The replication origin of Azotobacter vinelandii

The putative replication origin of Azotabacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistanc e cartridge. The resulting plasmids could be isolated and labelled by South ern hybridisation with the antibiotic resistance cartridge as probe and als o visualised by electron microscopy. These plasmids integrated into the chr omosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 20 0-bp DNA fragment was sufficient to allow the replication of pBR322 in an E scherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragm ent. The nucleotide sequence of the putative replication origin and its fla nking regions was determined. In the sequence of the 200-bp fragment many o f the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed i n A. vinelandii. Direct sequencing of the relevant genomic fragment was als o carried after amplifying it from. A. vinelandii chromosomal DNA by PCR. T his con; firmed that no rearrangements had taken place while the cloned fra gment was resident in E. coli. It was shown by hybridisation that the 200-b p chromosomal origin fragment of A. vinelandii was present in three other f ield strains of Azotobacter spp.