Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora

Abstract

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1.

FIG. 1.

PCR-ribotype fingerprints of E. amylovora strains isolated from tree fruits (Ea110, NZR5, CA263, OR6, and CA1R), hawthorn (IH3-1), and Rubus spp. (IL-5 and MR1) and three strains of E. pyrifoliae (Ep1/96, Ep4/97, and Ep16/96) isolated from Asian pear.

FIG. 2.

Organizational structure of 16S-23S rRNA ITS found in E. amylovora and E. pyrifoliae. Six rRNA operons were sequenced from each strain. The organizational structure of the spacers is indicated graphically. Regions C1, C2, and C3 are conserved regions of 58 to 60 bp, 98 to 99 bp, and 20 bp, respectively; region V is a variable 25-bp sequence; and region O is an optional 139-bp sequence. All X alleles contain a tRNA^Glu gene, and all Y alleles contain tandem tRNA^Ile and tRNA^Ala genes. X⁺ indicates alleles that contain tRNA^Glu genes with an XbaI restriction site. The number of copies of each allele is listed for each strain along with the primer (from 1 = 5SFlank1 to 6 = 5SFlank6) paired with the primer SpacerF, used to amplify the alleles given in parentheses.

FIG. 3.

Phylogenetic dendrogram based on a comparison of partial groEL gene sequences for Erwinia spp. and a set of bacteria closely related to Erwinia. The percentages at the nodes indicate the level of bootstrap support based on a neighbor-joining analysis of 1,000 resampled data sets. E. carotovora was used as the outgroup.

FIG. 4.

Physical and restriction map of 35,904-bp plasmid pEP36 isolated from E. pyrifoliae strain Ep1/96. (A) Genetic map of pEP36. Solid black bar (▪) indicates nucleotide homology to plasmid pEA29 isolated from E. amylovora strain Ea88. The forward-hatched box (▨), open box (□), and reverse-hatched box (▧) indicated regions of pEP36 with nucleotide similarities to the Y. pestis strain CO92 genome, E. amylovora plasmid pEA29 isolated from Rubus species, and an S. flexneri SHI-2 pathogenicity island, respectively; all nucleotide similarities reported are greater than 80%. The location of insertion elements IS285 and IS3 are shown by a dotted line (••••) and double line (=), respectively. The position of the transposon Tn5394 is designated by a dashed line (---). Potential ORFs greater than 375 nt and putative genes inferred from sequence data or with matches in GenBank are indicated by directional arrows. ORFs conserved between pEP36 and E. amylovora plasmid pEA29 are indicated in bold lettering. A key to the ORFs is found in Table 2. (B) Restriction map of pEP36. Numbers to the right of the restriction enzyme are indicative of the number of restriction sites found for each enzyme on the plasmid. Only nonmethylated ClaI sties are reported.

FIG. 5.

Physical map comparing the organization of the origin of replication for plasmids pEP36 and pEA29 isolated from E. pyrifoliae strain Ep1/96 and E. amylovora strain Ea88, respectively. ORFs and their orientation are indicated by large directional arrows (). Incomplete ORFs are indicated by flecked arrows (). The position and organization of discrete families of repeats are indicated by various symbols: dotted vertical lines (¦) indicate an 8-bp repeat (ATTCTGGG) originally observed in pEP36, line arrows (→) designate a series of 3 to 14 8-bp repeats previously described in pEA29 (38). Inverted triangles (▸◂) show the position of an inverted repeat; the right repeat (◃) is incomplete in pEA29. Open arrows (➯) indicate the position of two 23-bp repeats found on pEP36 and of a single copy found on pEA29. The nine iterons are designated by solid vertical lines (|). Solid arrows () indicate three 20-bp repeats found on pEP36 and on pEA29. Numbers found at either end of the drawing are the position in nucleotides of the segment depicted. The length of each segment is indicated in base pairs to the right of the drawing. An identical BamHI site is used to orient the plasmids. Maps are not to scale.

FIG. 6.

Genetic maps of E. pyrifoliae plasmid pEP2.6 from strain Ep1/96 and of E. amylovora plasmid pEA2.8 from strain IL-5 and plasmid pEA1.7 from strain IH3-1. Regions of nucleotide similarity between pEP2.6 and pEA1.7 (78%) and between pEP2.6 and pEA2.8 (84 to 89%) are indicated by a striped bar (▤) and a checked bar (), respectively. The ORF coding for the RNA1 modulator protein was found on both pEP2.6 and pEA1.7 (ORF 4 on both plasmids). ORF 2 from plasmid pEA2.8 was similar to a 13.8-kDa hypothetical protein encoded on plasmid ColE1 in E. coli (accession number NP_040360). ORF 4 from pEA2.8 had 98% similarity to a beta-lactamase protein that provides resistance to ampicillin. No other ORFs had identity to any existing GenBank entries.