Molecular identification of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector

Three new Acetobacter strains were isolated from vinegar. By plasmid profil ing they were recognized as genotypically different from each other. Sequen cing of the genes for 16S and 23S rRNA and DNA-DNA hybridization of total D NA against DNA of all type strains of Acetobacter identified Acetobacter st rains JK2 and V3 as A. europaeus, and Acetobacter strain JK3 as A. intermed ius. In contrast to the type strain of A. europaeus (DSM 6160), A. europaeu s JK2 and V3 do not require acetic acid for growth and can be successfully transferred between media with and without acetic acid. This phenotypic cha racteristic enables convenient handling of both strains in genetic studies. Plasmid pJK2-1 from A. europaeus JK2 was used as the basis for shuttle pla smid construction with the aim of developing an efficient vector system for these strains. The entire nucleotide sequence of pJK2-1 was determined. Hi gh amino acid identities were found for three open reading frames: Rep (rep lication protein); Dinj1 (DNA damage inducible enzyme); and Dinj2 proteins. A recombinant plasmid pUCJK2-1 (5.6 kb) consisting of the entire plasmid p JK2-1 and the entire plasmid pUC18 was successfully used in transformation experiments. Plasmid pJT2 (5.8 kb) was constructed from pUCJK2-1 with the a im of reactivating the lacZ' gene.