Identification of plasmid and Bacillus subtilis chromosomal recombination sites used for pE194 integration

Abstract

The plasmid pE194 (3.7 kilobases) is capable of integrating into the genome of the bacterial host Bacillus subtilis in the absence of the major homology-dependent RecE recombination system. Multiple recombination sites have been identified on both the B. subtilis chromosome and pE194 (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). The B. subtilis chromosomal recombination sites were recovered by genetic cloning, and these sites were studied by nucleotide sequence analysis. Recombination had occurred between regions of short nucleotide homology (6 to 14 base pairs) as indicated by comparison of the plasmid and the host chromosome recombination sites with the crossover sites of the integration products. Recombination between the homologous sequences of the plasmid and the B. subtilis genome produced an integrated pE194 molecule which was bounded by direct repeats of the short homology. These results suggest a recombination model involving a conservative, reciprocal strand exchange between the two recombination sites. A preferred plasmid recombination site was found to occur within a 70-base-pair region which contains a GC-rich dyad symmetry element. Five of seven pE194-integrated strains analyzed had been produced by recombination at different locations within this 70-base-pair interval, located between positions 860 and 930 in pE194. On the basis of these data, mechanisms are discussed to explain the recombinational integration of pE194.Several intramolecular and intermolecular genetic recombination events which do not require extensive nucleotide sequence homology have been described for Bacillus subtilis (18,23,25,34). These rare events are independent of the major homologous DNA recombination system, the RecE pathway, of B. subtilis and produce stable rearrangements in the cellular genetic material. Often these genetic rearrangements are accompanied by changes in the phenotype of the host cell and provide a means of evolving new traits, such as the stable acquisition of antibiotic resistance determinants. The integration of plasmid pE194 into the B. subtilis genome (19) represents an example of recE-independent intermolecular recombination. Integration of pE194 places the ermC antibiotic resistance determinant into the B. subtilis chromosome. This gene is subsequently inherited as a chromosomal marker.pE194 is a 3.7-kilobase (kb) multicopy plasmid, originally isolated from Staphylococcus alureus (22), that was stably introduced into B. slubtilis by genetic transformation (13). The entire nucleotide sequence is known for pE194 (20). The ermC gene encodes an inducible 29-kilodalton product which is responsible for mediating resistance to the macrolidelincosamide-streptogramin B (MLS) group of antibiotics (45). pE194 is known to be naturally temperature sensitive for replication (39), being unable to replicate autonomously at growth temperatures exceeding 43°C. The plasmid-encoded trans-acting RepF protein is required for initiation of pE194 replication (16,44), which is tho...

Sequence specificity of Bacillus subtilis DNA gyrase in vivo

“…Currently, much has become known about the nucleotide sequences involved in the illegitimate intermolecular (Bashkirov, Khasanov & Prozorov, 1987;Bashkirov, Stoilova-Disheva & Prozorov, 1988;Dempsey & Dubnau, 1989) or intramolecular (Lopez et al, 1984;Peijnenburg, Bron & Venema, 1988) in vivo recombination in Bacillus subtilis. As an initial step toward investigating the possible involvement and contribution ofB.…”

Localization of the replication origin of plasmid pE194

“…3, also contains a GC-rich dyad symmetry element (positions 876 to 902), and an adjacent 7-bp tandem repeat r5'-T(G)TGTCCA'-3] is present in three copies (positions 905 to 924). This interval in pE194 (860 to 930) is also a major recombination site for the low-frequency RecE-independent integration of pE194 into the B. subtilis chromosome (2,7). We hypothesize that the recombination site may be part of the origin of pE194 replication, possibly the site of RepF interaction.…”

“…pBD232 consists of pE194 carrying a 1.3-kilobase fragment derived from the B. subtilis chromosome. A unique SmaI recognition site within the pBD232 plasmid was fortuitously created at the crossover junction derived from in vivo recombination with the B. subtilis chromosome (2) The plasmid constructions were initially used to transform Escherichia coli to ampicillin resistance, and the predicted structures of pBD406 and pBD408 were verified by restriction mapping and Southern hybridizations (data not shown). These constructs were subsequently used to transform B. subtilis recE4 strains to chloramphenicol resistance.…”

Plus-origin mapping of single-stranded DNA plasmid pE194 and nick site homologies with other plasmids

“…This segment contains the RepF protein, which is required for replication (14,38), and the plus origin. More precise measurements of the plus origin, previously reported, were either in conflict or not entirely conclusive (6,33,38). To identify unambiguously a minimal region of pE194 containing the plus origin, segments of the minimal replicon (Fig.…”

“…However, the data supporting these implications and more detailed analyses of the origin of replication are inconclusive, even controversial (6,33,38). Since pE194 is a prototype representing an important family of plasmids in gram-positive bacteria (the plus origins of only two types of plasmids have been analyzed), we characterized the pE194 plus origin.…”