VIEW ARTICLE | DOI: 10.1094/MPMI-5-301
Cloning and Characterization of a Pectate Lyase Gene from the Soft-Rotting Bacterium Pseudomonas viridiflava. C. -H. Liao. Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118 U.S.A. K. Sasaki, G. Nagahashi, and K. B. Hicks. Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118 U.S.A. MPMI 5:301-308. Accepted 27 February 1992. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1992.
Pseudomonas viridiflava is a soft-rotting pathogen of harvested vegetables that produces an extracellular pectate lyase (PL) responsible for maceration of plant tissue. A pel gene encoding PL was cloned from the genome of strain SJ074 and efficiently expressed in Escherichia coli. After a series of deletion subclonings and analysis by transposon mutagenesis, the pel gene was located in a 1.2-kb PstI-BglII genomic fragment. This fragment appears to contain a promoter at the PstI end required for pel gene expression. The PL produced by pectolytic E. coli clones is identical to those produced by strain SJ074 and by other strains of P. viridiflava in terms of molecular weight (42 kDa) and pI (9.7). A mutant of strain SJ074, designated MEI, which had Tn5 specifically inserted in the pel locus was constructed by site-directed mutagenesis. The MEI mutant produced 70- to 100-fold less PL than the wild type and failed to cause tissue maceration in plants. PL production and soft-rot pathogenicity in MEI and in a Pel‾ mutant previously isolated from strain SF312 were restored to the wild-type level by complementation in trans with the cloned pel gene. By using the 1.2-kb fragment as a probe, pel homologs were detected in four bacteria that are pathologically unrelated to P. viridiflava. These include three pathovars of P. syringae (pv. lachrymans, pv. phaseolicola, and pv. tabaci) and Xanthomonas campestris pv. malvacearum. No DNA fragments showing homology to pel of P. viridiflava were detected in genomic digests prepared from two strains of soft-rot erwinias. The pel genes of Pseudomonas and Xanthomonas appear to be conserved and may have evolved independently from the pel family of Erwinia.