Advertisement

Molecular and General Genetics MGG

, Volume 179, Issue 2, pp 377–385 | Cite as

Amplification of fumarate reductase synthesis with λfrdA transducing phages and orientation of frdA gene expression

  • Stewart T. Cole
  • John R. Guest
Article

Summary

Thermoinducible lysis-defective derivatives of λfrdA phages (λG1F and λG40F) carrying the fumarate marate reductase gene of Escherichia coli, inserted in each of two possible orientations, were used to amplify fumarate reductase synthesis and study the aerobic repression of frdA gene expression. Anaerobic induction of lysogens containing λfrdA cI ts QS phages increased the fumarate reductase activities by up to 5 times the normal anaerobic level. Aerobic repression of frdA expression was overcome during aerobic induction and reductase activities up to twice the normal anaerobic level were produced. The rates and extents of amplification were dependent on the orientation of frdA in the λcI ts QS derivatives but not in the corresponding λcIN phages. Assays for the frdA-linked ampC gene product, β-lactamase, indicated that the intact ampC gene is not incorporated in the λfrdA phages.

Autoradiographic analysis of the polypeptides synthesized by U.V.-irradiated bacteria infected with λfrdA phages confirmed the identification of a polypeptide (Mr=72,000) as the frdA gene product (the large subunit of fumarate reductase). No precursor form was detected but another polypeptide (Mr=26.000) encoded by the 4.9 kb R.HindIII fragment was detected. The corresponding gene (g26) could be the structural gene (frdB) for the small subunit of fumarate reductase. The directions of transcription of the cloned frdA and g26 genes were deduced for the transducing phages λG1F (λfrdAr) and λG40F (λfrdA1) and were shown to be anticlockwise on the conventional Escherichia coli genetic map.

Keywords

Polypeptide Fumarate Reductase Activity Large Subunit Small Subunit 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Ambler RP, Scott GK (1978) The partial amino acid sequence of the penicillinase coded by the E. coli plasmid pR6K. Proc Natl Acad Sci USA 75:3732–3736Google Scholar
  2. 2.
    Cole ST (1979) Biochemical and genetic studies of the fumarate reductase of Escherichia coli K12. PhD Thesis. Sheffield UniversityGoogle Scholar
  3. 3.
    Cole ST, Guest JR (1978) Studies with artificially-constructed fumarate reductase transducing phages (λfrdA). Soc gen Microbiol Quart 6:42–43Google Scholar
  4. 4.
    Cole ST, Guest JR (1979) Amplification and aerobic synthesis of fumarate reductase in ampicillin-resistant mutants of Escherichia coli K12. FEMS Microb Letts 5:65–67Google Scholar
  5. 5.
    Cole ST, Guest JR (1979) Production of a soluble form of fumarate reductase by multiple gene duplication in Escherichia coli K12. Eur J Biochem 102:65–71Google Scholar
  6. 6.
    Cole ST, Guest JR Genetic and physical characterization of lambda transducing phages (λfrdA) containing the fumarate reductase gene of Escherichia coli K12. Mol Gen Genet In the pressGoogle Scholar
  7. 7.
    Dambly C, Couturier M (1970) A minor Q-independent pathway for the expression of the late in phage lambda. Mol Gen Genet 113:244–250Google Scholar
  8. 8.
    Dickie P, Weiner JH (1979) Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli. Can J Biochem 57:813–821Google Scholar
  9. 9.
    Di Rienzo JM, Nakamura K, Inouye M (1978) The outer membrane proteins of Gram-negative bacteria: biosynthesis, assembly and functions. Annu Rev Biochem 47:481–532Google Scholar
  10. 10.
    Grundström T, Jaurin B, Edlund T, Normark S Physical mapping and expression of plasmids carrying the wild-type and mutant alleles of the ampA and ampC genes. J Bacteriol. In the pressGoogle Scholar
  11. 11.
    Haddock BA, Jones CW (1977) Bacterial respiration. Bacteriol Rev 41:47–99Google Scholar
  12. 12.
    Hopkins AS, Murray NE, Brammar WJ (1976) Characterization of λtrp transducing bacteriophages made in vitro. J Molec Biol 107:549–569Google Scholar
  13. 13.
    Jones RW, Garland PB (1977) Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Biochem J 164:199–211Google Scholar
  14. 14.
    Kröger A (1978) Fumarate as terminal acceptor of phosphorylative electron transport. Biochim Biophys Acta, 505:129–145Google Scholar
  15. 15.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London New Biol) 227:680–685Google Scholar
  16. 16.
    Lambden PR, Guest JR (1976) Mutants of Escherichia coli K12 unable to use fumarate as an anaerobic electron acceptor. J Gen Microbiol 97:145–160Google Scholar
  17. 17.
    Linn T, Goman M, Scaife JG (1979) Studies on the control of the genes for transcription and translation in Escherichia coli K12. I. tufB and rplA,K have separate promoters. J Molec Biol 130:405–420Google Scholar
  18. 18.
    Lowry OH, Rosebrough NJ, Farr DL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  19. 19.
    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  20. 20.
    Moir A, Brammar WJ (1976) The use of specialised transducing phages in the amplification of enzyme production. Mol Gen Genet 149:87–99Google Scholar
  21. 21.
    Murray K, Murray NE (1975) Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease III of Haemophilus influenzae and restriction endonuclease I of Escherichia coli. J Mol Biol 98:557–564Google Scholar
  22. 22.
    Murray NE, Manduca de Ritis P, Foster LA (1973) DNA targets for the E. coli K restriction system analysed genetically in recombinants between phages phi80 and lambda. Mol Gen Genet 120:261–280Google Scholar
  23. 23.
    Normark S, Burman LG (1977) Resistance of Escherichia coli to penicillins: Fine-structure mapping and dominance of chromosomal beta-lactamase mutations. J Bacteriol 132:1–7Google Scholar
  24. 24.
    Ogawa T, Tomizawa J (1968) Replication of bacteriophage DNA. I. Replication of DNA of lambda phage defective in carly functions. J Mol Biol 38:217–225Google Scholar
  25. 25.
    Signer ER (1969) Plasmid formation: A new mode of lysogeny by phage lambda. Nature (London New Biol) 223:158–160Google Scholar
  26. 26.
    Spencer ME, Guest JR (1973) Isolation and properties of fumarate reductase mutants of Escherichia coli. J Bacteriol 114:563–570Google Scholar
  27. 27.
    Spencer ME, Guest JR (1974) Proteins of the inner membrane of Escherichia coli: Changes in composition associated with anaerobic growth and fumarate reductase amber mutation. J Bacteriol 117:954–959Google Scholar
  28. 28.
    Sutcliffe JG (1978) Nucleotide sequence of the ampicillin resistance gene of the E. coli plasmid pBR322. Proc Natl Acad Sci USA 75:3737–3741Google Scholar
  29. 29.
    Ward DF, Murray NE (1979) Convergent transcription in bacteriophage λ: Interference with gene expression. J Mol Biol 133:249–266Google Scholar
  30. 30.
    Wimpenny, JWT, Cole JA (1967) The regulation of metabolism in facultative bacteria. III. Ihe effect of nitrate Biochim Biophys Acta 148:233–242Google Scholar

Copyright information

© Springer-Verlag 1980

Authors and Affiliations

  • Stewart T. Cole
    • 1
  • John R. Guest
    • 1
  1. 1.Department of MicrobiologyUniversity of SheffieldSheffieldEngland

Personalised recommendations