elgii B69, in which at least 5 NRPS-related
biosynthetic gene clusters were found within its 7,981,270 bp long scaffold . Further inspection revealed that several NRPS genes located in scaffolds 3 and 43 were probably related with pelgipeptin biosynthesis. The gaps between and within these two scaffolds were filled by sequencing PCR products. These efforts resulted in a complete NRPS gene cluster (plp), harbouring eight open reading frames (ORFs), which could be assigned to pelgipeptin biosynthesis. These ORFs (designated plpA-plpH) were transcribed in the same direction (Figure1B). Upstream of the plp locus, two genes (ORF2 and ORF3) encoding proteins with similarities to heparinase II/III family proteins
learn more (YP_003243728 and YP_003243727, respectively) were transcribed in the same direction and were considered not to be involved in pelgipeptin production. Further upstream, a third ORF (ORF1), with TGA stop codon within ORF2, was found to encode a protein with high similarity to short-chain dehydrogenases/reductases (ZP_08509633) and was also considered not involved in the pelgipeptin biosynthesis. Downstream of the plpF gene, four genes encoding putative ABC transporter proteins were found. PlpG and PlpH, shared 72% and 69% identities with PmxC and PmxD, respectively, which were considered MM-102 responsible for the secretion of polymyxin produced by P. polymyxa. This transport activity may be needed for the transport of pelgipeptin out of the cell, Thiamet G and therefore, the gene products were attributed to pelgipeptin biosynthesis. The other two genes (ORF4 and ORF5) encoding putative nitrate/sulphonate/bicarbonate ABC transporter proteins were transcribed in
the opposite direction and were considered less likely to be involved in pelgipeptin production, although further evidence will be required before this can be decided unequivocally. The putative ORFs and the genetic organisation of the chromosomal region containing these sequences are depicted in Figure1B. Genes encoding NRPS As shown in Figure1B, three NRPS genes, plpD plpE, and plpF, are present in the plp cluster, and these genes encode proteins with estimated molecular masses of 171.8, 951.3, and 122.9 kDa, respectively. The modules and selleck compound domains of pelgipeptin synthetase were analysed as described in the “Materials and methods” section above. PlpD, containing four domains (C-A-T-C) (Figure1B), had an N-terminal C domain, which shared 43% identity with the starter C domain of PmxE . The amino acid predicted specific for the A domain of PlpD was 2,4-diaminobutyric acid (Dab) (Table1). The presence of a starter C domain in PlpD, and the specificity of the module for Dab are both consistent with this module providing the first amino acid of the pelgipeptin peptide, and therefore the fatty acid side chain should be connected to the peptide at this residue .