Molecular, Genetic, and Biochemical Analysis of Phage Lambda Lysis

Abstract

Spanins are required for outer membrane disruption, the final step in phage lysis of Gram-negative hosts. Recently spanins were shown to be fusogenic and it was proposed that spanins function by fusing the inner and outer membrane. This work uses a genetic approach to probe the functional domains of the λ spanins Rz and Rz1 by selecting for lysis-defective alleles. Our selection showed single missense mutations clustered within subdomains essential to other membrane fusion systems, including coiled-coil domains and a proline-rich region. Surprisingly, most products of lysis defective alleles were normal for accumulation and complex formation. This suggests that a majority of the mutations blocked function at a downstream step, e.g. membrane fusion.

To gain insight into spanin function, we selected for spontaneous suppressors that restored plaque formation to lysis defective alleles. Strikingly, regardless of the site of inactivating mutation, the second site rescuing mutations clustered within a coiledcoil domain near the cytoplasmic membrane. These changes encoded polar insertions into the hydrophobic core and were not allele specific. Furthermore, suppressor mutants were defective for Rz accumulation and exhibited a defect in lysis morphology. Instead of identifying point-to-point contacts, a global suppression pattern was indicated. This suggests that destabilization of the membrane-proximal segment of the Rz coiled-coil can rescue function for lysis defective alleles of Rz or Rz1 at cost of normal saltatory function.

Lastly, it is not known how λ causes lysis from the poles of E. coli. To address this, we use time-lapse microscopy to monitor the activity and subcellular localization of lysis proteins in the seconds prior to lysis. Results exclude the endolysin and spanin and indicate that the holin, which initiates lysis, also controls the site of lysis.