Identification of Escherichia coli Host Genes That Influence the Bacteriophage Lambda (λ) T4 rII Exclusion (Rex) Phenotype

Abstract

The T4rII exclusion (Rex) phenotype is the inability of T4rII mutant bacteriophage to propagate in hosts (Escherichia coli) lysogenized by bacteriophage lambda (λ). The Rex phenotype, triggered by T4rII infection of a rex⁺ λ lysogen, results in rapid membrane depolarization imposing a harsh cellular environment that resembles stationary phase. Rex "activation" has been proposed as an altruistic cell death system to protect the λ prophage and its host from T4rII superinfection. Although well studied for over 60 years, the mechanism behind Rex still remains unclear. We have identified key nonessential genes involved in this enigmatic exclusion system by examining T4rII infection across a collection of rex⁺ single-gene knockouts. We further developed a system for rapid, one-step isolation of host mutations that could attenuate/abrogate the Rex phenotype. For the first time, we identified host mutations that influence Rex activity and rex⁺ host sensitivity to T4rII infection. Among others, notable genes include tolA, ompA, ompF, ompW, ompX, ompT, lpp, mglC, and rpoS They are critical players in cellular osmotic balance and are part of the stationary phase and/or membrane distress regulons. Based on these findings, we propose a new model that connects Rex to the σ^S, σ^E regulons and key membrane proteins.

Keywords: Rex; bacteriophage T4rII; bacteriophage lambda; membrane proteins; sigma factors.

Figure 1

Upon infection by T4rII, RexA binds to RexB in a 2:1 ratio, activating RexB pore formation and cation efflux. This depolarizes the cell membrane.

Figure 2

(A) Plasmid pHA1 carries rexA and rexB on a high-copy backbone derived from pUC19. (B) pHA2 carries rexA and rexB with a transposable element Tn10 to randomly transpose the cassette into the host genome.

Figure 3

Omps may play a role in Rex, as their deletions affect Rex activity. In particular, ΔompF, ΔompL, ΔompT, ΔompW, and ΔompX attenuate Rex activity. (A) Rex activity is the ability of pHA1-delivered rex genes to reduce plating in the presence or absence of RII. Activity is derived by taking the inverse of the relative EOP of the sample in rex⁺ conditions (plasmid) compared to rex^- conditions (no plasmid). Maximal Rex activity is the complete attenuation of T4rII (rII^-) plating in presence of the rex pHA1 in E. coli BW25113 (wt). Absence of Rex activity is full plating of T4 (rII⁺) in presence of the rex⁺ pHA1 in E. coli BW25113 (wt). (B) Omps mediate ion transport as regulated by rpoE and rpoS.

Figure 4

Several other key membrane proteins may play a role in Rex, as their deletions affect Rex activity. In particular, ΔtolA completely restores T4rII plating, thereby eliminating Rex activity. (A) Rex activity is the ability of pHA1-delivered rex genes to reduce plating in the presence or absence of RII. Activity is derived by taking the inverse of the relative EOP of the sample in rex⁺ conditions (plasmid) compared to rex^- conditions (no plasmid). Maximal Rex activity is the complete attenuation of T4rII (rII^-) plating in presence of the rex pHA1 in E. coli BW25113 (wt). Absence of Rex activity is full plating of T4 (rII⁺) in presence of the rex⁺ pHA1 in E. coli BW25113 (wt). (B) These major membrane proteins are upregulated by rpoE.

Figure 5

Regulatory small RNAs and rpoS may interact with Rex, as rex genes are able to inhibit T4 (rII⁺) plating if rpoS is deleted. (A) Rex activity is the ability of pHA1-delivered rex genes to reduce plating in the presence or absence of RII. Activity is derived by taking the inverse of the relative EOP of the sample in rex⁺ conditions (plasmid) compared to rex^- conditions (no plasmid). Maximal Rex activity is the complete attenuation of T4rII (rII^-) plating in presence of the rex pHA1 in E. coli BW25113 (wt). Absence of Rex activity is full plating of T4 (rII⁺) in presence of the rex⁺ pHA1 in E. coli BW25113 (wt). (B) rpoE and rpoS are regulated by ClpXP activity and respective regulatory small RNAs.

Figure 6

Model of T4 or T4rII infection in a rex⁺ cell. Upon infection, rpoE/rpoS pathways are activated, depending on the presence of RII. Rex activation may be trigged by TolA, resulting in a physiological cellular shunt into a stationary-like phase. Blue cylinders indicate membrane proteins; green ovals indicate small regulatory RNAs involved in rpoS expression; red ovals indicate regulatory RNAs involved in rpoE expression; yellow boxes indicate small RNAs involved in the expression of membrane proteins. Small circles next to a protein indicate, firstly, its involvement in the infectivity of T4 (yellow), T4rII (blue), both (gray), or neither (X); secondly, postulated interaction with RexA and/or RexB only in the presence of RII (T4) (yellow), only in the absence of RII (T4rII) (blue), or regardless of RII presence (either phage) (gray). Pointed arrowheads indicate stimulation or upregulation, while bars indicate inhibition or downregulation. IM, inner membrane; OM, outer membrane.