Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1990 Sep;172(9):4988–4995. doi: 10.1128/jb.172.9.4988-4995.1990

Molecular cloning, nucleotide sequence, and marker exchange mutagenesis of the exo-poly-alpha-D-galacturonosidase-encoding pehX gene of Erwinia chrysanthemi EC16.

S Y He 1, A Collmer 1
PMCID: PMC213154  PMID: 2168372

Abstract

The pehX gene encoding extracellular exo-poly-alpha-D-galacturonosidase (exoPG; EC 3.2.1.82) was isolated from a genomic library of the pectate lyase-deficient Erwinia chrysanthemi mutant UM1005 (a Nalr Kanr delta pelABCE derivative of EC16) by immunoscreening 2,800 Escherichia coli HB101 transformants with an antibody against exoPG protein. The cloned pehX gene was expressed highly from its own promoter in E. coli, and most of the enzyme was localized in the periplasm. The nucleotide sequence of pehX revealed the presence of an amino-terminal signal peptide and an open reading frame encoding a preprotein of 64,608 daltons. The cloned pehX gene was insertionally inactivated with TnphoA and used to mutate the chromosomal pehX gene of E. chrysanthemi AC4150 (Nalr) and CUCPB5006 (Nalr Kans delta pelABCE) by marker exchange mutagenesis. Analysis of the resulting mutants, CUCPB5008 (Pel+ Peh-) and CUCPB5009 (Pel- Peh-), indicated that exoPG can contribute significantly to bacterial utilization of polygalacturonate and the induction of pectate lyase in the presence of extracellular pectic polymers. CUCPB5009 retained a slight ability to pit polygalacturonate semisolid agar and macerated chrysanthemum pith tissues when large numbers of bacteria were inoculated.

Full text

PDF
4988

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barras F., Thurn K. K., Chatterjee A. K. Resolution of four pectate lyase structural genes of Erwinia chrysanthemi (EC16) and characterization of the enzymes produced in Escherichia coli. Mol Gen Genet. 1987 Sep;209(2):319–325. doi: 10.1007/BF00329660. [DOI] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Collmer A., Schoedel C., Roeder D. L., Ried J. L., Rissler J. F. Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase. J Bacteriol. 1985 Mar;161(3):913–920. doi: 10.1128/jb.161.3.913-920.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Guiseppi A., Cami B., Aymeric J. L., Ball G., Creuzet N. Homology between endoglucanase Z of Erwinia chrysanthemi and endoglucanases of Bacillus subtilis and alkalophilic Bacillus. Mol Microbiol. 1988 Jan;2(1):159–164. doi: 10.1111/j.1365-2958.1988.tb00017.x. [DOI] [PubMed] [Google Scholar]
  8. Hinton J. C., Sidebotham J. M., Gill D. R., Salmond G. P. Extracellular and periplasmic isoenzymes of pectate lyase from Erwinia carotovora subspecies carotovora belong to different gene families. Mol Microbiol. 1989 Dec;3(12):1785–1795. doi: 10.1111/j.1365-2958.1989.tb00164.x. [DOI] [PubMed] [Google Scholar]
  9. KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
  10. Keen N. T., Dahlbeck D., Staskawicz B., Belser W. Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli. J Bacteriol. 1984 Sep;159(3):825–831. doi: 10.1128/jb.159.3.825-831.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Keen N. T., Tamaki S. Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli. J Bacteriol. 1986 Nov;168(2):595–606. doi: 10.1128/jb.168.2.595-606.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Payne J. H., Schoedel C., Keen N. T., Collmer A. Multiplication and Virulence in Plant Tissues of Escherichia coli Clones Producing Pectate Lyase Isozymes PLb and PLe at High Levels and of an Erwinia chrysanthemi Mutant Deficient in PLe. Appl Environ Microbiol. 1987 Oct;53(10):2315–2320. doi: 10.1128/aem.53.10.2315-2320.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Plastow G. S. Molecular cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia chrysanthemi B374. Mol Microbiol. 1988 Mar;2(2):247–254. doi: 10.1111/j.1365-2958.1988.tb00026.x. [DOI] [PubMed] [Google Scholar]
  15. Reverchon S., Huang Y., Bourson C., Robert-Baudouy J. Nucleotide sequences of the Erwinia chrysanthemi ogl and pelE genes negatively regulated by the kdgR gene product. Gene. 1989 Dec 21;85(1):125–134. doi: 10.1016/0378-1119(89)90472-1. [DOI] [PubMed] [Google Scholar]
  16. Ried J. L., Collmer A. Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. Appl Environ Microbiol. 1985 Sep;50(3):615–622. doi: 10.1128/aem.50.3.615-622.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ried J. L., Collmer A. Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, and Erwinia carotovora subsp. atroseptica. Appl Environ Microbiol. 1986 Aug;52(2):305–310. doi: 10.1128/aem.52.2.305-310.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Roeder D. L., Collmer A. Marker-exchange mutagenesis of a pectate lyase isozyme gene in Erwinia chrysanthemi. J Bacteriol. 1985 Oct;164(1):51–56. doi: 10.1128/jb.164.1.51-56.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tamaki S. J., Gold S., Robeson M., Manulis S., Keen N. T. Structure and organization of the pel genes from Erwinia chrysanthemi EC16. J Bacteriol. 1988 Aug;170(8):3468–3478. doi: 10.1128/jb.170.8.3468-3478.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Thurn K. K., Chatterjee A. K. Single-site chromosomal Tn5 insertions affect the export of pectolytic and cellulolytic enzymes in Erwinia chrysanthemi EC16. Appl Environ Microbiol. 1985 Oct;50(4):894–898. doi: 10.1128/aem.50.4.894-898.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 1983;2(3):411–417. doi: 10.1002/j.1460-2075.1983.tb01438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Witholt B., Boekhout M., Brock M., Kingma J., Heerikhuizen H. V., Leij L. D. An efficient and reproducible procedure for the formation of spheroplasts from variously grown Escherichia coli. Anal Biochem. 1976 Jul;74(1):160–170. doi: 10.1016/0003-2697(76)90320-1. [DOI] [PubMed] [Google Scholar]
  23. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  24. Zucker M., Hankin L. Regulation of pectate lyase synthesis in Pseudomonas fluorescens and Erwinia carotovora. J Bacteriol. 1970 Oct;104(1):13–18. doi: 10.1128/jb.104.1.13-18.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES