Cloning, Sequencing, and Characterization of the Iturin A Operon

Abstract

Bacillus subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames, ituD, ituA, ituB, and ituC. The ituD gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene, ituA, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene, ituB, and the fourth gene, ituC, encode 609-and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by B. subtilis ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are D-Ser3L-Asn, while in iturin A these amino acids are inverted (i.e., D-Asn3L-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that ituD, ituA, and ituB have high levels of homology to the counterpart genes fenF (79%), mycA (79%), and mycB (79%), respectively. Although the overall level of homology of the amino acid sequences encoded by ituC and mycC, the counterpart of ituC, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of xynD of B. subtilis 168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of xynD imply that horizontal transfer of this operon occurred. When the promoter was replaced by the repU promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.Many Bacillus subtilis strains produce a small peptide(s) with a long fatty moiety, the so-called lipopeptide antibiotics. The peptide portions of these compounds contain ␣-amino acids with a D configuration and are produced nonribosomally with templates of the multifunctional peptide synthetases. As in the synthesis of the peptide antibiotic gramicidin S, the peptide chain grows in a defined sequence by moving on the template of the multifunctional peptide synthetase (18,35,44). On the basis of the structural relationships, the lipopeptides that have been identified in B. subtilis are generally classified into three groups: the surfactin group (27), the plipastatin-fengycin group (16,41,42), and the iturin group (17). The members of the surfactin and plipastatin-fengycin groups are ...

Isolation, Structural Characterization, and Properties of Mattacin (Polymyxin M), a Cyclic Peptide Antibiotic Produced byPaenibacillus kobensis M

“…This is not consistent with other studies using Tn917 for iturin and fengycin biosynthesis genes (40,41). In those studies, the transposon disrupted the nonribosomal peptide synthetase genes and blocked the antimicrobial peptide synthesis completely.…”

Structural analysis of a non-ribosomal halogenated cyclic peptide and its putative operon from Microcystis: implications for evolution of cyanopeptolins

“…according to the amino acid activated) (Challis et al, 2000). For Bacillus subtilis it has been proposed that LGT of large NRPS gene clusters between strains is responsible for the very similar gene clusters present (Tsuge et al, 2001). Investigating the sequence variation in mcy genes of various Microcystis strains, Mikalsen et al (2003) suggested that frequent recombinations between imperfect repeats (i.e.…”

Iturin A is the principal inhibitor in the biocontrol activity ofBacillus amyloliquefaciensPPCB004 against postharvest fungal pathogens

“…Bacillus amyloliquefaciens is closely related to B. subtilis of which several strains have been reported to be effective in the control of plant pathogens (Asaka and Shoda 1996;Yoshida et al 2001;Yu et al 2002) with the production of antibiotics in the lipopeptide family (Yu et al 2002;Koumoutsi et alAntibiotics in the family iturin, represented by iturin A, mycosubtilin and bacillomycin are most commonly studied for their biocontrol activity. These are heptapeptides with a b-amino fatty acid that exhibit strong antifungal activity (Thimon et al 1995;Duitman et al 1999;Tsuge et al 2001;Moyne et al 2004). Iturin A specifically shows a strong antibiotic activity with a broad antifungal spectrum, making it an ideal potential biological control agent with the aim of reducing the use of chemical pesticides in agriculture (Hsieh et al 2008).…”