Genetic Analysis of the Lambda Spanins Rz and Rz1: Identification of Functional Domains.

Coliphage lambda proteins Rz and Rz1 are the inner membrane and outer membrane subunits of the spanin complex-a heterotetramer that bridges the periplasm and is essential for the disruption of the outer membrane during phage lysis. Recent evidence suggests the spanin complex functions by fusing the inner and outer membrane. Here, we use a genetics approach to investigate and characterize determinants of spanin function. Because is entirely embedded in the +1 reading frame of , the genes were disembedded before using random mutagenesis to construct a library of lysis-defective alleles for both genes. Surprisingly, most of the lysis-defective missense mutants exhibited normal accumulation or localization , and also were found to be normal for complex formation Analysis of the distribution and nature of single missense mutations revealed subdomains that resemble key motifs in established membrane-fusion systems, , two coiled-coil domains in Rz, a proline-rich region of Rz1, and flexible linkers in both proteins. When coding sequences are aligned respective to the embedded genetic architecture of within , genetically silent domains of correspond to mutationally sensitive domains in , and vice versa, suggesting that the modular structure of the two subunits facilitated the evolutionary compression that resulted in the unique embedded gene architecture.