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. 1996 May;62(5):1500–1506. doi: 10.1128/aem.62.5.1500-1506.1996

Stable transformation of the gram-positive phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus with several cloning vectors.

M J Laine 1, H Nakhei 1, J Dreier 1, K Lehtilä 1, D Meletzus 1, R Eichenlaub 1, M C Metzler 1
PMCID: PMC167925  PMID: 8633849

Abstract

In this paper we describe transformation of Clavibacter michiganensis subsp. sepedonicus, the potato ring rot bacterium, with plasmid vectors. Three of the plasmids used, pDM100, pDM302, and pDM306, contain the origin of replication from pCM1, a native plasmid of C. michiganensis subsp. michiganensis. We constructed two new cloning vectors, pHN205 and pHN216, by using the origin of replication of pCM2, another native plasmid of C. michiganensis subsp. michiganensis. Plasmids pDM302, pHN205, and pHN216 were stably maintained without antibiotic selection in various strains of C. michiganensis subsp. sepedonicus. We observed that for a single plasmid, different strains of C. michiganensis subsp. sepedonicus showed significantly different transformation efficiencies. We also found unexplained strain-to-strain differences in stability with various plasmid constructions containing different arrangements of antibiotic resistance genes and origins of replication. We examined the effect of a number of factors on transformation efficiency. The best transformation efficiencies were obtained when C. michiganensis subsp. sepedonicus cells were grown on DM agar plates, harvested during the early exponential growth phase, and used fresh (without freezing) for electroporation. The maximal transformation efficiency obtained was 4.6 x 10(4) CFU/microgram of pHN216 plasmid DNA. To demonstrate the utility of this transformation system, we cloned a beta-1,4-endoglucanase-encoding gene from C. michiganensis subsp. sepedonicus into pHN216. When this construction, pHN216:C8, was electroporated into competent cells of a cellulase-deficient mutant, it restored cellulase production to almost wild-type levels.

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Selected References

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  1. Baer D., Gudmestad N. C. In vitro cellulolytic activity of the plant pathogen Clavibacter michiganensis subsp. sepedonicus. Can J Microbiol. 1995 Oct;41(10):877–888. doi: 10.1139/m95-121. [DOI] [PubMed] [Google Scholar]
  2. Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Clark M. C., Lawrence C. H. Characterization of a plasmid in isolates of Corynebacterium sepedonicum. Can J Microbiol. 1986 Aug;32(8):617–622. doi: 10.1139/m86-116. [DOI] [PubMed] [Google Scholar]
  5. Dunny G. M., Lee L. N., LeBlanc D. J. Improved electroporation and cloning vector system for gram-positive bacteria. Appl Environ Microbiol. 1991 Apr;57(4):1194–1201. doi: 10.1128/aem.57.4.1194-1201.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. GOTO M., OKABE N. Cellulolytic activity of phytopathogenic bacteria. Nature. 1958 Nov 29;182(4648):1516–1516. doi: 10.1038/1821516a0. [DOI] [PubMed] [Google Scholar]
  7. Kurusu Y., Kainuma M., Inui M., Satoh Y., Yukawa H. Electroporation-transformation system for coryneform bacteria by auxotrophic complementation. Agric Biol Chem. 1990 Feb;54(2):443–447. [PubMed] [Google Scholar]
  8. Laine M. J., Zhang Y. P., Metzler M. C. IS1237, a repetitive chromosomal element from Clavibacter xyli subsp. cynodontis, is related to insertion sequences from gram-negative and gram-positive bacteria. Plasmid. 1994 Nov;32(3):270–279. doi: 10.1006/plas.1994.1066. [DOI] [PubMed] [Google Scholar]
  9. Meletzus D., Bermphol A., Dreier J., Eichenlaub R. Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382. J Bacteriol. 1993 Apr;175(7):2131–2136. doi: 10.1128/jb.175.7.2131-2136.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Meletzus D., Eichenlaub R. Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and development of a cloning vector. J Bacteriol. 1991 Jan;173(1):184–190. doi: 10.1128/jb.173.1.184-190.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Metzler M. C., Zhang Y. P., Chen T. A. Transformation of the gram-positive bacterium Clavibacter xyli subsp. cynodontis by electroporation with plasmids from the IncP incompatibility group. J Bacteriol. 1992 Jul;174(13):4500–4503. doi: 10.1128/jb.174.13.4500-4503.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mogen B. D., Oleson A. E. Homology of pCS1 Plasmid Sequences with Chromosomal DNA in Clavibacter michiganense subsp. sepedonicum: Evidence for the Presence of a Repeated Sequence and Plasmid Integration. Appl Environ Microbiol. 1987 Oct;53(10):2476–2481. doi: 10.1128/aem.53.10.2476-2481.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Strobel G. A. A phytotoxic glycopeptide from potato plants infected with Corynebacterium sepedonicum. J Biol Chem. 1970 Jan 10;245(1):32–38. [PubMed] [Google Scholar]
  14. Strobel G. A. Biological Activity of a Phytotoxic Glycopeptide Produced by Corynebacterium sepedonicum. Plant Physiol. 1968 Oct;43(10):1673–1688. doi: 10.1104/pp.43.10.1673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Strobel G. A. Purification and Properties of a Phytotoxic Polysaccharide Produced by Corynebacterium sepedonicum. Plant Physiol. 1967 Oct;42(10):1433–1441. doi: 10.1104/pp.42.10.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wohlleben W., Arnold W., Bissonnette L., Pelletier A., Tanguay A., Roy P. H., Gamboa G. C., Barry G. F., Aubert E., Davies J. On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette. Mol Gen Genet. 1989 Jun;217(2-3):202–208. doi: 10.1007/BF02464882. [DOI] [PubMed] [Google Scholar]
  17. Yoshihama M., Higashiro K., Rao E. A., Akedo M., Shanabruch W. G., Follettie M. T., Walker G. C., Sinskey A. J. Cloning vector system for Corynebacterium glutamicum. J Bacteriol. 1985 May;162(2):591–597. doi: 10.1128/jb.162.2.591-597.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

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