ppGpp-Dependent Negative Control of DNA Replication of Shiga Toxin-Converting Bacteriophages in Escherichia coli

Abstract

bThe pathogenicity of enterohemorrhagic Escherichia coli (EHEC) strains depends on the production of Shiga toxins that are encoded on lambdoid prophages. Effective production of these toxins requires prophage induction and subsequent phage replication. Previous reports indicated that lytic development of Shiga toxin-converting bacteriophages is inhibited in amino acidstarved bacteria. However, those studies demonstrated that inhibition of both phage-derived plasmid replication and production of progeny virions occurred during the stringent as well as the relaxed response to amino acid starvation, i.e., in the presence as well as the absence of high levels of ppGpp, an alarmone of the stringent response. Therefore, we asked whether ppGpp influences DNA replication and lytic development of Shiga toxin-converting bacteriophages. Lytic development of 5 such bacteriophages was tested in an E. coli wild-type strain and an isogenic mutant that does not produce ppGpp (ppGpp 0 ). In the absence of ppGpp, production of progeny phages was significantly (in the range of an order of magnitude) more efficient than in wild-type cells. Such effects were observed in infected bacteria as well as after prophage induction. All tested bacteriophages formed considerably larger plaques on lawns formed by ppGpp 0 bacteria than on those formed by wild-type E. coli. The efficiency of synthesis of phage DNA and relative amount of lambdoid plasmid DNA were increased in cells devoid of ppGpp relative to bacteria containing a basal level of this nucleotide. We conclude that ppGpp negatively influences the lytic development of Shiga toxin-converting bacteriophages and that phage DNA replication efficiency is limited by the stringent control alarmone.

Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages

“…Prophage induction experiments were performed as described previously (Nowicki et al, 2013). Phage lytic development was provoked in lysogenic bacteria, growing at 37 uC in LB medium at A 600 0.2, by addition of 1 mg mitomycin C ml 21 .…”

“…Lytic development of lambdoid phages was studied in one step growth experiments in phage-infected bacteria, as described previously (Nowicki et al, 2013). Briefly, bacteria were grown in LB medium supplemented with MgSO 4 and CaCl 2 (to a final concentration of 10 mM each) at 37 uC to A 600 0.2.…”

“…To estimate the efficiency of lysogenization, we used the procedure described in detail previously (Nowicki et al, 2013), with slight modifications. Briefly, host bacteria were cultured to A 600 1.0 in LB medium supplemented with MgSO 4 and CaCl 2 (to final concentrations of 10 mM each) at 37 uC with shaking.…”

“…The rationale for testing this parameter stems from previous observations that indicated sensitivity of DNA replication of different Stx phages to environmental or physiological stresses, like starvation, oxidative stress or temperature variation Nejman-Faleń czyk et al, 2012;Nowicki et al, 2013).…”

Shiga toxins and stx phages: highly diverse entities

“…Consequently, there are studies aimed at detecting conditions that repress phage induction. Nejman et al (2011) have shown that plasmids derived from stx phages are not able to replicate in amino acid-starved bacteria, and Nowicki et al (2013) have studied the mechanism responsible for the inhibition of stx phage replication under amino acid starvation, identifying the role of the ppGpp alarmone. Nejman-Faleń czyk et al (2012) have suggested that reducing food consumption during illness, or even fasting, and providing minerals and citrate could be an option to manage STEC infections as they found that these conditions can delay and diminish the efficiency of phage particle formation.…”