Expression of Clostridium thermocellum endoglucanase gene in Lactobacillus gasseri and Lactobacillus johnsonii and characterization of the genetically modified probiotic lactobacilli.
Endoglucanase A from Clostridium thermocellum resistant to pancreatic proteinase was selected out of a range of microbial cellulases expressed in lactobacilli. Two Lactobacillus-E. coli expression vectors, harboring the endoglucanase gene from C. thermocellum under the control of its own promoter (pSD1) and the Lactococcus lactis lac A promoter (pSD2), were constructed separately. Intestinal Lactobacillus strains, L. gasseri and L. johnsonii, were electrotransformed with pSD1 and pSD2, and the stability of each plasmid was evaluated. The endoglucanase activities of 0.722 and 0.759 U/ml were respectively found in culture medium of L. gasseri and L. johnsonii containing pSD1, and of 0.407 U/ml in medium of L. gasseri harboring pSD2. When the probiotic characteristics such as acid-tolerance, bile-salt tolerance, and antibiotic susceptibility were investigated, L. gasseri and L. johnsonii were resistant to low pHs of 2 and 3. Also, L. johnsonii was bile-salt resistant in the presence of 0.5% oxgall and porcine bile extract. L. johnsonii and L. gasseri showed a rather homogeneous resistant pattern against tested antibiotics. Both strains were resistant to amikacin, bacitracin, gentamicin, streptomycin, kanamycin, and colistin.
Cho
J S
JS
Department of Animal Science and Technology, College of Agriculture and Life Sciences, Seoul National University, Suweon, 441-744, Korea.
Choi
Y J
YJ
Chung
D K
DK
D000818:Animals; D001646:Bile; D002480:Cellulase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004352:Drug Resistance, Microbial; D010450:Endopeptidases / Q000494:pharmacology; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007778:Lactobacillus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D019936:Probiotics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D002480:Cellulase; D010450:Endopeptidases