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Journal of Bacteriology, November 2001, p. 6265-6273, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6265-6273.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cloning, Sequencing, and Characterization of the Iturin A
Operon
Kenji
Tsuge,
Takanori
Akiyama, and
Makoto
Shoda*
Chemical Resources Laboratory, Tokyo
Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama
226-8503, Japan
Received 19 March 2001/Accepted 7 August 2001
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-Ser
L-Asn, while in iturin A these
amino acids are inverted (i.e., D-Asn
L-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.
*
Corresponding author. Mailing address: Chemical
Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta,
Midori-ku, Yokohama 226-8503, Japan. Phone: 81-45-924-5274. Fax:
81-45-924-5276. E-mail: mshoda{at}res.titech.ac.jp.
Journal of Bacteriology, November 2001, p. 6265-6273, Vol. 183, No. 21
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6265-6273.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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