The impact of bacteriophage genomics

Stephen McGrath1,2,
, Gerald F Fitzgerald1,2,3,4 and Douwe van Sinderen2,3

The discovery of (bacterio)phages revolutionised microbiology tive analyses to be performed, resulting in more coherent
and genetics, while phage research has been integral to classification schemes and providing insights into evolu-
answering some of the most fundamental biological questions tionary processes [1,2,3]. The role of prophage-encoded
of the twentieth century. The susceptibility of bacteria to virulence factors in infectious bacteria has been firmly
bacteriophage attack can be undesirable in some cases, established [4,5], as has the ability of bacteriophages to
especially in the dairy industry, but can be desirable in others, contribute to bacterial genome diversity and to facilitate
for example, the use of bacteriophage therapy to eliminate horizontal gene transfer between bacteria [6,7,8]. At the
pathogenic bacteria. The relative ease with which entire applied level this information has provided insights into
bacteriophage genome sequences can now be elucidated has specific processes in bacteriophage lifecycles that present
had a profound impact on the study of these bacterial parasites. biotechnological opportunities for novel developments
in fermentation technology and new biotherapeutics
Addresses (Figure 1).
1
National Food Biotechnology Centre, University College, Cork, Ireland
2
Alimentary Pharmabiotic Centre, University College, Cork, Ireland This review focuses on the nascent applications borne out
3
Department of Microbiology, University College, Cork, Ireland
4
Department of Food and Nutritional Sciences, University College,
of the wealth of bacteriophage genomic information with
Cork, Ireland specific reference to bacteriophage defence systems


e-mail: [email protected] developed in the dairy industry and medical applications.

Fermentation technology
Current Opinion in Biotechnology 2004, 15:1–6
Members of the lactic acid bacteria (LAB), such as
This review comes from a themed issue on lactococci, lactic streptococci and lactobacilli, are com-
Food biotechnology monly used on an industrial scale for the production of
Edited by Oscar Kuipers
fermented foods such as cheese and yogurt. The suscept-
0958-1669/$ – see front matter ibility of these bacteria to infection by bacteriophages
ß 2004 Elsevier Ltd. All rights reserved. that are omnipresent in the dairy environment can have
serious economic consequences for the dairy industry.
DOI 10.1016/j.copbio.2004.01.007
This concern has provided the impetus for researchers to
study bacteriophage–host interactions in LAB, with a
Abbreviations view to developing ways to ultimately halt bacteriophage
LAB lactic acid bacteria proliferation. In fact, the economic consequences of fer-
PCR polymerase chain reaction
ori origin of replication
mentation failure have been the driving force that has
resulted in dairy bacteriophages becoming one of the
most thoroughly investigated phage systems with respect
Introduction to genome data [1,9,10]. The availability of these data
Since their discovery bacteriophages have been at the prompted Desiere et al. [10] to propose a new taxonomic
forefront of molecular biological research, both as model classification concept for bacteriophages that infect
systems and as biological tool boxes for the study and Gram-positive bacteria with a low G þ C content, based
manipulation of bacterial genes. The publication of the on the genetic organisation of the structural gene module.
complete genome of the bacteriophage fX174 in 1977
heralded a new era in molecular biology. The age of Labrie and Moineau [11] conducted an extensive com-
genomics was born and for the first time researchers parative analysis of all the genome sequence data available
had unravelled the complete set of genetic instructions for members of the three main species of bacteriophage
to make a biological entity. To date, there are over 100 encountered in dairy environments. Using this informa-
completed bacteriophage genomes available in public tion they developed a rapid polymerase chain reaction
databases, with the tally being added to almost daily. (PCR)-based method for the identification and character-
The genomic era has re-focussed researchers attention to isation of bacteriophage in dairy samples. The approach
the importance of bacteriophages, not only out of aca- involves multiplex PCR utilising three sets of oligonucleo-
demic curiosity, but mainly because of their contribution tide primers, each set being specific for the three genetic-
to bacterial virulence and evolution, and their potential ally distinct phage groups infecting Lactococcus lactis.
for treating bacterial infections. At the most fundamental
level, the availability of relatively large numbers of com- Recombinant phage resistance systems have been devel-
plete genome sequences has allowed detailed compara- oped for use in LAB since the early 1990s (for recent

www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:1–6

COBIOT 120
2 Food biotechnology

Figure 1

Bacteriophage genomics

Dairy industry Science Medicine

Virulence

Molecular Phage Genome Phage

tools identification evolution therapy

Phage-resistance Population Phage-derived

systems genetics therapeutics

Strain Bacterial strain Bacterial genome

identification/selection improvement diversity

Current Opinion in Biotechnology

Schematic to illustrate areas that have benefited from knowledge of bacterial genomics.

reviews see [12,13]). The first of these systems, termed Klaenhammer [16] recently used comparative computa-
phage-encoded resistance (Per), involved the cloning of a tional analyses of the genomes of six S. thermophilus
putative lactococcal phage ori [14]. It was demonstrated bacteriophages to identify conserved genetic targets for
that when supplied in trans on a high copy number vector, the construction of antisense RNA expression strategies.
normal lytic phage development was arrested in the Utilizing PCR with oligonucleotide primers complemen-
lactococcal cell, thereby allowing the cells to grow in tary to conserved S. thermophilus phage sequences, a
the presence of otherwise lethal amounts of phage. Sev- vector was constructed which contained a putative con-
eral other phage-encoded resistance systems have been served ori as well as expressing antisense mRNA com-
reported for use in lactococci, including Streptococcus plimentary to a putative helicase gene. When introduced
thermophilus and Lactobacillus casei [12,13]. The use of to S. thermophilus this plasmid was found to confer sig-
antisense mRNA technology for the control of bacter- nificant levels of phage-resistance against six out of nine
iophage proliferation in LAB has also been the focus of industrial phage isolates tested.
several reports over the 1990s. This strategy involves
cloning of a target gene in the reverse orientation relative McGrath et al. [17] have developed a set of criteria that
to an active promoter. The resulting antisense mRNA enable the identification of potential phage-encoded
produced is assumed to form stable hybrids with the phage-resistance genes in prophage genome sequences
target mRNA, thus inhibiting translation through inef- of disparate bacterial species. Furthermore, utilising
fective ribosome loading and/or increased sensitivity to PCR with oligonucleotide primers designed on the basis
RNA-degrading enzymes. One of the more effective of conserved lactococcal bacteriophage DNA sequences,
applications of this technology was reported by McGrath three separate phage resistance systems were constructed.
et al. [15]; in this study the replication module genes of the The expression of one such gene sie2009, identified on the
lactococcal bacteriophage Tuc2009 were targeted. This genome of the temperate lactococcal bacteriophage
strategy provided significant levels of phage resistance to Tuc2009, was demonstrated to render a lactococcal strain
the lactococcal host against several bacteriophages from completely insensitive to infection by all phages of an
the same industrially significant P335 species. Sturino and industrially problematic species that were tested.

Current Opinion in Biotechnology 2004, 15:1–6 www.sciencedirect.com

 The impact of bacteriophage genomics McGrath, Fitzgerald and van Sinderen 3

Martin et al. [18] have constructed a food-grade strain of discovery and widespread introduction of antibiotics
L. casei that is completely insensitive to fA2 infection. A effectively sidelined research into the application of
copy of the fA2 repressor-encoding gene was integrated bacteriophages as antibacterial agents in the western
into the chromosome of the L. casei strain, thus rendering world. However, in recent years it has become apparent
it completely immune to fA2 infection during milk that antibiotics alone are not sufficient to stem the rising
fermentation. This ingenious system might also be used tide of antibiotic resistance in infectious bacteria and the
for the stable integration of other recombinant DNA scientific community is once again looking to bacterio-
fragments. It consists of two separate parts: a delivery phages for the treatment and prevention of these proble-
vector for the integration of recombinant DNA and a matic strains. In light of the wealth of information now
clearing plasmid for the elimination of non food-grade available relating to bacteriophages there is a renewed
DNA. The delivery system contains a nonreplicative alacrity about their potential, as evidenced by many recent
heterologous replication origin and antibiotic resistance reviews [24–27,28,29]. An impressive example of the
markers surrounded by two directly oriented six sites, a potential of phage therapy was reported by Biswas et al.
multiple cloning site where passenger DNA can be [30]. The problematic infectious bacterium vancomycin-
inserted, the integrase-encoding gene, and the attP site resistant Enterococcus faecium (VRE) was successfully trea-
of the L. casei phage fA2. The clearing system provides a ted in a mouse model. The authors reported that a single
plasmid-borne gene encoding b-recombinase, which cat- dose of fENB6, a bacteriophage isolated from raw sew-
alyses intramolecular deletions of DNA sequences age, was sufficient to rescue all mice suffering from an
located between the two six sites and results in the otherwise fatal VRE-induced bacteremia. Cerveny et al.
elimination of all non food-grade DNA. [31] adopted a similar approach to the treatment of Vibrio
vulnificus induced septicemia, also in a mouse model.
The process of ripening contributes significantly to the
development of the characteristic aroma, flavour and A recent report has demonstrated the role played by
texture of matured cheeses. Ripening is a complicated human pharangeal cells in the induction of lysogenised
process that is influenced by many factors; however, bacteriophage from Toxþ Streptococcus pyogenes cells [32].
autolytic starter strains are known to positively contribute These induced prophage may subsequently infect and
to the process by releasing intracytoplasmic peptidases. integrate their DNA into the genome of Tox cells,
The positive correlation between autolysis and lysogeny converting them to Toxþ. This process of in vivo phage
was first established in S. thermophilus [19]. O’Sullivan et al. conversion represents a process by which bacteriophage
[20] measured the release of the intracellular enzyme can have a direct impact on the mammalian cell, and
lactate dehydrogenase from 31 strains of Lactococcus indicates that further research is needed to elucidate the
using a laboratory-scale cheese manufacturing assay. In possible side effects of bacteriophage therapy.
a parallel experiment, the genomes of these strains were
examined for the presence of prophage integrase seq- Analysis of bacteriophage genome sequences has allowed
uences using conserved primer sequences from known researchers to single out specific phage-encoded enzyme
lysogenic phage. The results demonstrated that the lytic activities and to incorporate them into more targeted
behaviour of lactococcal starter strains significantly cor- forms of treatment. A most apt example of this type of
relates with the presence of prophage sequences, indicat- application has been reported for the identification and
ing the potential to use this PCR-based screen in the treatment of Bacillus anthracis, the causative agent of
selection of starter bacteria for industrial use. Several anthrax [33]. It was demonstrated that a lysin protein
reports throughout the 1990s investigated the potential isolated from the fg bacteriophage of B. anthracis was
application of bacteriophage-derived cell-lysis systems in capable of rapidly killing both vegetative cells and ger-
the acceleration of cheese ripening, with generally favour- minating spores of the bacteria. Several other recent
able results [20–23]. Other phage-derived molecular reports from Fischetti and co-workers [34–37] have out-
tools, such as integration vectors, transduction and gene lined the efficacy with which bacteriophage-derived lysin
expression systems, have also been developed for use in enzymes can be used to eliminate problematic bacteria
LAB and have been reviewed recently [13]. such as S. pyogenes, Streptococcus pneumonia and group A
streptocci from the mucous membranes of mice. Sim-
Bacteriophage and biotherapeutics ilarly, Zimmer et al. [38] demonstrated that a murein
The discovery of bacteriophages in the pre-antibiotic hydrolase isolated from the Clostridium perfringens phage
world of the early 1900s led to a period of optimism in f3626 specifically killed all tested strains of the food-
which it was hoped that these new bacteriolytic agents borne disease-associated bacterium C. perfringens. Other
could be employed in the treatment and prevention of clostridia and bacteria of other genera remained generally
bacterial disease. Following this initial enthusiasm, poorly unaffected, indicating the potential of this technology for
controlled studies and the indiscriminate use of bacter- novel biocontrol measures in food, feed and complex
iophages in place of vaccination and hygiene measures microbial communities. An elegant bacteriophage-
resulted in discouraging outcomes. The subsequent derived alternative to the utilization of bacteriophage

www.sciencedirect.com Current Opinion in Biotechnology 2004, 15:1–6

4 Food biotechnology

lytic functions in the treatment of infection has recently to researchers. In silico comparisons and predictions can
been described [39]. This work outlines the use of the only take us so far, and the practical task of examining
well-characterised M13 phagemid system to deliver the individual gene transcription through protein expression
lethal gef and chpBK genes to cells of Escherichia coli. The and, ultimately, phenotype attribution still remains.
authors demonstrated that phage delivery of these lethal These questions have recently begun to be addressed,
agents resulted in a significant reduction in blood bacter- however. Ventura et al. [43] have demonstrated the suit-
ial titers in a bacteremic mouse model of infection. ability of transcription mapping to validate in silico pre-
Hoeprich et al. [40] have recently described a novel dictions of gene function and expression for the prototype
system for the treatment of hepatitis B infection of mam- S. thermophilus phage sfi21. The temporal transcription
malian cells. This system harnesses the DNA-packaging pattern of the phage was studied during its lytic life cycle
pRNA of the Bacillus subtilis bacteriophage f29 to deliver and confirmed previous predictions regarding the reg-
hammerhead ribozymes to the cell. These ribozymes ulatory circuits. The authors propose that similar analyses
specifically cleave the poly A signal of hepatitis B virus of the phage–host interaction under distinct conditions
mRNA, thus inhibiting replication. (i.e. establishment or maintenance of lysogeny and induc-
tion of the lytic cycle from the prophage state) will further
Another interesting bacteriophage-derived biotechnol- increase our understanding of cryptic aspects of the phage
ogical application is the exploitation of the integrase life cycle.
enzymes of lysogenic bacteriophages for the engineering
of mammalian cells. Stoll et al. [41] reported the con- Several recent articles have outlined the use of microarray
struction of a vector that expressed the integrase of the analyses to examine the contribution of prophage DNA to
lactococcal bacteriophage TP901-1 in human cells. interstrain genetic variability and host-cell phenotype for
Furthermore, the ability of the integrase to facilitate several pathogenic bacteria [44–48]. Conversely, Ventura
efficient intramolecular integration on a transfected plas- and co-authors [49,50] have published transcription and
mid substrate in the human cell environment was also microarray data relating to the contribution of prophage-
demonstrated. The ability of the E. coli bacteriophage encoded gene expression in two strains of lactobacilli
fHK022-derived integrase to integrate plasmids into the (Lactobacillus johnsonii and Lactobacillus plantarum
human cell nucleus when expressed both in cis and in WCFS1). Both of these strains are human commensals
trans has recently been demonstrated [42]. Figure 2 and are associated with probiotic function. The data
outlines bacteriophage-encoded enzymes and DNA presented raised the intriguing possibility of prophage-
sequences with biotechnological applications. encoded probiotic functions, leading the authors to pos-
tulate that prophages may contribute genes (encoding
Beyond bacteriophage genomics either virulence or probiotic function) that increase the
As in other areas of biology, the generation of large survival fitness of the lysogenic clone in its specific
amounts of DNA sequence data presents new challenges ecological niche [8].

Figure 2

Lysogenic life cycle Lytic life cycle

Lysogeny Replication Morphogenesis Packaging Lysis

Repressor Sie genes ori sequences Replication genes pRNA Holin Lysin
Phage resistance Phage resistance Phage resistance Phage resistance Therapeutic Therapeutic Therapeutic

Integrase
Bacterial and mammalian
cell engineering
Current Opinion in Biotechnology

Examples of phage genes/DNA sequences with biotechnological applications. The diagram represents a typical template bacteriophage genome
with genes involved in specific stages in phage development being grouped into modules (coloured boxes). The specific genes/DNA sequences
within these modules are indicated (bold text) along with the application (plain text).

Current Opinion in Biotechnology 2004, 15:1–6 www.sciencedirect.com

 The impact of bacteriophage genomics McGrath, Fitzgerald and van Sinderen 5

Conclusions 7. Canchaya C, Fournous G, Chibani-Chennoufi S, Dillmann ML,

Brüssow H: Phage as agents of lateral gene transfer.
It is clear that the relative ease with which entire gen- Curr Opin Microbiol 2003, 6:417-424.
ome sequences can now be deciphered has contributed 8. Canchaya C, Proux C, Fournous G, Bruttin A, Brüssow H:
inordinately to our understanding of bacteriophage life  Prophage genomics. Microbiol Mol Biol Rev 2003, 67:238-276.
cycles. The dairy industry has reaped the rewards of this This comprehensive review addresses the impact of prophages on
bacterial genotype and phenotype. All published bacterial genomes were
research, as evidenced by the development of technol- screened for prophage sequences and the role of phage-encoded genes
ogies for rapid phage detection, promoting phage- in the adaptation of the lysogenic bacterium to specific ecological niches
is discussed.
resistance and accelerated cheese ripening. The broader
scientific community has only recently realized the poten- 9. Brüssow H: Phages of dairy bacteria. Annu Rev Microbiol 2001,
55:283-303.
tial of unique bacteriophage-encoded enzymes for bio-
10. Desiere F, Lucchini S, Canchaya C, Ventura M, Brüssow H:
technological applications. One exciting example of this  Comparative genomics of phages and prophages in lactic acid
is the use of either whole bacteriophage particles or bacteria. Antonie Van Leeuwenhoek 2002, 82:73-91.
This article outlines a comparative analysis of the genomes of phages
specific phage-encoded proteins in the treatment of infecting the LAB. The authors noted a consistency in the gene order of
bacterial disease. A great deal more research is required the structural gene module of the phage sequences analysed, prompting
to understand the action of bacteriophages and their them to propose a new classification scheme for these tailed phage. The
evolution of these phage is also discussed.
encoded protein products in mammalian systems and
11. Labrie S, Moineau S: Multiplex PCR for detection and
how this environment in turn effects their interaction identification of lactococcal bacteriophages. Appl Environ
with the bacterial cell. The outlook is promising, how- Microbiol 2000, 66:987-994.
ever, and the elucidation of further bacteriophage gen- 12. McGrath SM, van Sinderen D, Fitzgerald GF: Bacteriophage-
omic sequences in conjunction with gene expression  derived genetic tools for use in lactic acid bacteria.
Int Dairy J 2002, 12:3-15.
analyses will add to both our antibacterial arsenal as well In this article the authors outline the development of novel bacteriophage-
as our molecular tool box for the manipulation of prokar- derived genetic tools for the manipulation of LAB.
yotic and eukaryotic cells. 13. Coffey A, Ross RP: Bacteriophage-resistance systems in dairy
 starter strains: molecular analysis to application.
Antonie Van Leeuwenhoek 2002, 82:303-321.
Acknowledgements This paper discusses the problem of bacteriophage infection of dairy
Stephen Mc Grath is the recipient of an Embark Initiative postdoctoral fermentation and the preventative measures employed. Specific empha-
fellowship from the Irish Research Council for Science, Engineering sis is placed on recently developed recombinant DNA technologies that
and Technology. Douwe van Sinderen is the recipient of a Science may be of use.
Foundation of Ireland investigatorship award (02/IN1/B198).
14. Hill C, Miller LA, Klaenhammer TR: Cloning, expression, and
sequence determination of a bacteriophage fragment
encoding bacteriophage resistance in Lactococcus lactis.
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