Identification of a holin encoded by the Streptomyces aureofaciens phage µ1/6; functional analysis in Escherichia coli system.

An open reading frame encoding an 88 amino acid protein was present downstream of the previously characterized endolysin of Streptomyces aureofaciens phage µ1/6. Structural analysis of its sequence revealed features characteristic for holin. This open reading frame encoding the putative holin was amplified by polymerase chain reaction and cloned into the expression vector pET-21d(+). Synthesis of the holin-like protein resulted in bacterial cell death but not lysis. The holµ1/6 gene was able to complement the defective λ S allele in the nonsuppressing Escherichia coli HB101 strain to produce phage progeny. This fact suggests that the proteins encoded by both phage genes have analogous function, i.e. the streptomycete holin induces nonspecific lesions in the cytoplasmic membrane, through which the λ endolysin gains an access to its substrate, the cell wall. The concomitant expression of both S. aureofaciens holµ1/6 and λ endolysin in E. coli resulted in abrupt cell lysis. This result provided further evidence that the product of holµ1/6 gene is a holin. [ABSTRACT FROM AUTHOR]