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Journal of Bacteriology, August 1998, p. 4199-4211, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Oligohistidine Tag Mutagenesis of the lambda  Holin Gene

David L. Smith and Ry Young*

Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128

Received 30 January 1998/Accepted 13 June 1998

Holins are a diverse group of small integral membrane proteins elaborated by bacteriophages to lyse bacterial hosts and effect release of progeny phages in a precisely timed manner. Recently, the holin S gene of phage lambda  was overexpressed and the holin protein was purified to homogeneity by means of an oligohistidine tag procedure and immobilized metal affinity chromatography (D. L. Smith, D. K. Struck, J. M. Scholtz, and R. Young, J. Bacteriol. 180:2531-2540, 1998). Numerous locations within the S gene were tested as sites for an oligohistidine-tag-encoding insertion which preserves holin function. The lysis phenotypes of these alleles, expressed from moderate-copy-number transactivation plasmids, were characterized. A striking class of mutants, previously referred to as early-dominant, have been found to have severe lysis defects but are fully functional in the presence of wild-type protein. Results presented here reveal that the early-dominance phenotype is independent of S107 inhibitor function. The results provide insight into the mechanism of hole formation and indicate that, while oligomerization is required in the pathway to hole formation, a nucleation event may also be required.

* Corresponding author. Mailing address: Department of Biochemistry and Biophysics, MS2128, Texas A&M University, College Station, TX 77843-2128. Phone: (409) 845-2087. Fax: (409) 862-4718. E-mail: ryland{at}.tamu.edu.

Journal of Bacteriology, August 1998, p. 4199-4211, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Xu, M., Struck, D. K., Deaton, J., Wang, I.-N., Young, R. (2004). A signal-arrest-release sequence mediates export and control of the phage P1 endolysin. Proc. Natl. Acad. Sci. USA 101: 6415-6420 [Abstract] [Full Text]  
  • Wang, I.-N., Deaton, J., Young, R. (2003). Sizing the Holin Lesion with an Endolysin-{beta}-Galactosidase Fusion. J. Bacteriol. 185: 779-787 [Abstract] [Full Text]  
  • Grundling, A., Manson, M. D., Young, R. (2001). Holins kill without warning. Proc. Natl. Acad. Sci. USA 10.1073/pnas.151247598v1 [Abstract] [Full Text]  
  • Gründling, A., Smith, D. L., Bläsi, U., Young, R. (2000). Dimerization between the Holin and Holin Inhibitor of Phage lambda. J. Bacteriol. 182: 6075-6081 [Abstract] [Full Text]  
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  • Grundling, A., Manson, M. D., Young, R. (2001). Holins kill without warning. Proc. Natl. Acad. Sci. USA 98: 9348-9352 [Abstract] [Full Text]  


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