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Lysis-lysogeny coexistence: prophage integration during lytic development.

ABSTRACT: The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic pathways. Although traditionally thought to be mutually exclusive, increasing evidence suggests that this lysis-lysogeny decision is more complex than once believed, but exploring its intricacies requires an improved resolution of study. Here, with a newly developed fluorescent reporter system labeling single phage and E. coli DNAs, these two distinct pathways can be visualized by following the DNA movements in vivo. Surprisingly, we frequently observed an interesting "lyso-lysis" phenomenon in lytic cells, where phage integrates its DNA into the host, a characteristic event of the lysogenic pathway, followed by cell lysis. Furthermore, the frequency of lyso-lysis increases with the number of infecting phages, and specifically, with CII activity. Moreover, in lytic cells, the integration site attB on the E. coli genome migrates toward the polar region over time, leading to more spatial overlap with the phage DNA and frequent colocalization/collision of attB and phage DNA, possibly contributing to a higher chance for DNA integration.

SUBMITTER: Shao Q

PROVIDER: S-EPMC5300877 | biostudies-other | 2017 Feb

REPOSITORIES: biostudies-other

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Lysis-lysogeny coexistence: prophage integration during lytic development.

Shao Qiuyan Q Trinh Jimmy T JT McIntosh Colby S CS Christenson Brita B Balázsi Gábor G Zeng Lanying L

MicrobiologyOpen 20160817 1

The infection of Escherichia coli cells by bacteriophage lambda results in bifurcated means of propagation, where the phage decides between the lytic and lysogenic pathways. Although traditionally thought to be mutually exclusive, increasing evidence suggests that this lysis-lysogeny decision is more complex than once believed, but exploring its intricacies requires an improved resolution of study. Here, with a newly developed fluorescent reporter system labeling single phage and E. coli DNAs, t ...[more]

PMID: 27530202

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Project description:Lactobacillus reuteri has the potential to be developed as a microbial therapeutic delivery platform because of an established safety profile, health-promoting properties, and available genome editing tools. Here, we show that L. reuteri VPL1014 exhibits a low mutation rate compared to other Gram-positive bacteria, which we expect will contribute to the stability of genetically modified strains. VPL1014 encodes two biologically active prophages, which are induced during gastrointestinal transit. We hypothesized that intracellularly accumulated recombinant protein can be released following bacteriophage-mediated lysis. To test this, we engineered VPL1014 to accumulate leptin, our model protein, inside the cell. In vitro prophage induction of recombinant VPL1014 released leptin into the extracellular milieu, which corresponded to bacteriophage production. We also employed a plasmid system that does not require antibiotic in the growth medium for plasmid maintenance. Collectively, these data provide new avenues to exploit native prophages to deliver therapeutic molecules.IMPORTANCE Lactic acid bacteria (LAB) have been explored as potential biotherapeutic vehicles for the past 20 years. To secrete a therapeutic in the extracellular milieu, one typically relies on the bacterial secretion pathway, i.e., the Sec pathway. Overexpression of a secreted protein can overload the secretory pathway and impact the organism's fitness, and optimization of the signal peptide is also required to maximize the efficiency of the release of mature protein. Here, we describe a previously unexplored approach to release therapeutics from the probiotic Lactobacillus reuteri We demonstrate that an intracellularly accumulated recombinant protein is released following prophage activation. Since we recently demonstrated that prophages are activated during gastrointestinal transit, we propose that this method will provide a straightforward and efficient approach to deliver therapeutics in vivo.

Project description:The lytic cassette of Lactococcus lactis prophage TP712 contains a putative membrane protein of unknown function (Orf54), a holin (Orf55), and a modular endolysin with a N-terminal glycoside hydrolase (GH_25) catalytic domain and two C-terminal LysM domains (Orf56, LysTP712). In this work, we aimed to study the mode of action of the endolysin LysTP712. Inducible expression of the holin-endolysin genes seriously impaired growth. The growth of lactococcal cells overproducing the endolysin LysTP712 alone was only inhibited upon the dissipation of the proton motive force by the pore-forming bacteriocin nisin. Processing of a 26-residues signal peptide is required for LysTP712 activation, since a truncated version without the signal peptide did not impair growth after membrane depolarization. Moreover, only the mature enzyme displayed lytic activity in zymograms, while no lytic bands were observed after treatment with the Sec inhibitor sodium azide. LysTP712 might belong to the growing family of multimeric endolysins. A C-terminal fragment was detected during the purification of LysTP712. It is likely to be synthesized from an alternative internal translational start site located upstream of the cell wall binding domain in the lysin gene. Fractions containing this fragment exhibited enhanced activity against lactococcal cells. However, under our experimental conditions, improved in vitro inhibitory activity of the enzyme was not observed upon the supplementation of additional cell wall binding domains in. Finally, our data pointed out that changes in the lactococcal cell wall, such as the degree of peptidoglycan O-acetylation, might hinder the activity of LysTP712. LysTP712 is the first secretory endolysin from a lactococcal phage described so far. The results also revealed how the activity of LysTP712 might be counteracted by modifications of the bacterial peptidoglycan, providing guidelines to exploit the biotechnological potential of phage endolysins within industrially relevant lactococci and, by extension, other bacteria.

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Lysis-lysogeny coexistence: prophage integration during lytic development.

Publications

Lysis-lysogeny coexistence: prophage integration during lytic development.

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