Signal Transduction by a Death Signal Peptide Rodger Novak, Emmanuelle Charpentier, Johann S. Braun, Elaine Tuomanen  Molecular Cell  Volume 5, Issue 1, Pages 49-57 (January 2000) DOI: 10.1016/S1097-2765(00)80402-5

Figure 1 Schematic of the vex, pep, and vnc Loci Organization of the pneumococcal gene loci encoding the putative ABC transporter Vex, the peptide Pep27, and the two-component regulatory system VncR–VncS. Putative promoters of the ABC transporter Vex (indicated by Pvex1 and Pvex3) are at positions −26 and 1930. Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)

Figure 2 Secretion of Pep27 (A) Subcellular localization of Pep27. Immunoblot analysis of Pep27 using polyclonal anti-Pep27 antibodies (1:2000). Pep27 was detected at 3 kDa in the cytoplasm and the supernatant of R6. Pep27 was nondetectable in the supernatant of the loss-of-function mutants vex1 or vex3. (B) Immunoblot analysis of RecA. The cytoplasmic protein RecA was nondetectable in the supernatants of the parent strain R6 and the loss-of-function mutants vex1 and vex3. Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)

Figure 3 Bactericidal Activity of Pep27 (A) Effect of the synthetic peptide Pep27 on viability: parent strain R6, loss-of-function mutant vncS, mutant vncR, mutant vex1, and mutant vex3. Cultures in the early exponential phase of growth (106 cfu/ml) were treated with 0.1 mM of Pep27, and viability was determined at 1 hr. (B) Dose-dependent effect of Pep27 on the growth of R6. Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)

Figure 4 Activity of Pep27 in the Absence of Autolysin Effect of synthetic Pep27 (100 μM) on the morphology of the autolysin-deficient strain Lyt-4-4 (A) without peptide and (B) with peptide. Lyt-4-4 grows in chains with a length of up to 30–40 pneumococci. Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)

Figure 5 Requirements for Bactericidal Activity of Pep27 (A) Effect of loss of function of vex3 and vncS on bactericidal activity of vancomycin. Parent strain R6 (square), vex3 mutant (circle), and vncS mutant (diamond). Cultures in the early exponential phase of growth (107 cfu/ml) were treated with 10 × MIC of vancomycin (5 μg/ml), and bacterial viability was followed for 6 hr. (B) Western blot analysis of autolysin preparations of parent strain R6, and the loss-of-function mutants vncS, vncR, vex3, and vex1. A polyclonal anti-autolysin (LytA) antibody (1:1000) was used. (C) Functional assay of autolytic activity. Autolysin preparations of parent R6 (square), loss-of-function mutant vex3 (diamond), and vex1 (circle) were added to cultures of the autolysis-defective strain Lyt-4-4 at an OD620nm of 0.3. 10 × MIC of penicillin (0.1 μg/ml) was added to all samples, and lysis was followed over 6 hr. Lyt-4-4 served as a negative control (triangle) (addition of penicillin without autolysin). Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)

Figure 6 Bactericidal Activity of Pep27 during the Stringent Response Activity of Pep27 in nongrowing bacteria. Inhibition of protein synthesis of R6 (diamond) by leucine deprivation results in growth arrest. The effect of addition of 10 × MIC of penicillin (circle) (0.1 μg/ml), 100 μM peptide Pep27 (square), and a combination of penicillin and Pep27 (triangle) to the leucine-deprived R6 was monitored over 6 hr. Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)

Figure 7 The Relationship of VncS Expression to Autolysis Effect of overproduction of VncS on morphology and autolysis. (A) The strain ΔvncS(pMU1328ωcom AP-vncS) grows like the parent strain R6: diplococci or short chains with up to 6–8 pneumococci (magnification 1000×). (B) Induction of transcription of vncS leads to chain formation in the ΔvncS(pMU1328ωcom AP-vncS) strain (magnification 1000×). Molecular Cell 2000 5, 49-57DOI: (10.1016/S1097-2765(00)80402-5)