DNA cloning in Lactobacillus helveticus by the exconjugation of recombinant mob-containing plasmid constructs from strains of transformable lactic acid bacteria.
A system developed for the genetic transfer of plasmids between strains of nontransformable bacteria (P. Langella, Y. le Loir, S. D. Ehrlich and A. Gruss, 1993, J. Bacteriol., 175, 5806-5813) by the specific inclusion of a mobilization (mob) region into a nonconjugative shuttle vector was used successfully to deliver the genetic determinants for beta-glucanase, beta-glucuronidase, and green fluorescent protein to Lactobacillus helveticus. Expression of two of the genes could be detected in the new host. Data suggested that resolution of cointegrates into components could release the original recombinant plasmid or generate a cointegrate deletion. All the recombinant plasmids were segregationally unstable in Lb. helveticus and there was some evidence for structural instability. Intrinsic instability in the mob-containing vector was reduced by replacing the duplicated pBluescript polylinker with that from pUC19. Sites at which cointegrate formation could occur were localized at two distinct tracts close to the D-loop that forms at the primosome during plasmid replication.
Thompson
J K
JK
Department of Food Science (Food Microbiology), Rural Development for Northern Ireland, UK. keith.thompson@dardni.gov.uk
McConville
K J
KJ
Nicholson
C
C
Collins
M A
MA
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins; D007778:Lactobacillus / Q000235:genetics*; D008164:Luminescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006026:Glycoside Hydrolases; D005966:Glucuronidase; C028020:licheninase