Characterization of the Dual Start Motif of a Class II Holin Gene

Holins are small membrane proteins that, at a genetically programmed time in a bacteriophage infective cycle, allow bacteriolytic enzymes, or endolysins, to escape to the periplasm and to attack the cell wall. Most holins fall into two sequence classes, I and II, based on the number of potential transmembrane domains (three for class I and two for class II). The prototype class I holin gene, S lambda, has a dual start motif and encodes not only the effector holin, Slambda105, but also an inhibitor, Slambda107, with a Met-Lys ...extension at the terminus. The prototype class II holin gene of phage 21, S 21, begins with the motif Met-Lys-Ser-Met ..., and a potential RNA secondary structure overlaps the Shine-Dalgarno sequence. Here, we demonstrate that (i) two protein products are elaborated from S 21, S2171 and S2168; (ii) the shorter product is required for lysis; (iii) the longer product, S2171, inhibits S 21 function; and (iv) the Lys-2 residue is important for the inhibitor function. Moreover, the RNA stem-loop structure is involved in the downregulation of S2171 synthesis. However, our results suggest that, in S 21, different segments of the single consensus Shine-Dalgarno sequence serve the two translational starts. These results show that the dual start motifs of class II holin genes are functionally homologous to those of class I holin genes.