Cloning and characterization of a pectate lyase gene from the soft-rotting bacterium Pseudomonas viridiflava.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Liao
C H
CH
Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia, PA 19118.
Sasaki
K
K
Nagahashi
G
G
Hicks
K B
KB
D002212:Capsicum / Q000382:microbiology; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics; D005816:Genetic Complementation Test; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D010368:Pectins / Q000378:metabolism; D010946:Plants, Medicinal; D011133:Polysaccharide-Lyases / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D011198:Solanum tuberosum / Q000382:microbiology; D014158:Transcription, Genetic; D014774:Virulence
D004251:DNA Transposable Elements; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase