ORIGINAL PAPER J. TrcÏek á P. Raspor á M. Teuber Molecular identi®cation of Acetobacter isolates from submerged vinegar production, sequence analysis of plasmid pJK2-1 and application in the development of a cloning vector Received: 14 June 1999 / Revision received: 27 August 1999 / Accepted: 17 September 1999 Abstract Three new Acetobacter strains were isolated from vinegar. By plasmid pro®ling they were recog- nized as genotypically dierent from each other. Sequencing of the genes for 16S and 23S rRNA and DNA±DNA hybridization of total DNA against DNA of all type strains of Acetobacter identi®ed Acetobacter strains JK2 and V3 as A. europaeus, and Acetobacter strain JK3 as A. intermedius. In contrast to the type strain of A. europaeus (DSM 6160), A. europaeus JK2 and V3 do not require acetic acid for growth and can be successfully transferred between media with and without acetic acid. This phenotypic characteristic en- ables convenient handling of both strains in genetic studies. Plasmid pJK2-1 from A. europaeus JK2 was used as the basis for shuttle plasmid construction with the aim of developing an ecient vector system for these strains. The entire nucleotide sequence of pJK2-1 was determined. High amino acid identities were found for three open reading frames: Rep (replication pro- tein); Dinj1 (DNA damage inducible enzyme); and Dinj2 proteins. A recombinant plasmid pUCJK2-1 (5.6 kb) consisting of the entire plasmid pJK2-1 and the entire plasmid pUC18 was successfully used in transformation experiments. Plasmid pJT2 (5.8 kb) was constructed from pUCJK2-1 with the aim of reacti- vating the lacZ¢ gene. Introduction Processes which enable the production of vinegar with high productivity are performed by submerged technol- ogy in acetators (Ebner 1982). Sievers et al. (1992) isolated and described the new species Acetobacter europaeus as a main component of the micro¯ora in that kind of industrial bioreactor in Europe. The type strain of A. europaeus and all investigated strains from Ger- man and Swiss vinegar factories have a strict require- ment of acetic acid for growth (Sievers et al. 1992). Lately, the new species A. intermedius was isolated from Kombucha beverage as well as from industrial acetators and described (Boesch et al. 1998). A. intermedius does not need acetic acid for growth (Boesch et al. 1998). The productivity and maximum concentration of acetic acid was reported to be increased 2- and 1.4-fold by transformation of A. aceti subsp. xylinus with a multi-copy recombinant plasmid harboring an aldehyde dehydrogenase gene from A. polyoxogenes (Fukaya et al. 1989). However, the maximum concentration of acetic acid produced by transformants was improved only to 96.6 g/l, which is lower than the natural pro- ductivity (139 g/l) of A. europaeus (Fukaya et al. 1989; Sievers and Teuber 1995). Therefore, this technological parameter could be improved additionally by multiply- ing the aldehyde dehydrogenase gene in A. europaeus. For such experiments a vector system is needed for introducing genes into A. europaeus. Vectors are also prerequisite tools for studying resistance to acetic acid and phages, both important features of the vinegar- producing strains. No vector systems for A. europaeus or A. intermedius are known. A. europaeus strains JK2, V3 and A. inter- medius JK3, all described in this work, are easily culti- vated without acetic acid, can be successfully introduced back into media with acetic acid and ethanol, and pos- sess plasmids which can be used for development of plasmid vectors. Therefore the aim of this work was to develop a transformation procedure and to construct Appl Microbiol Biotechnol (2000) 53: 289±295 Ó Springer-Verlag 2000 J. TrcÏek á P. Raspor (&) Biotechnical Faculty, Department of Food Science and Technology, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, Slovenia e-mail: Peter.Raspor@bf.uni-lj.si Tel.: +386-61-1231161 Fax: +386-61-274092 M. Teuber Laboratory of Food Microbiology, Department of Food Science, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zurich, Switzerland