Walking the fine line between intracellular and membrane activities of antibacterial peptides

Analogs of pyrrhocoricin, a proline-rich antibacterial peptide with a potential therapeutic use, show multiple actions on bacterial cells. We used a dual-fluorochrome membrane viability assay to provide evidence that the lead drug candidate, Pip-pyrr-MeArg dimer derivative, kills bacteria better than the native peptide due to an improved activity on bacterial membranes. This assay was also instrumental in documenting that activity on bacterial membranes and toxicity to human cells can be correlated, and the predominant mode of action can be changed from intracellular DnaK inhibition to membrane disintegration. Similar analyses with an alanine-scan on pyrrhocoricin identified Lys3 as a crucial player to interaction with bacterial membranes, three prolines in mid-chain position as being responsible for maintaining structural integrity and Asp2, Tyr6, Leu7, and Arg9 as putative contact points to the D-E helix of the bacterial target protein DnaK.     

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.