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

Abstract

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 Rz1 is entirely embedded in the +1 reading frame of Rz, 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 in vivo, and also were found to be normal for complex formation in vitro. Analysis of the distribution and nature of single missense mutations revealed subdomains that resemble key motifs in established membrane-fusion systems, i.e., 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 Rz1 within Rz, genetically silent domains of Rz1 correspond to mutationally sensitive domains in Rz, and vice versa, suggesting that the modular structure of the two subunits facilitated the evolutionary compression that resulted in the unique embedded gene architecture.

Suppressor Analysis of the Fusogenic Lambda Spanins

“…For Rz suppressors, densitometry of the ϳ36-kDa species showed diminished accumulation compared to that of the parental mutant (Table 3). This supports the prediction that such suppressors are causing a local disruption in Rz structure within the juxtamembrane to generate since the truncation of six residues (residues 55 to 60) from the C terminus of Rz1 does not block lysis (12). A simple explanation is that a polar substitution at position 54 is compensated by an increase in hydrophobicity at position 56, suggesting that a nonpolar character at the C terminus of Rz1 has a role in spanin function.…”

“…One of the most frequently isolated suppressors was S20P, which falls within the predicted transmembrane domain (TMD). Replacement of the native Rz TMD with an artificial TMD had no effect on Rz function, strongly indicating that the Rz TMD acts as a nonspecific membrane anchor (12). It has been demonstrated that proline introduces flexible kinks within TMDs (24,25).…”

“…This instability may be due to a defect in formation of a secondary or tertiary structure. Supporting this idea, Rz L72F and Rz G143R have been shown to be severely defective in accumulation (12). Rz L72F and Rz G143R affect the a and c positions of coiled coils within CC1 and CC2 (Fig.…”

“…Strains, plasmids, and primers used in this study are listed in Tables 4 and 5. Bacterial cultures were grown on Luria broth (LB) as described previously (12). Phage plating was done on tryptone broth (TB) agar plates with molten TB agar as described previously (7).…”

“…The substitution of polar residues (including Cys) into the hydrophobic core of a dimeric coiled coil would be expected to reduce stability (18)(19)(20). Allele-specific proteolysis of Rz has been reported previously (8,12), suggesting that perturbations in the juxtamembrane coil would be detectable by Western blotting.…”