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. 1985 May;162(2):708–714. doi: 10.1128/jb.162.2.708-714.1985

Isolation and characterization of Tn5 insertion mutants of Erwinia chrysanthemi that are deficient in polygalacturonate catabolic enzymes oligogalacturonate lyase and 3-deoxy-D-glycero-2,5-hexodiulosonate dehydrogenase.

A K Chatterjee, K K Thurn, D J Tyrell
PMCID: PMC218908  PMID: 2985544

Abstract

Mutants of Erwinia chrysanthemi EC16 deficient in the polygalacturonate catabolic enzymes oligogalacturonate lyase (Ogl-) and 3-deoxy-D-glycero-2,5-hexodiulosonate (ketodeoxyuronate) dehydrogenase (KduD-) were obtained by Tn5 mutagenesis using the R plasmid pJB4JI. Ogl- Exu+ (Exu+, D-galacturonate utilization) and KduD- Exu- strains macerated potato tuber tissue and utilized glucose, glycerol, and gluconate, but they did not utilize polygalacturonate, unsaturated digalacturonate, or saturated digalacturonate. Genetic and physical evidence indicated that the Ogl- mutants and a KduD- recombinant contained a single copy of Tn5 and that Tn5 (Kmr) was linked to the mutant phenotypes. In the Ogl+ parents, basal levels of oligogalacturonate lyase were present in glycerol-grown cells and induced levels were present with saturated or unsaturated digalacturonate, while oligogalacturonate lyase was undetectable under similar conditions in Ogl- strains. Pectate lyase, polygalacturonase, and ketodeoxyuronate dehydrogenase were induced in an Ogl- strain by 3-deoxy-D-glycero-2,5-hexodiulosonate and by the enzymatic products of unsaturated digalacturonate but not by the digalacturonates. The KduD- strains lacked the dehydrogenase activity but in the presence of the digalacturonates produced higher levels of pectate lyase, polygalacturonase, and oligogalacturonate lyase than the KduD+ parents did. In the KduD- strains, pectate lyase and oligogalacturonate lyase were induced by unsaturated digalacturonate in a "gratuitous" manner, suggesting an intracellular accumulation of the inducer(s). We conclude that an intermediate(s) of the ketodeoxyuronate pathway induces pectate lyase, polygalacturonase, oligogalacturonate lyase, and ketodeoxyuronate dehydrogenase in E. chrysanthemi.

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Selected References

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  1. Blanco C., Mata-Gilsinger M., Ritzenthaler P. Construction of hybrid plasmids containing the Escherichia coli uxaB gene: analysis of its regulation and direction of transcription. J Bacteriol. 1983 Feb;153(2):747–755. doi: 10.1128/jb.153.2.747-755.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chatterjee A. K. Acceptance by Erwinia spp. of R plasmid R68.45 and its ability to mobilize the chromosome of Erwinia chrysanthemi. J Bacteriol. 1980 Apr;142(1):111–119. doi: 10.1128/jb.142.1.111-119.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chatterjee A. K., Starr M. P. Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity. J Bacteriol. 1977 Dec;132(3):862–869. doi: 10.1128/jb.132.3.862-869.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chatterjee A. K., Starr M. P. Genetics of Erwinia species. Annu Rev Microbiol. 1980;34:645–676. doi: 10.1146/annurev.mi.34.100180.003241. [DOI] [PubMed] [Google Scholar]
  5. Chatterjee A. K., Thurn K. K., Feese D. A. Tn5-Induced Mutations in the Enterobacterial Phytopathogen Erwinia chrysanthemi. Appl Environ Microbiol. 1983 Feb;45(2):644–650. doi: 10.1128/aem.45.2.644-650.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chumley F. G., Menzel R., Roth J. R. Hfr formation directed by tn10. Genetics. 1979 Apr;91(4):639–655. doi: 10.1093/genetics/91.4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collmer A., Bateman D. F. Impaired induction and self-catabolite repression of extracellular pectate lyase in Erwinia chrysanthemi mutants deficient in oligogalacturonide lyase. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3920–3924. doi: 10.1073/pnas.78.6.3920. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Collmer A., Whalen C. H., Beer S. V., Bateman D. F. An exo-poly-alpha-D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi. J Bacteriol. 1982 Feb;149(2):626–634. doi: 10.1128/jb.149.2.626-634.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davé B. A., Vaughn R. H., Patel I. B. Preparation, separation and degradation of oligouronides produced by the polygalacturonic acid transeliminase of Bacillus pumilus. J Chromatogr. 1976 Jan 21;116(2):395–405. doi: 10.1016/s0021-9673(00)89909-4. [DOI] [PubMed] [Google Scholar]
  10. Farber J. M., Idziak E. S. Detection of glucose oxidation products in chilled fresh beef undergoing spoilage. Appl Environ Microbiol. 1982 Sep;44(3):521–524. doi: 10.1016/s0315-5463(82)72428-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fogarty W. M., Ward O. P. Pectinases and pectic polysaccharides. Prog Ind Microbiol. 1974;13:61–119. [PubMed] [Google Scholar]
  12. Jorgensen R. A., Rothstein S. J., Reznikoff W. S. A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. Mol Gen Genet. 1979;177(1):65–72. doi: 10.1007/BF00267254. [DOI] [PubMed] [Google Scholar]
  13. KILGORE W. W., STARR M. P. Catabolism of galacturonic and glucuronic acids by Erwinia carotovora. J Biol Chem. 1959 Sep;234:2227–2235. [PubMed] [Google Scholar]
  14. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  15. LUH B. S., PHAFF H. J. End products and mechanism of hydrolysis of pectin and pectic acid by yeast polygalacturonase (YPG). Arch Biochem Biophys. 1954 Jul;51(1):102–113. doi: 10.1016/0003-9861(54)90458-0. [DOI] [PubMed] [Google Scholar]
  16. Lee J. H., Heffernan L., Wilcox G. Isolation of ara-lac gene fusions in Salmonella typhimurium LT2 by using transducing bacteriophage Mu d (Apr lac). J Bacteriol. 1980 Sep;143(3):1325–1331. doi: 10.1128/jb.143.3.1325-1331.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Moran F., Nasuno S., Starr M. P. Extracellular and intracellular polygllacturonic acid trans-eliminases of Erwinia carotovora. Arch Biochem Biophys. 1968 Feb;123(2):298–306. doi: 10.1016/0003-9861(68)90138-0. [DOI] [PubMed] [Google Scholar]
  18. Moran F., Nasuno S., Starr M. P. Oligogalacturonide trans-eliminase of Erwinia carotovora. Arch Biochem Biophys. 1968 Jun;125(3):734–741. doi: 10.1016/0003-9861(68)90508-0. [DOI] [PubMed] [Google Scholar]
  19. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  20. PREISS J., ASHWELL G. Polygalacturonic acid metabolism in bacteria. I. Enzymatic formation of 4-deoxy-L-threo-5-hexoseulose uronic acid. J Biol Chem. 1963 May;238:1571–1583. [PubMed] [Google Scholar]
  21. PREISS J., ASHWELL G. Polygalacturonic acid metabolism in bacteria. II. Formation and metabolism of 3-deoxy-D-glycero-2, 5-hexodiulosonic acid. J Biol Chem. 1963 May;238:1577–1583. [PubMed] [Google Scholar]
  22. Portalier R., Robert-Baudouy J., Stoeber F. Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon. J Bacteriol. 1980 Sep;143(3):1095–1107. doi: 10.1128/jb.143.3.1095-1107.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ritzenthaler P., Mata-Gilsinger M., Stoeber F. Construction and expression of hybrid plasmids containing Escherichia coli K-12 uxu genes. J Bacteriol. 1980 Sep;143(3):1116–1126. doi: 10.1128/jb.143.3.1116-1126.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ritzenthaler P., Mata-Gilsinger M., Stoeber F. Molecular cloning of Escherichia coli K-12 hexuronate system genes: exu region. J Bacteriol. 1981 Jan;145(1):181–190. doi: 10.1128/jb.145.1.181-190.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ritzenthaler P., Mata-Gilsinger M. Use of in vitro gene fusions to study the uxuR regulatory gene in Escherichia coli K-12: direction of transcription and regulation of its expression. J Bacteriol. 1982 Jun;150(3):1040–1047. doi: 10.1128/jb.150.3.1040-1047.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shepard H. M., Polisky B. Measurement of Plasmid copy number. Methods Enzymol. 1979;68:503–513. doi: 10.1016/0076-6879(79)68039-4. [DOI] [PubMed] [Google Scholar]
  27. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  28. Starr M. P., Chatterjee A. K., Starr P. B., Buchanan G. E. Enzymatic degradation of polygalacturonic acid by Yersinia and Klebsiella species in relation to clinical laboratory procedures. J Clin Microbiol. 1977 Oct;6(4):379–386. doi: 10.1128/jcm.6.4.379-386.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsuyumu S. Inducer of pectic acid lyase in Erwinia carotovora. Nature. 1977 Sep 15;269(5625):237–238. doi: 10.1038/269237a0. [DOI] [PubMed] [Google Scholar]
  30. Van Gijsegem F., Toussaint A. In vivo cloning of Erwinia carotovora genes involved in the catabolism of hexuronates. J Bacteriol. 1983 Jun;154(3):1227–1235. doi: 10.1128/jb.154.3.1227-1235.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. WARREN L. Thiobarbituric acid spray reaction for deoxy sugars and sialic acids. Nature. 1960 Apr 16;186:237–237. doi: 10.1038/186237a0. [DOI] [PubMed] [Google Scholar]
  32. Zink R. T., Kemble R. J., Chatterjee A. K. Transposon Tn5 mutagenesis in Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica. J Bacteriol. 1984 Mar;157(3):809–814. doi: 10.1128/jb.157.3.809-814.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

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