Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis

Avirulent Erwinia carotovora subsp, carotovora CGE234-M403 produces two typ es of bacteriocin. For the purpose of cloning the bacteriocin genes of stra in CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-ri f-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mu tant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric i nterlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA seq uence of a contiguous 1,280-bp region were determined. One complete open re ading frame (ORF), designated ORF2, was identified within the sequenced fra gment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A non coding region and a putative promoter were located between ORF1 and ORF2. D ownstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 am ino acids, which showed high homology with Yersinia enterocolitica Yrp, a r egulator of enterotoxin CY-ST) production; Escherichia coli host factor 1, required for Q(beta)-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, w ere transferred into E. coli DH5, Plasmid pBYL1 was reisolated and transfer red into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-we ight bacteriocin.