TSS; the numbers in parenthesis indicate the position to the TSS according to the start codon of the gene locate immediately downstream. Start codon for jag, gidB, parA and parB is shown in bold and the putative parS sequence located upstream gidB is highlighted with grey.