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Journal of Bacteriology, March 1999, p. 1953-1957, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification and Cloning of an Erwinia carotovora subsp. carotovora Bacteriocin Regulator Gene by Insertional Mutagenesis

Duen-Yau Chuang,¹ Ampaabeng G. Kyeremeh,¹ Yuichi Gunji,^1, Yoshiyuki Takahara,² Yoshio Ehara,³ and Toshio Kikumoto^1,*

Institute of Genetic Ecology, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577,¹ Central Glass Co. Ltd., Chemical Research Center, 2805 Imafuku-nakadai, Kawagoe, Saitama 350-1151,² and Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555,³ Japan

Received 6 August 1998/Accepted 8 January 1999

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Q-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.

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^* Corresponding author. Mailing address: Institute of Genetic Ecology, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Phone: (022) 217-5682. Fax: (022) 263-9845. E-mail: kikumoto{at}bansui.ige.tohoku.ac.jp.

Present address: Kantounousan Co. Ltd., Nasu-machi, Nasu-gun 325-0001, Japan.

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Journal of Bacteriology, March 1999, p. 1953-1957, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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