1This simulated fermentation profile is based on the following assumptions:

Culture generation time = 0.5 hours

Phage replication time = 0.5 hours

Final desired pH after 3 hours = 5.2

Average phage burst size = 50

than the one cited in the example above, or that are present initially at much higher levels, may inactivate the culture even faster. Conversely, if the culture itself is more resistant to the phages present in the plant, or if the phage levels are low when its host is introduced into the cheese environment, then the effects of that phage may be much less severe.

Ever since the phage problem was first recognized (seventy years ago!), manufacturers and scientists have developed strategies to prevent or at least minimize the incidence and impact of phage infections. In fact, phages and the problems they cause cheese manufacturers have provided the driving force not only for much of the scientific research on dairy starter cultures, but also for the development of a highly sophisticated starter culture industry. Research on bacteriophages has advanced to a remarkable extent in the past two decades due to advances in molecular biology and biotechnology by groups in Australia, New Zealand, the Netherlands, United Kingdom, the United States and Switzerland.

Several control strategies have evolved from these phage research programs, and are now part of most cheese manufacturing operations. However, before describing these approaches, it is important to note that the first requirement of any phage control plan is to ensure that the starter culture propagation environment, as well as the cheese production areas, be well sanitized and that phage entry points are as restrictive as possible. Phages are sensitive to hypochlorite and heat, and can, therefore, be inactivated from equipment and culture media. Phages move about via whey aerosols, air, and personnel, so these represent important control points. The bottom line is that, unless a cheese manufacturer practices excellent sanitation, has appropriate air handling systems in place (e.g., HEPA filters, positive pressure in culture rooms, etc.), segregates whey processing to separate areas, and limits personnel traffic, more hightech phage control measures will have little chance of being effective.

Even when good sanitation programs are in place, phages cannot be eliminated from the cheese manufacturing environment. After all, wherever there are growing lactic acid bacteria, there will be phages that attack them. Given that reality, the culture industry offers a number of products designed to minimize or prevent phage problems. These include: (1) phage inhibitory media; (2) phage-resistant cultures; and (3) culture rotation programs.

The bulk culture tank is the first place where phage propagation can occur.Tradition-ally, milk or whey was used as the culture medium, and although good growth of lactic starter bacteria could be obtained, phages were also well-suited to this simple medium. Phage-inhibitory media, in contrast, are also milk- or whey-based media, but, in addition to some added nutrients and growth factors, they also contain phosphate and citrate salts.These salts bind calcium, which is required by phages for adsorption to host cells (the first step in the infection process). There are some phages, however, that are unaffected by the lack of calcium and replicate fine in phage-inhibitory media. Thus, despite its widespread use, this approach has only a modest overall effect on phage control. Moreover, this medium only affects phage propagation within the bulk tank, and not later, during cheese manufacture.

Dairy microbiologists have long searched for lactococci and other lactic acid bacteria that naturally resist bacteriophages. Although such strains exist, in many cases, these isolates were often found to lack other relevant characteristics necessary for cheese production. For example, they either grew too slowly in milk, produced poor-quality cheese, or eventually became phage sensitive. An alternative approach that has been more successful has been to isolate spontaneous phage-resistant mutants. These mutants or variants can be obtained by repeated exposure of cheese production strains to the predominant phage present in a given environment.To ensure that fermentative properties have not been altered, the mutants must be re-evaluated for acid production rates while exposed to phages and under conditions mimicking those that occur during cheese manufacture (the so-called Heap-Lawrence procedure, named after two New Zealand researchers).

The most powerful and effective means of developing phage-resistant cultures has been to exploit the innate ability of certain strains to resist lytic phage infections. Several mechanisms have been identified that are responsible for the phage resistance phenotype in lactic acid bacteria. In general, phage-resistant strains block infection at one of several points in the lytic cycle (Figure 5-10).In some strains,phage adsorption fails to occur, due either to modifi cation of cell wall attachment sites or interference by polysaccharides. Once adsorption occurs, phages ordinarily inject DNA into the host cell to initiate phage DNA replication.This step is inhibited in some cells, although the mechanism is not clear. Following injection of phage DNA, the host cell can attack that DNA via expression of restriction enzymes that hy-drolyze DNA at specific restriction sites.

Finally, if the phage has survived all of these host defense systems, its replication can still be inhibited via a mechanism known as abortive infection. Although some phages may still be released, and the infected cell is killed, the burst size is significantly reduced, and the impact on the remaining cells is minimized.

The discovery in the 1980s and 1990s of the mechanisms described above coincided with the observation that genes coding for most of these traits in lactococci were located on plas-mids.Thus, it was possible to mobilize the relevant plasmid from one lactic acid bacterium to another, and to confer a phage resistance phe-notype into the recipient strain. This strategy was first used, in 1986, to develop a phage-resistant cheese-making strain of L. lactis subsp. lactis.The plasmid, pTRK2030, actually encoded for several phage-resistant mechanisms (at the time, the researchers were aware of only a restriction system). Because conjugation, a natural means of genetic exchange, was used to effect gene transfer, the phage-resistant transconjugant strain required no regulatory approval. Once introduced into a cheese plant, it performed extremely well.

The application of this and other natural phage resistance mechanisms to improve starter cultures quickly followed. If one plas-mid is good, two (or more) reasoned the Klaen-hammer group, would be better. Thus, a stacking strategy was developed in which a single strain contained different plasmids, each coding for unique phage resistance properties. Alternatively, the different plasmids can be separately introduced into a single strain, yielding multiple isogenic strains harboring a unique phage resistant plasmid.These strains can then be used in a rotation program (see below) to

Figure 5-10. Lytic cycle of lactic bacteriophages and steps at which phage resistance mechanisms function.

Figure 5-10. Lytic cycle of lactic bacteriophages and steps at which phage resistance mechanisms function.

prevent any one phage from reaching high enough levels to cause problems.

As noted above, it is inevitable that a phage capable of infecting a given starter culture strain will eventually emerge in a cheese plant.The infecting phage may be naturally occurring, or more likely, is the result of DNA modification, mutation, or a gene acquisition event. There is, after all, no other phage-host relationship that is more dynamic than that which occurs in a large cheese plant. Every day, there may be as many as 1018 bacteria growing in such a plant, all serving as potential hosts for phages.Thus, staying a step or two or three ahead of the phage is no easy matter, and engineering more durable and longer-lasting phage resistance into lactic acid bacteria is a major challenge.The availability of genome information (for both the host cells and their phages) in the hands of clever microbiolo-gists has made it possible to deploy powerful strategies for development of new phage-resistant strains (Box 5-9).

Despite the opportunity for phages to find and infect host cells, the only scenario that would result in a particular phage actually affecting a fermentation would be if the phage were initially present at reasonably high levels. Phages can only replicate, and therefore increase in number and reach danger levels, when they infect a suitable host. If the host cell is not present, the phage will not last long in that environment, especially when good

Box 5—9. How to Outsmart a Virus

It is remarkable that the main biological problem that confronts the modern and sophisticated fermented dairy foods industry is viruses, nature's simplest life form. Given the discovery in the 1980s that some lactic acid starter culture bacteria are armed with natural anti-viral defense systems, one might think that controlling these viruses, or bacteriophages, would be a relatively easy task. However, this is clearly not the case, as "new" bacteriophages continue to emerge, much to the frustration of cheese manufacturers.

The inhibition of milk fermentations by bacteriophages was first described by New Zealand researchers in 1935 (Whitehead and Cox).Although phage infections of starter culture bacteria were initially only occasionally reported, such occurrences became more common as the size of the cheese industry grew.As previously noted, many innate phage-resistant mechanisms, and the genes responsible for those mechanisms, have been identified in lactococci and other lactic acid bacteria and subsequently used to construct new phage-resistant strains. However, the conferred resistance has generally been effective only for the short-term, because new virulent phages invariably appear within a relatively short time. Given the parasitic nature of bacteriophages (i.e.,they can only replicate on suitable hosts) and the incredible opportunity for phage-host interactions, it is really no surprise that wherever lactic acid bacteria live and grow, phages capable of infecting those bacteria will eventually arise. This realization has inspired researchers, especially the Todd Klaenhammer group at North Carolina State University, to develop novel and quite clever strategies for combating these phages and protecting starter cultures from phage infections.These "intelligent" approaches (also referred to as "artificial," since they do not exist naturally) rely on molecular trickery to either prevent phage adsorption or to short-circuit the phage lytic cycle and thereby block phage replication.

Pips and Pers

The first stage of the phage infection process occurs when a phage recognizes and then attaches to a specific site on the surface of the host cell. One such receptor site, referred to as a phage infection protein, or pip, has been identified in Lactococcus lactis subsp. lactis C2 (Geller et al., 1993; Garbutt et al., 1997). Infection by phage c2 does not occur in cells lacking an intact copy of pip. Since mutations in the pip gene do not appear to have any other phenotypic effect on the cell, engineered pip-defective cells would be insensitive to phages that depend on Pip for binding. Recently, similar results were obtained for another class of lactococcal phages that do not recognize pip, but instead bind to membrane proteins called receptor-binding proteins (Dupont et al., 2004).

Replication of the phage genome occurs soon after a phage has infected a host cell. Replication begins at a unique location within the phage genome,the so-called origin of replication (or

Box 5—9. How to Outsmart a Virus (Continued)

ori site), which is characterized by several direct and inverted repeated sequences.The ori region, therefore, serves as the recognition site for the DNA replication machinery. If, however, there were multiple "decoy" copies of the ori region within the cell, then the DNA replication system would, in theory, be diluted out and phage genome replication would be reduced. Indeed, this approach, called phage-encoded resistance, or Per, was shown by Hill et al. (1990) to work in Lactococcus lactis. In this case, a phage genome fragment (containing the ori region) from phage ^50 was introduced into a phage-sensitive strain via a multi-copy plasmid vector. The resulting transformant had a phage-resistant phenotype, and, as predicted, the greater the expression of the plasmid, the more phage resistant was the cell.

Suicide Traps

For a given phage to inhibit a fermentation, that phage must first infect a host cell, replicate inside that cell and make more phage particles, and then lyse the cell to release the fully assembled and infectious phage. If the lytic cycle is somehow blocked prematurely, then viable phage particles will not be formed or released. Although these naturally occurring abortive infection systems (Abi) are widespread in lactic acid bacteria, it is also possible to contrive or engineer such a system by genetic means.This strategy, envisioned by O'Sullivan et al. (1996) and Djord-jevic et al. (1997), involved construction of a plasmid that contained lactococcal-derived genes encoding for a restriction/modification system (LlaIR) positioned downstream from a promoter region obtained from a lactococcal phage (^31).When strains harboring this plasmid were attacked by phage, the restriction enzyme encoded by LlaIR was induced and expressed, which then lysed host DNA, leading to cell death.Thus, the single infected cell dies, but phage propagation is blocked. Or, as the authors eloquently stated,"infected cells . . . undergo programmed cell death in an altruistic fashion," sparing the remaining population from a more severe phage infection (Djordjevic et al., 1997).

Antisense mRNA

When DNA is transcribed into mRNA, the relevant transcription signals are present on only one of the two strands of DNA, the coding or sense strand.The non-coding or antisense strand is not transcribed. If, however, the transcription elements (consisting of a promoter and terminator regions) are positioned around the antisense strand, the latter will also be transcribed.Assuming enough of the antisense RNA transcripts are produced, they will hybridize with the sense mRNA, preventing translation and protein synthesis. Phage replication can be reduced by targeting genes encoding for essential phage proteins, although in lactococci, this approach has been met with mixed results (Kim et al., 1992;Walker and Klaenhammer, 1998). However, by combining antisense technology with a Per approach, it was possible to obtain "explosive" amplification of the number of antisense ori transcripts and achieve a more significant reduction in phage proliferation (McGrath et al., 2001;Walker and Klaenhammer, 2000).

Phages strike back

As noted above, the phage-host relationship is extremely dynamic.There may easily be more than 1015 cells in a large cheese plant and just as many viable phages. Even if the frequency of spontaneous mutations within the phage population is low (< 10-9), many "new" phages will certainly appear on a regular basis. Some of these mutations will confer an ability to infect a previously resistant cell. However, given the nature of the phage infection, DNA packaging, and cell burst processes, co-mingling of host and phage DNA is also likely to occur via recombination-type events.

Box 5—9. How to Outsmart a Virus (Continued)

Thus, lytic phages that have acquired host or prophage DNA will emerge within phage populations. In some cases, it turns out, the acquired DNA provides the phage with the means necessary to counter or circumvent the resistance of the host cell. For example, in one well-known study in which a once resistant L. lactis commercial starter culture strain (LMA12-4) harboring a phage resistance plasmid (pTR2030) had become phage-sensitive, it was shown that the phage (^50)had acquired a methylase gene that was carried by the host cell (Sanders et al., 1986;AIatossava and Klaenhammer, 1991). Since methylases modify host DNA so that it is not degraded by host restriction enzymes, the presence of this methylase gene in phage ^50 en-abIes this phage to seIf-methyIate its DNA and thereby evade restriction by the host ceII.


AIatossava,T., and T.R. Klaenhammer. 1991. Molecular characterization of three small isometric-headed bacteriophages which vary in their sensitivity to the lactococcal phage resistance plasmid pTR2030.AppI. Environ. Microbiol. 57:1346-1353. Djordjevic, G.M., D.J. O'Sullivan, S.A.Walker, M.A. Conkling, and T.R. Klaenhammer. 1997.Triggered-suicide system designed as a defense against bacteriophage.J. Bacteriol. 179:6741-6748. Dupont, K.,T.Janzen, F. K.Vogensen,J.Josephsen, and B. Stuer-Lauridsen. 2004. Identification of Lactococcus lactis genes required for bacteriophage adsorption.Appl. Environ.Microbiol. 70:5825-5832. Geller, B.L., R.G. Ivey,J.E.Trempy, and B. Hettinger-Smith. 1993. Cloning of a chromosomal gene required for phage infection of Lactococcus lactis subsp. lactis C2.J. Bacteriol. 175:5510-5519. Garbutt, K.C., J. Kraus, and B.L. Geller. 1997. Bacteriophage resistance in Lactococcus lactis engineered by replacement of a gene for a bacteriophage receptor. J. Dairy Sci. 80:1512-1519. Hill, C., L.A. Miller, and T.R Klaenhammer. 1990. Cloning, expression, and sequence determination of a bacteriophage fragment encoding bacteriophage resistance in Lactococcus lactis. J. Bacteriol. 172:6419-6426.

Kim, S.G., and C.A. Batt. Antisense mRNA-mediated bacteriophage resistance in Lactococcus lactis.Appl.

Environ. Microbiol. 57:1109-1113. O'Sullivan, D.J., S.A.Walker, and T.R Klaenhammer. 1996. Development of an expression system using a lytic phage to trigger explosive plasmid amplification and gene expression. Biotechnology 14:82-87. McGrath, S., G.F. Fitzgerald, and D. van Sinderen. 2001. Improvement and optimization of two engineered phage resistance mechanisms in Lactococcus lactis.Appl. Environ. Microbiol. 67:608-616. Sanders, M.E., RJ. Leonhard, W.D. Sing, and T.R. Klaenhammer. 1986. Conjugal strategy for construction of fast acid-producing, bacteriophage-resistant lactic streptococci for use in dairy fermentations.Appl. Environ. Microbiol. 52:1001-1007. Walker, S.A., and T.R Klaenhammer. 1998. Molecular characterization of a phage-inducible middle promoter and its transcriptional activator from the lactococcal bacteriophage ç>31. J. Bacteriol. 180:921-931. Walker, S.A., and T.R Klaenhammer. 2000.An explosive antisense RNA strategy for inhibition of a lactococcal bacteriophage.Appl. Environ. Microbiol. 66:310-319. Whitehead, H.R., and G.A. Cox. 1935.The occurrence of bacteriophage in starter cultures of lactic streptococci. N. Z.J. Sci.Technol. 16:319-320.

sanitation is practiced. This rationale forms the basis of the culture rotation programs that are widely used in the cheese industry. A given culture, which may contain only one or two, but as many as six different strains, will be used for a day or two, then followed by a different culture containing strains with different phage sensitivity patterns.Thus, phages that infect one or more of the strains present in the initial culture may have begun to become established in the plant, but absent their host cells (which were rotated out), these phages will quickly die out and decrease in number. Rotation programs can be supplemented by various phage monitoring procedures in which phage titers (i.e., phage concentrations) are enumerated or estimated. This allows cheese manufacturers to continue using the same culture (therefore, minimizing product variation) and only make a switch when the phage levels reach a particular threshold.

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