Summary
Lytic activity of the φX174E (lysis) protein had previously been localized to the amino terminal 51 amino acids (a.a.) of the molecule (Blasi and Lubitz 1985). This E gene lytic activity has here been further localized to the amino terminal 29 a.a., a region of the protein which is thought to just span the cell membrane (Young and Young 1982). φX174 E gene fusions to both the lacZ gene and the chloramphenicol acetyl transferase (CAT) gene resulted in fusion proteins with lytic activity. Fusion to a third protein, trpE, did not result in lytic activity. These results support a model of oligomerization of the φX174 E protein for lytic activity since both β-galactosidase and CAT exist as tetramers in their native state. A difference in the composition of the charged amino acids at the cytoplasmic boundary between the various fusion proteins could also account for these results, since these amino acids may play a role in proper anchoring of the E protein in the cell membrane. In a spontaneous E gene mutant, which introduces a proline residue at position 9 of the E protein, lytic activity of the E protein was decreased, but not abolished. The presence of the helix-breaking proline at this position may interfere with insertion of the lysis protein into the cell membrane, leading to the decreased functional activity of the protein.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alton NK, Vapnek D (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282: 864–869
Barrell BG, Air GM, Hutchinson CA III (1976) Overlapping genes in bacteriophage φX174. Nature 264: 34–41
Bassford PJ, Silhavy TJ, Beckwith JR (1979) Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm. J Bacteriol 139: 19–31
Blasi U, Lubitz W (1985) Influence of C-terminal modifications of φX174 lysis gene E on its lysis-inducing properties. J Gen Virol 66: 1209–1213
Buckley KJ (1985) Regulation and expression of the φX174 lysis gene. Ph. D. thesis. University of California, San Diego
Friesen JD, An G, Fiil N (1983) The lethal effect of a plasmid resulting from transcriptional readthrough of rplJ from the rplKA operon on Escherichia coli. Mol Gen Genet 189: 275–281
Germino J, Bastia D (1983) The replication initiator protein of plasmid R6K tagged with β-galactosidase shows sequence-specific DNA-binding. Cell 32: 131–140
Henrich B, Lubitz W, Plapp R (1982) Lysis of Escherichia coli by induction of cloned φX174 genes. Mol Gen Genet 185: 493–497
Hutchinson CA III, Sinsheimer RL (1966) The process of infection with bacteriophage φX174. X. Mutations in a φX174 lysis gene. J Mol Biol 18: 429–447
Lieb M (1966) Studies on heat-inducible lambda bacteriophage. J Mol Biol 16: 149–163
Lindqvist BH, Sinsheimer RL (1967) Process of infection with bacteriophage φX174. XIV. Studies on macromolecular synthesis during infection with a lysis-defective mutant. J Mol Biol 28: 87–94
Lubitz W, Harkness RE, Ishiguro EE (1984) Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage φX174. J Bacteriol 159: 385–387
Maratea D, Young K, Young R (1985) Deletion and fusion analysis of the phage φX174 lysis gene E. Gene 40: 39–46
Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digested restriction fragments. Gene 19: 269–276
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Sanger F, Coulson AR, Freidmann T, Air GM, Barrell BG, Brown NL, Fiddes JC, Hutchinson CA III, Slocombe PM, Smith M (1978) The nucleotide sequence of bacteriophage φX174. Mol Biol 125: 225–246
Shultz J, Silhavy TJ, Berman ML, Fill N, Emr SD (1982) A previously unidentified gene in the spc operon of Escherichia coli K12 specifies a component of the protein export machinery. Cell 31: 227–235
Shaw WV (1971) Comparative enzymology of chloramphenicol resistance. Ann NY Acad Sci 182: 234–242
Silbavy TJ, Shuman HA, Beckwith J, Schwartz M (1977) Use of gene fusions to study outer membrane protein localization in Escherichia coli. Proc Natl Acad Sci USA 74: 5411–5415
Spindler KR, Rosser DSE, and Berk AJ (1984) Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli. J Virol 49: 132–141
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268
von Heijne G (1981) Membrane proteins: the amino acid composition of membrane-penetrating segments. Eur J Biochem 120: 275–278
Yanofsky C, Platt T, Crawford IP, Nichols BP, Christie GE, Horowith H, van Cleemput M, Wu AM (1981) The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucl Acid Res 9: 6647–6669
Young KD, Young R (1982) Lytic action of cloned φX174 gene E. J Virol 44: 993–1002
Zalkin H (1973) Anthranilate synthetase. Adv Enzymol 38: 1–39
Zipser D (1963) A study of the urea-produced subunits of β-galactosidase. J Mol Biol 7: 113–121
Author information
Authors and Affiliations
Additional information
Communicated by A. Böck
Rights and permissions
About this article
Cite this article
Buckley, K.J., Hayashi, M. Lytic activity localized to membrane-spanning region of ϕX174 E protein. Molec Gen Genet 204, 120–125 (1986). https://doi.org/10.1007/BF00330198
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00330198