Summary
The possibility of raising the expression level of a heterologous gene inLactococcus lactis by exploiting the principle of translational coupling was investigated. For this purpose, theEscherichia coli lacZ gene was transcriptionally fused to a short open reading frame (ORF) of lactococcal origin. A Shine-Dalgarno (SD) sequence was introduced at the boundary of the two ORFs. In a series of otherwise identical plasmids, the relative positions of the translational stop codon of the upstream ORF and the translational start codon of the downstream ORF (lacZ) were varied. The expression oflacZ gradually increased as the stop and start codons were placed in closer proximity. A concomitant switch from translational interference to translational coupling was observed. Best results were obtained with partially overlapping stop and start codons. It is concluded that the principle of translational coupling offers good possibilities to increase the level of heterologous gene expression inL. lactis.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523
Bolivar F, Rodriguez RL, Greene PJ, Betlach MV, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning inEscherichia coli. J Mol Biol 138:179–207
Casadaban MJ, Chou J, Cohen SN (1980) In vitro gene fusions than join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins:Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980
Chopin A, Chopin MC, Moillo-Batt A, Langella P (1984) Two plasmid-determined restriction systems inStreptococcus lactis. Plasmid 11:260–263
Das A, Yanofsky C (1984) A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair.Nucleic Acids Res 12:4757–4768
De Smit M, van Duin J (1990) Control of prokaryotic translational initiation by mRNA secondary structure. Prog Nucleic Acid Res Mol Biol 38:1–35
De Vos WM (1987) Gene cloning and expression in lactic streptococci. FEMS Microbiol Rev 46:281–295
Gatenby AA, Rothstein SJ, Nomura M (1989) Translational coupling of the maize chloroplastatpB andatpE genes. Proc Natl Acad Sci USA 86:4066–4070
Hager PW, Rabinowitz JC (1985) Translational specificity inBacillus subtilis. In: Dubnau DA (ed) The molecular biology of the Bacilli, vol II. Academic Press, Orlando, pp 1–31
Harms E, Higgins E, Chen JW, Umbarger HE (1988) Translational coupling between theilvD andilvA genes ofEscherichia coli. J Bacteriol 170:4798–4807
Kok J, Van der Vossen JMBM, Venema G (1984) Construction of plasmid cloning vectors for lactic streptococci which also replicate inBacillus subtilus andEscherichia coli. Appl Environ Microbiol 48:726–731
Kok J, Leenhouts KJ, Haandrikman AJ, Ledeboer AM, Venema G (1988) Nucleotide sequence of the cell wall proteinase gene ofStreptococcus cremoris Wg2. Appl Environ Microbiol 54:231–238
Lindahl L, Archer RH, McCormick JR, Freedman LP, Zengel JM (1989) Translational coupling of the two proximal genes in the S10 ribosomal protein operon ofEscherichia coli. J Bacteriol 171:2639–2645
Little S, Hyde S, Campbell CJ, Lilley RJ, Robinson MK (1989) Translational coupling in the threonine operon ofEscherichia coli K-12. J Bacteriol 171:3518–3522
Ludwig W, Seewaldt E, Klipper-Balz R, Schleifer KH, Magrum L, Woese CR, Fox GE, Stackebrandt E (1985) The phylogenetic position ofStreptococcus andEnterococcus. J Gen Microbiol 131:543–551
Miller J (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Oppenheim DS, Yanofsky C (1980) Translational coupling during expression o£ the tryptophan operon ofEscherichia coli. Genetics 95:785–795
Rottlander E, Trautner TA (1970) Genetic and transfection studies withBacillus subtilis phage SP50. J Mol Biol 108:47–60
Schoner BE, Hsiung HM, Belagaje RM, Mayne NG, Schoner RG (1984) Role of mRNA translational efficiency in bovine growth hormone expression inEscherichia coli. Proc Natl Acad Sci USA 81:5403–5407
Schümperli D, McKenney K, Sobieski DA, Rosenberg M (1982) Translational coupling at an intercistronic boundary of theEscherichia coli galactose operon. Cell 30:865–871
Sprengart ML, Fatscher HP, Fuchs E (1990) The initiation of translation inE. coli: apparent base pairing between the 16S rRNA and downstream sequences of the mRNA. Nucleic Acids Res 18:1719–1723
Stanssens P, Opsomer C, Mckeown YM, Kramer W, Zabeau M, Fritz HJ (1989) Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers. Nucleic Acids Res 17:4441–4454
Tabor S, Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84:4767–4771
Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Microbiol 29:807–813
Tinoco I, Borer PN, Dengler B, Levine MD, Uhlenbeck OC, Crothers DM, Gralla J (1973) Improved estimation of secondary structure in ribonucleic acids. Nature New Biol 246:40–41
Van de Guchte M, van der Vossen JMBM, Kok J, Venema G (1989) Construction of a lactococcal expression vector: expression of hen egg white lysozyme inlactococcus lactis subsp.lactis. Appl Environ Microbiol 55:224–228
Van der Lelie D, van der Vossen JMBM, Venema G (1988) Effect of plasmid incompatibility on DNA transfer toStreptococcus cremoris. Appl Environ Microbiol 54:865–871
Van der Vossen JMBM, van der Lelie D, Venema G (1987) Isolation and characterization ofStreptococcus cremoris Wg2-specific promoters. Appl Environ Microbiol 53:2452–2457
Author information
Authors and Affiliations
Additional information
Communicated by H. Böhme
Rights and permissions
About this article
Cite this article
van de Guchte, M., Kok, J. & Venema, G. Distance-dependent translational coupling and interference inLactococcus lactis . Molec. Gen. Genet. 227, 65–71 (1991). https://doi.org/10.1007/BF00260708
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00260708