Exclusion of T4 phage by the hok/sok killer locus from plasmid R1

Abstract

The hok (host killing) and sok (suppressor of killing) genes (hok/sok) efficiently maintain the low-copynumber plasmid R1. To investigate whether the hok/sok locus evolved as a phage-exclusion mechanism, Escherichia coli cells that contain hok/sok on a pBR322-based plasmid were challenged with T1, T4, T5, T7, and phage. Upon infection with T4, the optical density of cells containing hok/sok on a high-copy-number plasmid continued to increase whereas the optical density for those lacking hok/sok rapidly declined. The presence of hok/sok reduced the efficiency of plating of T4 by 42% and decreased the plaque size by ϳ85%. Single-step growth experiments demonstrated that hok/sok decreased the T4 burst size by 40%, increased the time to form mature phage (eclipse time) from 22 to 30 min, and increased the time to cell lysis (latent period) from 30 to 60 min. These results further suggest that single cells exhibit altruistic behavior.Recent advances in the study of killer loci and phage exclusion suggest that the two areas may be closely related (22,29,39 (11,15,24,26,35) by killing plasmid-free daughter cells. Each killer system contains a gene that encodes a protein toxin and a gene that inhibits the toxin from being expressed (15). Antisense mRNA killer systems use fold-back inhibition and a small unstable antisense mRNA to prevent translation of the toxin in plasmid-bearing cells (44). A halt in transcription of antisense mRNA due to plasmid loss leads to a rapid decline in its concentration, allowing expression of the killer peptide (14). Proteic killer systems overexpress a labile antitoxin that masks the toxin. A halt in synthesis of antitoxin due to plasmid loss leads to a decline in antitoxin concentration, allowing the toxin to poison the cell.Plasmid loss is not the only event that can prevent transcription of antisense mRNA and induce suicide systems. Temperature shock, amino acid deprivation (1), antibiotics (e.g., rifampin [16]), and phage infection all cause a sudden change in protein synthesis (23). This suggests that cell killing might be induced during phage infection. Furthermore, restriction-modification systems are generally believed to have evolved to inhibit phage infection; however, some plasmid-borne restriction-modification systems have been shown recently to mediate plasmid stability by postsegregational killing (22). This also suggests that plasmid-stabilizing killer loci may also function to exclude phage and might explain why the chromosome contains several killer loci (such as relB Phage-exclusion systems (other than superinfectivity) have been found in phage, plasmids, and cryptic phage elements (29,42). The best-studied systems are exclusion of T4 rII mutants by rex of lysogens (2, 42), exclusion of T4 by lit of the cryptic DNA element e14 (3, 42), exclusion of polynucleotide kinase and RNA-ligase mutants of T4 by prr of cryptic DNA element prr (41, 46), and exclusion of T7 by pif of plasmid F (10, 29). The rex system consists of two proteins, RexA and RexB, which form a membrane comp...

Conditionally lethal genes associated with bacterial plasmids

“…Interestingly, gef and ref analogues are conserved in several bacterial species including E. coli B and C , Agrobacterium and Rhizobium, suggesting that these genes play an important role in the physiology of the cell (Poulsen et al, 1989). It has been shown recently that hoklsok genes are involved in phage exclusion of bacteriophage T 4 and this mechanism could play an important role in the evolution of hoklsok-like genes found on bacterial chromosomes (Pecota & Wood, 1996).…”

Within-host competition selects for plasmid-encoded toxin–antitoxin systems

“…Several laboratory experiments have demonstrated that this type of competition can influence the outcome of plasmid and virus evolution (Bull & Molineaux 1992;Turner & Chao 1998). Moreover, findings of antagonistic interactions between horizontally transmitted elements suggest that within-host competition plays an important role in their evolution in natural environments (Molineux & Spence 1984;Pecota & Wood 1996;Engelberg-Kulka et al 1998).…”

The phage abortive infection system, ToxIN, functions as a protein–RNA toxin–antitoxin pair

“…17). One suggested function of TA systems is to reduce phage infection; in fact, the hok/sok loci from E. coli plasmid R1 excludes phage T4 (26), and the chromosomal TA system, mazEF, protects against phage P1 (27). The toxIN genes abort the infections of different phages and function in a number of different genera; therefore, chromosomal or plasmid-encoded representatives across 3 TA classes [proteic (mazEF), RNA antisense (hok/sok), and protein-RNA (toxIN)] can function to limit phage infection.…”