1887

Abstract

SUMMERY:

Phytopathogenic bacteria cause tissue maceration by secretion of pectinolytic enzymes such as pectate lyase (PL). Sequencing of overlapping genomic fragments from subsp. established the organization of a 7·5 kbp region encoding PL isoenzymes. Two intergenic regions of 656 and 645 bp separate three enzyme coding regions of 1125 bp exhibiting approximately 80% positional identity. The promoters of each of the three genes contain a segment with high homology to the binding sequence of the KdgR transcription repressor, implying similar mechanisms of gene regulation in the two bacterial species. Separate expression of the genes in the -pT7-7 system and purification of their products yielded PLs at 7-33 mg (I culture) with greater than 95% purity. Availability of the recombinant enzymes allowed determination of the kinetic differences amongst the PL isoforms, PL1, PL2 and PL3. The results show that PL is not strictly confined to depolymerization of pectate since each isoenzyme more readily degrades 31 % esterified pectin. Addition of isoenzyme combinations revealed no synergism with respect to degradation of pectate or 31% esterified pectin. However, addition of enzyme combinations containing PL3 enhanced the activity towards 68% esterified pectin, against which individual PL activities were low, by up to 64%. These data suggest that the combination of PL isoenzymes extends the range of pectic substrates which the bacterium can degrade.

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1995-04-01
2021-10-22
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References

  1. Barras F., van Gijsegem F., Chatterjee A. K. 1994; Extra cellular enzymes and pathogenesis of soft-rot Erwinia . Annu Rev Phytopathol 32:201–234
    [Google Scholar]
  2. Boyd C., Keen N. T. 1993; Characterization of the prt A and prt B genes of Erwinia chrysanthemi EC16. Gene 133:115–118
    [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:6950–6958
    [Google Scholar]
  4. Brooks A. D, He S. Y, Gold S., Keen N. T., Collmer A., Hutcheson S. W. (1990); Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chysanthemi ECl6 and characterization of the enzyme product. J Bacterio l72:6950–6958
    [Google Scholar]
  5. Collmer A., Keen N. T. 1986; The role of pectic enzymes in plant pathogenesis. Annu Rep Phytopathol 24:383–409
    [Google Scholar]
  6. Condemine G., Robert-Baudouy J. 1991; Analysis of an Erwinia chrysanthemi gene cluster involved in pectin degradation. Mol Microbiol 5:2191–2202
    [Google Scholar]
  7. Condemine G., Hugouvieux-Cotte-Pattat N., Robert-Baudouy J. 1986; Isolation of Erwinia chrysanthemi kduD mutants altered in pectin degradation. J Bacteriol 165:937–941
    [Google Scholar]
  8. Cornish-Bowden A., Eisenthal R. 1978; Estimation of Michaelis constant and maximum velocity from the direct linear plot. Biochim Biophys Acta 523:268–272
    [Google Scholar]
  9. Favey S., Bourson C., Bertheau Y., Koutoujansky A., Boccara M. 1992; Purification of the acidic pectate lyase and nucleotide sequence of the corresponding gene (pel A) of Erwinia chrysanthemi strain 3937. J Gen Microbiol 138:499–508
    [Google Scholar]
  10. George H. L., Mount M. S., Berman P. M. 1991; Cellular localization and characterization of pectic enzymes of Erwinia carotovora subsp. atroseptica Phytopathology 81:134–139
    [Google Scholar]
  11. Harley C. B., Reynolds R. P. 1987; Analysis of Escherichia coli promoter sequences. Nucleic Acids Res 15:2343–2357
    [Google Scholar]
  12. Hugouvieux-Cotte-Pattat N., Robert-Baudouy j. 1992); Analysis of the regulation of the pel BC genes in Erwiniu chrysanthemi 3937. Mol Microbio 6:2363–2376
    [Google Scholar]
  13. Irwin D. C., Spezio M., Walker L. P., Wilson D. B. 1993); Activity studies of eight purified cellulases: specificity synergism and binding domain effects. BiotEchnol Bioeng 42:1002–1013
    [Google Scholar]
  14. Koutoujansky A. 1987; Molecular genetics of pathogenesis by soft-rot erwinias. Annu Rep Phytopathol 25:405–430
    [Google Scholar]
  15. Koutoujansky A., Diolez A., Boccara M., Bertheau Y., Andro T., Coleno A. 1985; Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes. EMBO J 4:781–785
    [Google Scholar]
  16. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  17. Lei S.-P., Lin H.-C., Wang S.-S., Callaway J., Wilcox G. 1987; Characterization of the Erwinia carotovora pel B gene and its product pectate lyase. J Bacteriol 169:4379–4383
    [Google Scholar]
  18. Lei S.-P., Lin H.-C., Wang S.-S., Wilcox G. 1988; Characterization of the Erwinia carotopora pel A gene and its product pectate lyase A. Gene 62:159–164
    [Google Scholar]
  19. McMillan G. P., Johnstone D. J., Pérombelon M. C. M. 1992; Purification to homogeneity of extracellular polygalacturonase and isoenzymes of pectate lyase of Erwinia carotovora subsp. atroseptica by column chromatography. J Appl Bacteriol 73:83–86
    [Google Scholar]
  20. Nasser W., Reverchon S., Condemine G., Robert-Baudouy J. 1994; Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis. J Mol Biol 236:427–440
    [Google Scholar]
  21. Nasuno S., Starr M. P. 1966; Polygalacturonase of Erwinia carotovora . J Biol Chem 241:5298–5306
    [Google Scholar]
  22. Pérombelon M. C. M., Kelman A. 1980; Ecology of the soft rot Erwinias . Annu Rev Phytopathol 18:361–387
    [Google Scholar]
  23. Plastow G. S. 1988; Molecular cloning and nucleotide sequence of the pectin methyl esterase gene of Erwinia chrysanthemi B 374. Mol Microbiol 2:247–254
    [Google Scholar]
  24. Reeves P. J., Whitcombe D., Wharam S., Gibson M., Allison G., Bunce N., Barallon R., Douglas P., Mulholland V., Stevens S., Walker D., Salmond G. P. C. 1993; Molecular cloning and characterization of 13 out genes from Erwinia carotopora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram-negative bacteria. Mol Microbiol 8:443–456
    [Google Scholar]
  25. Renard C. M. G. C., Searle-van Leeuwen M. J. F., Voragen A. G. J., Thibault J.-F., Pilnik W. 1991a; Studies on apple protopectin. II. Apple cell wall degradation by pure poly-saccharidases and their combinations. Carbohydr Polymers 14:295–314
    [Google Scholar]
  26. Renard C. M. G. C., Schols H. A., Voragen A. G. J., Thibault J.-F., Pilnik W. 1991b; Studies on apple protopectin. III. Characterization of the material extracted by pure polysaccharidases from apple cell walls. Carbobydr Pobmers 15:13–32
    [Google Scholar]
  27. Reverchon S., Van Gijsegem F., Rouve M., Koutoujansky A., Robert-Baudouy J. 1986; Organization of a pectate lyase gene family in Erwinia chrysanthemi . Gene 49:215–224
    [Google Scholar]
  28. Rexová-Benková L., Markovic O. 1976 Pectic enzymes. In Advances in Carbohydrate Chemistry and Biochemistry 33 pp 323–385 Edited by Tipson R. S., Horton D. Academic Press; New York::
    [Google Scholar]
  29. Ried J. L., Collmer A. 1986; Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp atroseptica . Appl Environ Microbiol 52:305–310
    [Google Scholar]
  30. Rogers H. J., Harvey A., Lonsdale D. M. 1992; Isolation and characterization of a tobacco gene with homology to pectate lyase which is specifically expressed during microsporogenesis. Plant Mol Biol 20:493–502
    [Google Scholar]
  31. Rombouts F. M., Pilnik W. 1980 Pectic enzymes. In Economic Microbiology. Microbial Enumes and Bioconversions 5 pp 228–282 Edited by Rose A. H. Academic Press; New York::
    [Google Scholar]
  32. Saarilahti H. T., Heino P., Pakkanen R., Kalkkinen N., Palva I., Palva E.T. 1990; Structural analysis of the peh A gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora . Mol Microbiol 4:1037–1044
    [Google Scholar]
  33. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: A Laboratory Manual, 2nd edn Harbor New York: Cold Spring Harbor Laboratory: Cold Spring Harbor Laboratory;
    [Google Scholar]
  34. Sauvage C., Expert D. 1994; Differential regulation by iron of Erwinia chrysanthemi pectate lyases : pathogenicity of iron transport regulatory (cbr) mutants. Mol Plant-Microbe Interact 7:71–77
    [Google Scholar]
  35. Schoedel C., Collmer A. 1986; Evidence of homology between the pectate lyase-encoding pel B and pel C genes in Erwinia chrysanthemi . J Bacteriol 167:117–123
    [Google Scholar]
  36. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
    [Google Scholar]
  37. Studnicka G. M. 1987; Nucleotide sequence homologies in control regions of prokaryotic genomes. Gene 58:45–57
    [Google Scholar]
  38. Tamaki S. J., Gold S., Robeson M., Manulis S., Keen N. T. 1988; Structure and organization of the pel genes from Erwinia chrysanthemi EC16. J Bacteriol 170:3468–3478
    [Google Scholar]
  39. Trollinger D., Berry S., Keen N. T. 1989; Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153. Mol Plant–Microbe Interact 2:17–25
    [Google Scholar]
  40. Varghese J. N., Garrett T. P. J., Colman P. M., Chen L., Høj P. B., Fincher G. B. 1994; Three-dimensional structures of two plant β-glucan endohydrolases with distinct substrate specificities. Proc Natl Acad Sci USA 91:2785–2789
    [Google Scholar]
  41. Varner J. E., Lin L.-S. 1989; Plant cell wall architecture. Cell 56:231–239
    [Google Scholar]
  42. Vieira J., Messing J. 1982; The pUC plasmids, an M13 mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
    [Google Scholar]
  43. Vincken J.-P., Beldman G., Voragen A. G. J. 1994; The effect of xyloglucans on the degradation of cell-wall-embedded cellulose by the combined action of cellobiohydrolase and endoglucanases from Trichoderma viride . Plant Physiol 104:99–107
    [Google Scholar]
  44. von Heijne G. 1985; Signal sequences The limits of variation. J Mol Bio 184:99–105
    [Google Scholar]
  45. Weber J. 1976; Untersuchungen über Zellwandgehalt undZusammensetzung der Kartoffelknollen. Biochem Physiol Pflanzen 169:589–594
    [Google Scholar]
  46. Wegener C., Bartling S., Thomsen K. K., Olsen O. 1994; Preferential degradation of middle lamellae by an Erwinia pectate lyase. Abstract 7th International Symposium on Molecular Plant-Microbe Interactions 26: June–1 July 1994 University of Edinburgh; UK:
    [Google Scholar]
  47. Wing R. A., Yamaguchi J., Larabell S. K., Ursin V. M., McCormick S. 1989; Molecular and genetic characterisation of two pollen expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia . Plant Mol Biol 14:17–28
    [Google Scholar]
  48. Yoder M. D., Keen N. T., Jurnak F. 1993; New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science 260:1503–1506
    [Google Scholar]
  49. Yoshida A., Matsuo Y., Kamio Y., Izaki K. 1992; Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovoraEr. Biosci BiotEch Biochem 56:1596–1600
    [Google Scholar]
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