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

Abstract

The complete sequence of plasmid pEA29 from Erwinia amylovora strain Ea88 consists 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 proteins with similarities to known proteins from other bacteria were identified along with two open reading frames related to hypothetical proteins found in GenBank and six open reading frames with no similarities to existing GenBank entries. Predicted products of open reading frames with similarity to the thiamine biosynthetic genes thiO, thiG, and thiF; a betT gene coding for choline transport; an msrA gene for the enzyme methionine sulfoxide reductase; 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-containing 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 different from those for pEA29 isolated from Rubus (raspberry) strains. All Rubus derivatives of pEA29 contained a point mutation that eliminated a PstI site 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 the length of a KpnI fragment. An insertion sequence, ISEam1, was detected in one Rubus strain, and transposon Tn5393 was detected in three apple strains in two separate locations on the plasmid. Plasmid-cured strains exhibited reduced virulence and modified colony morphology on minimal medium without thiamine, indicating that some of the genes in pEA29 play a role in the physiology or metabolism of E. amylovora.

FIG. 1

Potential ORFs at least 375 nt in length and putative genes inferred from sequence data found in plasmid pEA29 from E. amylovora strain Ea88. The unique BamHI site and all PstI restriction sites are shown (positions in base pairs are shown in parentheses). Arrows indicate the positioning of the ORFs. A key to the ORFs is found in Table 2.

FIG. 2

Organization of the origin of replication for plasmid pEA29 in E. amylovora Ea88. (A) Sequence upstream from the PstI site at nt position 26719, through the unique BamHI site, and into the repA gene. Relevant restriction sites within the region are indicated in bold lettering. Solid arrows indicate repeating regions. An AT-rich region is designated by the dotted line. (B) Alignment of the repeating sequences indicated in panel A. Nucleotide patterns conserved across the majority of the repeats are indicated in bold. With the exception of repeat number 4, repeats were either 22 or 11 bp long. The 11-bp repeats are truncated versions of the longer repeats.

FIG. 3

Restriction and physical maps and sequence data for a region of pEA29 from E. amylovora Ea88 with homology to regions in Tn2501 and Tn2502. Triple lines () indicate sequenced regions of Tn2501. (A) Physical and genetic map of Tn2501 compared to those of the partial res site and resolvase (tnpR) gene found on pEA29 (nt 18084 to 18785). Positions for the transposase gene (tnpA), res site, and resolvase gene (tnpR) of Tn2501 are indicated by the hatched box, white box, and latticework box, respectively. Gene orientation is indicated by arrows. Restriction sites are as follows: BamHI (B), BglII (Bg), HindIII (H), SalI (S), SmaI (Sm), and SstI (Ss). Inverted repeats for Tn2501 are indicated by IR-L (left) and IR-R (right). (B) Sequence alignment of the res regions of Tn2501, Tn2502, and pEA29 (nt 18084 to 18188). Dyad symmetry regions consistent with Tn2501 and Tn2502 res sites I, II, and III are boxed, as is the corresponding pEA29 sequence. Areas where the sequence is in agreement with Tn2501 are indicated by asterisks.

FIG. 4

Surface and cross section of immature pears stab inoculated with wild-type and plasmid-cured E. amylovora strain Ea110 and incubated for 7 days at room temperature under high humidity. (A and C) Pears inoculated with strain Ea110⁻; (B and D) pears inoculated with strain Ea110. Pears inoculated with plasmid-cured strains exhibited less water soaking and ooze production, and symptom expression was delayed and remained localized around the site of inoculation. Pears inoculated with wild-type strains exhibited extensive water soaking and a proliferation of ooze droplets (arrow), and necrosis developed rapidly and extended beyond the inoculation site.

FIG. 5

(A) Comparison of pEA29 PstI and KpnI restriction enzyme maps for E. amylovora strains Ea88 and MR1 from pear and raspberry, respectively. The maps were normalized to the unique BamHI restriction site. (B) Sizes in kilobases of the restriction fragments shown in the restriction maps above. (C) Map of a variable region of pEA29 found in two raspberry strains of E. amylovora. The locations of the 1,264-bp sequence of pEA29 found in raspberry strains MR1 and 2-95 and the corresponding 1,890-bp sequence found in pear strain Ea88 are shown. Shaded bars indicate that the sequences flanking the variable region are identical in the three strains. KpnI sites (K) (nt positions 2011 and 13642) used to confirm the fragment size variation are shown. Dotted arrows indicate the locations of primers AJ484 and AJ485, used to detect length variation in this region. Plasmid pEA29 in strain MR1 is 629 bp smaller than the 28,185-bp plasmid found in Ea88. The substituted sequence starts at bp 9635 and returns to Ea88 (pEA29) homology at nt 11525. A hypothetical ORF (ORF 7; solid arrow) and the restriction sites for PstI (P), SalI (S), and methylated ClaI (C^m) are missing in pEA29 of strains MR1 and 2-95.

FIG. 6

Fragment length polymorphism in KpnI digests of pEA29 from seven strains of E. amylovora. Lanes 1 to 9 contain the 1-kb Ladder Plus, strains Ea88, Ea110, MR1, 2-95, W2, DM22, BCN20, and a λ HindIII ladder, respectively. The 11.7-kb fragment found in strains Ea88 and Ea110 (lanes 2 to 3) is slightly smaller in strains MR1 and 2-95 (lanes 4 to 5). The 4.1-kb KpnI fragment in strains Ea88, Ea110, and MR1 (lanes 2 to 4) is a 5.4-kb fragment in strain 2-95 (lane 5) and a 10.8-kb fragment in strains W2 and DM22 (lanes 6 to 7). The 11.7-kb KpnI fragment found in the strains shown in lanes 2 to 7 is an 18.5-kb fragment in strain BCN20 (lane 8). Digests were analyzed on a 1% (wt/vol) agarose gel in 0.5× TBE buffer stained with ethidium bromide.

FIG. 7

Map of ISEam1 found in plasmid pEA29 of E. amylovora strain 2-95. (A) Genetic and physical map of ISEam1 and its location within pEA29. Boxed elements indicate a 100-bp direct repeat found prior to the insertion site (nt position 15692) and at the 3′ end of ISEam1. Genes for transposase and a hypothetical (hyp.) ORF are indicated with arrows. Restriction sites are as follows: SphI (Sp), SmaI (Sm), EcoRV (E), PvuII (P), ClaI (C), and DraI (D). (B) Nucleotide sequence of ISEam1. Underlining indicates the position and nucleotide sequence of the transposase gene tnpR. A dotted line indicates the 100-bp direct repeat region. (C) Alignment of the 100-bp direct repeat located in the pEA29 sequence upstream from the insertion site for ISEam1 and the repeat found in ISEam1 at the 3′ end.