Table 3.
Assessing if Antimicrobial peptides (AMPs) fused to gpD can complement for lambda infectivity and if the expression of the construct is toxic to E. coli cells.
| Strain 594[pcIpR-D-fusion-timm] Plasmid Construct a | Relative Culture Viability at 42 °C (D-fusion Toxicity) b | EOP c of λimm434 Dam123 at 42 °C on Assay Strain d |
|---|---|---|
| p613: Dcoe | 0.65 | 0.56 |
| p613*: Dcoe- (67 bp deletion within D) e | 0.79 | <0.0001 |
| p614: His-Dcoe (11AA N-terminal) | 0.09 | <0.0001 f |
| p614*: His- Dcoe-:*(Pro48Glu mutation in D) | 0.87 | <0.0001 |
| p615: Dcoe-HNP1 (35AA C-term.) | 0.83 | <0.0001 f |
| p626: HNP1- Dcoe (36AA N-term.) | 1.00 | <0.0001 f |
| p616: Dcoe-HBD3 (50AA C-term.) | <0.0001 | <0.0005 |
| p624: HBD3- Dcoe (53AA N-term.) | 0.78 | <0.0001 f |
| p627: Dcoe-LL37 (45AA C-term.) | 0.0002 | g |
| p617: LL37-Dcoe (46AA N-term.) | 0.93 | <0.0001 f |
| p618: Dcoe-DEFB126-Δ (84AA C-term.) | 0.0001 | g |
| p622: DEFB126-ΔC- Dcoe (85AA N-term.) | 0.85 | <0.0001 f |
| p619: His-TAGZ-Dcoe-TEV-LL37 (11AA N-, 55AA C-term.) | 0.0003 | <0.000 1f |
| p620: LL37-TEV- Dcoe-His (50AA N-, 12AA C-term.) | 0.87 | <0.0001 f |
| p628: Dcoe-HD5 (40AA C-term.) | 0.0034 | <0.0001 f |
| p621: HD5- Dcoe (41AA N-term.) | 0.77 | <0.0001 f |
| p625: Dcoe-PR39 (48AA C-term.) | <0.0001 | g |
| p623: PR39-Dcoe (49AA N-term.) | 0.97 | <0.0001 f |
| p674: Dcoe-YML (17AA C-term.) | 0.05 | ≥0.90 h |
| p675: D(wild type)-YML (17AA C-term.) | 0.88 | 0.93 |
| p676: YML-Dcoe (15AA N-term.) | 1.00 | 0.06 |
a The AMPs include α- and β-defensins and cathelicicins, with construction details indicated in Table 1 and in Methods. The amino acid fusions were made either at NH2-, or COOH-terminal end of D. b Efficiency of plating (EOP) of the 594[pcIpR-D-fusion-timm] E. coli cells. Cell titers obtained at 42 °C (where the cloned D-fusion gene is fully expressed) were divided by cell titers for the same strain incubated in parallel at 30 °C, where the D-fusion gene expression is repressed. This assay measures the relative cell viability change upon cellular expression of the D-fusion. c Complementation assay: EOP of a lysate of phage λimm434Dam123 with a conditional nonsense mutation in D plated on assay strain 594[pcIpR-D-fusion-timm] host cells grown at 42 °C compared to the plating of the same phage on TC600 cells. The lysate of λimm434-Dam123 was diluted 10−1, 10−2, 10−3, and 10−4 in buffer and 5 µL aliquots of the dilutions were spotted in triplicate on agar overlay plates made with 3.0 mL LB top agar and 0.3 mL of a stationary culture of cells grown up in LB-Amp50. When the spots dried on the plates at room temperature they were inverted and incubated overnight at 41–42 °C. d In parallel assays, plating efficiency was measured to determine if the expressed D-fusion proteins negatively complemented for the ability of D to form infectious phage particles: (i) strain TC600 supE was transformed with each of the plasmids and the plating efficiency of λimm434Dam123 was measured, or (ii) the 594 transformants shown were infected with the D+ phages λimm434cI or λimm434(18,12)P22 (where lambda genes O-P were substituted by genes 18–12 of phage P22). In both (i) and (ii) none of the D-fusions negatively complemented for growth of the imm434 phages on cells induced for expression of the cloned D-fusion gene. e The D sequence in p613* comprises the first 50 AA (codons intact) plus the first base of codon 51, then 87 codons (AAs) of scrambled sequence terminating in TAG stop, so it is 27 AA longer than the encoded 110 AAs of gpD (before the NH2-terminal methionine is removed). f Faint phage lysis spots were seen for the 10−1 dilution spots, but no individual plaques were observed. g There was too much killing of cells in the overlay to determine if the phage could be complemented. h These cells supported high-level complementation to λimm434 Dam123 when agar overlay plates were incubated at 37 °C, 39 °C and 41–42 °C (Table S1); approximating the phage titer on TC600 supE host cells at 39 °C.