Cloning of the egl gene of Pseudomonas solanacearum and analysis of its role in phytopathogenicity.
The egl gene of Pseudomonas solanacearum was cloned on a cosmid and expressed in Escherichia coli. Restriction endonuclease mapping, transposon mutagenesis, and subclone analysis showed that the egl gene was located on a 2.7-kilobase XhoI-SalI P. solanacearum DNA fragment. Immunoabsorption experiments and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the egl gene encodes the 43-kilodalton endoglucanase that is the major excreted endoglucanase of P. solanacearum. In E. coli, the egl gene appeared to be expressed from its own promoter, but its product was restricted to the cytoplasm. The cloned egl gene was mutagenized with Tn5 and used to specifically mutate the chromosomal egl gene of P. solanacearum by site-directed mutagenesis. The resultant mutant was identical to the wild-type strain in production of extracellular polysaccharide and extracellular polygalacturonase as well as several other excreted proteins but produced at least 200-fold less endoglucanase. This mutant strain was significantly less virulent on tomato than the wild-type strain in plant bioassay experiments. Virulence of the endoglucanase-deficient strain was restored to near wild-type levels by complementation in trans with the cloned egl gene, indicating that the egl gene is important but not absolutely required for pathogenesis.
Roberts
D P
DP
Department of Microbiology, University of Georgia, Athens 30602.
Denny
T P
TP
Schell
M A
MA
D002480:Cellulase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000382:microbiology*; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D002480:Cellulase