Abstract
Virtually all studies of phage infections investigate bacteria growing exponentially in rich media. In nature, however, phages largely encounter non-growing cells. Bacteria entering stationary phase often activate well-studied stress defense mechanisms that drastically alter the cell, facilitating its long-term survival. An understanding of phage-host interactions in such conditions is of major importance from both an ecological and therapeutic standpoint. Here, we show that bacteriophage T4 can efficiently bind to, infect and kill E. coli in stationary phase, both in the presence and absence of a functional stationary-phase sigma factor, and explore the response of T4-infected stationary phase cells to the addition of fresh nutrients 5 or 24 h after that infection. An unexpected new mode of response has been identified. “Hibernation” mode is a persistent but reversible dormant state in which the infected cells make at least some phage enzymes, but halt phage development until appropriate nutrients become available before producing phage particles. Our evidence indicates that the block in hibernation mode occurs after the middle-mode stage of phage development; host DNA breakdown and the incorporation of the released nucleotides into phage DNA indicate that the enzymes of the nucleotide synthesizing complex, under middle-mode control, have been made and assembled into a functional state. Once fresh glucose and amino acids become available, the standard lytic infection process rapidly resumes and concentrations of up to 1011 progeny phage (an average of about 40 phage per initially present cell) are produced. All evidence is consistent with the hibernation-mode control point lying between middle mode and late mode T4 gene expression. We have also observed a “scavenger” response, where the infecting phage takes advantage of whatever few nutrients are available to produce small quantities of progeny within 2 to 5 h after infection. The scavenger response seems able to produce no more than an average of one phage per originally available cell, and few if any further progeny are produced by cells in this mode even if fresh nutrients are made available later.
Highlights
Bacteriophage infection has traditionally been studied using bacterial hosts growing exponentially, with active aeration, in one of a few well-studied media
Patterns of T4 Infection of E. coli in Stationary Phase: To explore T4 infection of starved E. coli, a 48 h old culture of ZK126 grown in M9 supplemented with glucose and casamino acids (CAA) was infected at a range of different multiplicity of infection (MOI)
At lower MOIs, no phage are produced until fresh nutrients are provided, but the infected cells respond and produce phage very rapidly when glucose and CAA are re-added
Summary
Bacteriophage infection has traditionally been studied using bacterial hosts growing exponentially, with active aeration, in one of a few well-studied media. The resurgence of interest in therapeutic, prophylactic, and agricultural phage applications, as well as growing awareness of the substantial environmental impact of phages, make it increasingly important to study the details of phage-host interactions under conditions that are more similar to those encountered in natural environments. Such exploration is especially important since the emergence of multi-drug resistant pathogenic bacteria has become a major public health concern (World Health Organization [WHO], 2014). Such studies are relevant as close relatives of this carefully studied model organism are ubiquitous in nature and have been very widely used in cocktails for treating enteric infections (Brüssow, 2005; Sarker et al, 2012; Kutter et al, 2014; Sarker et al, 2016)
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