Virus Background and GRAS

Bacteriophages, often referred to as phages, are viruses that infect bacteria [1, 2]. The phage genetic material/genome is comprised of either DNA or RNA and may be either single or double-stranded [1, 2]. The genome is encapsulated within a protein coat referred to as the capsid, often polyhedral in shape, and can be attached to a tail with fibers involved in attachment to specific bacterial surfaces [1, 2]. Phages infect bacteria by inserting their genome into the bacterial cell [1, 2]. After the phage genome enters the cell, the phage then enters into one of two life cycles, the lytic (virulent phage) or lysogenic (temperate phage) life cycle [1–4]. The lytic life cycle begins by replication, packaging, and assembly of many new phage particles, followed by lysis of the bacterial cell resulting in the release of these phages [1–4]. The lysogenic life cycle inserts the phage genome into the bacterial cell’s chromosome, and remains dormant as a prophage until the cell encounters harsh/stressed conditions resulting in excision of the phage genome followed by replication, packaging, assembly, and lysis with the release of many new phage particles [1–4].

There are twelve known lactococcal phage species, however only three phage species dominate the industrial phage ecology and remain a primary concern for dairy fermentations using L. lactis [4–6]. The P335 species, one of the three species, is comprised of both virulent and temperate phages [4–6]. Of particular interest of this species are the virulent phages, because of their immediate negative impact upon dairy fermentations [6]. A common motif within the phage genome between both the virulent and temperate phages of this species is a genetic switch region (lambda-like) comprised of a leftward and rightward bidirectional promoter and the auto-regulatory repressor genes cI and cro [4–6]. The CI repressor is transcribed from the leftward promoter, and represses expression from the rightward promoter, and subsequent genes required for the lytic life cycle [4–6]. While the Cro repressor is transcribed form the rightward promoter, and represses expression form the leftward promoter, and subsequent genes required for the lysogenic life cycle [4–6]. These genes are regulated and expressed upon intial entry of the cell [4].

The lactococcal phage Φ31 is a lytic phage member of the P335 species [5, 6]. The 31.9 Kb Φ31 phage genome contains the aforementioned genetic switch region, but lacks the integrase gene and attachment site expressed from the leftward promoter and lacks repression from CI on the rightward promoter resulting in the lytic nature of the phage [5, 6]. The genetic switch region of Φ31 is conserved through other lytic members of the P335 species, with high sequence similarity [5, 6]. In the early stages of lactococcal infection by Φ31 expression is up regulated from the rightward promoter [5, 6]. Additionally, expression is also up regulated from a middle promoter, within the Φ31 phage genome [7]. Early expression from these two Φ31 phage promoters during lactococcal infection makes them excellent candidates for use in a Φ31 phage detection method and triggered defense system. Moreover, the conserved genetic switch region of the P335 lytic phages has the potential for use in detection and triggered defense against all lytic members of the P335 species, due to the high sequence similarity.

To date, no genetically engineered starter cultures have been approved for use in the USA by the FDA, and specific criteria for approval has yet to be established [8, 9]. This lack of approval is commonly attributed to a lack of consumer knowledge and understanding [9]. Because of the countless foreseeable benefits using genetically engineered starter cultures and the amount of increased potential profits in industrial cheese production, scientist continue research into the area and remain optimistic for future approval [9]. Generally recognized as safe (GRAS) is a status given by the USDA for allowable food additives. L. lactis is a GRAS organism, having FDA approval for food-grade applications [10]. Because the promoters and all subsequent genetic elements incorporated within this project are of lactococcal origin, it is a reasonable assumption that they should rationally meet the requirements of being considered GRAS, making this a promising model for P335 lytic phage detection and defense in future approved genetically engineered starter cultures [10].

Reference:

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2. bacteriophage | virus. In: Encycl. Br. http://www.britannica.com/science/bacteriophage. Accessed 26 Aug 2015
3. Samson JE, Moineau S (2013) Bacteriophages in Food Fermentations: New Frontiers in a Continuous Arms Race. Annu Rev Food Sci Technol 4:347–368. doi: 10.1146/annurev-food-030212-182541
4. Brüssow H (2001) Phages of Dairy Bacteria. Annu Rev Microbiol 55:283–303. doi: 10.1146/annurev.micro.55.1.283
5. Madsen SM, Mills D, Djordjevic G, et al. (2001) Analysis of the Genetic Switch and Replication Region of a P335-Type Bacteriophage with an Obligate Lytic Lifestyle on Lactococcus lactis. Appl Environ Microbiol 67:1128–1139. doi: 10.1128/AEM.67.3.1128-1139.2001
6. Durmaz E, Madsen SM, Israelsen H, Klaenhammer TR (2002) Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor. J Bacteriol 184:6532–6544.
7. Djordjevic GM, Klaenhammer TR (1997) Bacteriophage-triggered defense systems: phage adaptation and design improvements. Appl Environ Microbiol 63:4370–4376.
8. Hansen EB (2002) Commercial bacterial starter cultures for fermented foods of the future. Int J Food Microbiol 78:119–31. doi: 10.1016/S0168-1605(02)00238-6
9. Soccol CR, Pandey A, Larroche C (2013) Fermentation Processes Engineering in the Food Industry. CRC Press
10. Djordjevic GM, O’Sullivan DJ, Walker SA, et al. (1997) A triggered-suicide system designed as a defense against bacteriophages. J Bacteriol 179:6741–6748.