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Microbiology and Molecular Biology Reviews, September 2004, p. 560-602, Vol. 68, No. 3
1092-2172/04/$08.00+0 DOI: 10.1128/MMBR.68.3.560-602.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Phages and the Evolution of Bacterial Pathogens: from Genomic Rearrangements to Lysogenic Conversion
Harald Brüssow,1* Carlos Canchaya,1 and Wolf-Dietrich Hardt2*
Nestlé, Research Center, Vers-chez-les-Blanc, Lausanne,1
Institute for Microbiology, D-BIOL, ETH Zürich, Zürich, Switzerland2
Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving. This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA. Many prophages from bacterial pathogens encode virulence factors. Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen. These prophages behave like "swarms" of related prophages. Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes. Prophages also contribute to the diversification of the bacterial genome architecture. In many cases, they actually represent a large fraction of the strain-specific DNA sequences. In addition, they can serve as anchoring points for genome inversions. The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.
In the early days of molecular biology, phages were studied intensively for their own sake and as simple model systems. In the following years, many molecular biologists shifted their attention from phages and bacteria to higher organisms. Currently, we are witnessing a renaissance of phage research. The stage has been set by a recent shift toward ecology-oriented phage research covering the impact of phages on subjects as diverse as the cycling of matter in the oceans and bacterial pathogenesis. In fact, phages that carry key virulence factors were discovered long ago. Two examples are phages gamma and C1, which encode key virulence factors of Corynebacterium diphtheriae and Clostridium botulinum (11, 85). However, progress in the molecular biology of higher organisms and the continued advances in bacterial research have now opened the door to studies of "host-pathogen interactions" at a new level of detail. For some time, this research had focused on a few selected standard strains or on the molecular mechanism of selected toxins. The discovery of the cholera toxin phage CTX
(233) was one of the landmarks of this new phage research. It was becoming increasingly clear that phages play an important role in the evolution and virulence of many pathogens (Table 1). The interest in phage research was further fueled by the plethora of prophage sequences, which were discovered as a by-product of bacterial genome sequencing. The analysis of these sequences revealed that phages affect the bacterial genome architecture. In addition, phages are important vehicles for horizontal gene exchange between different bacterial species and account for a good share of the strain-to-strain differences within the same bacterial species (Fig. 1) (67, 136). In fact, two-thirds of all gamma-proteobacteria and low-G+C gram-positive bacteria harbor prophages (46, 47); these include Escherichia coli O157 Sakai (171) and Salmonella spp. (26). An example is shown in Fig. 1: the genomes from two Streptococcus pyogenes strains belonging to two different M serotypes and associated with different disease types were aligned at the DNA sequence level. All major genome differences were attributed to prophage sequences.
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FIG. 1. Genome comparisons of S. pyogenes strains. A dot plot alignment of the genomes from a U.S. M3 S. pyogenes strain MGAS315 (horizontal) against a U.S. M18 S. pyogenes strain MGAS8232 (vertical axis) (A) or a Japanese M3 S. pyogenes strain SSI-1 (vertical axis) (B) is shown. The position and name of the prophages are indicated by boxes on the axes and by thin horizontal and vertical lines crossing the dot plot. The dot plot was done with the Dottup program (http://www.emboss.org/); the word size was 15, the output format was Postscript, the program was run in the direct and reverse directions, and the figures were combined. The numbers and ticks on the axes give the scale for the genomes in base pairs.
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The early studies had indicated that some prophages carry additional cargo genes (termed morons or lysogenic conversion genes), and recent genomic analyses have revealed many more examples. These morons are not required for the phage life cycle. Instead, many morons from prophages in pathogenic bacteria encode proven or suspected virulence factors. They are postulated to change the phenotype or fitness of the lysogen. As a consequence, phages have emerged as prime suspects in the adaptation of pathogens to new hosts and the emergence of new pathogens or epidemic clones. This has led to a conceptual shift from host-pathogen interactions to host-pathogen-phage interactions.
It is important to note that lysogenic conversion is only one of at least five different ways by which temperate phages affect bacterial fitness: (i) as anchor points for genome rearrangements, (ii) via gene disruption, (iii) by protection from lytic infection, (iv) by lysis of competing strains through prophage induction, and (v) via the introduction of new fitness factors (lysogenic conversion, transduction).
We begin our review with a brief introduction to concepts of bacterial evolution and the evolution of pathogens, and we highlight the role of phages in shaping the bacterial genome architecture. Then we review the mechanisms of phage evolution and the mechanisms which allow phages to modify the host bacterium. A special focus is placed on lysogenic conversion, which is illustrated with seven portraits of human pathogens containing prophages that encode important virulence factors. Lysogenic conversion is thought to have a great impact on the evolution of pathogenic bacteria and results in a very interesting situation of bacterium-phage coevolution.
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EVOLUTION OF BACTERIAL PATHOGENS
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Before we start with the discussion of phages, we provide a conceptual framework by giving a brief introduction to basic concepts of the evolution of pathogenic bacteria.
Medical Microbiology: Adaptation over a Limited Timescale
Bacterial evolution started long before the emergence of animals. In fact, bacteria were the first, and for some time the only, inhabitants of Earth. Therefore, early evolution involved competition, genetic exchange, and selection only between bacteria. One can assume that phages also took part in this early phase of evolution. As discussed below, many of the virulence factors found in contemporary pathogens might actually date back to this time. Multicellular eukaryotes evolved only during the past 1 billion years, and mammals proliferated massively only during the past 65 million years (244). Human-restricted pathogens such as S. pyogenes, Shigella spp., and the human-adapted Salmonella strains must have become adapted to their hosts in 1 million years or less (the time frame of human evolution). This is approximately the timescale for the separation of E. coli strain K-12 from Shigella flexneri. It should be noted that the traditional bacterial nomenclature can be misleading. The Shigella genus attribution roots back to the historical recognition of shigellosis as a distinct medical entity. Modern taxonomy and comparative genomics would classify S. flexneri at best as a subspecies within E. coli. The emergence of S. flexneri as a human pathogen thus occurred at the level of the outmost twigs of the bacterial phylogenetic tree.
Based on bacterial genome analyses, the very idea of a phylogenetic tree as a description for bacterial evolution has recently come under heavy attack. It was proposed that horizontal gene transfer has occurred at such a level that the tree analogy should be replaced by a web analogy. However, also the validity of the web arguments has recently been challenged (60). Without going into this hotly debated area, we insist that most of the current evidence for the involvement of phages in shaping bacterial genomes, bacterial fitness, and host-pathogen interactions deals with events at this lowest taxonomy level. Figure 2A illustrates this for the comparison between E. coli K-12 and Shigella. The two genomes differ in DNA insertions (including many prophages), loss of genes (discussed below), genome inversions, and translocations (some are flanked on one side by prophage sequences). Most prophages found in these two genomes differ in their DNA sequence, suggesting that they were acquired (and then often degraded) after the separation of the two bacterial lineages. However, at the DNA sequence level both genomes could still be aligned over essentially the entire chromosomes (Fig. 2A). Figure 2B illustrates this for two even more closely related bacterial strains. They belong to the same O serotype of E. coli, the recently emerged food-borne pathogen O157:H7. Molecular evidence suggested that these strains evolved from an enteropathogenic O55 E. coli strain, perhaps over the last few decades (see below), and the prominent role of prophages for strain differentiation can be directly gleaned from the dot plot alignment (Fig. 2B).
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FIG. 2. Genome comparisons of E. coli strains. (A) Dot plot alignment of the laboratory E. coli strain K-12 (horizontal axis) against the S. flexneri strain 2a (vertical axis). Shigella is treated here as a subspecies of E. coli. (B) Dot plot alignments of a Japanese (horizontal axis) against a U.S. (vertical axis) Shiga-toxin producing O157:H7 E. coli strain. The prophages and prophage-like elements are indicated on the axes by boxes and by thin horizontal and vertical lines crossing the dot plot.
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Many of the concepts derived from the studies of human-pathogen-phage interactions will most probably also apply to phage-bacterium coevolution in other ecological niches. However, we cannot exclude that the view derived from pathogenic interactions is skewed by two facts. First, humans are evolutionary newcomers. Therefore, the time for adaptation to this new niche (i.e., balancing the pathogen-host interaction) has been relatively short and will be enriched for short-term adaptation processes. Second, pathogens attacking mammals and birds confront a formidable defense barrier generated by the adaptive immune system. Not surprisingly, numerous virulence factors from human and veterinary pathogens target this evolutionarily recent resistance system.
From Virulence to Fitness Factors
How do bacteria adapt to the life-style of a pathogen? At first glance, mammalian hosts are ecological niches like any other. However, in some respects they are difficult niches. In addition to the "normal" challenges encountered in nonliving ecological niches, mammals have defenses, which have been shaped by coevolution with microbes. These defenses include simple physical barriers like the dead cell layers of the skin and the mucus-covered epithelia, as well as the elaboration of antimicrobial peptides, iron-sequestering mechanisms, and immune responses. The factors and mechanisms that pathogens have evolved to circumvent these defenses are termed virulence factors. Virulence factors come in a great variety of forms and include factors that neutralize defenses of the host and factors that help to engage, subvert, or destruct host cells. Generally, the interaction of a pathogen with the host is a multistage process, which includes searching for an entry site, targeting a place for multiplication in the body of the host, and becoming persistent in the original host or finding ways to reach the next host. Survival or multiplication in the environment and transmission to the next host (which might include insect vectors) certainly affect the overall success of a pathogen. However, many bacterial functions enhancing fitness in these stages of the life cycle are not considered bona fide virulence factors. For this reason, we classify these factors, together with the bona fide virulence factors, as fitness factors. A fitness factor is thus broader in its scope than a virulence factor and is not limited to pathogenic bacteria. It could be defined in the following terms. (i) A gene, factor or allele is found in successful representatives of a bacterial species. Successful clones are those that outcompete their kin. (ii) Disruption of this gene reduces the chances for multiplication of the bacterium in its specific environment. (iii) Complementation (reintroduction of the fitness factor) restores the capacity for this multiplication.
A conceptual division of fitness factors into the three classes of survival, defensive, and offensive factors is illustrated in Fig. 3. Fitness factors can evolve "vertically" by gene duplication, mutation, and even gene disruption. However, quite long periods are needed to achieve results, especially when a complex combination of genes is required for a new niche. Combination of new gene constellations by a permutation principle is, in contrast, readily achieved by horizontal gene transfer (i.e., conjugation, DNA uptake, phage transduction, or lysogenic conversion). In this review, we discuss the role of phages in these processes. Several temperate phages which carry bacterial fitness factors in their genomes are discussed in detail. In these very interesting cases, the prophage-lysogen interaction is in fact a two-way process since the evolutionary success of prophages and their lysogens are intimately linked. In many cases it is not clear which partner exploits which in this relationship. Actually, much as the phylogentic relationships between bacteria changed from linear (trees) to two-dimensional displays (webs), we might also soon see web-like ecological relationships which define the interplay among phages, lysogens, and mammalian hosts.
Evolutionary Origin of Fitness Factors
Where do the virulence factors and fitness factors found in contemporary pathogens come from? Certainly, some of the virulence factors have evolved recently as a result of the ongoing "arms race" between higher eukaryotes and bacteria. However, many may stem from the coevolution of bacteria with unicellular eukaryotes during the past 1 billion years. This is supported by the notion that numerous virulence factors attack host cell proteins, molecules, and structures present in all eukaryotic cells. For example, there is a large number of toxins specific for heterotrimeric G-proteins, small G-proteins, or actin. Many membrane-disrupting toxins might also belong to this group. It is plausible that these virulence factors originally served to paralyze unicellular eukaryotic predators and were later adopted by animal-pathogenic bacteria. In fact, some virulence factors of animal pathogens are still active against unicellular eukaryotes. For example, the type III secretion system (specifically the type III effector protein ExoU) of Pseudomonas aeruginosa mediates killing of the slime mold Dictyostelium discoideum (188) but also plays a key role in the interaction with mammalian hosts (83, 102). Similarly, toxins injected into host cells via the Legionella pneumophila Icm/Dot type IV secretion system seem to be active in both mammalian cells and amoebae (110, 161).
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MOLECULAR MECHANISMS SHAPING BACTERIAL EVOLUTION
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Bacterial evolution requires the modification of "old" functions and the development of new ones. Nucleotide exchange, insertion, and deletion are the most frequent events. Mutation rates in bacteria are generally in the range of 10–6 to 10–9 per nucleotide per generation. In addition, module exchange between different genes, gene disruptions, and deletions occur at appreciable frequency. These mechanisms are common to all living organisms and allow modification of existing functions to optimize fitness in an existing niche or to adapt to a new niche. In contrast to many higher eukaryotes, bacteria have no sexual life cycles to facilitate the exchange of alleles within a population. In bacteria, this function is fulfilled by horizontal gene transfer: in this way, entire functional units can be imported from other sources, which are not restricted by species barriers. The transferred DNA can range in size from less than 1 to more than 100 kb. It can encode entire metabolic pathways or complex surface structures. These genes can be taken up as naked DNA or transferred in the form of plasmids, conjugative transposons, or phages. Here, we focus on the role of phages in horizontal gene transfer.
Comparative Genomics: Macroscopic Effects of Phages on Genome Structure
For a number of bacterial species, more than one genome sequence is available in the National Center for Biotechnology Information database. This allows us to investigate the genetic differences between strains within a bacterial species when taking a bird's eyes view of whole genome comparisons. There are also completed genome sequences from different bacterial species belonging to the same genus. These data thus permit a similar first glimpse of the evolution of bacterial species at the genomic level.
Intraspecies Bacterial Genome Comparisons
Colinear genomes.
When the genomes of two bacterial strains belonging to the same species were aligned, two different outcomes were observed. One type of dot plot alignment shows essentially a straight line across the entire genome length, indicating the matching of closely related DNA sequences. The colinear alignment is illustrated for a gram-positive pathogen in Fig. 1. The genomes of two S. pyogenes strains yielded a straight diagonal line when aligned in the dot plot display (Fig. 1) (203). The line is interrupted by small regions of nonalignment. In the depicted cases, most of the conspicuous gaps represent prophage sequences. The relative contribution of the different mobile DNA elements (prophages, transposons, integrative plasmids, pathogenicity islands, and IS elements) varies from one bacterial system to the next. In S. pyogenes and E. coli genome comparisons, most of the gaps are caused by prophages. In other systems, the proportion of prophages among the mobile DNA elements is less prominent (e.g., Streptococcus agalactiae) (216). Differences were seen even between strains belonging to the same species: the comparison of S. aureus strain NCTC 8325 and MSSA-476 showed prophages as the main determinants of difference (National Center for Biotechnology Information unfinished genomes) while the comparison between strains N315 and MW2 revealed transposons and small pathogenicity or genomic islands as major contributors of diversity (7). This important contribution of mobile DNA to the strain-specific DNA was confirmed by microarray analysis to occur in a number of pathogenic bacteria (e.g., S. pyogenes, S. agalactiae, S. aureus, and E. coli) (84, 172, 203, 216), but also in gut commensal bacteria (Lactobacillus johnsonii) (226).
Rearranged bacterial genomes.
The other type of intraspecies comparison also shows a high level of DNA sequence identity across the two compared genomes, but the two genomes are not longer colinear. The degree of rearrangement varies substantially. Some comparisons showed only one inversion, like the intraserovar comparison between the two Salmonella enterica serovar Typhi sequences (Fig. 4A), (66), or two inversions, like the two M3 serotypes of S. pyogenes (Fig. 1B). The interserovar comparison between S. enterica serovar Typhimurium and Typhi demonstrated multiple inversions and a less close DNA sequence relatedness (Fig. 4B). Even more complicated genomic rearrangements were observed between different strains of Yersinia pestis (65) or the plant pathogen Xylella fastidiosa (223). The rearrangements are in many cases probably the consequence of homologous recombination between repeat sequences in the bacterial genome. Sometimes these are duplicated bacterial genes (com genes in the M3 S. pyogenes strains) or genes which occur naturally in multiple copies (rRNA genes in S. enterica serovar Typhi). However, in some cases prophages with limited DNA sequence identity (S. pyogenes [Fig. 1B and see below]) or essentially duplicated prophages (X. fastidiosa) have served as anchoring points for homologous recombination reactions leading to major genomic rearrangements. In fact, in the plant pathogen X. fastidiosa, five of the six deduced recombination sites of three genome inversions were located in prophages (45, 223). The two sequenced X. fastidiosa strains represent different pathovars specialized for different plant species. It is currently unknown whether the genome rearrangement represents an adaptation of the bacteria to the different plant hosts. Recently, it was demonstrated that artificial chromosome inversions can significantly modulate the fitness of Lactococcus lactis (43). This supports the notion that the phage-mediated genome inversions observed in some genomes can indeed affect the fitness of the lysogen.
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FIG. 4. Genome comparisons of S. enterica strains. (A) Dot plot alignment of the S. enterica serovar Typhi strain Ty2 (horizontal axis) versus serovar Typhi strain CT18 (vertical axis). The positions of prophages and prophage remnants are indicated by boxes. The two rRNA gene clusters which served as anchoring points for genome rearrangement are marked with a tick. (B) Dot plot alignment of the S. enterica serovar Typhi strain CT18 (horizontal axis) versus S. enterica serovar Typhimurium strain LT2 (vertical axis). The locations and names of the prophages are given.
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Interspecies Bacterial Genome Comparisons
When the genomes of two different species belonging to the same bacterial genus were aligned, different outcomes were observed. On one hand, some genomes aligned perfectly across the entire genome length (e.g., Mycobacterium tuberculosis and M. bovis [Fig. 5A ]; note the absence of prophagelike elements in these two genomes) or aligned except for positions occupied by mobile DNA elements (Listeria monocytogenes-L. innocua [Fig. 5B]). Still other genomes from two different species belonging to the same bacterial genus aligned over a major part of the two genomes, but only at a lower level of DNA sequence identity (e.g., Bacillus cereus-B. anthracis and Staphylococcus aureus-S. epidermidis [Fig. 5C]).
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FIG. 5. Genome comparisons of gram-positive bacteria belonging to different species. The four panels compare two species from the high-G+C-content gram-positive bacterial genus Mycobacterium (M. tuberculosis versus M. bovis [A]) and two species from three different genera of low-G+C-content gram-positive bacteria, namely, Listeria (L. innocua versus L. monocytogenes [B]), Staphylococcus (S. aureus versus S. epidermidis [C]), and Streptococcus (S. pyogenes versus S. agalactiae [D]). In panels B and C, the positions of the prophages are indicated by boxes at the axes and thin lines crossing the dot plot. Further mobile elements are circled (transposon Tn916, restriction-modification genes R-M, and pathogenicity island PI).
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Finally, several genomes from different bacterial species belonging to the same genus yielded only small segments of DNA sequence alignment either scattered over the two genomes or in an X-like constellation (S. pyogenes-S. agalactiae [Fig. 5D]). Within the Streptococcus genus, S. pyogens, S. pneumoniae, S. agalactiae, and S. mutans did not have longer segments of DNA sequence similarity whereas S. pyogenes and S. equi are sister species which were still closely related at the DNA sequence level (J. Parkhill, unpublished data). This observation illustrates gradients of relatedness between bacteria. A similar pattern was seen in the Lactobacillus genus: L. johnsonii and L. gasseri showed DNA sequence similarity over most of their genomes, while L. johnsonii and L. plantarum had only seven small segments of DNA sequence similarity. At the protein level, about 20 conserved clusters comprising about 500 genes were identified and a cross-like organization of these conserved clusters was seen in a two-genome alignment (J. Boekhorst et al., unpublished data). Comparative genomics will probably soon assist bacterial taxonomy in the definition of the higher taxa at the genus and family levels. Overall, there is often less evidence for phage involvement in genome diversification in interspecies than in intraspecies alignments. Presumably this is due to the transient nature of prophage presence and the fast decay of defective prophages in bacterial genomes (see below).
Bacterial Genomes Lacking Prophages
In a number of sequenced bacterial pathogens, no prophages were detected (e.g., Helicobacter pylori, Campylobacter jejuni, Streptococcus pneumoniae, Mycobacterium leprae, and different Mycoplasma and Chlamydia spp.). Various reasons might account for this observation. Despite the lack of prophages in three sequenced S. pneumoniae strains, prophages are present in 76% of all clinical isolates of S. pneumoniae (189). This shows that the choice of bacterial strain for a sequencing project can result in nonrepresentative observations. Another case is presented by Mycoplasma spp. Mycoplasma phages are known, but the intracellular location of these bacterial pathogens might reduce the chances for lysogenic conversion, and lysogeny might therefore be a rare event in this group of pathogens. Similar arguments apply to Chlamydia spp. A further case is illustrated by H. pylori. To our knowledge, no H. pylori-specific phages have been described in the literature. Maybe nobody has looked for phages, but we cannot exclude the possibility that certain bacteria have eliminated prophages or that the peculiar habitat of a bacterium (e.g., the human stomach for H. pylori) is not favorable for phage infections. M. leprae represents another special case since its genome shows evidence of dramatic gene losses. Mobile DNA might have been a prime target for deletion.
In conclusion, the absence of prophages might simply reflect the chance event in the choice of a strain for sequencing. However, the fact remains that prophages may not be required for the evolution of a pathogenic life-style in every bacterial species.
General Considerations
Bacterial viruses (phages) depend for a number of functions on the energy production and biosynthetic activities of their host bacteria. This obligate dependence of viruses on host activity led many biologists to deny organismal status to viruses. This distinction between living and nonliving biological material is more of philosophical than of biological interest. Biochemically, viruses are composed of the same building blocks as their host cells and their genomes consist of nucleic acids, although sometimes in configurations (e.g., double-stranded RNA) not commonly encountered in cellular genomes. Furthermore, phages and bacteria are linked by a long history of coevolution. The time dimension of this coevolution can at present not be defined. We simply do not know when bacterial viruses evolved, i.e., whether they pre-date modern bacteria and represent remnants of former cellular life-forms that lost the competition with the modern forms of cellular life and persisted only as dependant nuisances to modern life. Some phages may have originated from assemblages of host genes that split billions of years ago from bacterial genomes, escaped from cellular control, and now lead a selfish life. Other phages might have originated recently. Most importantly, there is ample evidence for continued exchange of genetic elements between phages, bacterial genomes, and various other mobile genetic elements. This is illustrated below, and it explains the sometimes fuzzy distinction between phages, plasmids, and pathogenicity islands and the chimeric nature of some extant phages.
Phages have no fossil record and no molecular clock. DNA sequence analysis and sequence comparisons between extant phages are currently our only tools to look back into phage evolution. However, this view is blurred for two reasons. First, phage sequencing is a latecomer in the genomics revolution, and we currently have only about 200 complete phage genomes. This number may seem large, but actually it is extremely small considering that phages outnumber bacteria in many environments by a factor of 10 and represent, with approximately 1031 tailed phage particles, numerically the largest share of biological material on Earth (240). Up to 107 particles/ml were found in ocean water and sediment (30, 36, 176, 238, 240). Considering these vast numbers of phages and the number of bacteria coexisting in these niches, it has been estimated that 1025 phage infections are initiated every second worldwide (177); this has probably occurred for the last 3 billion years.
Second, we have no consensus model for phage evolution. Random sequencing of viral DNA in different environments ("metagenome" analysis [29, 30]) argues in favor of 2 billion undiscovered phage open reading frames, thus representing a large fraction of the total DNA sequence space (195). In addition, phage taxonomy is currently in turmoil. The official International Committee for Taxonomy of Viruses (ICTV) taxonomy based on phage morphology and genome organization was challenged by a taxonomy based on individual modules (135), a phage proteome tree (196), or elements of horizontal evolution revealed in the head structural gene cluster of phages (187). Originally, "lambdoid" phages were defined by their ability to form recombinant hybrids with phage lambda DNA (42, 212). In the meantime, this original definition has been extended on various occasions to include phages having DNA or protein sequence similarity to phage lambda or any other lambdoid phage. As in the bacterial phylogeny discussion, models of vertical and horizontal evolution and combination of the two have been proposed (106). Even if we add the about 200 prophage sequences identified in bacterial genomes to the phage database, the small number of phage sequences on which these models are based makes them tentative at best. Therefore, coverage of phage genomes is extremely low, while the number of phages is likely to be very large and phage evolution seems to be extremely fast. Therefore, it is no surprise that the phages analyzed so far show great variability.
Phage and Prophage Genomics: Evidence for the Modular Theory of Phage Evolution
The availability of more than 200 phage and 200 prophage genomes has facilitated the study of phage evolution by using the techniques developed for bacterial genome comparisons. Figure 6A shows a dot plot alignment for two prophages from two different S. aureus strains, N315 and Mu50A. Once again we see a familiar pattern for an intraspecies comparison: a long straight line interrupted by small regions of nonalignment. However, the type of nonalignment in prophages differs from the nonalignments in closely related bacterial genomes. In the bacterial case the gaps are frequently insertions of mobile DNA elements, while in prophage alignments the gaps are mainly DNA replacements. A DNA segment, found in one phage, is replaced in another phage by a sequence-unrelated DNA segment that frequently fulfils the same or a related function. These modular exchanges in phages had already been described by electron microscopy heteroduplex analysis of phages from enterobacteria in the pregenomics era and became the basis of one of the most popular hypotheses on phage evolution, the modular theory (24, 109, 212, 237). According to that theory, it makes no sense to speak about the evolutionary history of an entire phage genome. The genomes from lambdoid coliphages, for example, can be subdivided into 11 modules, each representing an independent genetic functional unit (head or tail genes, integration and excision; homologous recombination, and so forth). Each functional unit is represented by several alleles (modules). Only modules have an evolutionary history, which can be traced back over longer timescales. In particular, ancestral functions such as the building of a phage head or the phage tail may have a vertical evolutionary history. The order of modules on the phage genome map is fairly well conserved, while different alleles of the modules can be freely assorted (Fig. 7). This gives phage genomes a substantial genomic variability. Actually, one could describe the prophage from different E. coli isolates as a "swarm" of lambda genomes integrated at different chromosomal sites and sharing variable amounts of genome segments on a relatively random basis. It is currently not very clear how many different alleles exist for each module in lambdoid coliphages. Heteroduplex mapping and phage sequencing suggest that in this phage group there are perhaps about 10 different alleles for each module (48), but this might be a serious underestimation due to the incomplete sampling of lambdoid coliphages from the environment. Nevertheless, phage genomics (e.g., in lambdoid coliphages [125]) has largely confirmed the modular theory of phage evolution.
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FIG. 7. Dot plot matrix for the lambda-like prophages from E. coli strain O157:H7 VT-2 Sakai. The genomes of the lambda-like prophage from the Sakai strain were plotted versus themselves and phage lambda. At the bottom and on the right axis, the prophage names are given as in the original publication. Some prophage genomes were inverted to correct for the distinct orientation of the prophage genomes located on the right and left sides of the terminus of replication.
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Mechanism of Phage Module Exchange: Illegitimate versus Homologous Recombination
Today, it is widely accepted that phages evolve mainly via the exchange of modules. However, the genetic mechanisms (illegitimate versus homologous or even site-specific recombination) driving this process are still a matter of debate (Fig. 8).
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FIG. 8. Molecular models for module exchange between phages. (A) Module exchange by illegitimate recombination. (B) Module exchange by repeated homologous recombination. This results in blocks of lower sequence identity at the border between modules. The more recombinations occur, the less easily these blocks can be recognized. (C) Module exchange by recombination via conserved linker sequences. (D) Identical (100% identity) sequence elements in different regions of two unrelated phages (177). (E) Example of a conserved linker sequence in a lambdoid phage.
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The Pittsburgh phage group argues for the dominance of illegitimate recombination between phages infecting a wide range of bacteria. Illegitimate recombination takes place at random sites between different phages. However, most of these recombination events occur within open reading frames, change the phage genome size beyond useful limits, or disrupt gene clusters, rendering the recombinant phage nonfunctional. Only a few recombination events lead to viable phage. These "productive" recombinations most probably occur in intergenic regions, which do not disrupt functional modules. In this model, order is only the consequence of selection, discarding all unsuccessful recombination events that do not lead to viable phages (105, 125, 177). Furthermore, module exchange (at low frequency) is not restricted to specific families of tailed phages or phages infecting the same bacterial species (108).
Which enzymatic functions are driving illegitimate recombination? It has been suggested that special recombination systems catalyzing illegitimate recombination may exist (107). DNA restriction enzymes encountered on injection of phage DNA into a new host bacterium can efficiently fragment the phage genome. Normally these fragments are nonfunctional and are degraded. However, at a certain low frequency, these fragments might recombine with prophages present in the bacterial chromosome. It is unclear whether this mechanism is important in nature and which enzymatic functions might be involved.
Alternatively, DNA nonhomologous end-joining mechanisms present in the bacterial cytosol might play a role. DNA fragments could originate from different phages coinfecting the same bacterium, plasmids and DNA fragments originating from the host bacterial chromosome, or foreign DNA taken up from the environment. In this case, end-joining DNA ligase activities would be required to "assemble" these DNA fragments to yield new "mosaic" DNA assemblies. Some of the reassembled DNA fragments might turn out to be functional phages. DNA ligases of bacterial and phage origin have been known for a long time and are standard tools in the molecular biology laboratory. It is interesting to speculate whether this process might be enhanced by Ku-like proteins, which catalyze the recruitment of DNA ligase to DNA ends and ligation (68). Ku homologs have recently been identified in bacteria (68, 235, 236), and the Gam subfamily of Ku-like proteins is present in bacteria (Campylobacter spp., Neisseria spp., Haemophilus spp., E. coli O157:H7, and S. enterica serovar Typhi) and bacteriophage Mu (56).
Other observations argue that homologous recombination can also drive module exchange between phages (53, 155). This is supported by the presence of conserved linker sequences between modules of lambdoid coliphages, which could facilitate the exchange of DNA segments. Conserved linkers may also drive the transfer of modules between different phage families or even chromosomal loci. The sopE module (a moron [see below]) in Salmonella spp. has been studied in considerable detail. It has been found in P2-like prophages, lambdoid prophages, and the bacterial chromosome (Fig. 9). The surrounding sequences are very different, but the border regions flanking the sopE module on either side have considerable sequence similarity (157, 178) (Fig. 9). This suggests that the sopE cassette has been transferred between these loci by homologous recombination between flanking sequences (Fig. 8B and C). Similar observations have been made with dairy phages (35), and a recent study of moron gene cassettes in P2-like phages from E. coli lends further support to this notion (170). The moron gene cassettes were highly diverse in length, DNA sequence, and encoded gene products. However, the vast majority of these gene cassettes were flanked by conserved sequences (Fig. 8C). Therefore, these gene cassettes could travel between phages by homologous recombination (170).
Which recombination systems might facilitate module exchange by homologous recombination? The enzymes might be provided by the host bacterium (RecA), or they could be phage encoded (lambda red). Future laboratory work will have to determine which enzymatic functions of the phages and the host bacteria are involved.
In silico analyses of prophages, phage remnants, and functional phages have clearly demonstrated that all tailed phages have a common gene pool. At this stage, it is difficult to discern whether homologous or illegitimate recombination is more important. Many arguments seem to favour illegitimate recombination (Fig. 8A). Extensive alignments between prophages have identified phage pairs that share only rather limited segments of DNA identity, as demonstrated by the comparison of the S. aureus prophages Mu50B and ETA from different strains (Fig. 6B). The apparent units of DNA exchange between phages are small and might cover a few adjacent genes, only a single gene, or even a gene fragment encoding a single protein domain. The great variability seen in many phage genome alignments seems to exclude the use of a few predetermined conserved linker sites for recombination. Illegitimate recombination occurs with much lower frequency than homologous recombination and would thus require longer periods or very large populations of phages to materialize. The estimated size of the global phage population (approximately 1031 particles) makes even unlikely recombination events observable, provided that they are conferring a sufficiently strong selective advantage to the recombinant phage. In view of the vast global number of phage particles and their fast evolution, the current database represents the phage population only poorly. Therefore, many phages with intermediate sequence similarity to the existing ones (Fig. 8B) have simply not yet been identified. If one takes these "missing links" into account, one could envision that even the transfer of one phage module into an entirely different phage could be the result of a long sequence of homologous (not illegitimate) recombinations (compare Fig. 8A and B). Much more work is needed to settle this question. However, it is very likely that all types of recombination (illegitimate, homologous, and site specific) will contribute to some extent to the module exchange between phages.
It can be taken for granted that modular exchange reactions diversify phage and prophage genomes at an enormous frequency; even closely related strains of the same species almost never harbor 100% identical prophages. For example, for the recently emerged food pathogen E. coli O157:H7, a Japanese strain and a U.S. strain were sequenced. The two strains could be perfectly aligned at the DNA sequence level (Fig. 2B), in accordance with the hypothesis that they split quite recently from a common ancestor strain resembling E. coli O55 strains. Most of the differences were prophage related (Fig. 2B). Both genomes contain a prodigious number of lambda-like prophages (11 in the Sakai strain). However, despite the very short evolutionary distance separating the two strains, only one lamboid prophage pair could be aligned over the entire length without any modular exchanges (prophage Sp3 and CP-933K). Also, the numerous lambdoid prophages within the Sakai strain differed substantially from each other, and not a single one is an exact copy of the other (Fig. 7). The acquisition of new prophages seems to be a rapid process, as indicated by the sequential appearance of a specific set of prophages in S. pyogenes strains collected by hospital laboratories over the last 70 years. Historical surveys revealed the emergence of highly virulent S. pyogenes strains over the last half century that was correlated with the acquisition of a specific set of prophages (14).
Loss and Decay of Prophages
Prophages seem to be only transient passengers on the bacterial chromosomes, at least when seen on an evolutionary timescale. Theoretical arguments suggested a series of events leading from the accumulation of mutations to massive loss of prophage DNA and ultimate disappearance of the prophage (46, 136). This scenario is backed by a number of observations. Many prophages are no longer inducible: only one of the many lambda-like prophages in E. coli O157 EDL933 and none in strain Sakai could be induced. In two different Lactobacillus species, none of the multiple prophages could be induced (225). In some cases, inactivating point mutations were identified by bioinformatic analysis, e.g., introduction of stop codons into the replisome organizer gene and the portal protein-encoding gene in S. pyogenes prophages SF370.2 and SF370.3 (67) or inactivation of the N antitermination genes in the lambdoid coliphages from O157 (136). However, prophage inactivation is not a universal process, as demonstrated by an M3 serotype S. pyogenes strain in which all five prophages could be induced, although with different inducers and at different efficiencies (10).
Also, the frequent observation of prophage remnants (E. coli K-12, S. flexneri, Lactococcus lactis, and the S. enterica serovar Typhimurium sopE2 and sspH2 loci [Fig. 10 ]) could be easily explained by an ongoing prophage decay process. If the prophage decay process is slow with respect to bacterial speciation, one would expect the conservation of closely related prophage remnants in different bacterial isolates from a given species. A few closely related prophage remnants were detected in S. pyogenes (45) and E. coli (47). However, they are the exceptions and are not even widely distributed in the investigated species. In fact, even closely related bacteria generally do not share prophage remnants: the remnants in E. coli K-12 and S. flexneri (Fig. 2A), which diverged from K-12 perhaps just 1 million years ago, are distinct, as was the case when K-12 was compared to other sequenced E. coli strains. This observation suggests that the average time for acquisition and subsequent loss of prophages is shorter than the timescale of strain differentiation within a bacterial species.
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FIG. 10. Prophages and phage remnants in the S. enterica serovar Typhimurium strain LT2. (Top) Prophage remnants encoding the type III effector proteins SspH2 and SopE2. Phage-like genes in the vicinity of sspH2 and sopE2 are highlighted in blue. Selected genes are annotated. (Bottom) Genome maps of the four LT2 prophages. The modular structure of the prophages is indicated by the color code and the annotated brackets under the prophage genome maps. Proven or suspected fitness and virulence genes are colored in black.
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Role of Phages in Bacterial Evolution
It is conceivable that bacteria evolve with two gears. The slow mode (often in 1-million-year range) is based on the usual mechanisms of vertical evolution mediating a step-by-step genetic adaptation to their approximate environment.
Horizontal gene transfer can be regarded as the fast mode of evolution (timescale of years to decades). New sets of genes are acquired by transduction, transposition, transformation, and, last but not least, lysogenization with phages. Most of these gains might be ephemeral, and the genes are as easily gained as lost (134, 137). What counts is a momentary selective advantage over competing bacteria, especially in environments that are quickly changing. This changing environment can be the body surfaces of new host species that are rapidly proliferating in the ecosphere (e.g., humans) or settings with unusual host densities (animal farming or human urbanization). New industrial food preparation and health care techniques, international travel and transportation, and wars create enormous possibilities for exploitation by microbes that can invade these new niches. The impact of lateral gene transfer is so obvious in some of these settings (e.g., antibiotic resistance gene acquisition via plasmids and transposons in the hospital environment) that it would be surprising if it were restricted to these examples. The acquisition of mobile DNA can provide the necessary genetic material on a timescale that allows bacteria to quickly exploit these ecological "opportunities." Phage DNA fulfils a number of criteria for being an ideal vehicle for lateral gene transfer. Actually, some bacteria seem to use phages as gene transfer particles to shuttle pathogenicity islets (S. aureus phage 
80
[see below]) or random samples of chromosomal DNA (Bacillus subtilis prophage PBSX) (173).
In this model, different combinations of mobile DNA can be explored, and suitable combinations are maintained and further developed, leading to genotypes that suddenly fill old and newly created niches. In the context of pathogenic bacteria, we see these events as emerging infectious diseases (flesh-eating streptococci) or emerging food pathogens (E. coli O157). As discussed below, phages play a key role in these short-term adaptation processes.
The effect of this quick process on the long-term evolution of bacteria is less certain. Apparently only very small amounts of prophage DNA are fixed in the bacterial chromosome. However, it is a matter of speculation why phages do not accumulate to large numbers (or do so only in rare cases, e.g., E. coli O157). Are entire phages simply lost by excision reactions? Or are their genomes degraded so that they leave behind only a few remnants and possibly some fitness factors? The widespread occurrence of isolated phage-like integrase genes in bacterial chromosomes might be markers of previous prophage integration events (46, 67). However, only in some of the cases are a few phage-like genes (frequently repressor genes) found in the vicinity of these isolated integrase genes, which are sometimes still transcribed (224). At this stage, it is almost impossible to discern whether phage-encoded fitness factors might also have remained in the chromosome. Their phage origin cannot be recognized easily by simple sequence analyses. Some virulence genes or small genes clusters encoding fitness functions flanked by isolated phage genes might thus represent what remains after the prophages have decayed.
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PHAGE-MEDIATED GENE TRANSFER AND INACTIVATION
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Phage-mediated horizontal gene transfer occurs via transduction or lysogenic conversion. The global rate of phage-mediated genetic modification in bacteria has been estimated as being up to 20 x 1015 gene transfer events per s (39). In addition, bacterial gene disruption can occur by prophage integration into the bacterial genome.
Transduction
Generalized transduction is a "sloppy" feature observed with many bacteriophages. After the empty phage heads are completed, the phage DNA must be packaged. This process is quite accurate; however, DNA fragments of the host genome are packaged instead of the phage DNA at a finite frequency. This results in fully functional phage particles, which can attach and deliver the packaged DNA into suitable bacteria. Due to the absence of the phage DNA, this does not harm the bacterium. Instead, the injected foreign bacterial DNA can be incorporated into the genome. This is a typical example of phage-mediated horizontal gene transfer. Transducing phages have been observed in many bacteria including Salmonella spp. (200), Streptomyces spp. (38), and Listeria spp. (113).
Lysogenic Conversion
The acquisition of prophages would be an irrelevant process for the evolution of pathogenic bacteria if phages did not transfer useful genes to the lysogen. Some phage genes are known to increase the survival fitness of lysogens. The phage repressor and superinfection exclusion functions confer selective advantage to the lysogen by providing immunity against lytic infection. This was illustrated in a recent study of different S. enterica serovar Typhimurium strains harboring different sets of prophages. Lysogens like these often release low titers of phage (102 to 104 CFU/ml). These initially low titers of phage were sufficient to kick off an efficient decimation of a competing nonlysogenic strain (23). There are also some classical data that E. coli cells containing prophages (lambda, Mu, P2, and even cryptic prophages) grow quicker than nonlysogenic E. coli strains (68a, 68b, 141a).
It has been quite an exciting discovery that phages can also play an important role in the emergence of pathogens. This was recognized relatively early for toxins of Corynebacterium diphtheriae (diphtheria), Clostridium botulinum (botulism), Streptococcus pyogenes (scarlet fever), Staphylococcus aureus (food poisoning), and E. coli (Shiga toxin), which are all phage encoded. As far as we know, these genes do not play a role in the life cycle of the phages. The list of phage-encoded fitness factors is rapidly growing and now involves a wide range of different genes (Table 1). These factors include ADP-ribosyl transferase toxins, superantigens, lipopolysaccharide-modifying enzymes, type III effector proteins, detoxifying enzymes, hydrolytic enzymes, and proteins conferring serum resistance. In exceptional cases, phage tail genes seem to have developed dual functions and also serve as adhesion proteins for bacterial host attachment (Streptococcus mitis pblA and pblB genes). Many more prophage genes with sequence links to potential virulence factors were observed in the bioinformatic analysis of the genomes from bacterial pathogens, but experimental evidence for their role in bacterial pathogenicity is still largely lacking.
Another important lead was provided by the early observation that the toxin genes in Corynebacterium and S. pyogenes were located next to the phage attachment site (see Fig. 16). This specific location led to the hypothesis that these prophage genes actually represent bacterial genes that were acquired by a faulty excision process from a previous bacterial host. Sometimes these toxin genes still showed a clearly distinct G+C content, pointing to an unusual bacterial host as source for this DNA (78). However, to our knowledge, in no case was such a scenario demonstrated by direct experimental evidence. Fitness factors and "extra" genes of no known phage function are a relatively consistent finding in prophages from low-G+C-content gram-positive bacteria. "Extra" genes are not restricted to prophages from pathogenic bacteria but are also found in free-living bacteria and in bacterial commensals, where they are frequently located near the right attachment site, i.e., between the phage lysin gene and the transition zone from phage into bacterial DNA. Transcription studies of free-living dairy bacteria (Streptococcus thermophilus and Lactococcus lactis) (25, 224) and gut commensals (Lactobacillus plantarum) demonstrated that "extra" genes belong to the few constitutively transcribed prophage genes (225). Combined bioinformatic and transcription analysis revealed further extra genes between the phage repressor and the integrase gene near the left attachment site (226). In addition, isolated transcribed "extra" genes were found in the central part of the genome from prophages of low-G+C-content gram-positive bacteria (e.g., tRNA genes) (227).
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FIG. 16. S. pyogenes strain MGAS315 and its prophage- and chromosome-encoded virulence factors. (A) Genome map of the M3 S. pyogenes strain. The positions of the prophages are indicated on the outer circle by red boxes and their names. Proven and suspected virulence factors are located as red arrows on the inner circle and marked with their gene annotation. The origin of replication is located at the top. (B) Genome maps of three prophages. The modular structure of the prophage genome and the function of the modules are indicated. The virulence genes are marked in black (for details, see Table 3).
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In prophages from gram-negative bacteria, "extra" genes were also identified near both prophage DNA ends. Examples are O-antigen-modifying enzymes inserted between the phage integrase gene and the left attachment site in bacteriophage P22 (222) or genes inserted downstream of the phage tail genes (26). The location at both prophage ends has an intrinsic logic: an extension of these prophage transcripts would run into the bacterial DNA or as an antimessenger into the prophage DNA, in both cases preventing an accidental induction of the prophage via transcription of prophage DNA. Another frequent location for extra genes in lamboid prophages from gram-negative bacteria was next to the N- or Q-like antiterminator genes in the middle of the prophage genome (26). In these locations, accidental induction of the prophage is a real risk. Therefore, these extra genes were postulated to be flanked by an independent promoter and terminator structure. For this independently transcribed gene cassette, the Pittsburgh phage group coined the expression "moron" (more DNA [107]). No explicit predictions were made for the possible origin of these morons. Overall, the hot spots for insertion of "extra" genes are located at strategic positions, which minimize interference with the functions required during lysogeny and for lytic infection.
Gene Disruption
Gain of virulence genes is not the only mechanism by which pathogenicity develops. Pathogenic bacteria also develop from commensal bacteria by loss of genes. Shigella is a prominent example of the gain of virulence by loss of E. coli-specific genes, namely flagellar genes and cadA (5, 144). The latter encodes an enzyme required for cadaverine synthesis, and cadaverine has been shown to inhibit the Shigella enterotoxin function. On a smaller scale, prophages can cause single-gene loss when they integrate disruptively into host genes. A prominent locus of prophage integration involves the tRNA genes (41a), and many phage attachment sites carry the necessary DNA sequences to reconstitute a functional tRNA after prophage integration into the tRNA gene. Apparently, loss of tRNA genes could lead to fitness loss of the lysogen. However, not all prophage integration events reconstitute a functional tRNA gene. For example, two distinct Lactobacillus prophages share a nearly identical integrase gene but are located in two different tRNA genes at opposite locations on the bacterial genome. One prophage reconstitutes a functional tRNA, while the other apparently uses a secondary attachment site in a distinct tRNA gene of the same cell, which is disrupted by the integration event (226). Interestingly, both of these prophages, and many others, carry multiple tRNA genes that are transcribed from the prophage. This constellation might compensate for tRNA gene inactivation by prophages.
In other cases, prophages integrate into protein-encoding genes and lysogenization is linked to the loss of a protein function (negative lysogenic conversion phenotype). Well-characterized cases are the lipase- and the ß-toxin-negative phenotype due to the integration of prophage L54a and phi13, respectively, into the S. aureus genome (54). Even when prophages integrate into intergenic DNA, the ca. 40 kb of prophage DNA might disrupt the coordinated transcription of genes belonging to the same transcription unit (45). In conclusion, there are ample possibilities to explain how prophage integration can disrupt or modulate bacterial gene expression and thereby alter bacterial fitness or virulence.
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LYSOGENIC CONVERSION: ROLE OF PROPHAGE MORONS
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Morons of Temperate Phages, Prophages, and Phage Remnants in Pathogenic Bacteria
Morons are thought to enhance phage replication in times when the temperate phage is residing as a prophage in the chromosome of a bacterium. The effect is indirect since the moron-encoded functions enhance the fitness of the lysogen and improve the fitness of the phage only passively via its propagation with its host bacterium (67, 107). This hypothesis provides the theoretical framework for phage-mediated horizontal transfer of fitness factors between bacteria. It is thought that this mechanism is the most important one by which phages affect the evolution of pathogenic bacteria.
For the purpose of this review, we use the term "moron" for all extra genes present in prophage genomes which do not have a phage function but (may) act as fitness factors for the lysogen. Table 1 provides an overview of the known and putative morons of phages and phage-like elements found in pathogenic bacteria. Often, there is quite a complex interplay between phage and bacterial functions in order to express the moron properly and to provide a selective benefit for the lysogen.
Examples of Distribution and Function of Morons
To provide a benefit for the lysogens, a moron must fulfill several requirements.
(i) The moron has to be useful in the ecological niche of the lysogen. Clearly, a host cell invasion factor would be of little use for an extracellular pathogen. A moron can provide a benefit in three ways: it enhances the fitness of the lysogen in the bacterium's "old" ecological niche (in this case, it would allow the lysogen to outcompete the other inhabitants of this niche); its function allows the bacterium to counteract a sudden change occurring in its old niche (the former competitors are wiped out by the environmental change and the lysogen can proliferate); and its function allows the bacterium to conquer a new niche.
(ii) The expression of the moron function must be coordinated with the functions of the host bacterium. If the timing of moron expression is not well controlled, it cannot provide a benefit.
(iii) In some cases the moron function relies on the proper function of and interaction with other bacterial factors. This situation is somewhat similar to that in operons encoding entire metabolic pathways (138): one function makes sense only in the presence of the others. For example, in gram-negative bacteria, extracellular enzymes or toxins requiring a specialized export apparatus will be functional only if the lysogen provides the proper transport systems.
Prophages in S. enterica serovar Typhimurium.
Recently S. enterica serovar Typhimurium has emerged as an excellent example of the integration of moron function into the biology of the host bacterium. S. enterica serovar Typhimurium is the causative agent of Salmonella food poisoning. A wide variety of animals can be infected with S. enterica serovar Typhimurium when they ingest contaminated water or foodstuff. Infected animals excrete S. enterica serovar Typhimurium in the feces. The fecal bacteria contaminate water and foodstuff, where they can persist—and multiply under proper growth conditions— for days or weeks (239). Therefore, S. enterica serovar Typhimurium is adapted to two principally different ecological settings: niches in the environment and the intestines of different animal species (239).
More than 200 different S. enterica serovar Typhimurium strains have been identified. Typically, these strains cause epidemics for periods of about one decade, one specific strain dominates and causes a large percentage of all animal or human infections, while the other S. enterica serovar Typhimurium strains are isolated only rarely. Finally, the incidence of this epidemic strain declines and a new epidemic strain takes over. The exact reasons for this are unknown, but it is thought that slight strain-specific differences in virulence might play a role. This hypothesis is based on the observation that different S. enterica serovar Typhimurium strains have different combinations of fitness factor-encoding mobile genetic elements and phages. The recent identification of morons encoding fitness factors in several of the S. enterica serovar Typhimurium prophages has led to the hypothesis that phage-mediated reassortment of virulence factors and fitness factors is a key driving force in the optimization of the Salmonella-host interaction and the emergence of new epidemic clones (81, 156). Some of these moron-encoded functions have been studied in detail. These are discussed to illustrate how moron function can be precisely integrated with the functions of the host bacterium. A selection of S. enterica serovar Typhimurium fitness factors is given in Table 2.
Several S. enterica serovar Typhimurium prophages encode so-called type III effector proteins (Table 2). Type III effector proteins are a specific class of virulence factors which are injected by the bacterium into animal cells via a type III secretion system (TTSS; Fig. 11B). Inside the animal cell, these type III effector proteins manipulate signal transduction pathways. In this way, the bacterium can manipulate the host cell response for its own benefit. TTSS are found in several gram-negative bacteria living in close association with plants or animals (88, 119). Future work will have to address whether any of these bacteria besides Salmonella spp. may also harbor phages encoding type III effector proteins.
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FIG. 11. sopE moron of S. enterica serovar Typhimurium. (A) Chromosomal map of S. enterica serovar Typhimurium LT2. The chromosomal locations of the SPI-1 and SPI-2 TTSS, the type III effector proteins (inside the circle), and of the prophages and phage remnants (remn.) (outside the circle) are shown. (B) Role of the chaperone InvB in the transport of the type III effector proteins SopE, SopE2, SopA, and SipA. (Inset) General domain structure of type III effector proteins. N terminus, chaperone interaction and transport via the TTSS; rest of the effector protein, domains for manipulation of signaling cascades in eukaryotic cells. (C) Outline of the regulation of SPI-1 TTSS effector protein expression in S. enterica serovar Typhimurium.
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TTSS are of key importance in Salmonella pathogenesis (87). S. enterica serovar Typhimurium encodes two TTSS (not counting the flagellar apparatus). The TTSS encoded in Salmonella pathogenicity island 1 (SPI-1 TTSS [Fig. 11A]) is active during the early, gut-associated stages of the infection (234). In contrast, the SPI-2 TTSS is essential for the survival of S. enterica serovar Typhimurium inside phagocytic cells during later stages of systemic
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