Complete nucleotide sequence of ubiquitous plasmid pEA29 from Erwinia amylovora strain Ea88: Gene organization and intraspecies variation

The complete sequence of plasmid pEA29 from Erwinia amylovora strain Ea88 c onsists of 28,185 bp with a 50.2% G+C content, As deletions and insertions were detected in other derivatives of pEA29, its size actually varied from 27.6 to 34.9 kb. Thirteen open reading frames that encoded predicted protei ns with similarities to known proteins from other bacteria were identified along with two open reading frames related to hypothetical proteins found i n GenBank and six open reading frames with no similarities to existing GenB ank entries, Predicted products of open reading frames with similarity to t he thiamine biosynthetic genes thiO, thiG, and thiF; a betT gene coding for choline transport; an msrA gene for the enzyme methionine sulfoxide reduct ase; a putative methyl-accepting chemotaxis gene; an aldehyde dehydrogenase gene; an hns DNA binding gene; a LysR-type transcriptional regulator; and parA and parB partitioning genes were identified. A putative iteron-contain ing theta-type origin of replication with an AT-rich region and a gene for a RepA protein was identified. PstI and KpnI restriction patterns for pEA29 isolated from tree fruit strains of E. amylovora were homogenous and diffe rent from those for pEA29 isolated from Rubus (raspberry) strains. All Rubu s derivatives of pEA29 contained a point mutation that eliminated a PstI si te and a 1,264-bp region that replaced 1,890 bp of sequence found in pEA29 from strain Ea88, This change eliminated a second PstI site and increased t he length of a KpnI fragment. An insertion sequence, ISEam1, was detected i n one Rubus strain, and transposon Tn5393 was detected in three apple strai ns in two separate locations on the plasmid, Plasmid-cured strains exhibite d reduced virulence and modified colony morphology on minimal medium withou t thiamine, indicating that some of the genes in pEA29 play a role in the p hysiology or metabolism of E. amylovora.