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. 2011 Sep 6;2(5):e00179-11. doi: 10.1128/mBio.00179-11

FIG 2 .

FIG 2

Correction of the AAGC repeat in P174 results in active transcription of the blp locus and recovery of inhibitory activity. (A) Demonstration of the effect of the 4-bp repeat on the blpA ORF. The sequence starts at nt +433 from the blpA start codon for the predicted translated product. The 4-bp repeat sequence is underlined. Amino acids highlighted in light gray are found in the intact BlpA product; dark gray amino acids are found only in the disrupted BlpA protein. Predicted new stop and start codons resulting from the frameshift mutation are marked by boxes. (B) Diagramatic representation of construction of the on and off reporter strains in P174. P174 transformed with pE65 was selected on kanamycin to recover strains with plasmid integration. pE65 has the 5′ region of the non-repeat-containing blpA gene from P133 followed by divergent promoters driving blpA (PA) and blpI transcription (PI). PI drives lacZ expression. Integration at point 1 results in repair of the disrupted blpA ORF in P174, while integration at point 2 results in retention of the disrupted blpA gene. Promoters are shown with solid arrows signifying the direction of transcription. (C) Phenotypic confirmation of P174on-repaired and P174off demonstrating transcriptional activity on plates containing X-Gal with and without 100 ng/ml of BlpCR6 in strains with the full-length blpA ORF and dependence on exogenous BlpC in the strains with a disrupted blpA ORF. (D) P133 induces blp transcription in the P174off strain when X-Gal is included in the overlay. (E) BlpC6A secretion confirmed in strain 19Ablp133blpC6A using reporter strain P1802 and X-Gal in the overlay. 19Ablp133CR6 was unable to inhibit 19Ablp174 (F), while 19Ablp133C6A showed clear inhibition against 19Ablp174 in the overlay (G). (H) Inhibitory activity was noted against P537 in P174onΔlanC but not in P174offΔlanC.