Analysis of variable short-sequence DNA repeats on the 29 kb plasmid of Erwinia amylovora strains

The fire blight pathogen Erwinia amylovora has been specifically and sensit ively detected by PCR assays with primers derived from plasmid pEa29. The a mplified fragment of approximately 1 kb can vary in length for individual s trains, easily seen in a digest with restriction enzymes Sau3A or HpaII. DN A fragments from this variable region were cloned and DNA sequence analysis revealed short-sequence DNA repeat (SSR) motifs which were reiterated to v arious extents. The SSR units consisted of eight nucleotides (ATTACAGA), an d terminated with ATTA which is part of an SSR. The shortest repetition con sisted of four units and the longest one in Austrian E. amylovora strains w as 15 units. The number of SSR units was remarkably stable during propagati on of strains, but was occasionally changed when a strain was stressed by e xposure to antibiotics, copper sulphate or storage at low temperature. Chan ges in the SSR number could be due to adjustment in bacterial fitness to en vironmental pressure. We designed oligonucleotide PCR primers from DNA sequ ences adjacent to the SSR region of pEA29 for rapid analysis of SSR length variations. With this PCR assay, more than 130 strains were classified into at least 11 types based on the number of repeats. E. amylovora strains iso lated in Germany carried mostly six repeats in pEA29, which never changed u nder laboratory conditions. E. amylovora strains from Hungary and the Nethe rlands were quite divergent for the SSRs and further changes were sometimes observed after plating on agar medium. Homology search of nucleotide seque nce data libraries revealed similarities of the SSR motif to partition func tions of low copy number plasmids. Amino acid homology searches showed simi larity of the deduced amino acid sequence in the ORF adjacent to the SSR mo tif to replication proteins of plasmids. The SSR may play a role in regulat ion of plasmid replication and partition as assumed for iterons.