Oligohistidine tag mutagenesis of the lambda holin gene.

Abstract:

: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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Smith DL,Young R

doi

10.1128/JB.180.16.4199-4211.1998

subject

Has Abstract

pub_date

1998-08-01 00:00:00

pages

4199-211

issue

16

eissn

0021-9193

issn

1098-5530

journal_volume

180

pub_type

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