The pinholin of lambdoid phage 21: control of lysis by membrane depolarization

Abstract

The phage 21 holin, S(21), forms small membrane holes that depolarize the membrane and is designated as a pinholin, as opposed to large-hole-forming holins, like S(lambda). Pinholins require secreted SAR endolysins, a pairing that may represent an intermediate in the evolution of canonical holin-endolysin systems.

FIG. 1.

The absence of S²¹68 holin contributes to heterogeneity of plaque morphology. MDS12 tonA::Tn10 was used as a host for plating the indicated λ 21 hybrid phages. (A) λS²¹68R²¹; (B) λS²¹68_amR²¹; (C and D) replatings of the small and large plaques from panel B, respectively.

FIG. 2.

The S²¹68 holin triggers but does not allow release of cytoplasmic endolysins. (A) S²¹68 supports abrupt lysis with the SAR endolysin R²¹. Lysogens of MDS12 tonA::Tn10 growing logarithmically in LB medium at 30°C were thermally induced at an A₅₅₀ of 0.2 by aerating at 42°C for 15 min and at 37°C thereafter. Prophage symbols: ▪, λS²¹68⁺R²¹; ○, λS²¹68⁺R²¹_am; •, λS²¹68_amR²¹⁺; □, λS²¹68_am R^21am. (B) S²¹68 and S^λ are not functionally equivalent. Lysis-defective lysogens of MC4100 carrying a plasmid with the indicated alleles of holin-endolysin gene pairs were grown and induced as for panel A. The lysis genes on the plasmids are under the transcriptional control of the λ P_R′ promoter, which is activated by the Q protein supplied from the induction of the lysogen, as previously described (14). Symbols: ▪, λΔ(SR) and pTP2 (S²¹68R²¹); ○, λΔ(SR) and pS105 (S^λR^λ); ▾, λS_am7R⁺ and pTP4 (S²¹68R^21am), with the arrow indicating the time of addition of CHCl₃; □, λS⁺R_am and pTP3 (S²¹68_amR²¹). (C and D) Lysogens of MDS12 tonA::Tn10 bearing the indicated prophage and plasmid, the latter carrying an endolysin gene under tac promoter control, were grown and thermally induced as for panel A, except that isopropyl-β-d-thiogalactopyranoside was also added at time zero to induce the plasmid. (C) S²¹68 does not facilitate lysis by T4 E. •, λΔ(SR) and pJFT4E (e⁺); □, λS²¹68⁺R^21am and pJFT4E. CHCl₃ was added (arrow) to both cultures (• and □) at 65 min after induction. (D) S²¹68 allows abrupt lysis by the SAR endolysin P1 lyz. •, λS²¹68R^21am and pJFLyz (lyz⁺); ○, λS²¹68R^21am and pR^λ; ▪, Δ(SR) and pJFLyz; □, λΔ(SR) and pR^λ. To verify the expression of the endolysin gene, CHCl₃ was added (arrow) to two cultures (○ and □) at 75 min after induction.

FIG. 3.

Assessing the passage of a periplasmic marker through membrane lesions generated by S105 and S²¹68. MDS12 tonA::Tn10 λΔ(SR) lysogens bearing pTGS (torA-gfp-ssrA) and either pRE (vector) (A), pS105 (S105) (B), or pTP2 (S²¹68) (C) were grown in the presence of 0.2% arabinose for 100 min to induce Tor-GFP-SsrA fusion and then thermally induced. After 1 h, cells were collected by centrifugation, washed, and immediately examined under a Zeiss Axioplan 2 imaging fluorescence microscope.