Analysis of variable short-sequence DNA repeats on the 29 kb plasmid of Erwinia amylovora strains.
The fire blight pathogen E. amylovora has been specifically and sensitively detected by PCR assays with primers derived from plasmid pEa29. The amplified fragment of approx. 1 kb can vary in length for individual strains, easily seen in a digest with restriction enzymes Sau3A or HpaII. DNA fragments from this variable region were cloned and DNA sequence analysis revealed short-sequence DNA repeat (SSR) motifs which were reiterated to various extents. The SSR units consisted of 8 nucleotides (ATTACAGA), and terminated with ATTA which is part of an SSR. The shortest repetition consisted of 4 units and the longest one in Austrian E. amylovora strains was 15 units. The number of SSR units was remarkably stable during propagation of strains, but was occasionally changed when a strain was stressed by exposure to antibiotics, copper sulfate or storage at low temperature. Changes in the SSR number could be due to adjustment in bacterial fitness to environmental pressure. Oligonucleotide PCR primers were designed from DNA sequences adjacent to the SSR region of pEA29 for rapid analysis of SSR length variations. With this PCR assay, >130 strains were classified into at least 11 types based on the number of repeats. E. amylovora strains isolated in Germany mainly carried 6 repeats in pEA29, which never changed under laboratory conditions. E. amylovora strains from Hungary and the Netherlands were quite divergent for the SSRs and further changes were sometimes observed after plating on agar medium. Homology search of nucleotide sequence data libraries revealed similarities of the SSR motif to partition functions of low copy number plasmids. Amino acid homology searches showed a similarity of the deduced amino acid sequence in the ORF adjacent to the SSR motif to replication proteins of plasmids. It is suggested that the SSR may play a role in regulation of plasmid replication and partition as assumed for iterons.