Non-ribosomal peptide synthetases (NRPS) systems constitute a yet underexplored source of natural products, but genomics-based approaches open up new opportunities for a more efficient and rational exploration. Albicidin and cystobactamid are potent DNA gyrase inhibitors exhibiting very similar structures. They are produced by the sugarcane pathogenic bacterium Xanthomonas albilineans and the soi! bacterium Cystobacter sp., respectively. Their biosynthesis involves identical families of NRPS genes that have never been described to date in any other biosynthesis pathway. For example, adenylation domains exhibiting unusual specificity-conferring signatures activate para-aminobenzoate (pABA). Likewise, an asparagineactivating adenylation domain exhibiting an unusual 342-amino-acid insertion which harbors the ATP-binding motif SGGKD was proposed to be linked to the incorporation of cyanoalanine in albicidin. A genome-based survey was performed with bacterial sequences available on GenBank in order to identify new NRPS loci sharing features with albicidin and cystobactamid loci. As a result, 13 different loci encoding an adenylation domain containing the above-described 342- amino-acid insertion were identified in the genera Arenibacter, Algoriphagus, Candidatus, Clostridium, Leptolyngbya, Microvirga, Paenibacillus, Roseobacter, Ruegeria, Verrucomicrobiae and lobe/lia, respectively. Additionally, three different hitherto unknown NRPS loci encoding a pABA-activating adenylation domain were identified in the genera Actinoplanes, Janthinobacterium and Xanthomonas, respectively. Comparative analyses provide in silico insights on the functional organization of these newly discovered NRPS domains. Additionally, annotation of the unknown NRPS loci identified in this study indicates that they are involved in biosynthesis of entirely different molecules and are therefore promising candidates for further experimental studies aiming at discovering new natural products. (Texte intég