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.
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.
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 (typhoid-like) disease (
98). Expression of the SPI-1 and SPI-2 TTSS is strictly and inversely regulated. The SPI-1 TTSS is expressed when the bacteria grow in nutrient-rich high-osmolarity environments thought to simulate the intestinal lumen. SPI-2 is expressed when the bacteria replicate intracellularly in a vacuole with low ion concentrations and acidic pH (
98).
How does the SPI-1 TTSS induce diarrhea? The type III effector protein cocktail injected into gut cells via the SPI-1 TTSS is very complex and consists of at least 11 different proteins including SopB, SopE2, AvrA, SipB, SptP, SipC, SipA, SspH1, SopD, SlrP, SopA, and, in some strains, SopE (
87). Each of these effector proteins seems to manipulate a specific signaling pathway of the mammalian cell, which together provoke a strong intestinal inflammation. It is becoming increasingly clear that
Salmonella requires a fine-tuned delivery of the effector proteins into the intestinal cells of the infected animal. A similar picture emerges for the SPI-2 effector proteins. Overall, it is clear that acquisition of new type III effector proteins can alter virulence of
Salmonella spp. Specificity for certain host animals might also be an issue, because signaling pathways are thought to be wired slightly different in every animal. Many prophages have been identified in
Salmonella spp., and more are identified as additional
Salmonella genomes are sequenced. Most of them belong to the P2 family (SopEΦ, Fels-1, and Fels-2) or the lambda family (GIFSY-1, GIFSY-2, GIFSY-3, and P22), and many carry morons encoding TTSS effector proteins and other fitness factors (Fig.
10). These are discussed in detail, below.
(i) The sopE moron of SopEΦ.
SopEΦ is a P2-like phage. It has been identified and sequenced in
S. enterica serovar Typhimurium strain DT49/DT204. This strain caused a major epidemic in England and the former German Democratic Republic in the 1970s and 1980s (
156). In its tail and tail fiber region, SopEΦ carries a moron containing
sopE (
101,
157,
177). In SopEΦ lysogens, SopE is part of the effector protein cocktail that is injected into cells of the mammalian gut by the SPI-1 TTSS (
101,
242) (Fig.
11B). It has been speculated that lysogenic conversion with SopEΦ was an important step in the emergence of this epidemic strain (
156).
SopE is a so-called G-nucleotide exchange factor for RhoGTPases, central switches of mammalian cell physiology. For this reason, it is not surprising that SopE has profound effects once it is delivered into animal cells (
100). In order to function, the expression of
sopE must be tightly controlled and coregulated with the SPI-1 TTSS and the other effector proteins. This has been studied in considerable detail in the natural lysogen
S. enterica serovar Typhimurium SL1344 (Fig.
11C).
sopE expression depends on the SPI-1-encoded proteins SicA and InvF (Fig.
11C) (
70,
127,
218). The InvF/SicA complex is a positive regulator for the
sicAsipBCDA operon,
sopB and
sopE (
57-
59,
70,
218). This ensures coexpression of
sopE with the other type III effector proteins.
Recent work on the transport of SopE via the SPI-1 TTSS has identified a second mechanism ensuring proper functional integration of the
sopE moron. After production in the bacterial cytosol, SopE binds to the SPI-1 encoded chaperone InvB (
69,
114). Type III secretion chaperones such as InvB are small acidic proteins which bind to the N-terminal region of the effector proteins (
55,
175,
208). This interaction is required for proper recognition and transport by the TTSS (
19). Recent work on the chaperone InvB has revealed that in addition to SopE, it is the chaperone for the effector proteins SipA, SopE2, and SopA (Fig.
11B) (
31,
69,
69a,
114).
In conclusion, the proper function of the
sopE moron in
S. enterica serovar Typhimurium is ensured in two ways:
sopE is coregulated with other SPI-1 effector proteins by a common regulator, and its proper recognition and transport via the SPI-1 TTSS is ensured by the SPI-1-encoded type III chaperone InvB (Fig.
11). This is quite an interesting situation because neither the regulators nor the chaperone are encoded by SopEΦ;
sopE travels without this “dead freight.”
(ii) Other S. enterica serovar Typhimurium prophages: Fels-1, Fels-2, GIFSY-1, GIFSY-2, GIFSY-3, E34, and ST64B.
Besides SopEΦ, two other P2-like prophages (Fels-1 and Fels-2) have been identified in
S. enterica serovar Typhimurium strains (
2,
81) and several other
Salmonella spp. also harbor Fels-1- or Fels-2-like prophages (
117,
184,
194).
Fels-2 carries one moron of unknown function (
abiU [
26]). Three morons (
sodCIII,
nanH, and
grvA) have been spotted in Fels-1 from
S. enterica serovar Typhimurium LT-2:
sodCIII encodes a superoxide dismutase (
81). Superoxide dismutases are thought to protect bacteria from oxygen radicals, which are produced by macrophages (
64,
74).
nanH encodes a neuraminidase/sialidase, NanH. There is only circumstantial evidence for a role of
nanH in
Salmonella virulence: neuraminic acid-containing oligosaccharides are present on many animal cell surface glycoproteins and glycolipids, and neuraminidases are found in a variety of pathogenic bacteria such as
V. cholerae and
Clostridium perfringens (
44,
123). However, the function of neuraminidases in virulence has remained enigmatic. Similarly, it has remained unclear if
nanH can contribute to
Salmonella virulence. The function of the
grvA moron of Fels-1 has also remained unknown. Considering this large arsenal of putative fitness factor-encoding morons, it seems a bit surprising that lysogenic conversion of a
fels-1-lacking
S. enterica serovar Typhimurium mutant strain with Fels-1 did not enhance mouse virulence (
81). Each of the morons might make only a small contribution. However, one also has to consider that
S. enterica serovar Typhimurium can infect host animals as different as reptiles, birds, and mammals. It is conceivable that the morons enhance
S. enterica serovar Typhmurium fitness only in very specific host animals and not in laboratory mice. Yet another possibility is that research has focused too much on the
S. enterica serovar Typhimurium-animal interaction and that some of the morons might actually provide a selective advantage in the second niche of
S. enterica serovar Typhimurium: the environment.
The lambdoid GIFSY-1 prophage is present in serveral
S. enterica serovar Typhimurium strains. GIFSY-1 affects mouse virulence in certain
S. enterica serovar Typhimurium strains lacking GIFSY-2 (
79). The exact reasons for this are still unclear, but one can speculate that one of the GIFSY-1 morons might be involved. The
gogB moron of GIFSY-1 (Fig.
10 and
11A) is expressed under control of the master regulatory system
ssrAB of the SPI-2 TTSS (
81). Future work will have to confirm whether GogB is an effector protein of the SPI-2 TTSS. The GIFSY-1
gogA moron is similar to PipA of
S. enterica serovar Dublin and 98% identical to the
gtgA moron of GIFSY-2, but so far it has no known function (
81). Finally, GIFSY-1 includes the
ehly-1 moron (
26) and the
gipA moron, which encodes a putative transposase which enhances growth or survival in the Peyer's patches of the murine small intestine (
207).
The lambdoid GIFSY-2 prophage has been identified in
S. enterica Typhimurium strains LT2 and ATCC 14028 (Fig.
10 and
11A). Curing these strains from GIFSY-2 reduces virulence (
79,
80). GIFSY-2 carries a multitude of known or putative fitness factors (Fig.
10). The periplasmic CuZn-superoxide dismutase SodCI was the first GIFSY-2 virulence factor described (
79). Interestingly, most if not all
Salmonella strains already encode such an enzyme (via the
sodCII gene) in the chromosome. So why should the GIFSY-2-encoded enzyme enhance the fitness of a lysogen? Gene expression analyses have revealed that the chromosomal
sodCII gene is abundantly expressed in stationary-phase cultures in rich broth (RpoS control [
74]) but only scarcely expressed when the bacteria are growing inside phagocytes or an infected spleen (
220). In contrast,
sodCI is expressed abundantly and protects the
S. enterica serovar Typhimurium bacteria in this niche. In other words,
sodCI moron expression requires correct timing to confer a selective advantage to GIFSY-2 lysogens. However, the regulatory cascades are unknown. The GIFSY-2 moron
gtgE also contributes to
S. enterica serovar Typhimurium virulence (
111). Western blot analyses revealed that it is expressed during intracellular growth as well as in nutrient broth (
221). The
grvA moron carries a highly interesting gene, which has been classified as an “avirulence” factor (
112). Its sequence is not homologous to any other known gene. Overexpression and deletion of
grvA increased virulence (
112). The exact mechanism underlying this observation is still unclear. Nevertheless, this illustrates that proper regulation of moron expression is of great importance. The
sseI/gtgB/srfH moron encodes a type III effector protein for the SPI-2 TTSS (
150). It is expressed intracellularly under the control of the master regulator of the SPI-2 TTSS (
221,
243). This is somewhat reminiscent of the
sopE moron described above and should guarantee proper integration of this moron function with SPI-2. However,
sseI mutants did not show a virulence defect in mice (
111). Other GIFSY-2 morons have sequence similarity to known or suspected fitness factors including
ailT,
pipA,
msgA, and
gtgCD (Table
1). So far, their function has not been studied in detail.
GIFSY-3 harbors two morons with possible fitness function (
81,
150). The first,
pagJ, was first identified by virtue of its regulation by the PhoPQ system (
13,
93), a key regulator of
Salmonella virulence functions (Fig.
11C) (
92). The molecular function of
pagJ is unclear. The second moron contains
sspH1, a type III effector protein, which inhibits NF-κB-dependent production of inflammatory signals by infected animal cells (
99). The C-terminal domain of SspH1, which manipulates cell signaling, harbors a leucine-rich sequence motif and is similar to type III effector proteins from various pathogenic bacteria and to SlrP and SspH2 (see below) from
S. enterica serovar Typhimurium (
150). The N-terminal region of SspH1 shows similarity to an even larger family of type III effector proteins from
S. enterica serovar Typhimurium (SlrP, SspH1, SspH2, SseJ, SseI, SifA, SifB). This common N-terminal region harbors the signals for chaperone binding and transport via TTSS (
55,
150). Probably, these type III effector proteins use the same chaperone for transport. This chaperone has not yet been identified but could ensure the function of
sspH1 and its coordination with the TTSS and the other effectors (Fig.
11B).
Regulation of
sspH1 expression is quite unusual for a type III effector protein. It is not under control of the “master regulators” for either SPI-1 or SPI-2 expression. In fact, SspH1 can be transported via both TTSS (
150). Therefore, the
sspH1 moron might represent an evolutionary intermediate in the optimal integration of a moron into the functional networks of the host bacterium. Alternatively,
sspH1 might confer some (so far unknown) advantage during the extracellular and intracellular stages of the disease. In spite of these
pagJ and
sspH1 morons, a GIFSY-3-cured strain did not show a virulence defect in mice (
81). As discussed above, this does not mean that GIFSY-3 cannot enhance the fitness of certain
Salmonella strains if one looks at the right ecological niche (i.e., the right animal species). In fact, some data indicate that SspH1 contributes to virulence in calves (
151).
Other phages found in
S. enterica serovar Typhimurium strains include E34, ST64T, and ST64B (Table
1) (
159,
160). It is safe to assume that this is only the tip of the iceberg. The
Salmonella strains analyzed so far carry on average between two and six prophages, and the number of
Salmonella strains is enormous: there are at least 200 different
S. enterica subspecies 1 serovar Typhimurium strains, and Typhimurium is just 1 of more than 2,000 different subspecies 1 serovars. In addition, many strains seem to carry some prophage remnants. It will be a formidable task for the future to devise efficient strategies to identify these phages and analyze them.
(iii) Phage remnants encoding SopE2 and SspH2.
Two further type III effector proteins (SopE2 and SspH2) of
S. enterica serovar Typhimurium are encoded by phage remnants (Fig.
10). SopE2 is 70% identical to SopE and was detected in every analyzed
Salmonella strain (
155,
185). It contributes to intestinal inflammation in cows and mice (
98a,
249,
250). SopE2 is coregulated with the SPI-1 TTSS (
8,
209). The regulator has not been identified. SopE2 requires the same type III chaperone (InvB) for transport as SopE does (
69), and we can assume that functional integration of
sopE2 works along the same lines as described above for the
sopE moron (Fig.
11B).
SspH2 belongs to the same type III effector protein family as SspH1 does (see the discussion of GIFSY-3 above). Expression of
sspH2 is controlled by the SPI-2 master regulator
ssrAB (
150), and it is translocated into host cells via the SPI-2 TTSS. Disruption of either
sspH1 or
sspH2 alone did not have much of an effect, but an
sspH1 sspH2 double mutant showed reduced virulence in a calf infection model (
150,
151). This illustrates a common problem encountered in
Salmonella virulence studies. Often the bacteria have several redundant proteins to achieve one specific task (e.g., SspH1 and SspH2; SopE1 and SopE2). It turns out that one has to identify and inactivate all the redundant proteins before one can observe a virulence phenotype for a specific fitness factor.
In conclusion, the morons of the Salmonella prophages and phage remnants encode products with a variety of functions. Even though we have learned quite a bit about the regulation and functional integration of the phage morons encoding type III effector proteins, only little is known about the other morons. Nevertheless, the diversity of phages and moron functions among S. enterica serovar Typhimurium strains is striking. The diversity of moron functions indicates that many bacterial properties can be optimized to yield a selective advantage for the lysogen and to establish a prophage-lysogen coevolution. Interestingly, many Salmonella phage morons just slightly modify the fitness factor repertoire of the lysogen; i.e., they add one more type III effector protein to the cocktail injected into host cells via the SPI-1 or SPI-2 TTSS, or they encode one additional periplasmic superoxide dismutase besides the chromosomally encoded enzyme. In most cases, lysogenic conversion leads to an incremental change in the fitness of a given Salmonella strain. Nevertheless, this small competitive edge might be decisive in determining the next emerging epidemic clone.
It is quite striking that many prophages and phage remnants of Salmonella spp. encode type III effector proteins. After lysogenic conversion, these effector protein-encoding morons can modify fitness only if the lysogen has a TTSS, a suitable chaperone, and the correct regulators. None of these functions are encoded by the prophage. These restrictions represent a significant functional barrier, which limits the spread of the prophage between Salmonella spp. Most probably, these morons would be functional in only very few other bacterial species. This should result in an intimate coevolution of these type III effector-encoding phages with Salmonella spp. It will be an important task to devise experimental strategies to study this coevolution in detail.
V. cholerae toxin moron.
Vibrio cholerae, the causative agent of Asiatic cholera, is a gram-negative bacterial species. Of the >100 known
Vibrio serogroups, the two toxigenic serogroups “classical” O1 and O139 have been associated with epidemic cholera. In the environment,
V. cholerae is found in marine and estuarine habitats in association with copepods, planktonic species, insects, and water plants (
12). Therefore,
V. cholerae switches between two different ecological niches: the aquatic environment and mammalian and human hosts.
V. cholerae enters its host via the oral route, colonizes the small intestine, and secretes “exoproteins,” which cause severe secretory diarrhea.
The two human-pathogenic
V. cholerae serogroups (O1 and O139) have evolved by sequential acquisition of two key fitness factors: the toxin-coregulated pilus (TCP) and cholera toxin (CT) (Fig.
12A) (
27,
28). Both are encoded by phages or phage-like elements (
129,
233). Extensive epidemiological studies support the notion that the evolution of new toxigenic
V. cholerae clones (especially O139 strains) is still going on at breathtaking speed and includes repeated rearrangements of the CTXΦ element (
76).
CT, the principal virulence factor of
V. cholerae, is encoded by the bacteriophage CTXΦ (
233). CT is expressed in the host intestine as a classical AB toxin, and human volunteer studies using purified CT demonstrated that it accounts for the induction of diarrhea (
141). The B subunit of CT binds to enterocytes and transports the catalytic A subunit into the host cell cytoplasm. There, the A subunit triggers signaling cascades leading to rapid chloride and water efflux into the intestinal lumen, causing watery diarrhea, the hallmark of epidemic cholera. This diarrhea is thought to enhance fecal shedding and dissemination from host to host (see also below). In addition, CT might enhance bacterial survival in the intestine.
TCP is critical for intestinal colonization (
149). It is a type IV bundle-forming pilus, whose major subunit (TcpA) was identified in a screen for secreted virulence factors which are coregulated with CT (
214,
215). It is expressed in the human intestine and belongs to the major antigens in human infections (
18,
97). The genetic element encoding TCP (also termed VPI for “
V. cholerae pathogenicity island”) has been described as the genome of a filamentous phage (VPIΦ or TCPΦ [
129]), but the phage nature has been disputed recently (
63,
77). Beside TCP, this locus encodes accessory colonization factors and the ToxT, TcpP, TcpH, and TcpI regulatory proteins (Fig.
12B).
Proper regulation of virulence factor expression is important, as suggested by a number of mutational analyses, which demonstrated that certain regulators are required for virulence. Recently, it was discovered that TCP and CT expression are controlled by at least three different quorum-sensing systems present in
V. cholerae (
154). The importance of regulation is also illustrated by the observation that
V. cholerae isolated from fresh feces is more infectious than bacteria grown under laboratory conditions (
148).
The key virulence factors required for the host phase of the
V. cholerae life cycle (TCP and CT) are under the control of master regulators (ToxR, AphA, and AphB) encoded in the ancestral
Vibrio chromosome and by newly acquired DNA (VPI [TcpP and ToxT] [
139,
140] and RS1 [RstC] [
61] [Fig.
12B]). ToxR is regarded as the central virulence regulator, and a set of about 60 genes (termed the ToxR regulon) involved in colonization, toxin production, and bacterial survival within the host are coregulated in response to external stimuli such as temperature, pH, and osmolarity (
18,
202). ToxR belongs to an interesting family of transmembrane proteins with cytoplasmic transcription factor domains. ToxR has presumably evolved to control outer membrane (OM) synthesis in response to osmolarity. Later, expression of CT, TCP, and the regulators encoded in the TCP element (Fig.
12B) were plugged into this preexisting ToxR regulatory system. In any event, expression of the virulence factors is controlled by fairly complex signaling cascades in modern epidemic
V. cholerae strains, and key regulators are not encoded by the prophage.
CT has to be secreted into the intestinal lumen in order to exert its function. As with most other AB toxins, this occurs via type II secretion. Type II secretion is a multistep pathway, and transport across the inner membrane (IM) and (OM) occurs in separate steps (Fig.
12C). The A and B subunits of CT are synthesized as precursor proteins. These are transported into the periplasm and proteolytically processed by the ubiquitous Sec system. With the help of DsbA, the subunits are correctly folded and assembled into the AB
5 complex, which is subsequently transported across the OM via the
eps type II secretion system (
198). Besides CT, the
eps type II secretion system transports chitinases and proteases. Type II secretion systems are widely distributed: However, CT export is specific for the
eps type II system and does not function in other species (e.g.,
Pseudomonas aeruginosa,
Klebsiella oxytoca, and
Erwinia chrysanthemi) bearing different type II secretion systems (
152). In contrast, environmental
Vibrio spp. can secrete the CT B subunit, presumably via the
eps type II system (
152). It is thought that the
eps type II system is shared by all
Vibrio spp. and causes the secretion of proteins (chitinases and proteases) common to all vibrios. CT evolved to be specifically and efficiently transported by this system (
198). In other words, the CT moron of CTXΦ can be functional and provide a selective advantage only in vibrios. Only here are the proper regulators and transport systems available.
Intriguingly, the filamentous phage CTXΦ, which does not encode its own OM pore, also requires one component of the
eps system for its escape from the bacterium (secretion [Fig.
12C]). In
epsD mutants, the phage cannot be assembled (
62). This suggests that not only CTXΦ-encoded virulence factors but also phage viability require vibrios as the host bacteria. This is supported by the observation of lysogenic conversion of CT
−Vibrio mimicus strains by CTXΦ. The lysogens obtained in this study produced viable CTXΦ and secreted biologically active CT (
75). In addition, integration of CTXΦ into the
Vibrio chromosome requires host functions: the host-encoded recombinases XerC and XerD (
118). It is unclear whether XerCD-like recombinases from other bacterial species could also foster CTXΦ integration. If not, it would be another mechanism for functional restriction of CTXΦ to
Vibrio spp.
Shiga toxin-producing E. coli.
E. coli is a gram-negative enterobacterium which diverged from the
Salmonella lineage about 100 million years ago. Many contemporary
E. coli strains are benign intestinal commensals; but a few strains have evolved into pathogens. These pathogenic
E. coli strains are classified as enteropathogenic
E. coli, enterohemorrhagic
E. coli, Shiga-toxin producing
E. coli (STEC), enteroaggregative
E. coli, enterotoxigenic
E. coli, enteroinvasive
E. coli, or uropathogenic
E. coli based on their repertoires of virulence factors and the most common diseases associated with them. The vast majority of pathovar-specific virulence factors are encoded in horizontally acquired DNA fragments: pathogenicity islands, transposons, plasmids, and, last but not least, phages. The STEC strain O157:H7 is a prominent example of the last. During the past 20 years,
E. coli strain O157:H7 has evolved from a clinical novelty, first described in 1982, to a global public health concern (
147). O157:H7 is a mucosal pathogen that produces several virulence factors, with the principal one being a prophage-encoded Shiga toxin (Stx), an AB-type toxin that inhibits protein synthesis. A second important virulence factor found in O157:H7 strains is the TTSS. It is encoded in a pathogenicity island called the locus of enterocyte effacement, which is located adjacent to prophage 933L (
179). This locus is responsible for a specific pathological change in the infected intestinal mucosa, called the attaching and effacing lesion.
STEC causes diarrheal disease as well as more severe clinical manifestations, including hemorrhagic colitis and hemolytic-uremic syndrome. It is thought that the Shiga toxin Stx released by bacteria residing in the intestinal lumen is responsible for all these symptoms. The toxin traverses the intestinal epithelial barrier, enters the bloodstream, and damages vascular cells of the colon, the kidneys, and the central nervous system.
STEC strains are commonly found in the intestine of cattle and other ruminants, where they are associated with several O serotypes, including O157 (
131). Cattle are the principal environmental reservoir of STEC. However, only a subgroup of STEC within the bovine reservoir is capable of causing disease (
16). This might be linked to differences in the Stx expression characteristics (
193).
The available genetic evidence suggests that O157:H7 is a group of closely related strains, which have emerged during the past 50 years from the enteropathogenic
E. coli strain O55:H7 by a small number of genetic events (
201). Key events in this process were the replacement of the
rfb gene region (
213) followed by the sequential acquisition of first bacteriophage stx2 and then phage stx1 (Fig.
13A). The O157 strains are not monolithic. Some geographical variation was documented between O157 strains from Europe, the United States, and Australia (
130a). Variation occurred through the emergence of regional subclones showing distinct genetic polymorphisms. Genome diversity occurred through random drift and bacteriophage-mediated events. Many strains possess only the stx2 prophage. The
rfb region encodes the enzymes necessary for the synthesis of the O side chains of the bacterial lipopolysaccharides. The lipopolysaccharide is the dominant and highly variable surface molecule under strong immune selection pressure, and the
rfb locus is the target for frequent recombination and horizontal gene transfer.
Two bacteriophages encode the Shiga toxins Stx1 and Stx2, respectively. The stx2 phages sp5 and 933W from the two sequenced O157 strains Sakai and EDL933, respectively, are closely related at the DNA sequence level, share the genome organization of phage lambda (Fig.
13B), and are integrated into the same chromosomal site,
wrbA (
143,
180).
In contrast, the two stx1 phages sp15 and 993V from the two sequenced O157 strains share DNA sequence identity only across the nonstructural genes. Prophage sp15 closely resembles phage lambda over the structural genes (
246). In comparison,
E. coli strain Morioka V526 contained two nearly identical stx1 and stx2 prophages, which are closely related to sp5. The two Morioka stx prophages differed only over the region surrounding the distinct
stx genes (
199). The Stx2-encoding bacteriophage P27 from a clinical
E. coli isolate in Germany clearly differed from the corresponding prophages in the Sakai and EDL933 strains and was integrated into a different
E. coli gene,
yecE (
191). The P27 genome organization corresponded to a mixture of modules from a siphovirus (λ) and myovirus (Mu). A similar hybrid phage organization was previously observed in a serotype-converting prophage from
Shigella (
3,
4). Overall, the stx phages provide strong evidence for the shuffling of phage modules and morons between phages from different
E. coli strains.
Most differences between the genomes of the pathogenic
E. coli strain O157:H7 Sakai and the laboratory strain K-12 (
21,
171) are due to prophages (Sp1 to Sp18 in Sakai), prophage remnants resulting from phage genome truncations (e.g., e14, Rac, and Qin in K-12), and more distantly phage-related mobile DNA elements (SpLE1 to SpLE1 in Sakai) (Fig.
13C; see also Fig.
2B). A particularly striking feature was the sheer number of 18 prophages in the Sakai strain, covering P2-like, P4-like, Mu-like, and a large number of lambda-like prophages. Only two lambda-like prophages (sp5 and sp15) carried proven virulence factors (stx1 and stx2), but many other prophages contained potential virulence factors (e.g., sp4
sodC; sp6 candidate cytotoxin) (
171). Cytolethal distending toxin (
124) was detected in O157 strains, where it was flanked by phage genes related to phage P2 and lambda, suggesting another toxin-carrying prophage in STEC (
122).
Ohnishi et al. (
172) analyzed the whole-genome structure of eight O157 strains by whole-genome PCR scanning. While the chromosomal DNA was conserved between the strains, a high level of variation was observed for the prophages. The stx2 prophages differed widely between these strains and were integrated at distinct chromosomal sites. In conclusion, prophages are thus the most dynamic genetic elements in this recently emerged
E. coli lineage. The data suggest that prophages from contemporary O157 strains are the result of multiple, independent prophage acquisition events and that many modular exchanges between prophages must have occurred in O157 over the past few decades (see also Fig.
7).
A crucial issue for the understanding of the prophage-bacterium-host interaction is the regulation of the expression of the lysogenic conversion genes. The location of virulence genes in lambdoid coliphages next to the lambda
Q or lambda
N gene homologues provided first hints (Fig.
13B and
14A). These proteins are the phage lambda antitermination proteins and act in a cascade. The N protein associates with the RNA polymerase, and, assisted by several bacterially encoded Nus factors, it allows the polymerase to transcribe from the early p
R promoter in the genetic switch region into DNA replication genes and then the
Q gene. The Q protein, being itself an antiterminator, allows then transcription to continue into late genes (lysis and structural genes [Fig.
14A ]). Q binds DNA in the p
R′ promoter located directly downstream of the
Q gene (for a review, see reference
86).
In the majority of STEC strains, the
stx genes are located downstream of the
Q gene (Fig.
14A). Deletion of the
Q-pR′ region in the Stx2-encoding prophage φ361 abrogated the basic level of Stx2 toxin production by the lysogen. This result was somewhat surprising since previous studies had identified functional promoters immediately upstream of
stx2 (
211a) that were not touched by the deletion. Mitomycin-C treatment led to high toxin production in the lysogen while the mutant showed only an increase to basic toxin levels (
232). Apparently, in this prophage the Q function is not only necessary for transcription of the late phage genes but also important for
stx2 transcription (Fig.
14A). The effect was also reproduced in vivo (
89). Substantial amounts of Stx2 were measured in the stools of mice inoculated with the wild-type lysogen but not in those inoculated with the deletion mutant. In this lysogen the
stx gene is apparently entirely under the control of the phage, which is surprising since phage induction leads to the death of the lysogen and should thus reduce and not enhance the fitness of the lysogen. However, comparable amounts of wild-type and mutant cells were recovered from the gut (
232).
There is precedent to this situation in bacteriocin-producing bacteria. Bacteriocins are molecules that are lethal to other bacteria of the same or closely related species. They are produced by a subpopulation of the bacteria harboring that gene, and these bacteria are lysed in the process of releasing the bacteriocin. The death of a minority of a bacterial population therefore contributes to the survival advantage of the majority. Notably, there are also examples where bacteriocins were derived from prophages; e.g., the R- and F-type pyocins from
Pseudomonas aeruginosa correspond to phage tail gene clusters from a P2- and a lambda-like prophage, respectively (
165).
A functional promoter,
pStx1, was identified directly upstream of the Stx1 coding sequence. The activity of this promoter is regulated by the environmental iron concentration by a mechanism involving the iron-dependent Fur transcriptional repressor, which is thought to bind to a site near
pStx1 (
40,
41). In this scenario, the toxin is under the control of the bacterial cell. This hypothesis has an inherent logic. The lysogen, or, better, a minority of the lysogens, expresses Stx only in case of low iron concentration, which is a typical growth-limiting factor for intestinal bacteria. Teleologically, the expression of the toxin leads to intestinal hemorrhage, which then liberates iron from the blood cells released into the gut and causes resumed growth of the bacteria and hence Stx downregulation.
Prophage H-19B revealed a complex network of transcriptional regulation of the
stx1 genes (
168,
231). Stx1 concentrations were increased fivefold over basic levels by growth in low-iron medium. Mitomycin C induced the prophage and led to a remarkable 70-fold increase in the Stx1 titer. Deletion of
Q and
pstx1 had no effect on this titer, while deletion of
N compromised but did not abolish the Stx1 production. Only a combined deletion of several phage genes including
N and
Q reduced Stx1 to basic levels.
There was another important difference between Fur-regulated and mitomycin-induced Stx1 production. In low-iron media, Stx1 remained intracellular while mitomycin induction resulted in fast release of Stx1 into the supernatant. Stx1 has no export system in STEC and is released by the cells via lysis due to prophage induction (Fig.
14A). Interestingly, the phage lysis cassette is directly downstream of the
stx1 genes and is cotranscribed with them.
These observations are interesting for several reasons. First, the phage seems to compete with the bacterium for
stx1 regulation. Second, it suggests that Stx1 can be released only from lysing cells. Based on a number of recent reports, STEC might have found an elegant way around this suicidal Stx1 production (Fig.
14B). When
E. coli containing a genetically labeled H-19B prophage was introduced into mice, infectious virions are produced within the host intestine. The released phage was capable of converting other
E. coli strains within the gastrointestinal tract (
1). Apparently, specific mammalian host signals induce the Stx-encoding prophages. Candidate mammalian inducers were identified for prophage φ361: coculture of the STEC strain with human neutrophils and the neutrophile product hydrogen peroxide induced Stx2 production (
230). In vitro experiments verified that phage and toxin production by STEC was amplified in the presence of susceptible
E. coli cells (
89). When 37 intestinal
E. coli isolates were individually incubated with the STEC test strain, 3 strains produced significantly more toxin in a coculture with STEC than did STEC alone, while cocultivation with one strain led to significantly less toxin production. This toxin amplification by a susceptible
E. coli strain and toxin decrease by a resistant
E. coli strain was also demonstrated to occur in mouse intestine inoculated jointly with STEC and an
E. coli tester strain. In this way, the pathogenic bacteria pass the burden of toxin production to the harmless bystander member of the intestinal flora. This model has fascinating evolutionary and clinical implications. On the clinical side, Stx seems to play a role in the development of hemorrhagic lesions, but even in a single outbreak not all subjects excreting STEC strains experienced diarrhea and only a minority of the symptomatic individuals developed severe sequels like hemolytic-uremic syndrome (
163). It was recently suggested that the virulence of the isolates could be related to the clonal variability of the induced Stx prophages (podovirus versus siphovirus) (
163). Alternatively, the susceptibility of the resident intestinal
E. coli population could be a determining factor for clinical complications associated with STEC infections. Prophage induction was also obtained by exposure to antibiotics (
89). This could contribute to antibiotic-induced exacerbation of STEC infection in mice (
251) and in humans (
241).
From an evolutionary viewpoint,
stx genes may represent a recent acquisition by STEC strains, which have not yet recruited a protein secretion system for a nonsuicidal production of the toxin. Via the infection of bystander cells with the Stx-producing prophage, STEC can deploy the toxin without automatically lysing itself. In this way, even the possession of a toxin without a secretion system can confer a selective advantage. The selective advantage is deduced from the wide distribution of Stx prophages and high sequence conservation of the two
stx gene cassettes in STEC. However, the teleological interpretation of the STEC data should be treated with caution, since in cattle, which are the natural reservoir for STEC, the strains are carried asymptomatically in the intestine despite some (although more variable) Stx production. Virulence studies have been performed mostly in acute-infection models (see, e.g., reference
210), and it has remained unclear whether Stx toxins may help to shape the long-term coexistence of STEC with the bovine host. Even though the exact mechanism by which Stx toxins can provide a selective advantage has remained unclear, the Stx phages provide a formidable example of the rapid exchange of moron cassettes between phages from different
E. coli strains.
C. botulinum.
Clostridia are strictly anaerobic gram-positive bacteria which are ubiquitous in the environment. These organisms produce extremely resistant spores which sporulate under anaerobic conditions.
C. botulinum strains were originally defined by their ability to produce one of the closely related but antigenically distinct members (A, B, C1, D, E, F, or G) of the botulinum neurotoxin family. Later, it turned out that the botulinum neurotoxin producers are quite heterogeneous and belong to different groups of strains (groups I to IV) and even different species (
C. butyricum and
C. baratii) (
90,
94,
145,
146).
Human botulism is caused by the consumption of toxin-contaminated food. In other cases, the bacteria replicate within the human gut or sometimes in infected wounds, where they release the toxin in situ. The botulinum neurotoxins are expressed as ca. 150-kDa precursors lacking classical signal peptides (Fig.
15A). In vitro, release of the 150-kDa precursor from the cell is observed as a result of sporulation. Surprisingly, the exact mechanism of toxin release from the bacterium has not been studied in more detail. Once outside the cell, the botulinum neurotoxins are cleaved by bacterial or host proteases to yield the active toxin, consisting of 100-kDa H (heavy) and 50-kDa L (light) subunits. Often, the botulinum neurotoxins are complexed with hemagglutinins and Ntnh. This protects the toxin and facilitates its absorption through the gastric mucosa (
96,
183). The “complexing” proteins are encoded in the same gene cluster as the corresponding botulinum neurotoxins (Fig.
15B).
The H subunit targets the toxin to neuronal tissue, mediates neuron binding, and delivers the L subunit into the neuronal cell. The L subunit is a metalloprotease, which cleaves protein components of the neuroexocytosis apparatus. This leads to irreversible blockade of acetylcholine secretion at neuromuscular synapses, resulting in flaccid paralysis.
The botulinum neurotoxins A, B, and F are encoded in the chromosome, while G is plasmid encoded (
253) and C1 and D (and possibly E [
252]) are encoded by prophages (CEβ and DEβ [
11,
71,
72]). Surprisingly, sequence analysis of the botulinum neurotoxin C1 and D loci has remained restricted to the direct vicinity of the neutotoxin genes. There is a transcriptional start site 100 nucleotides upstream and a rho-independent terminator downstream of the toxin gene. Genes for both classes of “complexing” proteins and a gene for a regulator (
botR/C) were also present in the phage (
103,
217). Overall, the genetic organization of the botulinum neurotoxin C1 and D loci suggests that most regulators and cofactors required for proper toxin expression are encoded by the phage. Therefore, lysogenic conversion of a new strain or even another bacterial species should lead to “toxin conversion,” provided that the recipient strain can sporulate or release the toxin in some other way. Surprisingly little work has been done in recent years to further explore this important aspect of CEβ and DEβ biology.
The
C. botulinum lysogens can be cured easily, and cultures of the C1 and D toxin-producing strains release significant amounts of phage. The cured strains can be readily relysogenized, and curing-reinfection cycles may also occur in nature. This is backed by the identification of nontoxigenic or “low-toxin-producing” derivatives of certain
C. botulinum “toxotypes” (
73).
In addition, some phages carrying botulinum neurotoxins C1 and D were found to also encode a C3 toxin, an ADP-ribosyltransferase for Rho GTPases (
104,
162,
181,
182). This supports the notion that the neurotoxin phages in
C. botulinum behave like most other toxin-encoding phages in terms of moron exchange and variability.
Overall, much groundbreaking work has been done on the structure and the toxic mechanism of botulinum neurotoxins and ways to prevent and cure the disease. In sharp contrast, the complete sequences of the phages themselves are still not available in the public database. This is quite astonishing, considering that CEβ and DEβ were among the first toxin-converting phages ever discovered. Future work will have to address the genetic characteristics of these phages, their evolution, and also the issues of toxin gene expression and release of the toxin from the bacterium.
S. pyogenes.
S. pyogenes has fascinated medical microbiologists for nearly a century. It is a protean pathogen, and humans are its only reservoir. One-third of all humans are colonized with
S. pyogenes. The bacteria are commonly found in the throat and on the skin.
S. pyogenes strains harbour a large variety of fitness factors in the chromosome as well as on mobile genetic elements (Fig.
16A). Sequence analysis of 10 chromosomally encoded fitness factors (extracellular proteins) revealed a striking correlation with the M serotypes of these strains (
192). These observations suggest that the
S. pyogenes strains (M serotypes) have evolved as distinct pathovars, harboring a specific set of chromosomally encoded fitness factors. These data fit with the epidemiological observation of an association between the M serotype and certain pathological conditions in humans and mice (
158). On the other hand, a detailed analysis of DNA sequence polymorphism of the set of chromosomal virulence genes studied by Reid et al. (
192) revealed numerous likely horizontal transfer events between the different M strains, and the authors suspected that generalized phage transduction was responsible for at least some of these gene transfers.
S. pyogenes strains are polylysogenic organisms whose prophages constitute about 10% of the total genome of sequenced strains (up to two-thirds of the strain-specific genes [Fig.
1]). The prophages encode a wide variety of putative and established virulence factors (Table
3). Recently the sequences of four
S. pyogenes strains representing three different M serotypes were published (
14,
78,
164,
203). Each strain contained three to six seemingly complete prophage sequences closely related to phages known from the dairy field (
67). Only one of the three prophages in strain SF370 could be induced by mitomycin C treatment. The two noninducible prophages contained stop codons within essential genes (
67). In contrast, all five prophages from strain MGA315 could be induced (
10) and thus could actively participate in gene transfer between the strains. The comparative genomics of these prophages was recently reviewed (
46) and is not repeated here. In this review, we focus on the expression and role of the phage-encoded fitness factors in disease.
The vast majority of
S. pyogenes prophages encode one or two likely or experimentally proven virulence or fitness factors between the lysis cassette and the right phage attachment site (Fig.
16B). Similar to the situation in
Salmonella, the coexistence of multiple fitness factor-encoding prophages (polylysogeny) provides the opportunity for fast reassortment of fitness factors. Already the presently sequenced
S. pyogenes phages allow a substantial permutation of virulence factor combinations. This permutation might account for the temporal and geographical variability and the distinct disease pathologies seen between clinical isolates. Diseases range from mild pharyngitis to life-threatening toxic shock syndromes, covering many distinct pathological entities associated with otherwise very similar strains (
9).
Lytic induction of
S. pyogenes prophages has been studied in considerable detail. In bacterial growth media, phages were either spontaneously released from the lysogenic
S. pyogenes strain MGAS315 (two prophages) or induced by more or less physiological stimuli. Hydrogen peroxide, produced in vivo by attacking phagocytes, induced three prophages, while mitomycin C, which causes damage to the bacterial DNA, induced all five prophages, although with variable efficiency (
10). Mitomycin C might not be a natural inducer, but this type of induction of prophages is interesting for the understanding of paradoxical effects of antibiotics on bacterial infections. Fluoroquinolone antibiotics, which poison DNA topoisomerase II, induce not only Shiga toxin-encoding bacteriophages in
E. coli (see above) but also a mitogen-encoding bacteriophage in
Streptococcus canis (
121).
S. canis is a commensal bacterium in dogs and may cause various opportunistic infections. Notably, after the introduction of fluoroquinolones into canine veterinary medicine, the incidence streptococcal toxic shock syndrome and necrotizing fasciitis, clinically very similar to the corresponding syndromes caused by
S. pyogenes in humans, increased steadily (
186).
Less artificial stimuli can also induce
S. pyogenes prophages. Coculture of
S. pyogenes with human pharyngeal cells induced the phage and the production of the phage-encoded streptococcal pyrogenic exotoxin SpeC, Spd1, and a number of other bacterial proteins. A low-molecular-weight nonproteinaceous factor from the supernatant of the pharyngeal cells was the inducing factor (
33,
34). Spd1 and SpeC are encoded by adjacent genes on the same prophage. Virtually identical adjacent genes (
speC and
mf2; Mf2 and Spd1 are 98% identical) were detected in a prophage from the sequenced M1 strain.
Spd1 belongs to a family of DNases found in a variety of
S. pyogenes strains. Some of these proteins also function as bacterial superantigens (see below). Spd1 showed DNase activity but no superantigen activity (
34). It has a leader sequence and is secreted via the Sec pathway. The authors suggested that in vivo streptococcal phage induction occurs in the pharynx, where other strains of
S. pyogenes are likely to be present. Spd1 is secreted just before lysis of the induced lysogenic bacterial cell and could thus digest the bacterial DNA, which is spilled out after lysis. This would reduce the DNA-mediated viscosity of the tissue fluid and facilitate the spread of the released phage to the next target bacterium. Lysogenic conversion of Tox
−S. pyogenes in the pharyngeal flora by phages released from Tox
+ lysogens or free phage was demonstrated in mice (
32). Another function of the phage DNase is to liquefy pus, which contains substantial amounts of DNA from dying leukocytes. DNase would thus also increase the spread of the bacteria on the pharyngeal cells. Such a dual function of phage enzymes was also proposed for the phage hyaluronidase. This enzymatic activity is associated with a phage tail fiber protein. The enzyme apparently helps the phage to cross the hyaluronic acid-containing capsule surrounding
S. pyogenes during the adsortion process. From the fact that patients mount an immune response to this phage enzyme (
95), it was concluded that it might also assist bacteria in spreading along the connective tissue planes, which also contain hyaluronic acid.
The second phage protein induced by coculture with pharyngeal cells was SpeC, a member of a growing family of streptococcal and staphylococcal superantigens. These proteins simultaneously bind major histocompatibility complex (MHC) class II molecules and specific variable regions of T-cell receptors. In contrast to normal antigens presented by MHC class II, which activate 0.001 to 0.0001% of all T cells, superantigens activate up to 20% of all T cells. This results in massive proliferation and subsequent release of inflammatory cytokines. These factors are thought to cause the high fever and shock or autoimmune sequelae in some patients with streptococcal infections who develop acute rheumatic fever (ARF).
Epidemiologically, ARF is associated with M18
S. pyogenes serotypes. In the sequenced M18 strain, one prophage encodes the SpeL and SpeM superantigens, which are sequence-related to the SpeC and SpeK superantigens (
203). SpeL and SpeM cause the proliferation of blood cells, are pyrogenic, and, in combination with endotoxin, are lethal in rabbits. They stimulated T cells with three specific β-chains. Their transcription was enhanced in exponentially growing cells, and all ARF patients showed significantly elevated antibody titers to both proteins (
204).
Prophage 315.4 from the sequenced M3 strain had
speK and
sla genes in the lysogenic conversion region. The recombinant Sla protein had phospholipase A2 activity and structural similarity to a snake venom toxin (
14). It is the target for the antibody response in infected patients and may account for bleeding disorders, which accompany some invasive
S. pyogenes infections. The authors proposed a model where the contemporary highly virulent M3 strains are clonal and are the result of the sequential acquistion of three prophages (φ315.5, acquired circa 1920; φ315.2, acquired circa 1940, and φ315.4, acquired circa 1980), leading to the constellation of three particular superantigens (SpeA3, SSA, and SpeK) combined with Sla (
9). However, there is only circumstantial evidence, but no experimental proof, for contribution of these prophage borne morons to the high virulence of this strain.
A basic problem with all studies of
S. pyogenes virulence is that most of the time, these bacteria behave as commensals and do not cause disease. For this reason, much effort was spent in correlating virulence factor (i.e., superantigen) secretion with certain disease phenotypes. In 2000, Kotb and colleagues observed that the expression of the phage-encoded SpeA was either very low or undetectable in about half of the clinical M1
S. pyogenes isolates (
51). When these isolates were introduced into Teflon tissue chambers within mice for 5 days, the expression of SpeA was turned on and concomitantly the expression of SpeB was downregulated (
130). SpeB is a chromosomally encoded secreted cysteine protease. Interestingly, the same authors had previously observed an inverse relationship between SpeB expression and disease severity in clinical M1
S. pyogenes infections (
128). Isolates recovered from the chambers continued to produce SpeA for extended passages in vitro, suggesting a stable genetic switch for SpeA expression. In cases where in vitro SpeA expression was finally downregulated, SpeB expression was again turned on. Electrophoretic two-dimensional gel analysis of the secreted M1
S. pyogenes proteome, coupled with matrix-assisted laser desorption-ionization/time-of-flight mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of the secreted bacterial proteins, including several known virulence factors (
6). Deletion of the
speB gene or addition of a cysteine protease inhibitor inactivating SpeB yielded cells that revealed more than 150 spots in the secreted proteome, including the prophage-encoded Sda (streptodornase). A complex secreted proteome was also reported for the strain recovered from the mouse tissue chamber. The proteome included SpeA, representing a clear case of in vivo up-regulation of a phage-encoded virulence protein.
The regulation of gene expression in
S. pyogenes grown under different conditions was the focus of a series of studies from the Musser laboratory. At the extremes of its habitats,
S. pyogenes has to adapt to a range of temperatures extending from about 25°C on the superficial skin to 40°C or more in deep tissue infections. In microarray analysis, globally 9% of the genes from the M1 strain SF370 were differentially transcribed by organisms grown at 29°C compared with 37°C (
205). Genes from mobile DNA (mainly phages) belonged to the most prominently up-regulated genes at 29°C followed by transport and binding protein genes (Fig.
1B) (
45a,
46,
164).
During
S. pyogenes-phagocyte interaction, about 16% of the bacterial genes were differentially transcribed. The largest fraction (69 genes) represented hypothetical up-regulated genes. The next most prominent group comprised prophages (23 genes). Since most of the prophage genes showed increased transcription, the authors suggested that these phage genes play a role in the host-pathogen interaction (
229). The expression pattern of putative virulence and regulatory genes in
S. pyogenes strains recovered from pediatric patients presenting with pharyngitis was investigated. Notably, the most prominently up-regulated gene (with a 60-fold increase in transcript level) was the prophage
sda gene (
228).
Two regulators were identified that controlled the prophage moron expression. One was the two-component regulator CovR/S. When it was inactivated, a mucoid colony phenotype was observed in
S. pyogenes, associated with overexpression of the hyaluronic acid capsule. The phage-encoded DNase was more abundant in the strain (
91) since it was relieved from repressor binding to the
sda promoter (
153). The second identified transcriptional regulator was Rgg, which controlled the expression of the prophage-encoded genes
sda and
mf3 (
52).
However, the exact role of the up-regulated phage genes in
S. pyogenes cells making contact with the mammalian cells is not yet clear: do they directly benefit the propagation of the phage, or do they benefit the lysogen via a bacteriocin-like effect or the expression of phage-encoded fitness factors? In summary, it is fair to say that perhaps more than in any other system, the recent evolution of
S. pyogenes has been guided by bacteriophages. Even when making contact with the mammalian host,
S. pyogenes strains not only alter their gene expression pattern but—by lysogenization of bystander cells—also alter the genomes of commensal
S. pyogenes (
32). In addition, prophages change the bacterial genome while residing silently in the chromosome by serving as anchor points for homologous recombination leading to bacterial chromosome rearrangements independent of lysogenization. In contrast, the actual role of the prophage morons in the emergence of new epidemic strains with different disease characteristics is mainly speculative so far, and there is no direct evidence from animal experiments.
S. aureus.
S. aureus strains are gram-positive cocci which can grow under aerobic and anaerobic conditions. Their natural habitat is the nose and the skin of warm-blooded animals. A large fraction of the human population is colonized with this bacterium. These colonizing
S. aureus strains cause disease only rarely. Nevertheless,
S. aureus is one of the most frequent causes of bacterial infection in humans. Specific antibiotic-resistant strains cause epidemics in hospital settings, and one can distinguish between these “nosocomial” infections and community-acquired infections, which are caused by a much more diverse group of strains with different properties.
S. aureus can cause a wide range of diseases ranging from toxicoses such as food poisoning to invasive diseases. Many skin infections such as furunculosis, staphylococcal scalded skin syndrome, and wound infections are caused by this bacterium.
S. aureus strains encode a large variety of secreted toxins, and these toxins (Fig.
17A) (Table
4) are responsible for most of the clinical symptoms associated with the infections.
The production of some
S. aureus toxins was first linked to lysogeny over 40 years ago: a phage could convert nontoxigenic strains to alpha-hemolysin production (
20). However, it took more than 20 years after that discovery until a toxin gene was located on a staphylococcal phage by molecular means. The staphylococcal enterotoxin A gene,
sea, was mapped near the attachment site of the temperate phage PS42-D (
15). Southern hybridizations revealed that the
sea genes in staphylococcal strains were associated with a family of phages rather than with one particular phage. The potential medical importance of staphylococcal phage-encoded toxins has recently motivated a number of phage sequencing projects. Phage PVL encodes a bicomponent cytotoxin, the Panton-Valentine leukocidin. The two toxin genes
lukS and
lukF were located between the phage lysin gene and the right attachment site (
126). The two toxins assemble into pore-forming transmembrane complexes and lyse their target cells, human polymorphnuclear leukocytes (
82). Phosphorylation of LukS by protein kinase A was found to be required for the leukocytolytic activity (
167). Very similar toxin genes were found at the same location in a morphologically and molecularly distinct
S. aureus phage, SLT (Fig.
17B). Sequence comparison suggested that the entire region surrounding the attachment site was the result of a modular exchange between the two phages (
166).
S. aureus prophage PV83 also encodes a leukocidin, this time a
lukM-lukF gene combination (Fig.
17B). As in other staphylococcal phages, these toxin genes were located between the lysin gene and the right attachment site. The toxin genes in PV83 are flanked by a transposase gene, suggesting that this gene cassette was derived from a mobile DNA element (
254). The different
S. aureus phages showed a patchwork pattern of relatedness (
46), as predicted by the modular theory of phage evolution (
24).
The exfoliative toxin is an extracellular protein that underlies the scalded skin syndrome associated with
S. aureus infections (
133). The causative proteins are ETA and ETB. The former is encoded by prophage ETA, and the latter is encoded by a large plasmid. Again, the
eta gene is located downstream of the phage lysin gene. Notably, the ETA protein was found by Western blot analysis in the supernatant from the parental lysogen and in two
S. aureus strains lysogenized with ETA phage. Its toxic activity was demonstrated in mouse experiments (
245). Interestingly, ETA production was not stimulated by mitomycin C induction, suggesting that it is constitutively produced from its own promotor and neither regulated by the preceding lysis gene cassette nor up-regulated with phage replication during prophage induction (see above).
The recent sequencing of several
S. aureus strains confirmed and extended the observations from the phage-sequencing projects. The bacterial genome projects were motivated by the increasing antibiotic resistance demonstrated by clinical
S. aureus isolates. Methicillin-resistant
S. aureus is now the main etiological agent of nosocomial infection, and vancomycin, the only antibiotic effective against it, is no longer effective against all
S. aureus isolates. The methicillin-resistant strain N315 isolated in 1982 differed from the vancomycin-resistant strain Mu50 isolated in 1997 by less than 4% at the nucleotide level (
132). Most of the differences in genome structure were due to the insertion of Mu50-specific mobile genetic elements, including a Mu50-specific prophage. Two phages are very similar between the two strains: φN315 and φMu50A (Fig.
6A). The two prophages carry known virulence factors: a gene encoding enterotoxin P (the
sep gene), a superantigen involved in the symptoms of food poisoning, and a gene encoding staphylokinase (the
sak gene), suspected to be involved in the proteolytic destruction of host tissue. In addition, an M-like protein fragment is encoded by a gene preceding
sep. The virulence genes flank the phage lysis cassette on both sides. However, the two prophages are not identical. Especially over the lysogeny and early genes, the two prophages differed in numerous small modular exchanges (Fig.
6A).
One major difference between the two sequenced
S. aureus strains was prophage φMu50B. Strain N315 showed no prophage at this position. φMu50B shares segments of sequence relatedness with phage ETA, especially over the late genes, but lacks the ET toxin gene (Fig.
6B). In its place, it carries four genes of unknown function. A potential virulence gene was identified upstream of the integrase gene; it had similarity to a gene from the staphylococcal pathogenicity island SaPIn1. Notably, both prophages were flanked near their integrase gene by pathogenicity or genomic islands, suggesting a potential link between the acquisition of the two types of mobile DNA elements in strain Mu50.
A relationship between the two types of mobile DNA element was demonstrated in
S. aureus with the 15 kb-long pathogenicity island SaPI1 and the temperate staphylococcal phage 80α (
142,
197). SaPI1 carries the gene for the toxic shock syndrome toxin 1 (TSST-1) and an integrase gene. It is flanked by a 17-bp repeat, and it is excised and circularized and replicates autonomously. Notably, SaPI1 interferes with phage 80α growth and is encapsidated into special small phage 80α heads commensurate with its smaller DNA size. On phage-mediated transfer to a recipient organism, SaPI1 integrates at a specific attachment site via the SaPI1-encoded integrase. However, as with the P4 satellite phage and its P2 helper phage, phage 80α provides functions for excision, replication, and encapsidation of this pathogenicity island. This peculiar link with a phage ensures mobility to the pathogenicity island and may be responsible for the spread of TSST-1 among
S. aureus strains.
Also a community-acquired
S. aureus isolate, strain MW2, was sequenced (
7). It differed from strain N315 by numerous insertions, deletions, and gene replacements. The most obvious differences were near the origin of replication, including the DNA element encoding methicillin resistance (SCCmec). Further differences between the strains were linked to other mobile DNA elements: prophages, transposons, and a number of small genomic islands. MW2 contains two prophages: φSa2 and φSa3. φSa2 resembles
S. aureus phage φ12, but also carries the
lukS and
lukF genes in a constellation identical to that phage in SLT. φSa3 closely resembles phage PVL over most of their genomes, but the two phages differed in their content of virulence genes. Sa3 contains, in addition to
sak and
sea around the lysin gene, two new enterotoxin gene alleles,
seg2 and
sek2. The latter genes are encoded between repressor and integrase genes in the lysogeny module. Prophage Sa3ms from an unfinished
S. aureus genome sequenced at the Sanger Centre differed from Sa3 at only 14 positions (
211). The extensive patchwise sequence similarities between
S. aureus phages suggested that multiple recombination events between N315-, PVL-, PV83-, Sa3-, and φ13-like phages had occurred in the evolutionary history of these phages. In the Sa3ms lysogen, a single 1- and 1.7-kb long transcript was detected with a
sak and
sea probe, respectively. Previously, a promoter was demonstrated upstream of
sea (
22). Mitomycin C induction led to a marked increase in the number of these mRNAs, and transcription of two higher-molecular-weight forms of mRNA was observed that covered both genes. If the replication of the prophage was prevented by a mutation, no increase in transcription was observed, suggesting that the augmented transcription was the direct result of the increased phage DNA copy number. Also, the transcription of the
seg2 and
sek2 genes was increased by mitomycin C (
211). This result was surprising since these genes were cotranscribed from a promoter upstream of the
cI-like repressor gene. This constellation ensures constitutive expression during lysogeny in coliphages, but repression via the Cro repressor occurs after prophage induction.
A comparison of the different
S. aureus prophages revealed that the toxin genes are mobile DNA elements of their own and suggested that they are not stably associated with an individual prophage (
7). Microarray analysis also demonstrated an extensive variation in gene content among different strains of
S. aureus, with 22% of the genome comprising dispensable genetic material (
84). A total of 18 large regions of difference were identified, 10 of which encode virulence factors or antibiotic resistance genes. Apparently, lateral gene transfer has played a fundamental role not only in the evolution of
S. aureus prophages but also in that of their hosts.
C. diphtheriae.
C. diphtheriae is a strictly human-adapted gram-positive bacterium. It can cause local infections of the tonsils, pharynx, nose, and conjunctiva and systemic intoxications when the released toxin destroys the parenchyma of the heart, liver, kidneys, or adrenal glands. The diphtheria toxin (DT) is the major virulence factor of this pathogen, and the DT gene is carried by a family of closely related bacteriophages (Fig.
18).
DT is a classical AB toxin. The precursor-protein has a signal sequence for Sec-mediated protein secretion. Later, the pre-toxin is proteolytically processed to yield the mature AB toxin. DT is one of the best-characterized bacterial toxins, and we possess X-ray crystallographic structures for free DT and DT complexed with different substrates and a detailed knowledge of the mode of action of this toxin (reviewed in reference
115). The A subunit of DT is an ADP-ribosyltransferase which covalently modifies the elongation factor EF-2, thereby inhibiting chain elongation during protein synthesis. Detailed knowledge is also available for the cellular receptor of DT and the way in which DT is processed after entry into the cell. This knowledge has allowed the establishment of a transgenic mouse model for diphtheria, even though mice are naturally resistant to this disease (
50).
Diphtheria research has a long history. The discovery of DT goes back to Roux and Yersin in 1888. Subsequently, the work performed by Behring and Kitasato in 1891 and the discovery of the DT-overproducing strain PW8 in 1896 laid the ground for antitoxin therapy of and active immunization against this upper respiratory tract infection, respectively. The link of DT to lysogeny was established in the 1950s, when the nonlysogenic
C. diphtheriae strains C4 and C7 were shown to become toxicogenic after infection with the
tox+ corynephage beta but not with the
tox-lacking corynephage gamma. Work in the late 1960s established that DT expression was achieved from the integrated prophage as well as from an extrachromosomal replicating or nonreplicating phage DNA, suggesting that DT expression was not regulated with other phage genes. In the mid-1970s a restriction map of phage beta was elaborated which showed that the
tox gene was located next to the phage attachment site, making its independent regulation plausible. Earlier work had already established that
tox+ lysogens produce DT only in iron-depleted media (
174). The regulation occurred at the level of transcription. In the mid-1980s, a promoter directly upstream of the
tox gene was characterized; this was followed by the discovery of a diphtheria toxin repressor (DtxR) that binds to an operator directly upstream of the
tox gene. DtxR functions as an iron-dependent global regulator in
C. diphtheriae. Currently, at least 18 DtxR binding sites are known to occur in
C. diphtheriae, and they affect the expression of about 40 genes. There are fascinating analogies between the iron-regulated Fur repressor in
E. coli and its effect on Shiga toxin-encoding prophages and DtxR in
C. diphtheriae on DT-encoding prophages, despite the wide phylogenetic distance separating these bacteria (gram-negative proteobacteria versus high-G+C-content gram-positive bacteria). A recent review provides a detailed list of the classical papers on DT briefly mentioned in the preceding paragraph (
115).
C. diphtheriae phages have been poorly investigated. Most toxigenic
C. diphtheriae strains contain DNA sequences related to phage beta, but the
tox gene was also found to be associated with the distinct phages δ and ω (
37,
190). Interestingly, the
tox corynephage γ also contained the
tox gene. However, a 1.5-kb IS-like DNA element inserted near the 5′ end of the coding sequence prevented
tox translation (
140a). The recently published
C. diphtheriae genome sequence of a bacterial strain from the current diphtheria epidemic in Eastern Europe (
49) provided the first complete sequence of a
tox+ phage. Surprisingly, its genome organization resembled that of phages found in low-G+C-content gram-positive bacteria. In fact, several proteins still demonstrated weak sequence links with this group of phages. However, its closest sequence matches were with structural genes of
Brevibacterium phage BFK20. The
tox gene was found at the right prophage genome end. Interestingly, two additional candidate lysogenic conversion genes were detected at the left genome end between phage integrase and the attachment site, a tRNA gene (Fig.
18). Both prophage genome ends showed a decreased G+C content. A tRNA gene was also found downstream of the
tox gene, followed by what could be a further mobile DNA element. Overall, the bacterial genome sequence showed 13 regions with local anomalies in nucleotide composition. Seven of them were flanked by tRNA genes, and none were present in the two sequenced environmental corynebacteria. Many genes that could contribute to the pathogenicity of
C. diphtheriae are found in these genomic islands. Six regions contained phage-related genes. Unlike its closest sequenced pathogenic relative,
Mycobacterium tuberculosis,
C. diphtheriae appears to have recently acquired many genes necessary for survival, attachment, and virulence in the host.
M. tuberculosis contains only a few small prophage remnants that still have functions related to the excision of these elements (
17), and these were not associated with recognizable virulence factors. This difference may be a reflection of the different ecology:
M. tuberculosis is a predominantly intracellular pathogen and thus has less opportunity for genetic exchanges than does the extracellular
C. diphtheriae.