More recently, it was shown that the activity of PR-AMPs, including PR-39 was reduced when mutations were formed in the sbmA gene of E. coli. This gene is predicted to encode a component of an inner membrane transporter belonging to the ATP-binding-cassette superfamily of transporter proteins. It is hypothesized that this transporter is used to translocate PR-39 over the bacterial membrane. In support of this hypothesis, it was shown for Bac7, that the peptide was inactive against E. coli and S. enteritidis when the ATP-dependent transporter was inactivated through the use of the metabolic uncoupler DNP. Interestingly, smbA has been identified in several Gram-negative bacteria but not in Pseudomonas aeruginosa, a bacterial strain relatively resistant to PR-AMPs. In addition smbA has not been described in Gram-positive bacteria, which are generally also considered less susceptible to PR-39 and other PR-AMPs. After energy dependent uptake, PR-AMPs bind to DnaK as their intracellular target. DnaK belongs to the HSP70 family of chaperone proteins and binding of peptide interferes with normal protein folding in bacterial cells. For oncocin, an activityoptimized PR-AMP, it was shown that it binds with its N-terminal residues PPYLPR to the substrate binding site of DnaK. Interestingly PR-39��s N-terminal sequence contains an identical motif at the N-terminus indicating that it would bind similarly to DnaK. However, despite the binding evidence pointing towards a DnaK inhibiting function of PRAMPs, DnaK deficient E. coli was found equally susceptible as wild type E. coli to Bac7. This indicates that other unidentified targets apart from DnaK could be involved in the bactericidal mode of action of PR-AMPs. In our HBB2 experimental set-up, we observed some contrasting results to the general hypothesis on the antimicrobial working mechanism of PR-39. Firstly, our PR-39 peptides are as active against Gram positive CID 5380390 strains as against Gram negative bacteria. In addition we did not observe an effect of metabolic uncouplers DNP or CCCP on PR-39 activity, and finally we showed fast ATP leakage and loss of membrane potential upon incubation with PR-39.