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Project Overview

Bacteria are used for a number of important purposes within the bioprocess and food industries. One notable example is the fermentation of dairy products in order to create cheese and yogurt. One problem that practitioners of bacteria fermentation deal with is attack by bacteriophages—viruses that infect bacteria. Many different techniques are used to control phage infection, including prevention of phage contamination by routine cleaning of the fermentation areas and rotation of cultures, employment of phage resistant strains of bacteria, and efforts to minimize opportunities for the appearance of new strains of phage. With the rise of molecular biotechnology, it is possible to create new types of phage resistant bacteria. The dairy industry has funded research of this sort to creating phage resistant strains of lactic acid bacteria.

The 2015 Utah State University iGEM team is using synthetic biology to create a strain of Lactococcus lactis that is capable of detecting, reporting, and fighting against phage infection, specifically the bacteriophage P335 from the Siphoviridae family. These bacteria will work by using a promoter from the virus itself which will activate when the phage is present. A GFP or RFP that is functional in the L. lactis bacteria will report the presence of phage so that actions can be taken to counteract virus propagation. In the version where the bacteria are able to fight against the infection, a kill switch will cause the bacteria to lyse before the phage is able to fully form, preventing further damage to the culture.

In addition, another goal for the USU team is to test the viability of promoters and fluorescent proteins that were designed for lactic acid bacteria in L. lactis. With these parts we improved the characterization of previously existing parts such as (BBa_K1033221) by showing expression of different proteins with BBa_K1820015 and BBa_K1820018, or characterizing BBa_K1033225 with BBa_K1820016 and BBa_K1820019.

See our page for the interlab study HERE

Cheese Making

Cheese making requires a lot of different steps in order to produce a successful, great-tasting cheese. The process starts off with gallons and gallons of raw milk. Because raw milk contains many unwanted organisms, pasteurization is normally applied to the milk. From the pasteurizer, the milk travels through pipes and into a centrifuge where the milk is filtered and the protein to lipid content is standardized. Once all of the unwanted solids have been filtered out of the milk, the milk is placed in troughs where enzymes and bacteria, such as Lactococcus lactis, are added to create flavor and texture. At this point, the milk starts to curdle and the curds and whey become differentiated. The curds are cut with multiple blades and the product is stirred and cooked. Because cheese makers are only interested in the curds, the whey is separated from the curds. A critical step in the entire process is the salting of the curds. Cheese would be very bland if salt was not in the process; therefore, after separation, the curds are milled, where they are broken up into smaller pieces and then salted. From here the curds are poured into different sized molds, are pressed, and then ripened for a specified time since each cheese is different. Thus cheese has been made!

While many steps within this process are very important in cheese making, the most critical step is the addition of enzymes and bacteria, specifically rennet and lactic acid bacteria, respectively. As said previously, these organisms provide the different textures and flavors to the cheese. More importantly, the bacteria are used to ferment the milk by lowering the pH, which ultimately aids in the creation of a product that can actually be cut and molded into what we call cheese. Keep scrolling to discover more about lactic acid bacteria!

Lactic Acid Bacteria Background

Lactic acid bacteria hold an important role in the production of many foods. Their use has a fairly long history and began with an interest in their ability to preserve food. Raw, unprocessed milk has a shelf life of only a few hours while cheese, depending on the type, can survive for up to five years [1]. Lactic acid bacteria not only aid in the protection of foods from other microorganisms, but also help to preserve and add nutrients, flavors, and textures that many have come to love.

The primary feature of lactic acid bacteria is that they produce organic acids, in particular lactic acid, from the fermentation of sugars. This results in two main benefits. The low pH inhibits the growth of other microorganisms which helps preserve the food and also adds a tangy, lactic acid taste [2]. Lactic acid bacteria also produce bacteriocins which naturally have antimicrobial activity but are regarded worldwide as safe, since they occur naturally and are broken down by pancreatic and gastric acids. Nisin is currently the only one of these bacteriocins to reach commercial status, but others are bound to follow [1]. They also produce exopolysaccharides which improve the texture and water-binding qualities of cheeses. This is of particular interest in low fat cheeses where they have been shown to improve melting properties [1].

Lactic acid bacteria are used in a variety of foods, and specific varieties have been selected for based upon their ability to change the flavor, texture, and nutrition of these foods (see Tables 1 and 2). Their use in dairy is particularly widespread. A variety of lactic acid bacteria is used, but the most common is Lactococcus lactis which is used in the majority of dairy products (see Table 3). As lactic acid bacteria occur naturally in dairy products, it is no wonder that they have such widespread use there. This is quite a fortuitous event as milk contains a lot of nutrients that are beneficial (see Table 4), and here we have a natural system that helps to preserve that.

Virus Background

Bacteriophages, often referred to as phages, are viruses that infect bacteria [3,4]. The phage genetic material/genome is comprised of either DNA or RNA and may be either single or double-stranded [3,4]. 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 [3,4]. Phages infect bacteria by inserting their genome into the bacterial cell [3,4]. 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 [3-6]. 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 [3-6]. 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 [3-6].

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 [6-8]. The P335 species, one of the three species, is comprised of both virulent and temperate phages [6-8]. Of particular interest of this species are the virulent phages, because of their immediate negative impact upon dairy fermentations [8]. 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 [6-8]. 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 [6-8]. 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 [6-8]. These genes are regulated and expressed upon intial entry of the cell [6].

The lactococcal phage Φ31 is a lytic phage member of the P335 species [7,8]. 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 [7,8]. The genetic switch region of Φ31 is conserved through other lytic members of the P335 species, with high sequence similarity [7,8]. In the early stages of lactococcal infection by Φ31 expression is up regulated from the rightward promoter [7,8]. Additionally, expression is also up regulated from a middle promoter, within the Φ31 phage genome [9]. 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.

Phage Control

Virus infection is something that can affect any endeavor based on cell growth—from scientific research that uses cell cultures to dairy culturing. Such infections are likely to reduce output and affect the final quality of the desired product—in our case, cheese. In fact, phage infection of lactic acid bacteria (LAB) cultures is the leading cause of slow or incomplete fermentation for cheese makers [10]. Since virus particles are ubiquitous (bacteriophages are the single most abundant type of biological entities on the planet), [11] infections are likely to occur at some point in any lab or factory. If steps are not taken to remedy a situation where an infection has occurred, virus particles will be able to infect subsequent cultures in the same bioreactors and also to spread to other bioreactors or fermentation vats.

An understanding of how the phage infections occur in the first place is helpful to know how to best approach solving this problem. In the dairy industry, a culture may be infected by phage particles in raw milk, water used in cheese production, the factory environment, ingredients recycled from previous cultures, and from the bacteria that make up the starter cultures [12]. A large percentage of raw milk contains phage particles, and studies have indicated that many phage particles can survive pasteurization, thus some infection is unavoidable in any dairy production facility [13]. Once phage particles have entered a factory environment, they may be spread a number of ways, such as aerosolization and contamination of work surfaces and equipment. When some milk by-products like whey are kept and recycled in production, they may carry phage particles over from previous cultures, even if heat treatments are applied to the by-products [14]. Finally, there may be a number of prophages contained within the bacteria in the start cultures that can be induced to start replicating viron particles by certain environmental conditions in the factory [15].

There are a number of steps that may be taken to greatly reduce the likelihood of infection. The most basic way to prevent phage infection is proper sanitation. Consistent cleaning with effective sanitizers and disinfectants, occasional treatments with UV irradiation, and thermal treatments of raw or recycled materials and ingredients are all sanitation techniques used in dairy production plants [16]. Proper design of factory and equipment including such things as maintaining separate plant areas for specific processes, using surface materials that are easy to clean and maintain, keep air relatively sterile with filters, and preventing aerosolization is also an important way to prevent the spread of phage particles [17]. Since LAB bacteriophage are generally specific to individual species and strains, rotating cultures that are made up of different bacteria reduces the likelihood that phages will be able to infect subsequent cultures in a fermentation vat. Another technique that has been used to reduce phage infection is growing starter cultures in media that inhibits phage activity by binding chemicals that are necessary to complete the phage lytic cycle or by other means [18].

Each one of the above-mentioned means of reducing phage infections are somewhat effective, however there is no silver bullet that has been able to completely solve the problem. The root problem is that the phages themselves are very difficult to destroy. For example, heat treatments only reduce the number of phages present—they do not completely remove them from media and milk [19]. Phage particles are also able to bind to dust particles in the factory, and it is not clear how effective UV irradiation or fumigation are in eliminating these phage particles present in the air [20]. Most of the other treatments currently available have similar limitations—they only control and do not eliminate phage problems.

Genetically engineered strains, such as our own, have been the subject of intense study over the last quarter century as another promising approach to control phage infection. The difficulty is that legislation regarding Genetically Modified Organisms has not allowed the implementation of these bacteria in industrial starter cultures. Research continues, however, with the hope that when legislation changes to allow these strains to be used, there will be GMO bacteria ready to be used to benefit dairy production processes.

References:

1. Kongo, J. Marcelino. "Lactic Acid Bacteria as Starter-Cultures for Cheese Processing: Past, Present and Future Developments." Kongo, J. Marcelino. Lactic Acid Bacteria-R&D for Food, Health and Livestock Purposes. InTech, 2013.
2. Leroy, Frederic and Luc De Vuyst. "Lactic acid bactereia as functional starter cultures for the food fermentation industry." Trends in Food Science & Technology (2004): 67-78.
3. Haq IU, Chaudhry WN, Akhtar MN, et al. (2012) Bacteriophages and their implications on future biotechnology: a review. Virol J 9:9. doi: 10.1186/1743-422X-9-9
4. bacteriophage | virus. In: Encycl. Br. http://www.britannica.com/science/bacteriophage. Accessed 26 Aug 2015
5. 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
6. Brüssow H (2001) Phages of Dairy Bacteria. Annu Rev Microbiol 55:283–303. doi: 10.1146/annurev.micro.55.1.283
7. 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
8. 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.
9. Djordjevic GM, Klaenhammer TR (1997) Bacteriophage-triggered defense systems: phage adaptation and design improvements. Appl Environ Microbiol 63:4370–4376.
10. Marcó M.B., Moineau S., Quiberoni A. (2012). Bacteriophage and Dairy Fermenters. Bacteriophage 2:3 (July/August/September)): 149-158.
11. Emond E., Moineau S. (2007). Bacteriophage and food fermentations. In: McGrath S, van Sinderen D, eds. Bacteriophage: Genetics and Molecular Biology. Horizon Scientific Press/Caister Academic Press, 93-124.
12. Marcó M.B., Moineau S., Quiberoni A. (2012).
13. Suárez VB, Reinheimer JA. (2002). Effectiveness of thermal treatements and biocides in the inactivation of Argentinian Lactococcus lactis phages. J Food Prot 2002; 65:1756-9; PMID: 12430698.
14. Atamer Z, Ali y, Neve H, Heller KJ, Hinrichs J. (2011). Thermal resistance of bacteriophages attacking flavor-producing dairy Leuconostoc starter cultures. Int Dairy J; 21:327-34; http://dx.doi.org/10.1016/j.idairy.2010.11.005.
15. Canchaya C, Proux C, Fournous G, Bruttin A, Brüssow H. (2003). Prophage genomics. Microbiol Mol Biol; 67:238-276; PMID: 12794192; http://dx.doi.org/10.1128/MMBR.67.2.238-276.2003.
16. Marcó M.B., Moineau S., Quiberoni A. (2012).
17. Moineau S, Lévesque C. (2005). Control of bacteriophages in industrial fermentations. In: Kutter E, Sulakvelidze A, eds. Bacteriophages: biology and applications. CRC Press, Boca Raton, FL; 285-296
18. Sturino JM, Klaenhammer TR. Bacteriphage defense system and strategies for lactic acid bacteria. Adv Appl Microbiol; 56:331-78; PMID: 15566985; http://dx.doi.org/10.1016/S0065-2164(04)56011-2.
19. Atamer, et al (2009).
20. Marcó M.B., Moineau S., Quiberoni A. (2012).
21. Broome, M.C., I.B. Powel and G.K.Y. Limsowtin. "Starter Cultures: Specific Properties." Regisnki, H. Fuquay and P.F. J.W. & Fox. Encyclopedia of Dairy Sciences. London: Academic Press, 2003.
22. Cheese Production Image: http://arizonacheese.com/cheese_production_flow_chart.htm