Isolation, characterization, and sequence analysis of cryptic plasmids from Acinetobacter calcoaceticus and their use in the construction of Escherichia coli shuttle plasmids

Abstract

Three cryptic plasmids have been discovered in Acinetobacter calcoaceticus BD413. These three plasmids, designated pWM10 (7.4 kb), pWM11 (2.4 kb), and pWM12 (2.2 kb), exhibited extensive homology to one another, as shown by Southern blot hybridization and restriction site analysis data, and also hybridized with three plasmids having slightly different sizes detected in a second strain, A. calcoaceticus BD4. Plasmid pWM11 and a fragment of pWM10 were each subcloned into pUC19, yielding plasmids pWM4 and pWM6, respectively, and were used in a series of inter- and intraspecies transformation experiments. Both plasmids replicated as high-copy-number plasmids in A. calcoaceticus BD413, as well as in strains of Escherichia coli. However, when transformed into the oil-degrading strain Acinetobacter lwoffii RAG-1, both plasmids were maintained at low copy numbers. No modification of the plasmids was detected after repeated transfers between hosts. An analysis of a series of deletions demonstrated that (i) a 185-bp fragment of pWM11 was sufficient to permit replication of the shuttle plasmid in A. calcoaceticus BD413, (ii) the efficiency of transformation of A. calcoaceticus BD413 decreased according to the size of the deletion in the insert by up to 4 orders of magnitude, and (iii) the entire insert was required for transformation and replication in A. lwoffii RAG-1. The sequence of pWM11 contained several small (150- to 300-bp) open reading frames, none of which exhibited any homology to known DNA or protein sequences. In addition, a number of inverted and direct repeats, as well as six copies of the consensus sequence AAAAAAATA previously described for a cryptic plasmid from A. lwoffii (M. Hunger, R. Schmucker, V. Kishan, and W. Hillen, Gene 87:45-51, 1990), were detected. Cloning and expression of the alcohol dehydrogenase regulon from A. lwoffii RAG-1 were accomplished by using the Acinetobacter shuttle plasmid.