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Engineered bacteriophage-defence systems in bioprocessing

Key Points

  • Bacteriophages (phages) can devastate bacterial strains that are used in fermentations and bioprocesses.

  • Comparative genomic analyses can be used to streamline the construction of genetic systems that are designed to protect bioprocessing strains against phage attack.

  • Gene-silencing techniques, such as antisense-RNA targeting of essential phage-encoded genes (for example, DNA replication) can effectively inhibit the propagation of virulent phages.

  • Trans-dominant negative mutant proteins that are derived from phage-encoded genes can be used to sabotage the function of multimeric phage–protein complexes by a process called subunit poisoning.

  • The native role of some phage-encoded genes is to protect lysogens against superinfecting phages. Such genes can also be exploited to protect non-lysogenic bioprocessing cultures from superinfection (for example, superinfection exclusion or immunity).

  • Selected engineered systems have been composed of phage-encoded cis regulatory elements, such as phage origins of DNA replication or phage promoters that drive abortive or suicide systems in the infected cell (for example, phage-triggered suicide systems).

Abstract

Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment.

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Figure 1: Points of inhibition by specific phage-resistance mechanisms during a generalized phage lytic life cycle.
Figure 2: The proposed mechanism of antisense-RNA-mediated gene silencing.
Figure 3: The accumulation of phage DNA fragments over time.
Figure 4: Phage-triggered suicide from an inducible cassette encoding a DNA endonuclease.
Figure 5: The proposed mechanism of subunit poisoning in phage defence.

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Acknowledgements

General support for the research on engineered phage-defence systems at North Carolina State University has been provided by Danisco USA, Inc., the US Department of Agriculture/National Research Initiative Competitive Grants Programme and the North Carolina Dairy Foundation. The authors also thank Chr. Hansen Inc. for their support of J. Sturino during the preparation of this article.

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DATABASES

Entrez Genome

2972

7201

DT1

O1205

Sfi11

Sfi19

Sfi21

Entrez Genome Project

Bacillus subtilis

Escherichia coli

Lactococcus lactis

Streptococcus thermophilus

FURTHER INFORMATION

Todd R. Klaenhammer's homepage

Joseph M. Sturino's homepage

Glossary

Organoleptic

Being, affecting or relating to qualities (such as taste, colour, odour and feel) of a substance that stimulate the sense organs.

Backslopping

An artisanal practice whereby a small portion of a previous batch is used to inoculate subsequent batches. From the standpoints of safety and product consistency, backslopping is not recommended owing to the risk of transferring and enriching for pathogenic microorganisms or bacteriophages, respectively.

Starter culture

Concentrated preparations of viable microorganisms (usually strains of lactic acid bacteria or yeasts) that are added to bioprocessing systems to mediate the bioconversion of the substrate in an accelerated and more reproducible manner when compared to spontaneous fermentations. These preparations are product-optimized and are generally either freeze-dried or stored frozen.

Phage unrelated

Character of strains or species whereby the bacteria in question exhibit distinct phage-sensitivity profiles, meaning that they are attacked by different groups of phages.

Holin

Small, membrane-spanning protein that accumulates in the cytosolic membrane during the lytic life cycle of a phage. Holin proteins act as a gateway to the cell wall for the phage-encoded endolysin.

Endolysin

Phage-encoded muralytic enzyme that degrades bacterial cell-wall polymers. During the lytic life cycle, endolysin accumulates in the cytosol and can cross the cytosolic membrane by the cooperative action of the phage-encoded holin.

Efficiency of plaquing

(EOP). Calculated by dividing the phage titre (in plaque forming units (pfu) per ml) on the test strain (phage-resistant) by the phage titre in pfu per ml on the parent strain (phage-sensitive indicator).

Prophage

The latent form of a temperate bacteriophage in which its genome is integrated into the bacterial chromosome without causing disruption of the bacterial cell.

Lysogen

A bacterium that contains a prophage integrated into its genome. Lysogens can induce the prophage into a lytic developmental cycle and cause cell lysis to release progeny phage. In the lysogen, the prophage remains quiescent and is effectively replicated once with every chromosomal division of the bacterium.

Superinfection

Any phage infection that occurs after an earlier one; often describing a secondary infection of a lysogenic bacterium.

Amber mutation

A nonsense mutation that introduces a premature UAG translational stop codon in a gene.

Ochre mutation

A nonsense mutation that introduces a premature UAA translational stop codon in a gene.

Phage out

The event whereby the starter culture inoculated in a bioprocessing system has been decimated by the lytic activity of phages to such a degree that the bioconversion is abandoned. Vats of partially cultured milk that have 'phaged out' are normally discarded.

Phage-insensitive mutant

Phage-resistant mutants that are derived from a parent strain that are insensitive to the phage(s) in question. Phage-insentive mutants are generally the result of targeted phage-challenge assays, whereby a phage-sensitive parent strain is repeatedly challenged with a cocktail of two or more unrelated phages. This process is often referred to as phage hardening.

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Sturino, J., Klaenhammer, T. Engineered bacteriophage-defence systems in bioprocessing. Nat Rev Microbiol 4, 395–404 (2006). https://doi.org/10.1038/nrmicro1393

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