Oligohistidine tag mutagenesis of the lambda holin gene.
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.
Smith
D L
DL
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Young
R
R
D000483:Alleles; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli; D006639:Histidine / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009841:Oligonucleotides / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
D003062:Codon; D008565:Membrane Proteins; D009841:Oligonucleotides; C108624:S holin, bacteriophage lambda; D014764:Viral Proteins; D006639:Histidine