Peptide-Based Drug Design

Proline-Rich Antimicrobial Peptides 167
SbmA in peptide’s translocation, we devised a simple cytofluorimetric method
(see Subheadings 3.6 and 3.7) that couples the use of fluorescently-labelled
peptides with an impermeant quencher that can bleach the fluorescence of the
peptide molecules outside or on the surface of bacteria but not of those translocated
into the cytoplasm.
3.1. Set-Up of the PRP-Resistant Selection Conditions
1. Melt the agar medium (see Subheading 2.1) and let it cool in a water bath.
2. When the medium reaches a temperature of about 55◦C, divide it into aliquots and
add in each of them increasing concentrations of the PRP (from 1 to 10 �M inthe
case of Bac7-derived fragments). Stir or shake thoroughly to get a homogenous
peptide distribution.
3. Pour approximately 15–20 mL into a 10 cm Petri dish and let the agar solidify at
room temperature.
4. Plate an overnight suspension of the wild-type HB101 strain.
5. Check the growth of bacteria on the PRP-containing plates.
6. Determine the minimum concentration of the peptide able to completely inhibit
the growth of the wild-type strain. In our case, to inhibit the growth of the HB101
strain, an active fragment of Bac7, Bac7(1-35), had to be added to the plate at
10 �M.
3.2. Generation of E. coli Mutants Resistant to PRP
The following protocol for the isolation of mutants showing an increasing
resistance to PRPs is based on the method of J.H. Miller (16).
1. Collect 120 mL of a mid-log phase culture of E. coli HB101 (∼1 × 10 8 CFU/ml),
wash twice in Buffer 1 by centrifugation at 1000g for 10 min, resuspend the final
pellet in 2 mL of Buffer 1 and place it on ice.
2. Add 50 �L of MNNG stock solution to 1.95 mL of the cell suspension, and 50 �L
of Buffer 1 only to a control sample. Incubate all the samples at 37 ◦ C, with shaking,
for different times.
3. At the end of each incubation time, wash the sample with 5 mL of the Buffer 2 to
remove the mutagen, and resuspend the treated cells in 2 mL of the same buffer.
Plate the 10 −4 and 10 −5 dilutions of each mutagenized sample and the 10 −5 and
10 −6 dilutions of the control on agar medium to check the viability of the treated
cells. Dilute 1:20 each of the mutagenized samples in broth medium and incubate
overnight at 37 ◦ C with mild-shaking.
4. Plate the 10 −6 dilution of each of the overnight cultures, including the control,
on agar plates to monitor the presence of viable cells. To look for rifampicinresistant
(Rif r ) mutants, plate undiluted and a 10 −1 dilution of the control and a 10 −2
dilution for each mutagenized culture on agar plates with rifampicin (100 �g/mL).