Antirepression System Associated with the Life Cycle Switch in the Temperate Podoviridae Phage SPC32H

Abstract

Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho-and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction. B acteriophages (phages), which are natural viral predators of bacteria, multiply by infecting specific host bacteria. Although there is an additional type of phage-host relationship called "steady-state infection," which is exemplified by filamentous phages (1), phage genome replication generally occurs via two different developmental paths: the lytic cycle and the lysogenic cycle. In contrast to the lytic cycle, which results in immediate bursting of the host bacteria and the release of bacteriophage progeny, the lysogenic cycle involves the maintenance of the phage genome as a part of the host genome for several generations, typically by integrating into host chromosomes or, more rarely, by replicating as low-copy-number phage plasmids (2-4). The expression of genes necessary for progeny production and host cell lysis is tightly repressed by a phage regulatory system, but some physiological changes in the host induced by UV light irradiation or other DNA-damaging agents activate the lytic cycle by disabling the phage repressor. Phages fall into two categories: virulent phages that replicate strictly by the lytic cycle and temperate phages that can enter both the lytic cycle and the lysogenic cycle.The lytic switch following lysogenic development has been well studied in the temperate phage lambda. In the lambda lysogenic phase, phage CI repressors form dimers and bind to specific operators to prevent expression of lambda early genes and subsequent late genes (5, 6). Upon host DNA damage, the activated host RecA protein induces CI proteolysis in a manner similar to the inactivation of the host SOS response regulator LexA (7-9). CI proteolysis leads to the expression of early and late genes, resulting in lytic development. This mechanism illustrates how lambda and other similar phages exploit ...

Noncanonical DNA-binding mode of repressor and its disassembly by antirepressor

“…To evaluate the residues that were predicted to be involved in DNA binding in vivo, we designed site-directed mutants of Rep and evaluated their repression function in a dual-plasmid bioluminescence reporter assay (Table S4) (11). Salmonella cells harboring both a reporter plasmid that contained a luciferase reporter gene fused to the Rep-binding SPC32H_042 promoter (P 042 ) and an expression plasmid that expressed WT or mutant Rep proteins under an arabinose-inducible promoter were incubated in the presence or absence of arabinose (Fig.…”

“…S1). Previously, we reported a previously unidentified homolog of Ant from the temperate phage SPC32H, which is a novel Podoviridae phage (11).…”

“…To date, two mechanisms for the inactivation of the phage Rep in temperate phages have been described. The CI repressors of lambda phage form dimers to bind operators that control the expression of lambda genes in the lysogenic phage, and derepression allowing entry into the lytic cycle relies on the autoproteolysis of CI repressors (9)(10)(11). The second mechanism to control repressor activity uses the Ant.…”

Effects of Actin-Like Proteins Encoded by Two Bacillus pumilus Phages on Unstable Lysogeny, Revealed by Genomic Analysis

“…acteriophages (phages) are viral predators of bacteria that propagate by infecting the host (1) and, based on the mode of replication of the phage genome, they are classified as lytic or lysogenic phages. To maintain the lysogenic state, phage gene expression is regulated by the phage lysogenic regulatory system.…”

Core Lipopolysaccharide-Specific Phage SSU5 as an Auxiliary Component of a Phage Cocktail for Salmonella Biocontrol

“…The host receptors for phages in Gram-negative bacteria include many outer membrane proteins, flagella, pili, and lipopolysaccharides (LPSs) (16). In Salmonella phages, for example, phages bind to various structures on the bacterial surface, such as FhuA for ES18 (17), OmpC for Gifsy-1 and Gifsy-2 (18), BtuB for SPC35 (19), flagella for ⌽ (20), and LPSs for several Salmonella phages, including P22, Felix O1, and SPC32H (21)(22)(23).…”