Evolution of a gene for pathogenicity: endo-pectate lyase

Erwinia carotovora subsp. Carotovora (Ecc) and Erwinia carotovora subsp. Atroseptica (Eca) are plant pathogenic bacteria that cause soft rot disease of many plant species and blackleg disease of potatoes, respectively. Ecc and Eca attack plants by means of a group of extracellular plant tissue-degrading enzymes. which rapidly breaks down the pectic polymers that form a structurally important part of the plant cell wall, is considered central to soft rot pathogenesis. In this work, I isolated and studied the genes encoding this enzyme from Ecc and Eca. A clone library of Ecc strain EC14 was constructed using cosmid PLAFR3. This library contains 2,200 clones with an average insert size of 27 kilobases of DNA and included a proteolytic clone, five cellulolytic clones, and ten pectolytic clones. The proteolytic clone was used to complement a Tn5-induced protease mutant of Ecc; the complemented mutant was restored to near-wild type phenotype. Six of the pectolytic clones hybridized to a probe from a. previously isolated extracellular endo-pectate-pectate lyase gene from Ecc; one pectolytic clone had homology to a previously isolated clone encoding endo-polygalacturonase: three clones showed no relationship to either of the previously characterized Ecc pectolytic enzyme genes. A clone encoding the major endo-pectate lyase gene from Ecc was chosen for subcloning and further study. I used the plasmid vector pBR322 to construct a clone bank of Eca strain SRB; of the 1700 clones screened, five were pectolytic. Two of the Eca pectolytic. clones had homology to the Ecc endo-pectate lyase gene; upon examination, they proved to contain the same insert in opposite orientations. The Ecc endo-pectate lyase had a pI of 9. 5 and a molecular weight of 33,000; the analogous Eca endo-pectate lyase had a pI of 9.2 and a molecular weight of 31,000. Both enzymes required a divalent cation for activity (preferring Ca2+ over Mg2+ over Mn²⁺. The restriction endonuclease maps of the two clones did not have any tested sites in common. These differences suggest that although these two genes may have originated from a common ancestral gene, considerable divergence has taken place. I analyzed the fine structure of the Ecc endo-pectate lyase gene by DNA sequencing. The coding region of the gene is preceded by E. coli-type -10 and -35 sequences and encodes an unmodified protein of 281 amine acids. A typical secretion signal peptide is not present.

Name thesis:degree_name

Ph. D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Plant Pathology

Department dc:contributor.department

Plant Pathology

Grantor dc:publisher

Virginia Polytechnic Institute and State University

Year dc:date.issued

1987

Author dc:creator

• Allen, Caitilyn

Chair dc:contributor.committeechair

• Lacy, George H.

Committee members dc:contributor.committeemember

• Hooper, Gary P.
• Bates, Robert C.
• Johnson, John L.
• Tolin, Sue A.

Statement dc:rights

• In Copyright

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

en_US

Handle dc:identifier.uri

http://hdl.handle.net/10919/82610

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/82610