The amphibian temporins, amongst the smallest antimicrobial peptides (AMPS), are alpha-helical, amphipathic, hydrophobic and cationic and are active mainly against Gram-positive bacteria but inactive or weakly active against Gram-negative bacteria. Here, we report two novel members of the temporin family, named temporin-1Ee (FLINIAGVLSKLF(amide)) and temporin-1 Re (FLPGLLAGLL(amide)), whose biosynthetic precursor structures were deduced from clones obtained from skin secretion-derived cDNA libraries of the European edible frog, Pelophylax kl. esculentus, by 'shotgun' cloning. Deduction of the molecular masses of each mature processed peptide from respective cloned cDNAs was used to locate respective molecules in reverse-phase HPLC fractions of secretion. Temporin-1Ee (MIC = 10 mu M) and temporin-1Re (MIC = 60 mu M) were both found to be active against Gram-positive Staphylococcus aureus, but retaining a weak haemolytic activity. To our knowledge, Single-site substitutions can dramatically change the spectrum of activity of a given temporin. Compared with temporine-1Ec, just one chemically-conservative substitution (Val8 instead of Leu8), temporin-1 Ee bearing a net charge of +2 displays broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC = 40 mu M). These factors bode well for translating temporins to be potential drug candidates for the design of new and valuable anti-infective agents. (C) 2016 Elsevier Inc. All rights reserved.