A 'phase-shift' fusion system for the regulation of foreign gene expression by lambda repressor in gram-negative bacteria.
A 'phase-shift' translation fusion vector was constructed in which mutually compatible restriction sites BamHI, BclI and BglII are positioned in such a manner that the cut point is in a different reading frame, immediately following the ATG start codon and ribosome-binding site of the lambda cro gene. The lambda cro gene is expressed from promoter pR and controlled by a thermosensitive (cI857) lambda repressor. The usefulness of the expression vector was demonstrated using a galK gene lacking the ATG start codon and fusing this to the pR promoter and ATG start codon of the lambda cro gene, resulting in cI857-regulated expression of galactokinase. The vector is of general use for foreign gene expression in Escherichia coli when the target gene has a compatible cohesive end (5'-GATC-3') at the N terminus (provided, for example, by a BamHI linker). The lambda cI857-pR-cro-galK cassette was cloned into pJRD215, a wide-host-range plasmid and transferred by conjugation to a variety of Gram-negative bacteria. In all cases, thermosensitive regulation of galactokinase could be demonstrated, though the levels of induction varied considerably. These results show that the powerful lambda pR promoter and the efficient lambda repressor can be used to regulate expression of foreign genes in Gram-negative organisms other than E. coli.
Davison
J
J
Unit of Molecular Biology, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Heusterspreute
M
M
Chevalier
N
N
Brunel
F
F
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004274:DNA, Recombinant; D005686:Galactokinase / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D005814:Genes, Viral; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D003062:Codon; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D005686:Galactokinase