Bacteriophages in Industry

Michael J Callanan, North Carolina State University, Raleigh, North Carolina, USA Todd R Klaenhammer, North Carolina State University, Raleigh, North Carolina, USA
The biotechnology industry has benefited from the molecular knowledge and tools derived from the study of bacteriophage biology. For industries that rely on bacteria for bioprocessing, fermentation failure due to phage infection is a long-term and continuing problem.

Secondary article
Article Contents
. Introduction . Bacteriophages Molecular Workhorses for Biotechnology . Bacteriophages and Bioprocessing . Future Perspectives

Introduction
Bacteriophages are viruses of bacteria. As the most basic biological entities, phages have provided the molecular insight and tools required to exploit biological systems. The ability to manipulate biological systems has provided the platform for the current expansion in biotechnologybased industries. Traditionally, bacteriophages have been regarded as a nuisance in industrial processes that rely on bacterial fermentation. In particular, the dairy industry has struggled with phage-induced fermentation failure in the production of cheese, yogurt, and other fermented food products. The bacteria that comprise the starter cultures produce acids and other metabolites that contribute to the avour and consistency of the food products. Phages that are naturally present in the environment can attack the bacteria and slow or stop the fermentation process, resulting in reduced product quality and considerable nancial loss. Signicant eort has been dedicated by the dairy industry to combating phage infection.

Bacteriophages Molecular Workhorses for Biotechnology

The discoveries of restriction endonucleases, which cut DNA into fragments at specic sites, and of DNA ligase, which catalyses the cojoining of DNA molecules from any organism, have produced a biotechnological revolution. The ability to manipulate the hereditary molecule has provided the means to redesign cellular processes for commercial-scale synthesis of bioactive proteins and has given rise to the modern biotechnology industry. There are currently about 1600 biotechnology companies in the United States alone, with total revenues of $19.6 billion and sales of $13.4 billion (Ernst & Young, 1999). Therapeutics and health care products are the focus of the majority of the new biotechnology companies. In fact, it is becoming increasingly dicult to separate the biotechnology and pharmaceutical sectors since pharmaceutical companies have decided to adopt recombinant DNA approaches to new product development. The

synthesis of scarce biomolecules, such as hormones, growth factors, enzymes and antibodies, currently represents the most lucrative application of recombinant DNA technology. The exact contribution of bacteriophages in the synthesis of the myriad of new biotechnology products reaching the market is impossible to ascertain because of the need to protect the technology in this competitive sector. However, aside from elucidating the mechanics of the molecular biology that underpins the biotechnology industry, some of the most basic tools in recombinant DNA technology are based on phage genetics (see Figure 1). The recombining of DNA molecules from dierent organisms has been facilitated by phage enzymes, which are routinely employed in molecular biology. For example, the most common DNA ligase in use today, phage T4 DNA ligase, is an enzyme involved in phage DNA recombination and packaging (Richardson et al., 1968). Phage T4 itself is a member of the T-class of Escherichia coli phages (coliphages) and the study of bacteriophage genetics originated with seven T-class coliphages, designated T1 to T7. The T4 genome has been the most extensively characterized, although the smaller T7 genome has also provided many valuable genetic tools. Phage T7 has provided one of the most potent expression systems to achieve high-level production of recombinant proteins (Studier and Moatt, 1986). High levels of expression can be achieved by cloning a gene of interest downstream of the T7 promoter and introducing it into cells already expressing T7 RNA polymerase. A relatively small amount of T7 RNA polymerase, provided in trans from a cloned copy of T7 gene 1, is sucient to direct high-level transcription from a T7 promoter in a multicopy plasmid. With ecient translation, a target protein can potentially accumulate to greater than 50% of the total cell protein in 3 h or less. As an alternative, the pL promoter from coliphage l also oers precise control of gene expression (Caulcott and Rhodes, 1986). In the presence of the phage cI repressor protein, expression of the pL promoter is strictly repressed. Employing the temperature-sensitive mutant of the cI repressor means that simple exposure to a dierent external temperature inactivates the repressor and allows pL-directed transcription. These elegant systems have been
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Bacteriophages in Industry

(d) Packaging and release Transduction Cosmids Integration or transcription Chromosome Bacterial cell

(a) Lysogeny or lysis Integration systems Promoters, T7, SP6, pL RNA polymerase

Phage infection cycle Linear replication O Theta/RCR replication

(c) Capsid assembly Phage display

Capsid proteins

(b) DNA replication Vector construction DNA polymerase DNA ligase

Figure 1 Molecular tools and enzymes derived from the study of bacteriophage (see text for details). (a) Phage DNA is injected from the phage particle and either integrates in the chromosome (lysogeny) or enters the lytic cycle. (b) In the lytic cycle, phage DNA replicates as either a linear molecule or via theta/rolling circle replication (RCR) as a circular form. (c) Phage proteins redirect the hosts cellular machinery to produce the building blocks of the virion. (d) Phage DNA is packaged in the capsid of the phage particle and cell lysis is effected to release progeny phage.

widely exploited as genetic tools for expression of proteins in bioreactors. The other essential components of recombinant DNA technology are plasmid and cosmid vectors. Plasmids are self-replicating pieces of DNA that allow the recombinant DNA to be cloned, manipulated and transferred between dierent organisms. Bacteriophages provided the basis for many of the rst vectors that allowed the cloning and characterization of important genes from the chromosomes of both prokaryotic and eukaryotic organisms. In addition, the natural ability of coliphage l to mobilize nonphage DNA between bacteria, known as transduction, allowed the rst genetic maps of bacterial chromosomes to be determined. Transduction occurs because the phage DNA can integrate into the chromosome of the bacteria to become a prophage, a process known as lysogeny. Imprecise excision events of the lysogenized phage from the host chromosome result in chromosomal DNA being packaged in the capsid, the protein-based head of the phage particle (see Figure 2). The non-phage DNA can be introduced into a new host bacterium by infecting it with the phage. Elucidation of the mechanism of packaging DNA in the phage capsid facilitated the design of vectors known as cosmids (Collins and Hohn, 1978). Cosmids are
2

Capsid proteins Displayed peptide Phage DNA Gene for displayed peptide

Collar

Tail fibres

Figure 2 Schematic representation of a bacteriophage particle. The phage DNA is encapsulated in the capsid prior to the release of progeny phage from the cell by DNA packaging mechanisms. Insertion of foreign DNA into the gene encoding a capsid protein results in expression of the introduced peptide or protein on the surface of the phage.

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Bacteriophages in Industry

plasmid vectors that also encode the l packaging signals, or cos sites, which allow cloned DNA to be selectively packaged into phage heads. They have the advantage of accommodating up to 40 kb of DNA, which is invaluable for cloning large fragments and screening of genome libraries. Vectors based on plasmids are generally limited to DNA inserts of 10 kb or less. Therefore, a library constructed using a cosmid requires a signicantly smaller number of clones to completely encode the same amount of genetic material. Furthermore, the plasmid origin of replication means the cosmid vectors can also replicate intracellularly as plasmids, which is advantageous for preparation and manipulation of the cloned DNA. Current research in phage genetics continues to provide new tools with obvious biotechnological applications. For example, a novel expression system that exploits both a phage promoter and phage-based replication system was recently described for high-level protein production in Lactococcus lactis (OSullivan et al., 1996). The system relies on a high-copy-number plasmid encoding the origin of replication of lactococcal phage f31. When f31 infects a L. lactis strain harbouring this construct, the phage origin of replication produces an explosion of plasmid replication. Genes of interest are cloned downstream of a phageinducible promoter on the same plasmid and introduced into L. lactis. Upon phage infection, the gene of interest is amplied as part of the plasmid and expressed from the phage-inducible promoter. A 20-fold increase in production of a gene cloned downstream of the promoter was observed in L. lactis using this construct. The system has the advantage of being silent, or inactive, in the absence of phage infection. In addition, the protein product is released into the supernatant upon cell lysis by the inducing phage, reducing the number of manipulations required for product purication. This tightly controlled explosive expression system clearly demonstrates the enormous potential of phage-based tools. With the advent of whole-genome sequencing and analysis, phage P1 has supplanted the use of l-based vectors. P1-based vectors can encode even larger foreign DNA inserts (up to 200 kb) and are referred to as phage P1 articial chromosomes or PACs (Ioannou et al., 1994). Genomic libraries currently employed in the investigation of important organisms (including human, mouse, virus and bacteria) have been constructed as PACs. These PAC libraries have the added advantage over phage l of being maintained in eukaryotic cells, which allows the genomic library of a eukaryotic organism to be investigated in a eukaryotic background. In addition to PAC libraries, a novel system that allows dened nucleotide changes and specic chromosomal rearrangements in eukaryotic cells has been developed based on the Cre site-specic DNA recombinase of P1 (Sauer and Henderson, 1988). The Cre/ lox system has multiple potential applications and the genetic engineering of transgenic mice has particularly beneted from its use (Sauer, 1998).

One of the most exciting developments in the biotechnology eld of recent years has been the generation of phage display libraries (Smith, 1985). Phage display allows the presentation of peptides or, more recently, entire proteins on the surface of a phage particle (Figure 2). The phage library is screened (also known as biopanning) with antibodies, DNA or other proteins that can interact with the displayed protein or peptide. This interaction allows the separation and identication of ligands with high anity for the displayed protein. By presenting antibodybinding domains or combinatorial peptides on the surface of the phage, the technique has been used successfully to select for antibodies and enzyme inhibitors with increased anities for the target protein (Wilson and Brett Finlay, 1998). The main advantages of phage display libraries are the relatively small size of the phage particle for screening protocols and the physical link to the DNA contained within the phage (see Figure 2). The ability to select ligates from millions to billions of bioactive ligands provides a powerful tool for drug and target discovery in the biopharmaceutical industry. In conclusion, the emergence of the biotechnology industry has relied to a large degree on bacteriophage genetics, not just in research and development but also at the production level. The genes for important bioactive proteins have been cloned and characterized from human and other cells, using phage-based vectors, phage enzymes and phage display. Promoters isolated from phage genomes are employed to drive the expression of these genes in recombinant microorganisms. Future developments in this rapidly expanding industry will inevitably be linked to advances in bacteriophage genetics.

Bacteriophages and Bioprocessing

Microorganisms produce a wide range of economically important products. Commercial products include amino acids synthesized by Corynebacterium, antibiotics by Streptomyces, enzymes by Bacillus, acetic acid by Acetobacter, and lactic acid produced by species comprising the lactic acid bacteria, notably, Lactococcus, Lactobacillus, Oenococcus, Leuconostoc and Streptococcus thermophilus (Demain, 2000). All of these products require large-scale fermentation processes in order to be protable, and a bacteriophage infection can devastate any bacterial fermentation. The details of phage infections of industrial-scale processes are not generally published, but phages have been isolated following fermentation failure for all the commercially important bacteria listed above (Ogata, 1980; Sanders, 1987). Introduction of carefully controlled operating procedures and maintenance of aseptic conditions can usually control phage-related problems. One notable exception is the food industry, where the raw material is not sterile and
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Bacteriophages in Industry

the batch-type processes are particularly prone to phage contamination. Phage infections occur primarily in the largest component of the dairy fermentation industry: mesophilic cheese (cottage, cheddar, gouda) fermentations that rely on Lactococcus lactis. However, it is now apparent that phages can disturb the process and microbial ecology of many industrial food and beverage fermentations. Phages attacking Oenococcus oenos are suspected to cause periodic failures of the malolactate reaction during secondary fermentation of wine (Davis et al., 1985; Gindreau and Lonvaud-Funel, 1999). The industrial production of vinegar by Acetobacter is carried out either in trickling generators or as a submerged fermentation. Phages have been suggested as one reason for slowing of trickling generators and the complete loss of productivity in submerged fermentation. Isolation of phage particles from disturbed fermentations, both trickle and submerged, points to phages as the problem (Sellmar et al., 1992). More recently, investigations into the ecology of sauerkraut and pickle fermentations have revealed a number of phages, virulent for Leuconostoc mesenteroides, Leuconostoc fallax, Lactobacillus plantarum, and Pediococcus pentosaceus. Leuconostoc fallax phages representing the major two families, Siphoviridae and Myoviridae, appear to alter the composition and succession of bacteria involved in this natural fermentation process. Finally, the expansion of markets and increasing production of dairy food products that rely on lactic acid bacteria other than Lactococcus have been reected in heightened interest in the phages of Lactobacillus and Streptococcus. In the Yakult milk fermentation, a prophage lysogenizing Lactobacillus casei S-1 (Shirota) became obligately virulent after transposition of an insertion element into its genome (Shimizu-Kadota and Tsuchida, 1984). Curing the prophage from the L. casei starter culture permanently eliminated phage disturbances in the fermented Yakult milk process. Increased production of yogurt worldwide has been accompanied by a corresponding increase in bacteriophage attacks against the coccus component of the yogurt starter cultures, Streptococcus thermophilus. A number of these Streptococcus phages have been studied in detail and their complete genomes have been sequenced (Table 1). Sequence analysis revealed that the phages of S. thermophilus are highly related, composed of both virulent and temperate members, and appear to have been derived from one com-

mon ancestor (Desiere et al., 1998). It is interesting that Lactobacillus delbrueckii, the rod component of yogurt starter cultures, is not generally known to suer from bacteriophage attacks in industrial-scale fermentations. The industrialization of the manufacture of cheese and other fermented food products has accentuated the problem of phage-related fermentation failure. Phage infection of dairy starter cultures is common, devastating and without parallel in other fermentation industries. As a result, considerable eort has been devoted to the molecular characterization of the lactococcal phages that infect industrial starter strains. Phages isolated from dairy plants have been classied into 12 genetically distinct species (Jarvis, 1991). Three species (936, P355 and c2 species) are the main causes of disruption in dairy plants worldwide (Daly et al., 1996). Monoclonal antibodies, DNA probes and PCR methods are now available to rapidly identify these three common species. The results of identication eorts are applied to adjust starter rotations and to develop phage-resistant strains. The genomes of members of each species have been fully sequenced as part of the eort to combat these phages and provide important insights into phage genetics and phagehost relationships (Table 1). The relationship between bacteriophage and bacterial genome plasticity was observed early in phage biology. Of particular interest to dairy microbiologists is the relationship of the emergence of new industrial phages by acquisition of DNA from the bacterial host chromosome. It is clear that phages can acquire DNA from latent prophages in the chromosome, and this plays a signicant role in the emergence of new industrial phages (Durmaz and Klaenhammer, 2000). These observations concur with the proposed modular evolution of bacteriophages (Botstein, 1980). Phages appear to evolve not along lines of linear descent but rather from a family of interchangeable genetic elements, each of which carries a particular function and which are exchanged via recombination among phages with overlapping host ranges. The rapid appearance of phages resistant to anti-phage systems is a testament to the capabilities of this evolutionary process and conrms that phages will continue to be a problem in the bioprocessing industry. Phages are always present in dairy environments, and phage control relies on a variety of practical approaches. Modern food manufacturing facilities employ advanced

Table 1 Lactococccus and Streptococcus phages for which the genome has been sequenced Phage (species) S19/S21 f01205 fDT1 sk1 (936) r1t (P355) c2/bIL67 (c2) Host S. thermophilus S. thermophilus S. thermophilus L. lactis L. lactis L. lactis Reference Lucchini et al. (1999) Stanley et al. (1997) Tremblay and Moineau (1999) Chandry et al. (1997) van Sinderen et al. (1996) Lubbers et al. (1995); Schouler et al. (1994)

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Bacteriophages in Industry

factory designs with improved sanitation, adequate ventilation, incorporation of closed vats, direct vat inoculation, propagation of starter cultures in phageinhibitory media, culture rotation and the use of phageresistant starters. Even with these measures, phage infection remains a major cause of slow acid production in dairy fermentations. Slow acid development results in unpredictable processing schedules, decreased turnover of product in fermentation vats and ultimately considerable nancial loss. Historically, starter cultures were complex mixtures of many individual strains that were developed and propagated over many years of subculturing. With an increased understanding of microbiology, starter cultures were developed with single, paired or multiple microbial strains. In recent years the starter culture system rst described by Lawrence and Pearce (1972) has been favoured by commercial manufacturers (Daly et al., 1996). The system relies on multiple-strain starters that contain 25 carefully selected cultures used continuously without rotation. The single or paired cultures contain ecient acid-producing strains that reduce fermentation time, increase plant productivity and enhance the uniformity and product quality of larger-scale fermentations. When phages are detected during the cheesemaking process, the aected strain is removed and replaced with a new strain or a phage-resistant derivative. While this system is widely used and maintains reasonably uniform product quality, the limited availability of suitable replacement strains can restrict its long-term use. Replacement strains must be phage-unrelated and there is a nite
Recombinant defence mechanisms

number of commercially viable Lactococcus strains. In addition, single and paired strain starter cultures that are used continuously in fermentation plants are more susceptible to phage attack than their mixed-strain ancestors. For many years, bacteriophage-insensitive mutants (BIMs) were the only alternative to replacing a starter strain with another phage-unrelated strain. BIMs arise naturally during phage infection, most likely as a result of a change in the phage receptor on the surface of the bacterium. However, BIMs for particular strains can be problematic, since in many cases the mutant derivatives lack the fermentative capability of the parent strain. Recent advances in lactococcal genetics have expanded knowledge of natural bacteriophage defences and increased the ability to protect starter cultures from phage infection. In response to attacks by highly virulent bacteriophages encountered in the dairy environment, the lactococci have evolved multiple and elaborate phage defence mechanisms (Figure 3). The genes encoding these anti-phage systems are generally plasmid-encoded but some are located on the bacterial chromosome. The resistance mechanisms encode proteins that target specic points in the phage infection cycle and usually disrupt the timing or eciency of the process. The documented mechanisms of inhibition have been divided into four groups, based on their mode of action: adsorption inhibition, blocking of DNA injection, restriction/modication, and abortive infection (reviewed by Allison and Klaenhammer, 1998; Forde and Fitzgerald,
Natural defence mechanisms

Phage attachment to phage receptor Cell wall Phage DNA injection Phage transcription Phage DNA Phage RNA Antisense RNA prevents translation or destabilizes phage transcripts Antisense RNA Phage DNA replication

Bacteriophage-insensitive mutants mask the phage receptor and inhibit adsorption DNA injection is retarded R/M systems digest unmodified phage DNA

Phage

Plasmid Molecular decoys, plasmids encoding phage origins of replication compete for critical DNA replication proteins

Replication protein

Abortive infection systems act at various stages following DNA injection of the phage genome to inhibit DNA replication, retard packaging or limit progeny development

Figure 3 Naturally occurring and recombinant phage defence mechanisms.

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Bacteriophages in Industry

1999). Adsorption inhibition and blocking of DNA injection remain the least understood of the resistance mechanisms owing to the lack of knowledge regarding the cell wall and membrane receptors involved in phage adsorption and injection. The ability to interfere with phage adsorption and DNA injection has been correlated with a number of lactococcal plasmids, although the genetic determinants responsible for the interference have yet to be fully characterized at the molecular level. Restriction modication (R/M) systems are widely disseminated across bacteria and have been characterized at the molecular level. The restriction enzyme component of an R/M system cleaves DNA at a specic site and the modication component encodes a methylase activity that modies DNA at the restriction site. As noted above, the discovery of restriction enzymes was seminal in the development of recombinant DNA technology. These naturally serve to protect the cell from bacteriophage infection by hydrolysis of unmodied foreign DNA after injection. R/M mechanisms are widely distributed in Lactococcus and have the advantage of acting early in phage infection, which reduces the impact on cell viability. On the other hand, R/M systems are inherently leaky. Some phage DNA will escape restriction, and will replicate and yield modied progeny phages that are immune to restriction when they encounter the R/M system again. The last group of resistance mechanisms, abortive infection (Abi) systems, is a very diverse group of genetic elements that disrupt the phage cycle at some point following infection and the early stages of phage development. They are characterized by a high death rate of the host cell before the release of phage progeny. This can act as an eective trap to prevent the accumulation of phage in the culture medium. Of the 17 Abi systems studied to date, little is known about the exact molecular mechanism of action. Abi genes characteristically show a G 1 C content of 2627%, well below that of lactococci, indicating their acquisition via horizontal evolution. The point of interference has been identied for four Abi systems that act at or before phage DNA replication. Three others appear to retard phage DNA transcription and one may act on the cos ends of certain phages. The unique and multivariant action of Abi systems and lack of homology between the genes have clouded eorts to elucidate the mechanism of action of these anti-phage systems. To date, more than 40 dierent lactococcal plasmids encoding phage resistance systems have been reported (Moineau, 1999). Anti-phage barriers hold signicant commercial value and many of the plasmids incorporating these systems have been patented worldwide (Table 2). The ecacy of the various plasmid encoded phage resistance mechanisms varies considerably. In this regard, single defence mechanisms are quickly overwhelmed by resistant phages. It has been found that naturally occurring lactococcal strains isolated from dairy cultures usually harbour multiple plasmids encoding complementary
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Table 2 Examples of patented natural phage resistance plasmids in Lactococcus lactis Plasmid pTR2030 pCI528 pCI750 pFW094 pJW563 pHW393 pIL415 pSRQ700 pSRQ800
a

Resistance mechanism AbiAa, acts at or before phage DNA replication LlaI, restriction/modication system Ads, inhibition of phage adsorption AbiG, possibly eects phage transcription LlaAI, restriction/modication system LlaBI, restriction/modication system LlaDII, restriction/modication system AbiB, degradation of phage transcripts LlaDCHI, restriction/modication system AbiK, aects phage DNA replication or maturation

Abi, Abortive infection

defence systems. Stacking, or combining, of the phage resistance mechanisms occurs naturally in wild-type lactococci, presumably selected by the constant pressure of virulent phage encountered during years of selection in the phage-contaminated dairy fermentation environment. Many of the phage resistance plasmids found in lactococci can be mobilized by conjugation, and this property has been exploited to construct phage-insensitive strains harbouring multiple phage resistance plasmids. Two major problems can be encountered using this approach. First, there remains only a handful of industrially useful defence systems on plasmids that can be easily mobilized. Second, many industrial Lactococcus strains are highly refractory to the acquisition of new DNA and, once modied, strains can lose key functional attributes. In spite of these diculties, numerous Lactococcus starter strains have been modied to contain additional phage defenses and these have been successfully incorporated in starter culture rotation programmes throughout the United States and the rest of the world. The ease with which phages overcome natural phage defences encountered in the lactococci has prompted investigation of novel phage resistance mechanisms (Figure 3). A number of successful approaches have examined the phage genome itself as a source of potential resistance traits. For example, the phage origin of DNA replication was introduced in multiple copies on a plasmid into lactococcal cells, its presence retarded phage replication by acting as a decoy and titrating away phage replication proteins from the phage origin (Hill et al., 1990). Antisense RNA technology has also shown some promise in providing a barrier to phage attack (Chung et al., 1992; Kim and Batt, 1991; Walker and Klaenhammer, 2000). Antisense RNA relies on the articial expression in the cell of antisense RNA to an important phage gene. The antisense RNA facilitates degradation of transcripts or inhibits the translation to protein of the sense

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Bacteriophages in Industry

RNA strand by hybridizing to it and creating an inactive double-stranded RNA molecule. In contrast to mobilizing the plasmids encoding the natural defence mechanisms between starter strains, these new anti-phage strategies rely on recombinant DNA technology. In the current social and regulatory climate they are unlikely to nd immediate industrial application. The direct incorporation of genetically modied bacteria into the food chain remains a controversial and emotive topic. None the less, these novel anti-phage strategies have provided signicant insights into the fundamentals of the phage lytic cycle and phage host evolutionary relationships.

Future Perspectives
Recombinant DNA technology has been successfully exploited to synthesize important biomolecules primarily for the pharmaceutical industry. Commercial products generated through conventional biotechnology also have the potential to benet enormously from recombinant DNA technology (Demain, 2000). The Gram-positive bacterial species Bacillus, Corynebacterium, Lactobacillus and Streptomyces already produce enzymes, amino acids and life-saving antibiotics on an industrial scale. Other bacterial species, such as Pseudomonas and Clostridium spp, have the potential to provide many more industrially useful enzymes and biologically active proteins that cannot currently be produced on an industrial scale at a reasonable cost. Strain improvements of Bacillus and Streptomyces were achieved using random mutagenesis and subsequent rounds of selection for overproducing mutants. These crude methods can now be replaced by rened genetic techniques whereby precise manipulation of the bacterial chromosome can be performed to synthesize a desirable product in large quantities. Moreover, with genome sequencing and microarray technology the process of metabolic engineering, wherein a bacterial cells catabolism is redirected and ne-tuned to preferentially synthesize a selected end product, is now possible. Implementation of such exquisite control requires special tools. The availability of new molecular biology tools derived from bacteriophage will accelerate the employment of these microorganisms in the biotechnology industry. It should be pointed out that, through recombinant DNA technology, production of biomolecules is not limited to the organism in which the required genes are discovered. It is possible to use many combinations of desirable organisms as cell factories. Thus, the continued expansion of the biotechnology industry will be based on the ability to eectively, and protably, exploit microorganisms and to develop the phage-based tools necessary to accomplish this exploitation. The longevity of these microorganisms in bioprocessing applications will also depend on the phage defence strategies available to protect them.

A potentially exciting development relating to bacteriophages is the resurgence of interest in phage therapy (Barrow and Soothill, 1997). Initial studies in the use of phages to counteract bacterial infections 60 years ago failed to produce proper phage-based therapies for a number of reasons. The results of therapies were inconsistent because of the emergence of phage-resistant strains, the lack of highly virulent strains and the selection of phages with too narrow a strain specicity. Host-related factors, such as clearance of phages by defence mechanisms and liberation of endotoxins as a result of widespread bacterial lysis, also contributed to cessation of work in this area. The recent emergence of antibiotic-resistant microbes and increasing problems with allergic reactions to antibiotics have prompted a re-evaluation of phage-based therapy. In particular, experiments with bacteriophages investigating the prevention of E. coli-induced diarrhoea in calves and protection of skin grafts from Pseudomonas aeruginosa and Acinetobacter baumanii infections have proved promising (Barrow and Soothill, 1997). Moreover, proliferation of undesirable microorganisms occurs in many other situations that could be amenable to using bacteriophages as antibacterial agents. Contaminating pathogenic and spoilage organisms are a serious problem for the food industry. With increased consumer concern regarding food additives, phages represent a safe and natural biological control system to deal with contamination of food after processing. The build-up of biolms on food processing equipment and in medical devices such as catheters, prostheses and implants is of particular concern to the food and biomedical sectors. Incorporated within a biolm, bacteria are less accessible to antibiotics and chemical control and present a considerable challenge. It is possible in the future that phages will become part of our rst line of defence against bacteria in these situations, further increasing their importance in the food, agricultural, pharmaceutical and bioprocessing industries.

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Further Reading
Demain AL and Davies JE (1999) Manual of Industrial Microbiology and Biotechnology, 2nd edn. Washington, DC: American Society for Microbiology Press. Cairns J, Stent GS and Watson JD (1992) Phages and The Origins of Molecular Biology. New York: Cold Spring Harbor Press. Glick BR and Pasternak JJ (1994) Molecular Biology: Principles and Applications of Recombinant DNA. Washington, DC: American Society for Microbiology Press. Marks T and Sharp R (2000) Bacteriophages and biotechnology: a review. Journal of Chemical Technology and Biotechnology 75: 617. Santi E, Capone S, Mennuni C et al. (2000) Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics. Journal of Molecular Biology 296: 497508.

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