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Applied and Environmental Microbiology, December 2004, p. 7466-7473, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7466-7473.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cyclo(L-Leucyl-L-Prolyl) Produced by Achromobacter xylosoxidans Inhibits Aflatoxin Production by Aspergillus parasiticus

Pei-Sheng Yan,^1, Yuan Song,^1, Emi Sakuno,² Hiromitsu Nakajima,² Hiroyuki Nakagawa,¹ and Kimiko Yabe^1*

National Food Research Institute, Tsukuba, Ibaraki,¹ Faculty of Agriculture, Tottori University, Koyama, Tottori, Japan²

Received 15 March 2004/ Accepted 5 August 2004

Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(L-leucyl-L-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml^–1, as determined by the tip culture method. High concentrations (more than 6.0 mg ml^–1) of cyclo(L-leucyl-L-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(D-leucyl-D-prolyl) and cyclo(L-valyl-L-prolyl), whereas cyclo(D-prolyl-L-leucyl) and cyclo(L-prolyl-D-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(L-leucyl-L-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production.

Present address: Laiyang Agricultural University, Laiyang, Shandong, People's Republic of China.

Present address: College of Biological Science, China Agricultural University, Beijing, People's Republic of China.

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