Summary
A clone containing the gene encoding a pectolytic enzyme of Erwinia carotovora subsp. carotovora was selected as the one that showed maceration on a solid medium containing sodium polypectate. The gene was located on a 3.2-kb DNA fragment flanked by a BglII site and a Hin-dIII site. Via mini-Mudlac mutagenesis, a possible promoter site was located within the gene between the BglII site and the EcoRI site. The mRNA transcribed from the promoter was directed from the BglII site toward the EcoRI site, determined from the orientation of the inserted mini-Mudlac. The probable gene product was identified as a 78 kDa protein. The enzyme activity of the Escherichia coli clone was detected mainly in the periplasmic space. Potato tuber slices were not macerated by the E. coli clone and synthesis of the enzyme in E. coli was not regulated by the enzyme substrate, sodium polypectate.
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References
Andro T, Chambost JP, Kotoujansky A, Catteneo J, Bertheau Y, Barras F, van Gijsegem F, Coleno A (1984) Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase. J Bacteriol 160:1199–1203
Castilho B, Olfson P, Casadaban M (1984) Plasmid insertion mutagenesis and lac gene fusion with mini-Mu bacteriophage transposons. J Bacteriol 158:488–495
Chatterjee AK, Starr MP (1980) Genetics of Erwinia species. Annu Rev Microbiol 34:645–676
Cohen SN, Chang ACY, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114
Collmer A, Schoedel C, Roeder DL, Ried JL, Rissler JF (1985) Molecular cloning in Escherichia coli of Erwinia chrysanthemi genes encoding multiple forms of pectate lyase. J Bacteriol 161:913–920
Cupples D, Kelman A (1974) Evaluation of selective media for isolation of soft-rot bacteria from soil and plant tissue. Phytopathol 64:468–475
Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298
Keen NT, Dahlbeck D, Staskawicz B, Belser W (1984) Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli. J Bacteriol 159:825–831
Kotoujansky A, Diolez A, Boccara M, Berthea Y, Andro T, Coleno A (1985) Molecular cloning of Erwinia chrysanthemi pectinase and cellulase structural genes. EMBO J 4:781–785
Lei SP, Lin HC, Heffernan L, Wilcox G (1985) Cloning of the pectate lyase genes from Erwinia carotovora and their expression in Escherichia coli. Gene 35:63–70
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Messing J (1979) A multipurpose cloning system based on the single-stranded DNA bacteriophage M13. Recombinant DNA Technical Bulletin, N1H Publication No 79-99, vol 2, no 2, pp 43–48
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli osmotic shock and during the formation of spheroplasts. J Biol Chem 240:3685–3692
Reverchon S, van Gijsegem F, Rouve M, Kotoujansky A, Robert-Baudouy J (1986) Organization of a pectate lyase gene family in Erwinia chrysanthemi. Gene 49:215–224
Roberts DP, Berman PM, Allen C, Stromberg VK, Lacy GH, Mount MS (1986) Requirement for two or more Erwinia carotovora subsp. carotovora pectolytic gene products for maceration of potato tuber tissue by Escherichia coli. J Bacteriol 167:279–284
Stachel S, An G, Flores C, Nester EW (1985) A Tn3 lacZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium. EMBO J 4:891–898
Stack JP, Mount MS, Berman M, Hubbard JP (1980) Pectic enzyme complex from Erwinia carotovora: a model for degradation and assimilation of host pectic fractions. Phytopathol 70:267–272
Sykes RB, Matthew M (1979) Detection, assay and immunology of β-lactamases. In: Hamilton-Miller JMT, Smith JT (eds) β-Lactamases. Academic Press. New York, pp 17–49
Tsyumu S (1977) Inducer of pectic acid lyase in Erwinia carotovora. Nature 269:237–238
Tzeng KC, Hsu ST (1981) Identification and characterization of soft-rotting erwinia in Taiwan. Plant Protection Bull 23:77–85
van Gijsegem F, Toussaint A, Schoonejans E (1985) In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi. EMBO J 4:782–792
Vieira J, Messing J (1982) The pUC plasmids, and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Wang YM (1985) Cloning and expression of pectate lyase gene from Erwinia carotovora pv. carotovota. Thesis, National Chung Hsing University, Taiwan
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Hu, NT., Wang, YM., Lee, WY. et al. Characterization of a genomic clone of Erwinia carotovora subsp. carotovora TG3 encoding a pectolytic enzyme of apparent molecular weight 78 kDa. Molec Gen Genet 210, 294–298 (1987). https://doi.org/10.1007/BF00325697
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DOI: https://doi.org/10.1007/BF00325697