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Fig. 6 | BMC Microbiology

Fig. 6

From: Expression and evolutionary patterns of mycobacteriophage D29 and its temperate close relatives

Fig. 6

Toxicity of the highly expressed Pleft locus. a. At the top is shown the RNAseq profile of the L5 lysogen at the right end of the genome. Below are indicated the regions targeted for deletion using BRED (Δ1, Δ2, Δ3), only one of which (Δ3, green) could be constructed. Below that are segments of L5 cloned into several plasmids (pTM series), which are aligned with the genomes below. Horizontal dashed lines indicate large deleted segments, triangles indicate single nucleotide mutations, the star indicates a single nucleotide deletion, and the faded region in pTM9 reflects incomplete sequence verification of this locus. Constructs also contain the cloned L5 temperature-sensitive repressor and integration locus, which are not shown. To the right are shown ten-fold serial dilutions of M. smegmatis cultures carrying these plasmids, at 30 °C and 44 °C, grown for four days with kanamycin selection. Genome alignments show the positions of predicted genes (yellow arrows), stoperator and operator sites [with corresponding site numbers (Brown et al., 1997)], the L5 Pleft transcriptional start site (arrow) and promoter elements, and the highly expressed regions from the RNAseq data (blue arrow). Black lines indicate nucleotide sequence with alignment gaps, and the conservation track indicates levels of nucleotide identity across this locus. The size and position of probes used for Northern blots in panel B are labeled below (A-G). b. Northern blots were run with a positive control of denatured L5 end dsDNA (Northern probe A) (lane 1), RNA isolated from 30 min post induction (lane 2), 150 min post induction (lane 3), and the uninduced L5 thermo-inducible lysogen (lane 4), as well as an uninfected wildtype mc2155 control (lane 5). The probe used for each blot is represented by a letter at the top of each blot and correspond to the lines located at the bottom of the conservation profile which are similarly labeled in panel A. The cropped image of the ethidium bromide stained ssRNA ladder from the denaturing PAGE gel, prior to transfer of the nucleic acids to the membrane, is located at the extreme left of the panel (M)

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