A C B Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 Page 7 of 19 Microorganism Stenotrophomonus sp. LZ-1 E. coli ATCC 25922 Lethal conc. (CL) (µM) 1.52 (y = 0.7048x + 1.0129, R2 = 0.9988) 15.4 (y = 0.75x + 0.3033, R2 = 0.9805) Figure 4 Purification and characterization of nasonin-1. A. RP-HPLC showing retention time difference between the reduced and refolded products; B. MALDI-TOF MS of the refolded nasonin-1; C. Antibacterial activities of nasonin-1. deletion of 31 amino acids in each repeat. Similarly, a conserved phase 0 intron is also present in the amino- termini of bee and parasitoid hymenoptaecins, suggest- ing that these peptides diverged from a common ances- tor after speciation. Although hymenoptaecins are believed to be only pre- sent in Hymenoptera, our study reveals that similar peptides were also evolved by Dipteran Drosophila (Figure 6A). These fly-derived peptides display a chi- meric characteristic because they have an acidic propep- tides, as bee hymenoptaecins, but their mature peptides more resemble the repeat of nahymenoptaecin-2 in size. Moreover, Drosophila hymenoptaecins have lost their introns, consistent with the lineage-specific intron loss in these species [61]. All Drosophila hymenoptaecins clustering together in the tree (Figure 6B) indicates their monophyletic origin after separation of Diptera from Hymenoptera. As a unique region of nahymenoptaecin-2, the amino- terminal pronavicin shares detectable sequence identity (25-41.2%) to other known Pro-rich AMPs from insects such as drosocin [62], apidaecin [12] and formaecin [63] (Figure 6C). Thus we synthesized this peptide to verify its antibacterial activity. Results showed that pronavicin was able to inhibit the growth of all Gram-positive and Gram-negative bacteria used here with differential potency. It displayed the strongest inhibitory effect on the Gram-positive bacterium Bacillus megaterium (CL = 3.11 μM) (Table 1). At high micromolar concen- trations this peptide did not affect fungi and yeasts. Tachystatin-type AMPs ICK fold is an evolutionarily conserved structural motif shared by a large group of polypeptides with diverse sequences and bioactivities. For example, venomous ani- mals (e.g. spider and scorpion) usually employed this fold to develop their neurotoxins affecting channels’ functions [64,65] while horseshoe crabs, insects and some plants use ICK peptides as a part of their antibac- terial defense system [66-69]. Peptides belonging to this fold type exhibit antibacterial (plant ICK peptides), anti- fungal (tachystatin and Alo-3), and chitin-binding (tachystatin-A) activities. Three peptides (named naickin-1, -2 and -3) with the ICK structure were char- acterized for the first time from the N. vitripennis gen- ome that all share sequence similarity to an ICK peptide from A. mellifera (named amickin-1). All four peptides have a typical ICK cysteine arrangement pattern with a unique extended carboxyl-terminus rich in proline and positively charged residues. A phase 0 intron preceding the second cysteine is conserved between naickins and amickin-1 (Figure 7A), suggesting their homologous relationship. A typical cystine knot stabilized by three disulfide bridges can be observed in all these peptides, as revealed by comparative modeling using tachystatin- B1 [70] as a template. Figure 7B presents the model structure of naickin-1 (residues T2 to T40) which is