In this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceae family. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia coli strains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. In a previous study, we have shown that the unique posttranslational modification of MccE492 consists of a C-glucosylated linear trimer of N-(2,3 dihydroxybenzoyl)-L-serine (DHBS) linked to the C-terminal serine carboxylate (28). This modification is reminiscent of the catecholate siderophores, especially of salmochelin S4. Salmochelin S4 is derived from enterobactin (Ent), the cyclic trimer of DHBS, by addition of two -D-glucose (Glc) moieties linked to the DHBS units through C-glucosidic bonds (1). Moreover, we have previously shown that Ent is a precursor for the biosynthesis of MccE492 posttranslational modification (29). Siderophores are generally synthesized by the nonribosomal pathway. Indeed, the Ent gene cluster is necessary for Ent biosynthesis, and genes in the iroA locus are required for the conversion of Ent into salmochelins (5, 16). Consequently, siderophore-microcin MccE492 can be considered to be both ribosomally (peptide moiety) and nonribosomally (enterobactin moiety) synthesized, with glucose being the linker between the two moieties. Nonetheless, MccE492 can also be isolated from bacterial culture supernatants as an unmodified peptide, which lacks the siderophore moiety (u-MccE492), and intermediate forms , which were shown to be degradation products (29).Ten genes (mceABCDEFGHIJ) located on the bacterial chromosome of Klebsiella pneumoniae RYC492 (Fig. 1A) are required for MccE492 production, export, and posttranslational modification biosynthesis and the immunity of the producing strain (18), with four genes (mceC, mceD, mceI, and mceJ) being necessary for the acquisition of the siderophore moiety (22,29). MceC and MceD are responsible for the C glucosylation of Ent and the cleavage of one of the trilactone ester bonds of Glc-Ent, respectively. The MceIJ complex is required for attachment of the siderophore moiety (linear GlcEnt) to the C-terminal serine of the microcin precursor via an ester bond (23). The resulting posttranslational modification (siderophore moiety) enhances the antibacterial activity of MccE492, which is mainly directed against Escherichia coli, and MccE492 targets bacteria via the catecholate siderophore receptors (6, 28).
Anatoxin-a and homoanatoxin-a are potent neurotoxins produced by cyanobacteria such as Oscillatoria PCC 6506. Sequencing of the genome of this strain is underway, and we have identified a 29 kb DNA fragment containing a sequence called ks2 that we previously showed to be specific to Oscillatoria cyanobacteria producing anatoxin-a and homoanatoxin-a. Bioinformatic analysis of this 29 kb fragment revealed a cluster of genes, which were annotated. The function assigned to the products of eight contiguous genes, from anaA to anaH, provides a clue to the biosynthesis of anatoxin-a and homoanatoxin-a. Proline is first loaded on an acyl carrier protein and its five-membered cycle oxidized to the pyrroline oxidation state. This activated ring is then successively loaded on three polyketide synthase modules for elongation, reduction, cyclization, and methylation. The final step is the hydrolysis of the thioester with subsequent decarboxylation. GC-MS and NMR analyses of homoanatoxin-a produced by PCC 6506 using labeled precursors confirm that proline is very likely the starter of these polyketide synthases. Using specific PCR amplifications, we have also shown that the anaC, anaE, anaF, and anaG genes are always present in the genome of cyanobacteria producing anatoxin-a and homoanatoxin-a and absent in nonproducing strains. Histidine-tagged AnaC was purified to homogeneity and showed to catalyze the loading of proline on purified histidine-tagged AnaD that had been previously transformed into its holo form using the Bacillus subtilis Sfp phosphopantetheinyl transferase. All of these data provide strong evidence that we have successfully identified the gene cluster responsible for the production of anatoxin-a and homoanatoxin-a in Oscillatoria PCC 6506.
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