JOURNAL OF BACTERIOLOGY, Oct. 2008, p. 6290–6301
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 19
Phage-Associated Mutator Phenotype in Group A Streptococcus?
Julie Scott, Prestina Thompson-Mayberry, Stephanie Lahmamsi,
Catherine J. King, and W. Michael McShan*
Department of Pharmaceutical Sciences, The University of Oklahoma College of Pharmacy, P.O. Box 26901,
Oklahoma City, Oklahoma 73190
Received 28 September 2007/Accepted 15 July 2008
Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and
commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus
pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in
response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a
common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the down-
stream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage
excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and
expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low;
this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of
MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is
functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage
integrated into the 5? end of mutL correlates with a mutator phenotype (10?7to 10?8mutation/generation, an
approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains
[10?9to 10?10mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13
completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are
positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a
bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond
rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.
The ability to adapt to a changing environment is crucial to
the success of any species. The mutation rate in bacteria has
been estimated to be 0.003 mutation per genome (?5 ? 10?10
mutation per base) per replication (13), and therefore, a min-
imum population size is needed to ensure that that there are
rare variants that are resistant to an antibiotic, for example.
Accordingly, if the population density of a bacterial species is
low, then at typical mutation rates rare mutants may not arise,
leading to extinction.
A growing body of evidence indicates that bacteria from wild
populations often avoid population extinction by altering their
mutation rates. These strategies typically either reduce the
fidelity of DNA replication or alter DNA repair mechanisms,
resulting in a hypermutable state (49). As originally reported
by LeClerc et al., the incidence of mutators among clinical
isolates of pathogenic Escherichia coli and Salmonella enterica
was found to be much higher than anticipated (?1%), with
defects in DNA mismatch repair (MMR) being responsible for
this (29). Subsequent studies found examples in many bacterial
species; for example, 30% of Pseudomonas aeruginosa isolates
from cystic fibrosis patients and 57% of serogroup A epidemic
isolates of Neisseria meningitidis were found to exhibit a muta-
tor phenotype or be defective for MMR (18, 28, 43, 47). How-
ever, the appearance of mutator strains is not confined to
pathogenic bacteria, since the frequency of the defects was
essentially the same in commensal and pathogenic E. coli in the
survey performed by Matic and colleagues (36). The evidence
suggests that the frequency of mutators and thus the potential
for evolution in wild populations of bacteria may be signifi-
cantly different from the frequency of mutators and potential
for evolution in laboratory strains.
Prokaryotic MMR has been most intensively studied in E.
coli, where transient DNA hemimethylation patterns following
replication are used to discriminate between the template
strand and the newly synthesized strand containing the mis-
match. The required proteins MutS, MutL, and MutH mediate
MMR, recognizing the mismatch and cleaving the transiently
unmethylated strand, allowing removal of the region contain-
ing the erroneous base and repair by resynthesis of the strand
(35). Homologs of MutS and MutL appear to be universal;
however, outside the gram-negative bacteria, homologs of
MutH are not found. In gram-positive bacteria and eukaryotes,
discrimination between the template strand and the strand
needing repair does not appear to be based upon a transient
hemimethylation state but may be based upon localization of
MutS homologs by the DNA polymerase proliferating cell nu-
clear antigen (PCNA) clamp to base mismatches in newly
replicated DNA. Following MutL incision of the strand, nu-
clease Exo1 is recruited to perform 5?33? excision through
and beyond the site of the mismatch (25, 27, 51).
The genomes of temperate bacteriophages, upon integration
into a host chromosome, can alter the genotype and phenotype
of bacteria (6, 38). Sequencing and analysis of the genome of
group A streptococcus (GAS) (Streptococcus pyogenes) sero-
type M1 strain SF370 revealed the presence of several endog-
* Corresponding author. Mailing address: Department of Pharma-
ceutical Sciences, The University of Oklahoma College of Pharmacy,
P.O. Box 26901, Oklahoma City, OK 73190. Phone: (405) 271-6593.
Fax: (405) 271-7505. E-mail: William-McShan@ouhsc.edu.
?Published ahead of print on 1 August 2008.
enous bacteriophage genomes (prophages) (10, 12, 14). Pro-
phage SF370.4, integrated between mutS and mutL (10),
appears to be defective; the expected modules for integration,
lysogeny control, replication, and regulation are present, but
no identifiable genes for structural capsid proteins, host lysis,
or DNA packaging are present (Fig. 1A). Thus, it is unlikely
that this prophage could complete the lytic cycle and release
new virions. However, the phage-bacterium DNA junctions
(attL and attR) are intact, and direct sequence repeats define
the ends of the prophage genome, a requirement for integrase-
mediated integration and excision. For GAS genomes that lack
this prophage, a genetic structure and promoter analysis pre-
dicted that mutS and mutL are transcribed together on a poly-
cistronic message from a promoter located upstream of mutS.
Since both genes are required for MMR (20), the presence of
phage SF370.4 was expected to render the host defective for
MMR so that it lacked mutL expression, resulting in a fixed
mutator phenotype. However, here we show that in rapidly
growing cells or following DNA damage, S. pyogenes strain
SF370 expresses both mutS and mutL, while in stationary-
phase cells only mutS is expressed. Further, the differential
expression of mutL during growth results from the dynamic
excision and reintegration of the SF370.4 prophage. This al-
teration in prophage integrative states results in a unique and
sophisticated molecular mechanism to achieve a growth phase-
dependent mutator phenotype in S. pyogenes strain SF370.
MATERIALS AND METHODS
Bacterial strains and growth conditions. S. pyogenes SF370, originally isolated
from a wound infection, is a serotype M1 strain whose complete genome se-
quence has been determined (14) (Table 1). S. pyogenes NZ131 (? ATCC
BAA-1633) is a serotype M49 strain that lacks any phage between mutS and
mutL and was used as a source of phage-free DNA; its genome has also been
completely sequenced (GenBank accession no. CP000829). Strain MGAS10394
is a serotype M6 strain whose genome has been determined and contains a
prophage closely related to SF370.4 integrated into the same attachment site (2);
it was obtained from the American Type Culture Collection (ATCC BAA-946).
Strain JRS1 is a serotype M1 strain isolated from a case of streptococcal toxic
shock syndrome in Oklahoma City, OK, that lacks an SF370.4-like prophage, as
determined by DNA sequencing of the region (not shown). Bacteria were grown
in Todd-Hewitt broth (Difco) supplemented with 2% yeast extract (THY me-
dium) at 37°C; growth was monitored by determining the absorbance at 600 nm.
FIG. 1. mutS-mutL region of S. pyogenes SF370 and proposed mechanism of prophage SF370.4 excision. (A) Chromosomal region of the S.
pyogenes SF370 chromosome that contains prophage SF370.4, which is integrated between the flanking host genes mutS and mutL. (B) In the
absence of the prophage, a shared promoter is predicted to control mutS and mutL, as well as lmrP, ruvA, and tag, all of which are transcribed on
a polycistronic mRNA. The presence of prophage SF370.4 truncates this mRNA after mutS, silencing the downstream genes until cinA and recA,
each of which has its own promoters. As described in this report, activation of prophage SF370.4 leads to excision and release of the circular form
of its genome (C) and restoration of the prophage-free MMR operon (B). Excision of the prophage leads to transcriptional activation of mutL,
lmrP, ruvA, and tag, restoring MMR, Holliday junction resolution, and base excision repair. Transcriptionally active streptococcal genes are green,
and the predicted mRNAs are indicated by arrows below the open reading frames. The small arrows above the open reading frames indicate the
locations of the predicted promoters (46). Phage genes whose functions were predicted by homology to known genes are identified below the open
reading frames in panel A.
TABLE 1. Bacterial strains used in this study
SF370emm1 ?SF370.4 mutS?mutL
VOL. 190, 2008HOST GENE CONTROL BY GAS PHAGES 6291
Nucleic acid preparation and PCR. DNA was isolated from streptococci as
previously described (39, 44). The cells were harvested by centrifugation and
resuspended in 100 ?l Tris-EDTA buffer containing 50 U Streptomyces glo-
bisporus mutanolysin (Sigma-Aldrich, St. Louis, MO) and 5 mg lysozyme (Fisher
Scientific, Pittsburgh, PA), and the suspensions were incubated at 37°C for 30
min. Cell lysis was carried out by addition of 0.5 ml GES reagent (5 M guanidium
thiocyanate, 100 mM EDTA, 0.5% Sarkosyl), followed by vortexing and incuba-
tion on ice for 5 min. Lysis was quenched by addition of 0.25 ml of 7.5 M
ammonium acetate, vortexing, and incubation on ice for 10 min. DNA was
extracted by addition of 0.5 ml chloroform-isoamyl alcohol (24:1), which was
mixed by vortexing until a uniform emulsion formed. Samples were centrifuged
at full speed in a microcentrifuge for 10 min. The aqueous phase was retained,
and DNA was precipitated by addition of 0.6 volume of isopropyl alcohol.
Samples were centrifuged at 6,500 ? g for 1 min and washed with 70% ethanol
five times. Ethanol was removed by vacuum aspiration, and the pellets were dried
and resuspended in 100 ?l Tris-EDTA buffer containing 0.01 ?g RNase A. For
some experiments, cells in the growth media were harvested by centrifugation
and then stored overnight in RNAlater (Ambion) at 4°C before they were
processed as described above.
RNA was prepared using the RiboPure system for bacteria (Ambion) by
following the manufacturer’s recommended protocol. RNA samples were tested
for a lack of DNA contamination by PCR using primers specific for the variable
region of the emm gene (3). RNA samples were converted to cDNA using
Superscript II (Invitrogen) and random hexamer priming by following the man-
Detection of prophage excision. PCR amplification of DNA (PCR) or cDNA
(reverse transcription-PCR) sequences was performed using Taq DNA polymer-
ase (Invitrogen), the buffers supplied by the manufacturer, and the recom-
mended conditions. Primers were used to amplify specific internal regions of
mutS, mutL, and the mutS-mutL intergenic region (Table 2). The attP site from
the excised, circular prophage was amplified using specific primers (Table 2).
When the phage was excised from the chromosome and its DNA was circular-
ized, the attP primers generated a 486-bp PCR product. If the phage remained
in the host strain’s chromosome, no product was generated using the attP prim-
ers. Thermal cycling was performed by using initial denaturation at 94°C for 3
min, 35 cycles of denaturation at 94°C for 30 s, annealing at various temperatures
(depending on the primer melting temperature) for 30 s, and synthesis at 72°C
for 30 s, and a final extension at 72°C for 5 min.
Mitomycin C induction of MMR prophage. Mitomycin C induction of proph-
age was performed by using a modification of a previously described method
(40). A single colony of strain SF370 from a tryptic soy agar plate supplemented
with 5% sheep blood was used to inoculate THY broth, which was subsequently
incubated overnight at 37°C. The overnight culture was diluted 1:20 into 100 ml
fresh THY broth, and the culture was incubated at 37°C until early logarithmic
growth began (A600, 0.2). This culture was divided into two 50-ml cultures, and
one of the latter cultures was treated with mitomycin C from Streptomyces
caespitosus (Calbiotech, Spring Valley, CA) at a final concentration of 0.2 ?g/ml.
The cultures were incubated at 37°C for 1 h, and cells were harvested by cen-
trifugation at 1,000 ? g for 15 min at 4°C. The cell pellets were incubated at 65°C
for 15 min to inactivate endogenous DNases, and chromosomal DNA was iso-
lated as described above.
Kinetics of prophage excision during cell growth. Quantitative real-time PCR
was used to observe prophage SF370.4 excision kinetics in the strain SF370
chromosome during growth. A single colony was used to inoculate 5 ml THY
broth, which was then incubated at 37°C for 16 h. The overnight culture was
diluted 1:20 in fresh, prewarmed THY broth. The culture was incubated at 37°C,
and growth was monitored by determining the absorbance at 600 nm. Beginning
30 min postinoculation, samples were collected at 30-min intervals until 2 h
postinoculation and then at 15-min intervals. Samples (30 ml) were collected
when the culture density was low (A600, ?0.2), and 10-ml samples were collected
later. Cells were harvested by centrifugation (3,500 ? g for 10 min), suspended
in 1 ml RNAlater (Ambion), and stored at 4°C for 24 h. Total DNA was then
isolated as described above. Real-time PCR to detect phage SF370.4 attP, attB,
and attL was carried out with a Bio-Rad iCycler equipped with the real-time
optical fluorescent detection system using SYBR green PCR master mixture
(Bio-Rad Laboratories, Hercules, CA) and the primer pairs shown in Table 2.
The following program was employed for all PCRs: (i) an initial denaturation at
95°C for 3 min and (ii) 35 cycles consisting of denaturation at 95°C for 30 s,
annealing at 55°C for 30 s, and elongation at 72°C for 30 s. Following the final
cycle, a melting curve analysis was performed for each sample to verify that a
single product was produced. For each real-time PCR plate evaluated, primers
for the 16S rRNA subunit housekeeping gene were also included for each sample
for normalization of the data, and water blanks were used as negative controls.
To determine the linear range of amplification for this primer set, initial PCRs
were performed with serial dilutions of DNA containing from 150 to 0.015 ng as
previously described (32). It was determined that 10 ng of DNA per reaction
mixture was optimal under these conditions; accordingly, all DNA preparations
were diluted so that they contained 10 ng/?l DNA. The products of three
separate DNA isolations were analyzed by quantitative real-time PCR for all
primer pairs, and the results were averaged.
MutS and MutL protein expression. An overnight broth culture of strain
SF370 was diluted 1:20 into fresh, prewarmed THY broth and grown at 37°C.
Growth was monitored by spectrophotometry, and an early-logarithmic culture
was obtained (A600, 0.2). Samples were removed when the A600of the culture was
0.2, 0.3, 0.4, and 0.6, and the cells were quick-frozen in a dry ice-ethanol bath
after they were harvested. All samples were stored at ?80°C until they were
processed. After thawing, the cells were collected by centrifugation and resus-
pended in 0.5 ml lysis buffer (20% Tween 20, 150 mM NaCl, 50 mM Tris; pH 8.0)
supplemented with 0.05 ml of a protease inhibitor cocktail (Sigma). An equal
volume of zirconium beads was added, and the cells were lysed by mechanical
shearing using a bead beater (BioSpec Products, Bartlesville, OK). Shearing was
performed using a 30-s pulse followed by 1 min of cooling of each sample, which
was repeated five times. Cell debris and beads were removed by centrifugation,
and the cell lysate was treated with a Bio-Rad 2D clean-up kit by following the
manufacturer’s protocol. Fifteen micrograms of protein from each sample was
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, trans-
ferred to a nylon membrane by Western blotting, and probed with polyclonal
rabbit antibodies to either S. pyogenes MutS or MutL using standard protocols
(21). Rabbit antibodies were prepared by ProSci Incorporated, Poway, CA, using
synthetic peptides corresponding to predicted antigenic sites from either S.
pyogenes MutS (LEIGLTSRNKNAEN) or S. pyogenes MutL (IQENHTSLREL
GKY) as haptens. Antibody binding was detected using an amplified alkaline
phosphatase goat anti-rabbit immunoblot assay kit (Bio-Rad Laboratories, Her-
cules, CA) by following the manufacturer’s recommended protocol. The relative
TABLE 2. Oligonucleotide primers used in this study
Gene targetOligonucleotide primer
Phage SF370.4 attP5?CTCAGGAAAATACGACAAGG
Phage SF370.4 int5?CTGCGGTACGTTCAGTCATC
emm variable regionc
aPrimers used for quantitative real-time PCR.
bData from reference 3.
6292SCOTT ET AL. J. BACTERIOL.
intensities of the MutS and MutL bands were quantified using the ImageJ
software package (http://rsb.info.nih.gov/ij/).
Determination of the spontaneous mutation rates. A standard fluctuation test
(30, 48) was used to estimate the mutation rates of S. pyogenes MMR prophage-
containing strains SF370 and MGAS10394 and MMR prophage-free strains
JRS1 and NZ131. A THY broth culture of the strain to be tested was started
using an individual colony. After overnight incubation at 37°C, the cultures were
diluted into fresh media to obtain final cell densities of ?1,000 CFU/ml and
dispensed to obtain 31 separate 1-ml aliquots. After incubation for 24 h at 37°C,
one tube was used to determine the total number of CFU/ml by serial dilution,
and the contents of the remaining tubes were mixed with 3 ml of melted soft agar
(45°C) and poured onto THY medium plates containing 2 ?g/ml ciprofloxacin, a
DNA gyrase inhibitor. This concentration of antibiotic is 10 times the MIC (not
shown). The cultures were incubated for 2 to 4 days to allow growth of cipro-
floxacin-resistant colonies, and the number of resistant colonies per culture was
determined. The mutation rate (with confidence limits) was calculated using the
algorithm of Ma et al. (31), combined with the maximum likelihood estimation
technique (54) and implemented by the ft software package (P. D. Sniegowski,
University of Pennsylvania) (50). The results were plotted using the Prism4
software package. This experiment was repeated using the P0method of the
Poisson distribution with 10-fold dilutions of the mutator SF370 cultures, as
recommended by Rosche and Foster (48); this experiment gave similar estimates
for the mutation rates (not shown). The MIC of ciprofloxacin was determined
using the criteria recommended by the Clinical and Laboratory Standards Insti-
tute (Wayne, PA).
UV irradiation killing assay. Overnight cultures of strains SF370, MGAS10394,
JRS1, and NZ131 were harvested by centrifugation and resuspended in sterile
0.1 M MgSO4at a final absorbance at 600 nm of 0.5. A calibrated 254-nm
germicidal lamp (120 ?W/cm2) was prewarmed for 30 min prior to exposure
of the strains. For each strain, 5 ml of a resuspended culture was placed in a
sterile glass petri dish and exposed to the UV lamp. Since a homolog of
photolyase is present in the GAS genome, the UV light treatment was carried
out in a darkened room. At selected intervals (30, 60, 90, and 120 s), 1 ml was
removed and serially diluted 10-fold using 0.1 M MgSO4. Each dilution (2 ?l)
was spotted onto a THY agar plate and incubated in the dark at 37°C for 24 h.
Survey of GAS strains. One hundred S. pyogenes strains were randomly se-
lected from the laboratory collection of J. J. Ferretti at the University of Okla-
homa Health Sciences Center. This collection contains isolates obtained from
worldwide locations during the last three decades. The emm type of each strain
either had been previously determined serologically or was determined by per-
forming PCR with primers that amplify the variable region. Approximately 1 ?g
of chromosomal DNA from each strain was added to enough deionized water to
obtain a final volume of 0.15 ml. Sodium hydroxide (45 ?l of a 1 M stock
solution) was added to each sample, which was followed by heating at 65°C for
30 min. The solution was neutralized by addition of sodium acetate (pH 5) (65
?l of a 3 M stock solution). The samples were applied to a nylon membrane using
a slot blot apparatus, washed twice with 2? SSC (34), and fixed to the membrane
using UV light (1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate). A
nonisotopic DNA probe for the SF370.4 integrase gene was prepared using a
PCR DIG probe synthesis kit (Roche Diagnostics Corporation) by following the
manufacturer’s recommended protocol and reagents and using integrase-specific
primers. The nylon membranes containing the DNA samples were hybridized to
the probe and detected using a DIG DNA detection kit (Roche) by following the
recommended protocol. DNA from strains SF370 and NZ131 were used as
positive and negative controls, respectively. Strains giving positive results were
confirmed by performing PCR for the attL junction between the phage integrase
DNA sequencing and analysis. Automated DNA sequencing was performed at
the University of Oklahoma Health Sciences Center Laboratory of Microbial
Genomics. Prior to sequencing, PCR products were treated with shrimp alkaline
phosphatase and exonuclease I by incubation at 37°C for 60 min, followed by
inactivation of the enzymes by heating at 85°C for 15 min. Sequencing was
performed using the same primers that were used for PCR. In some cases, the
amplified PCR product was cloned for future study using the pGEM-T Easy
vector (26). Computer predictions for promoter elements were performed using
the Berkeley Drosophila Genome Project neural network promoter prediction
Nucleotide sequence accession number. The nucleotide sequence of the phage
SF370.4 attP region has been deposited in the GenBank database under acces-
sion no. AY684192.
Expression of MMR genes in GAS strain SF370 is related to
the growth state of the cells. In contrast to the MutS and MutL
genes in many eubacteria, the MutS and MutL genes in S.
pyogenes are genetically linked together under control of a
common promoter upstream of mutS and are thus predicted to
be transcribed together on a polycistronic mRNA (Fig. 1).
Additionally, three additional genes, lmrP, tag, and ruvA, are
predicted to be components of this polycistronic mRNA. In the
SF370 serotype M1 genome strain, as well as in several other S.
pyogenes genome strains, a prophage is integrated between
mutS and mutL; its attachment site overlaps the first five
codons of the mutL coding region. Thus, the integration of
phage SF370.4 at the 5? end of mutL should interfere with or
alter the transcription of this gene by separating it from the
remainder of the normal mRNA, potentially inactivating
MMR. Analysis of rapidly dividing S. pyogenes SF370 cells
(mid-logarithmic phase) showed, however, that mRNAs for
both mutL and mutS were transcribed (Fig. 2A, lanes 2 and 5,
respectively), which should allow a functional MMR system. In
contrast, when the cells reached stationary phase and stopped
dividing, mutS was still transcribed (lane 6) but mutL was not
FIG. 2. Expression of mutL is growth dependent in GAS strain
SF370. (A) cDNA from SF370 cells was synthesized from RNA iso-
lated at mid-logarithmic (ML) or stationary (ST) phase, and PCR
primers specific for mutS and mutL were used to amplify products
specific for each gene (the targeted region of each gene in a phage-free
chromosome is shown below the gels). The mutS message is detectable
in both rapidly growing and stationary-phase cells (lanes 5 and 6), but
mutL is expressed only in actively dividing cells (lane 2). Lanes 1 and
4, molecular weight standard (DNA kilobase ladder; Invitrogen); lane
2, mutL, mid-logarithmic cells; lane 3, mutL, stationary-phase cells;
lane 5, mutS, mid-logarithmic cells; lane 6, mutS, stationary-phase
cells. (B) Identification of the uninterrupted mutS-mutL mRNA in
SF370. Primers specific for the phage-free junction between mutS and
mutL amplify this region in SF370 cDNA obtained from actively di-
viding streptococci but not from genomic DNA. Lane 1, molecular
weight standard; lane 2, NZ131 cDNA (phage-free strain; positive
control); lane 3, SF370 mid-logarithmic cDNA; lane 4, SF370 chromo-
VOL. 190, 2008HOST GENE CONTROL BY GAS PHAGES6293
transcribed (lane 3), disabling MMR. The expression of mutL
during logarithmic growth suggested that either the prophage
was excised via integrase-mediated site-specific recombination,
restoring the phage-free genetic constellation, or the phage
genome harbored an alternative promoter for mutL that was
activated during growth.
If prophage SF370.4 undergoes integrase-mediated excision
during exponential growth, the event should restore a phage-
free mutS-mutL sequence. Using cDNA as a template, the
mutS-mutL intergenic region was readily detected by PCR
amplification of a 461-bp product in the prophage-free genome
of S. pyogenes strain NZ131 (Fig. 2B, lane 2), demonstrating
that the two genes occupy a common transcript. The presence
of the SF370.4 prophage in strain SF370 introduced over 13
kbp of intervening DNA that under typical PCR conditions
prevents similar amplification from chromosomal DNA iso-
lated from stationary-phase cells (Fig. 2B, lane 4). However,
the phage-free mutS-mutL intergenic product was present in
the SF370 cDNA from mRNA obtained from logarithmically
growing streptococci (Fig. 2B, lane 3); its identity was con-
firmed by DNA sequencing (attB) (Fig. 3B). The restoration of
the wild-type arrangement of the mutS and mutL genes and the
FIG. 3. The prophage SF370.4 chromosome excises from the host genome as a replicating circular molecule during exponential growth.
(A) PCR primers (arrows 1 and 2) are located so that the phage attP region may be amplified by PCR only when the phage genome is excised from
the bacterial chromosome and exists as free circular DNA. No product can be amplified from the integrated prophage in this reaction. Using DNA
isolated from cells grown to mid-logarithmic stage, the specific PCR product was identified by gel electrophoresis (lane 2), and DNA sequencing
confirmed the identity of the specific phage attP sequence. The orientation of the open reading frames matches the genome sequence. Lane 1,
molecular weight standard; lane 2, attP region from the circular phage genome amplified from SF370 DNA isolated during mid-logarithmic growth;
lane 3, chromosomal DNA isolated from a culture of strain SF370 after 18 h of growth at 37°C, showing no detectable attP PCR product.
(B) Sequences flanking the integrated phage-host genome junctions (attL and attR) (14), the phage-free mutS-mutL junction (attB), and the
circular phage genome attP site. The sequence shared by the phage and host genomes is underlined, and the initial amino acid residues of MutL
are indicated below the attB sequence. Phage DNA sequences are enclosed in a box.
6294SCOTT ET AL. J. BACTERIOL.
appearance of the associated mRNA transcript indicated that
prophage excision from the S. pyogenes SF370 genome had
occurred, allowing transcription from the mutS promoter to
continue through to the downstream gene, mutL.
The phage SF370.4 genome is excised during exponential
growth. The excision of phage SF370.4 should have released a
circular free form of the prophage genome via Campbell-type
homologous recombination (Fig. 3A). The attachment sites
linking the phage genome to the bacterial genome in S. pyo-
genes SF370 could be identified by the 21-bp direct repeat
(5?CAATAATGTTTGTCATAATTT3?) created by the inte-
grative joining of the two genomes at the ends of the linear
prophage (attL and attR) (Fig. 3B). The attL site encompassed
the first five codons of mutL. Circularization of the phage
genome upon excision brought the two ends together to create
attP and simultaneously restored the prophage-free attB site on
the bacterial genome (Fig. 3B). PCR performed with primers
designed to amplify only the attP region in the circular genome
(Fig. 3A) resulted in the predicted product when DNA from
logarithmically growing cells was used as the reaction template,
and DNA sequencing confirmed the specificity of the products
(attP) (Fig. 3B). Using these primers, it was not possible to
amplify a product from the integrated prophage. PCR per-
formed with chromosomal DNA isolated from cells grown for
18 h at 37°C, which were in deep stationary phase, resulted in
no product, indicating that most or all cells in the culture
contained the prophage in the integrated state (Fig. 3A,
Many prophages are induced by DNA-damaging agents,
such as UV light or mitomycin C, and it was reasoned that such
a challenge might promote population-wide induction of
prophage SF370.4 if the phage repressor was sensitive to cleav-
age following an SOS response, as seen in phage lambda.
Using PCR, the bacterial attachment site (attB) and the phage
attachment site (attP) were amplified from total DNA isolated
from mitomycin C-induced and uninduced mid-log-phase
SF370 following incubation for 1 h after treatment (Fig. 4).
Both the attB and attP PCR products (461 and 892 bp, respec-
tively) (Fig. 4) were strongly amplified when DNA from the
mitomycin C-induced cells was used; DNA sequencing con-
firmed the specificity of the products. By contrast, using an
equimolar template, the DNA from uninduced but logarithmi-
cally growing SF370 produced decreased amounts of the attB
product, and the attP PCR generated a secondary product that
was ?500 bp long (Fig. 4). This additional product was cloned
and sequenced, which showed that it was the amplification
product of a false priming site in an unrelated part of the
genome (not shown). Therefore, this secondary attP PCR
product appeared when the specific target (the circular phage
genome) was absent, as in the case of SF370.4 prophage-free
strain NZ131 (not shown), or when a mixed population of
integrated and episomal prophage was present in logarithmi-
cally growing cells (Fig. 4). When complete or nearly complete
prophage induction occurred after mitomycin C treatment, this
secondary product was not detectable.
Excision of prophage SF370.4 and expression of MutL are
growth dependent. The kinetics of prophage SF370.4 excision
and reintegration were determined by examining the appear-
ance and/or disappearance of the prophage-bacterium chro-
mosome junctions that reflect the integrated or episomal state
of the prophage DNA. The integrated prophage DNA is de-
fined by the left and right junctions with the bacterial chromo-
some (attL and attR, respectively); excision of the prophage
eliminates these sequences, while simultaneously creating the
prophage attP sequence and the bacterial attB sequence. As
shown in Fig. 5, attL began to disappear during very early
logarithmic growth, presumably about the time of DNA repli-
cation initiation. The disappearance of attL was coordinated
with the appearance of attB, the prophage-free constellation of
the bacterial chromosome. The episomal prophage-associated
attP site was detected within minutes after the appearance of
attB and the disappearance of attL, and it continued to be
present in the cell after attL reappeared. The simultaneous
presence of attP and attL during part of the growth cycle
(between approximately 120 and 180 min [Fig. 5]) indicated
that there was a mixed population of cells with both integrated
and episomal forms of the prophage, in agreement with the
results for the uninduced cells shown in Fig. 4. Further, the
continued increase in attP levels following the reappearance of
attL may have reflected an increased number of copies of the
extrachromosomal prophage during this period.
Prophage excision during early exponential growth pre-
dicted that the levels of MutL protein should increase rapidly
over the same interval. The amounts of MutS protein, by con-
trast, should have been constitutive and little affected by the
prophage. Total cytoplasmic proteins recovered from strain
SF370 cells over the course of exponential growth showed that
there was a high level of MutL expression very early (A600,
0.2), which decreased to nearly undetectable levels as the cul-
ture density increased as the culture reached stationary phase
FIG. 4. Mitomycin C treatment enhances prophage excision.
Equimolar amounts of chromosomal DNA from mitomycin C-induced
(?) or uninduced (?) strain SF370 were used as templates to amplify
attB and attP (the prophage-free chromosomal attachment site and the
free, circular prophage attachment site, respectively). DNA was iso-
lated 1 h postinduction. Both the attB- and attP-specific PCR products
were strongly amplified when the mitomycin C-induced cells were
used; DNA sequencing confirmed the specificity of the products. By
contrast, using an equimolar template and the uninduced SF370 DNA
resulted in amplification of decreased amounts of both products, and
the attP PCR generated a secondary product (indicated by an asterisk)
resulting from a false priming site in an unrelated part of the genome
(not shown). Thus, when the specific target (attP on the circular phage
genome) is absent (as it is in a prophage-free strain) (not shown) or
when a mixed population of integrated and episomal prophage is
present, this product can be amplified. Mitomycin C treatment of
strain SF370 results in disappearance of this secondary PCR product.
VOL. 190, 2008 HOST GENE CONTROL BY GAS PHAGES 6295
(Fig. 5). This loss is in contrast to the expression of MutL in the
prophage-free strain NZ131, which was not decreased during
logarithmic growth (not shown). The level of MutS, by con-
trast, was essentially constant over the same period. The results
strongly suggest that prophage SF370.4 is excised from its
attachment site in mutL early in exponential growth of a cell
culture, allowing the expression of MutL to be restored. As the
prophage returned to the integrated state, the levels of MutL
in the cell decreased until they were very low. This pattern of
MutL expression suggests that the cells alternate between a
mutator phenotype and a wild-type phenotype with respect to
A mutator phenotype and sensitivity to killing by UV irra-
diation are associated with phage SF370.4. The loss of MutL
expression following integration of prophage SF370.4 into the
S. pyogenes SF370 genome would indicate that a mutator phe-
notype was conferred upon its host. Using a modified Luria-
Delbru ¨ck fluctuation assay (30, 48), the mutation rate for spon-
taneous resistance to 10 times the MIC of ciprofloxacin, a
DNA gyrase inhibitor, was determined for strains SF370,
NZ131, MGAS10394, and JRS1. MGAS10394 is a serotype
M6 genome strain (2) harboring a closely related prophage
integrated into the same attB site as SF370.4, while JRS1 is a
serotype M1 clinical isolate from a case of streptococcal toxic
shock. Neither strain NZ131 nor strain JRS1 has a prophage
integrated into the mutL gene, and both strains should be wild
type for MMR. The mutation rates were estimated to be 3.3 ?
10?7and 3.2 ? 10?9mutation/generation for SF370 and
NZ131, respectively; thus, the mutation rate for SF370 was
almost 100-fold greater than the mutation rate for prophage-
free strain NZ131 and was consistent with a mutator pheno-
type (Fig. 6A). An association between a prophage integrated
into mutL and an increased mutation rate was also observed
for strain MGAS10394 (6.8 ? 10?8mutation/generation).
Strain JRS1, wild type for MMR, had a mutation rate of 5.3 ?
10?10mutation/generation (Fig. 6A).
The polycistronic mRNA containing mutS and mutL is also
predicted to contain the downstream gene ruvA, and transcrip-
tion of this gene would be interrupted by the presence of
SF370.4, resulting in increased sensitivity to killing by UV
irradiation (24). As shown in Fig. 6B, MMR prophage lysogen-
containing strains SF370 and MGAS10394 were at least 100
times more sensitive to killing by 254-nm irradiation than
SF370.4 prophage-free strains NZ131 and JRS1 with a 2-min
exposure. Both strains were analyzed in stationary phase, when
the differences should have been most pronounced due to the
integrative state of prophage SF370.4. Ideally, an isogenic pair
of strains with and without the prophage would be used for
analysis, but attempts to cure the prophage have not been
successful yet (unpublished results). However, comparison of
the previously described genomes of SF370 and MGAS10394
(14) with the recently completed genome sequence of NZ131
(W. M. McShan, J. J. Ferretti, T. Karasawa, A. N. Suvorov, S.
Lin, B. Qin, H. Jia, S. Kenton, F. Najar, H. Wu, J. Scott, B. A.
Roe, and D. J. Savic, submitted for publication) allowed ex-
amination of other genes that might influence the mutation
rate or UV sensitivity (mutS2, ruvB, etc.). No differences in the
gene products between SF370 and the other genome strains
were found, so it is probable that the difference in mutation
rates is due to the presence of prophage SF370.4.
Frequency of MMR prophages in GAS. Related prophages
integrating into the same attB site in mutL appear to be com-
mon genetic elements in S. pyogenes. Thirteen GAS genomes
have been completed so far; 12 of these genomes have been
published (2, 4, 5, 14, 19, 23, 41, 53, 55), and one has not been
FIG. 5. Induction of prophage SF370.4 and expression of MutL in rela-
tion to growth. (A) The induction of prophage SF370.4 occurs near the
beginning of exponential growth. Samples were removed from a culture of
strain SF370 at timed intervals during growth. DNA was extracted and ana-
of sequences specific for attP, attB, and attL relative to the 16S rRNA gene.
The appearance of attB and the disappearance of attL at around 100 min
delay in detection may have reflected the episomal prophage replication
leading to a higher copy number. By 150 min, prophage reintegration had
occurred, leading to the reappearance of attL and the disappearance of attB
and attP. (B) Expression of protein MutL occurred early in logarithmic
growth and diminished as stationary phase was approached, mirroring the
kinetics of phage SF370.4 excision and reintegration. Growth of GAS strain
for cytoplasmic protein analysis were taken when the culture density reached
approximately 0.2, 0.3, 0.4, and 0.6. After extraction, proteins (3 ?g) were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
of 0.2. The relative amount of MutL protein detected decreased to ?5% of
the maximum amount by the time that the cells reached stationary phase. A
of the predicted 95.5-kDa protein during growth of GAS strain SF370. Thus,
in contrast to MutL expression, MutS expression does not decrease as the
cells move from the logarithmic phase to the stationary phase.
6296SCOTT ET AL. J. BACTERIOL.
published (M49 strain NZ131 [submitted]). Prophages closely
related to SF370.4 have been found in six genomes (M2 strain
MGAS10270, M4 strain MGAS10750, M5 strain Manfredo,
M6 strain MGAS10394, and M28 strain MGAS6180). Al-
though each prophage is unique, the prophages share extensive
regions of identity or homology (Fig. 7). Particularly conserved
are the lysogeny and DNA replication regions, and, as is the
case for prophage SF370.4, none of the prophages contain
genes for phage structural proteins or host lysis. Therefore,
they all appear to have the potential to have a molecular
lifestyle similar to that of SF370.4, causing their hosts to switch
between a wild-type phenotype and a mutator phenotype. The
possible exception is serotype M5 strain Manfredo, which has
a deletion of 43 codons within int compared to the other strains
and thus may be defective for integration and excision. The
frequent appearance of SF370.4-like prophages in the genome
strains, all of which were chosen for sequencing because of an
association with severe human disease (2, 4, 5, 14, 19, 41, 53,
55), suggests that these prophages may be associated with hosts
having increased pathogenicity.
One hundred randomly selected clinical isolates having diverse
geographical origins and serotypes were screened for the pres-
ence of SF370.4-like phages by DNA hybridization to the phage
SF370.4 int gene. Twenty of these isolates were positive for the
presence of phage; these strains included serotype M1, M2, M18,
M31, M37, M49, M58, M78, and M123 strains and strains with
nontypeable serotypes (Table 3). The phage integrase hybridiza-
tion probe had no homology to any bacterial DNA in the
related phages in the other genome strains (using BLASTN with
a minimum e value of 10?4). Bacteriophage integrases are very
diverse at the protein level (1, 42), and given further variation at
the DNA level due to the degeneracy of the code, the phage
SF370.4 integrase probe can be expected to be a reasonable
reporter for phages using the mutL attachment site. To confirm
use of the mutL attB attachment site in the probe-positive strains,
PCR was performed using primers to amplify the mutL-integrase
junction (not shown). Therefore, both the results of genome se-
quencing and this brief survey indicate that SF370.4-related
phages are frequent genetic elements in S. pyogenes. The mech-
anism of dissemination of these phages among the various sero-
types remains unknown since none of the phages discovered by
genome sequencing have any identifiable late genes for DNA
packaging, capsids, or host lysis. Given the overall frequency of
overall prophage carriage as shown by the multiple examples
be generalized transduction. However, it is possible that there is
some unknown packaging mechanism, perhaps employing a
helper phage, that could generate infectious particles that could
spread the phages in a manner similar to packaging and dissem-
ination of the Staphylococcus aureus pathogenicity islands (57).
The evidence presented here shows that mutL expression in
S. pyogenes SF370 is controlled by a dynamic process involving
FIG. 6. Mutator phenotype associated with prophages integrated into mutL. (A) Calculated spontaneous rates of mutation (?) (mutation/
generation) to ciprofloxacin resistance of strains SF370 (serotype M1, MMR prophage positive), MGAS10394 (serotype M6, MMR prophage
positive), NZ131 (serotype M49, MMR prophage negative), and JRS1 (serotype M1, MMR prophage negative). Here, prophage carriage refers
to the presence or absence of phage SF370.4 or its close relative found in strain MGAS10394. For each strain, 30 parallel cultures were established
with ?1,000 CFU/culture, grown for 24 h at 37°C, and plated individually on selective media. After 48 to 96 h of incubation, colonies were
enumerated, and mutation rates with 95% confidence limits were calculated using the maximum likelihood estimation technique (31, 54).
Prophage-carrying strains SF370 and MGAS10394 both showed enhanced mutation rates compared to prophage-free strains NZ131 and JRS1.
(B) Enhanced sensitivity of MMR prophage strains SF370 and MGAS10394 to killing by UV irradiation. Strains SF370, MGAS10394, JRS1, and
NZ131 were exposed for 0 to 120 s to 258-nm light (120 ?W/cm2), and 10-fold dilutions were spotted onto an agar plate. Prophage-carrying strains
SF370 and MGAS10394 showed ?100-fold-greater killing than prophage-free strains JRS1 and NZ131, consistent with the inhibition of ruvA
expression. The protocol was performed in a darkened room to prevent photoreactivation.
VOL. 190, 2008 HOST GENE CONTROL BY GAS PHAGES6297
prophage excision and reintegration in response to growth,
activating MMR under conditions under which resources are
not limiting. Further, induction of the prophage may occur at
low population density since the highest levels of MutL expres-
sion were seen in early logarithmic growth phase, presumably
during the initiation of DNA replication. The results presented
here suggest that under the conditions used, there is a mixed
population of bacteria, with individual cells having the proph-
age in either the integrated or episomal state. This situation
indicates that the mutation rate of the population fluctuates
between the wild-type and mutator rates, and the penetrance
of the mutator phenotype could vary by availability of re-
sources, environmental insult, or initiation of DNA replica-
tion. The presence of the prophage in strains SF370 and
MGAS10394 correlates with a mutator phenotype compared
to MMR prophage-free strains NZ131 and JRS1. While it is
possible that other factors could be responsible for the mutator
phenotype in these strains, the simultaneous sensitivity to UV
irradiation in these strains, indicated by the inactivation of
ruvA following prophage integration, supports the hypothesis
that the presence of the prophage is the most direct explana-
tion for the increase in the mutation rate. The creation of
prophage-free isogenic mutants of these strains or the passage
of the prophages to new hosts should allow a definitive answer
to this question to be obtained. Further, the phenotypic
changes resulting from inactivation of the other genes sharing
the same mRNA with mutL and ruvA (tag and lmrP) have not
been explored yet, and the integration of prophage SF370.4
into the MMR operon probably creates a complex mutator
Excision during logarithmic cell division dictates that phage
SF370.4 must be able to replicate its genome to prevent elim-
ination from the population. Some temperate phages, such as
coliphage P1, replicate as a plasmid in the temperate state, and
phage SF370.4 may adopt a similar strategy when it is excised.
The center of the integrated phage SF370.4 genome contains a
region that is highly conserved in all of the related genome
prophages (Fig. 7). This section of the genome contains puta-
tive replicase and primase genes that are homologous to DNA
replication genes from plasmid pSt106 of Streptococcus ther-
FIG. 7. Genomic MMR-converting phages. The prophages from S. pyogenes genome strains SF370, MGAS10394, Manfredo, MGAS10750,
MGAS10270, and MGAS6180 that integrate into the same attB site at the 5? end of mutL are compared. In the upper panel, the insertions,
deletions, and base substitutions of the genomes are compared. The lower panel shows the levels of conservation of the genomes; black indicates
the highest level of similarity. No identifiable capsid, DNA packaging, or lysis genes are present in any phage, but all six prophages contain either
identical or highly conserved integration, control, and replication genes. The locations of several identifiable and conserved genes are indicated
to provide a reference. Scale, 2,000 bp/tick.
TABLE 3. GAS strains positive for SF370.4-related prophages
StrainSerotype OriginAssociated disease
Acute rheumatic fever
Acute rheumatic fever
6298 SCOTT ET AL.J. BACTERIOL.
mophilus (17). The lack of identifiable DNA packaging, struc-
tural, or lytic genes prevents the phage from entering a lytic
cycle, and so replication of the circular phage genome as an
autonomous element seems likely.
The excision of phage SF370.4 during exponential growth
may occur by inactivation of its predicted repressor by prote-
olysis or by allosteric interaction with some protein or metab-
olite expressed by rapidly dividing streptococci. Phage SF370.4
thus appears to have evolved to function as a genetic switch to
control a mutator phenotype, protecting rapidly growing cells
from unwanted genetic changes while allowing the accumula-
tion of random mutations, some of which might be adaptive
when resources become limiting, or from the acquisition of
new genetic material by horizontal transfer (37). Further, mi-
tomycin C treatment of strain SF370 stimulated the excision of
the prophage following the induction of an SOS-like response.
Such a response in S. pyogenes presumably leads to an in-
creased mutation rate through the induction of error-prone
DNA replication, as seen in E. coli (45, 56), and thus the
restoration of MMR following prophage induction by mitomy-
cin C may counteract this increase in the mutation rate. It is
unclear whether this balancing of error-prone DNA replication
with restoration of normal MMR activity is the result of direct
selection or is a circumstantial by-product of the evolution of
prophage SF370.4. That is, the induction of prophage by the
SOS response may be a genetic remnant from an ancestral
phage that responded to cellular damage like a typical temper-
ate prophage and entered the lytic cycle to escape from a
damaged host. In S. pyogenes, there may be some common
cellular signal during early logarithmic growth and induction of
the SOS pathway. The induction of the SOS response in E. coli
is controlled by induction of the RecA coprotease activity
leading to the autocatalytic cleavage of the LexA repressor. In
gram-positive bacteria, lexA equivalents have been found in
some species, such as Bacillus subtilis, but a gene homolog has
not been identified in the streptococci. A recent report iden-
tified a gene cassette that mediates the SOS response in Strep-
tococcus uberis (58). One product of this cassette (HdiR) ap-
pears to function as a LexA equivalent in this species, and
homologs of HdiR have been found in several other strepto-
coccal species, including one of the genome strains of S. pyo-
genes (MGAS10394). Closer examination showed that this
HdiR homolog is encoded on one of the temperate bacterio-
phages harbored by MGAS10394, and thus, while HdiR may
play some role in the SOS response in S. pyogenes, it seems
likely that a true LexA equivalent would be common to all
GAS genomes rather than carried sporadically on a mobile
element. A number of conserved hypothetical proteins con-
taining predicted helix-turn-helix motifs are encoded in all of
the sequenced S. pyogenes genomes, and it is possible that one
of these proteins may function as the LexA equivalent. Clearly,
this is a topic that needs more study in GAS.
A recent survey of the endogenous prophages found in the
sequenced bacterial genomes showed that 41% of these phages
are integrated into tRNA and transfer-messenger RNA genes,
31% are integrated into intergenic regions, and 28% are inte-
grated into open reading frames for genes (16). Prophage
site-specific integration occurs via a duplication between the
phage and the host chromosome, and when integration occurs
at gene targets, the duplication usually occurs at the 3? end of
the host target gene, leaving the target gene intact (via dupli-
cation); in at least one case, the duplication provides an alter-
native carboxy terminus for the specified protein (8, 9). By
contrast, phage SF370.4 integrates into the 5? end of mutL,
blocking its transcription. Integration at the 5? end of a host
gene has also been found in another S. pyogenes SF370 proph-
age (phage SF370.1 integrates at the 5? end of a dipeptidase
gene) (38), and prophages acting as regulatory elements may
be not uncommon in S. pyogenes. For example, in the strains
with published genomes, prophages are integrated in the 5?
regions of the gamma-glutamyl kinase gene proB, recX, a
HAD-like hydrolase gene, and a gene encoding a conserved
hypothetical protein (2, 5, 41, 53).
Bioinformatic analysis suggests that several genes down-
stream from mutL can be predicted to be additional compo-
nents of the polycistronic mRNA containing mutS and mutL;
these genes are Spy2120 (encoding a predicted integral mem-
brane protein related to the Lactococcus lactis multidrug ex-
porter encoded by lmrP), ruvA (encoding a Holliday junction
helicase subunit), and tag (encoding DNA-3-methyladenine
glycosidase I). The comX-dependent competence damage pro-
tein gene cinA and recA follow, completing a remarkable ge-
netic group of recombination and repair genes. The same gene
cluster is present in the genomes of group B streptococci and
Streptococcus mutans, although the lmrP homolog is missing in
S. mutans. This entire group of genes is responsible for a range
of DNA repair functions, and therefore, in addition to MMR,
the presence of phage SF370.4 may alter the expression of
several DNA repair systems. For example, ruvA mutants have
increased sensitivity to mutagens and an overall increased mu-
tation rate (33), while in tag mutants the cell’s sensitivity to
alkylating mutagens is increased (60). Finally, although this
operon is very similar in group B streptococci and S. mutans,
the specific attB DNA sequence is unique to GAS, and so it is
unlikely that prophage SF370.4 could integrate into these for-
eign species. This does not rule out the possible presence of
equivalent prophages in the other streptococcal species, al-
though none have been identified.
The endogenous bacteriophages of S. pyogenes are often
vectors for toxin genes and other virulence factors, but the
control of host gene expression (MMR) by a bacteriophage in
response to the bacterial growth state and via cycles of re-
peated excision and integration is completely novel. A variety
of bacterial stress responses that include mechanisms of induc-
ing spontaneous mutations in slowly growing or nongrowing
cells have been described as “adaptive mutations” (15). Some
adaptive responses in E. coli have been shown to be influenced
by environmental conditions (7), and the MutS?MutL?phe-
notype that results following integration of phage SF370.4 is
strikingly similar to the limitation of MutL during stationary
phase observed by Harris et al. in E. coli (22), suggesting that
such a phenotype may be generally advantageous in situations
where resources are limited. The nature of this advantage is
unclear, however, since constitutive expression of MutS is en-
ergetically unfavorable. In the case of S. pyogenes, this may be
due to lack of optimization of the phage integration site due to
its relatively recent evolutionary appearance, or alternatively,
the constitutive expression of MutS may contribute to main-
taining some level of discrimination for RecA-mediated home-
ologous recombination between divergent DNA sequences
VOL. 190, 2008 HOST GENE CONTROL BY GAS PHAGES6299
(59). It may well be that the observed system for control of
MMR by prophage SF370.4 is indeed close to optimal, balanc-
ing the different selective pressures on the various repair sys-
tems coordinated by this element.
The frequent occurrence of MMR defects in natural popu-
lations of bacteria indicates that the benefit of increased
mutability or the potential for horizontal genetic transfer (37)
has a sufficiently high selective value to balance the risk of
unfavorable mutations. Further, a bacterial species may un-
dergo successive rounds of loss and regain of MMR function.
The mutS and mutL genes from natural populations of E. coli,
for example, exhibit high sequence mosaicism derived from
diverse phylogenetic sources, while other housekeeping genes
do not (11). This mosaicism was interpreted as having arisen
from recurrent losses in MMR function, followed by reacqui-
sition by horizontal transfer from wild-type strains. The phage-
controlled system in S. pyogenes represents a sophisticated
molecular alternative that does not require rare spontaneous
mutations to inactivate MMR or the acquisition of exogenous
DNA to reinstate the system. Indeed, the conditional expres-
sion of MMR in S. pyogenes fulfills the prediction of LeClerc et
al. that “the ultimate pathogen would possess an elevated mu-
tation rate that is transient (or conditional), providing genetic
variation during the first few hours when the pathogen must
survive, invade, and colonize its host” (29). A conditional mu-
tator phenotype allows a bacterium to accumulate mutations
that may provide an advantage during periods of stress, com-
petition from other strains or species of bacteria, or limited
resources. Conversely, a nonconditional mutator would even-
tually accumulate too many mutations that would prove to be
deleterious to the cell. The ability to switch from mutator to
nonmutator allows a cell to take advantage of both situations,
ensuring its survival in the population. The specific selection
advantage that MMR-converting prophages confer on their
hosts and under what circumstances this occurs remain to be
discovered, as do their mechanisms of dissemination through
streptococcal populations. However, the widespread presence
of prophages related to SF370.4 that are integrated into mutL
in S. pyogenes strains suggests that these elements may confer
a significant survival advantage to these strains.
We thank Gorana Savic and Mona Balkis for expert technical help
and J. Iandolo, J. J. Ferretti, and D. J. Savic for insightful discussions.
J.S. was supported in part by a predoctoral fellowship award from
the American Foundation for Pharmaceutical Education. This study
was made possible by NIH grant P20 RR016478 from the INBRE
Program of the National Center for Research Resources, by NIH grant
P20 RR015564, and by NIH grant 1R15A1072718 to W.M.M.
The contents of this paper do not necessarily represent the official
views of NIH.
1. Argos, P., A. Landy, K. Abremski, J. B. Egan, E. Haggard-Ljungquist, R. H.
Hoess, M. L. Kahn, B. Kalionis, S. V. L. Narayana, L. S. Pierson III, N.
Sternberg, and J. M. Leong. 1986. The integrase family of site-specific
recombinases: regional similarities and global diversity. EMBO J. 5:433–440.
2. Banks, D. J., S. F. Porcella, K. D. Barbian, S. B. Beres, L. E. Philips, J. M.
Voyich, F. R. DeLeo, J. M. Martin, G. A. Somerville, and J. M. Musser. 2004.
Progress toward characterization of the group A streptococcus metagenome:
complete genome sequence of a macrolide-resistant serotype M6 strain.
J. Infect. Dis. 190:727–738.
3. Beall, B., R. Facklam, and T. Thompson. 1996. Sequencing emm-specific
PCR products for routine and accurate typing of group A streptococci.
J. Clin. Microbiol. 34:953–958.
4. Beres, S. B., E. W. Richter, M. J. Nagiec, P. Sumby, S. F. Porcella, F. R.
Deleo, and J. M. Musser. 2006. Molecular genetic anatomy of inter- and
intraserotype variation in the human bacterial pathogen group A strepto-
coccus. Proc. Natl. Acad. Sci. USA 103:7059–7064.
5. Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella,
M. Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, D. S. Campbell, T. M.
Smith, J. K. McCormick, D. Y. Leung, P. M. Schlievert, and J. M. Musser.
2002. Genome sequence of a serotype M3 strain of group A streptococcus:
phage-encoded toxins, the high-virulence phenotype, and clone emergence.
Proc. Natl. Acad. Sci. USA 99:10078–10083.
6. Bishai, W. R., and J. R. Murphy. 1988. Bacteriophage gene products that
cause human disease, p. 683–724. In R. Calendar (ed.), The bacteriophages,
vol. 2. Plenum Press, New York, NY.
7. Bjedov, I., O. Tenaillon, B. Ge `rard, V. Souza, E. Denamur, M. Radman, F.
Taddei, and I. Matic. 2003. Stress-induced mutagenesis in bacteria. Science
8. Campbell, A., S. J. Schneider, and B. Song. 1992. Lambdoid phages as
elements of bacterial genomes. Genetica 86:259–267.
9. Campbell, A. M. 1992. Chromosomal insertion sites for phages and plasmids.
J. Bacteriol. 174:7495–7499.
10. Canchaya, C., F. Desiere, W. M. McShan, J. J. Ferretti, J. Parkhill, and H.
Brussow. 2002. Genome analysis of an inducible prophage and prophage
remnants integrated in the Streptococcus pyogenes strain SF370. Virology
11. Denamur, E., G. Lecointre, P. Darlu, O. Tenaillon, C. Acquaviva, C. Sayada,
I. Sunjevaric, R. Rothstein, J. Elion, F. Taddei, M. Radman, and I. Matic.
2000. Evolutionary implications of the frequent horizontal transfer of mis-
match repair genes. Cell 103:711–721.
12. Desiere, F., W. M. McShan, D. van Sinderen, J. J. Ferretti, and H. Brussow.
2001. Comparative genomics reveals close genetic relationships between
phages from dairy bacteria and pathogenic streptococci: evolutionary impli-
cations for prophage-host interactions. Virology 288:325–341.
13. Drake, J. W., B. Charlesworth, D. Charlesworth, and J. F. Crow. 1998. Rates
of spontaneous mutation. Genetics 148:1667–1686.
14. Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C.
Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, H. Lai, S. Lin, Y. Qian, H. G.
Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton,
B. A. Roe, and R. McLaughlin. 2001. Complete genome sequence of an M1
strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658–4663.
15. Foster, P. L. 1999. Mechanisms of stationary phase mutation: a decade of
adaptive mutation. Annu. Rev. Genet. 33:57–88.
16. Fouts, D. E. 2006. Phage_Finder: automated identification and classification
of prophage regions in complete bacterial genome sequences. Nucleic Acids
17. Geis, A., H. A. El Demerdash, and K. J. Heller. 2003. Sequence analysis and
characterization of plasmids from Streptococcus thermophilus. Plasmid 50:
18. Giraud, A., M. Radman, I. Matic, and F. Taddei. 2001. The rise and fall of
mutator bacteria. Curr. Opin. Microbiol. 4:582–585.
19. Green, N. M., S. Zhang, S. F. Porcella, M. J. Nagiec, K. D. Barbian, S. B.
Beres, R. B. Lefebvre, and J. M. Musser. 2005. Genome sequence of a
serotype M28 strain of group A streptococcus: potential new insights into
puerperal sepsis and bacterial disease specificity. J. Infect. Dis. 192:760–770.
20. Harfe, B. D., and S. Jinks-Robertson. 2000. DNA mismatch repair and
genetic instability. Annu. Rev. Genet. 34:359–399.
21. Harlow, E., and D. Lane. 1999. Using antibodies: a laboratory manual. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
22. Harris, R. S., G. Feng, K. J. Ross, R. Sidhu, C. Thulin, S. Longerich, S. K.
Szigety, M. E. Winkler, and S. M. Rosenberg. 1997. Mismatch repair protein
MutL becomes limiting during stationary-phase mutation. Genes Dev. 11:
23. Holden, M. T., A. Scott, I. Cherevach, T. Chillingworth, C. Churcher, A.
Cronin, L. Dowd, T. Feltwell, N. Hamlin, S. Holroyd, K. Jagels, S. Moule, K.
Mungall, M. A. Quail, C. Price, E. Rabbinowitsch, S. Sharp, J. Skelton, S.
Whitehead, B. G. Barrell, M. Kehoe, and J. Parkhill. 2007. Complete ge-
nome of acute rheumatic fever-associated serotype M5 Streptococcus pyo-
genes strain manfredo. J. Bacteriol. 189:1473–1477.
24. Iwasaki, H., T. Shiba, A. Nakata, and H. Shinagawa. 1989. Involvement in
DNA repair of the ruvA gene of Escherichia coli. Mol. Gen. Genet. 219:328–
25. Kadyrov, F. A., L. Dzantiev, N. Constantin, and P. Modrich. 2006. Endonu-
cleolytic function of MutLalpha in human mismatch repair. Cell 126:297–
26. Kobs, G. 1995. pGEM-T vector: cloning of modified blunt-ended DNA
fragments. Promega Notes 55:28.
27. Lau, P. J., and R. D. Kolodner. 2003. Transfer of the MSH2.MSH6 complex
from proliferating cell nuclear antigen to mispaired bases in DNA. J. Biol.
28. LeClerc, J. E., and T. A. Cebula. 2000. Pseudomonas survival strategies in
cystic fibrosis. Science 289:391–392.
29. LeClerc, J. E., B. Li, W. L. Payne, and T. A. Cebula. 1996. High mutation
6300SCOTT ET AL. J. BACTERIOL.
frequencies among Escherichia coli and Salmonella pathogens. Science 274: Download full-text
30. Luria, S. E., and M. Delbru ¨ck. 1943. Mutations of bacteria from virus
sensitivity to virus resistance. Genetics 28:491–511.
31. Ma, W., G. V. H. Sandri, and S. Sarkar. 1992. Analysis of the Luria-Delbru ¨ck
distribution using discrete convolution powers. J. Appl. Prob. 29:255–267.
32. Malke, H., K. Steiner, W. M. McShan, and J. J. Ferretti. 2006. Linking the
nutritional status of Streptococcus pyogenes to alteration of transcriptional
gene expression: the action of CodY and RelA. Int. J. Med. Microbiol.
33. Mandal, T. N., A. A. Mahdi, G. J. Sharples, and R. G. Lloyd. 1993. Reso-
lution of Holliday intermediates in recombination and DNA repair: indirect
suppression of ruvA, ruvB, and ruvC mutations. J. Bacteriol. 175:4325–4334.
34. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a labo-
ratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
35. Marti, T. M., C. Kunz, and O. Fleck. 2002. DNA mismatch repair and
mutation avoidance pathways. J. Cell Physiol. 191:28–41.
36. Matic, I., M. Radman, F. Taddei, B. Picard, C. Doit, E. Bingen, E. Denamur,
and J. Elion. 1997. Highly variable mutation rates in commensal and patho-
genic Escherichia coli. Science 277:1833–1834.
37. Matic, I., C. Rayssiguier, and M. Radman. 1995. Interspecies gene exchange
in bacteria: the role of SOS and mismatch repair systems in evolution of
species. Cell 80:507–515.
38. McShan, W. M., and J. J. Ferretti. 2006. Bacteriophages and the host
phenotype, p. 229–250. In S. McGrath (ed.), Bacteriophages: genetics and
molecular biology. Horizon Scientific Press, Hethersett, Norwich, United
39. McShan, W. M., R. E. McLaughlin, A. Nordstrand, and J. J. Ferretti. 1998.
Vectors containing streptococcal bacteriophage integrases for site-specific
gene insertion. Methods Cell Sci. 20:51–57.
40. McShan, W. M., Y.-F. Tang, and J. J. Ferretti. 1997. Bacteriophage T12 of
Streptococcus pyogenes integrates into the gene for a serine tRNA. Mol.
41. Nakagawa, I., K. Kurokawa, A. Yamashita, M. Nakata, Y. Tomiyasu, N.
Okahashi, S. Kawabata, K. Yamazaki, T. Shiba, T. Yasunaga, H. Hayashi,
M. Hattori, and S. Hamada. 2003. Genome sequence of an M3 strain of
Streptococcus pyogenes reveals a large-scale genomic rearrangement in inva-
sive strains and new insights into phage evolution. Genome Res. 13:1042–
42. Nunes-Duby, S. E., H. J. Kwon, R. S. Tirumalai, T. Ellenberger, and A.
Landy. 1998. Similarities and differences among 105 members of the Int
family of site-specific recombinases. Nucleic Acids Res. 26:391–406.
43. Oliver, A., R. Canton, P. Campo, F. Baquero, and J. Blazquez. 2000. High
frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung
infection. Science 288:1251–1254.
44. Pitcher, D. G., N. A. Saunders, and R. J. Owen. 1989. Rapid extraction of
bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Micro.
45. Radman, M. 1975. SOS repair hypothesis: phenomenology of an inducible
DNA repair which is accompanied by mutagenesis. Basic Life Sci. 5A:355–
46. Reese, M. G. 2001. Application of a time-delay neural network to pro-
moter annotation in the Drosophila melanogaster genome. Comput.
47. Richardson, A. R., Z. Yu, T. Popovic, and I. Stojiljkovic. 2002. Mutator
clones of Neisseria meningitidis in epidemic serogroup A disease. Proc. Natl.
Acad. Sci. USA 99:6103–6107.
48. Rosche, W. A., and P. L. Foster. 2000. Determining mutation rates in bac-
terial populations. Methods 20:4–17.
49. Rosenberg, S. M. 2001. Evolving responsively: adaptive mutation. Nat. Rev.
50. Shaver, A. C., and P. D. Sniegowski. 2003. Spontaneously arising mutL
mutators in evolving Escherichia coli populations are the result of changes in
repeat length. J. Bacteriol. 185:6076–6082.
51. Shell, S. S., C. D. Putnam, and R. D. Kolodner. 2007. The N terminus of
Saccharomyces cerevisiae Msh6 is an unstructured tether to PCNA. Mol. Cell
52. Simon, D., and J. J. Ferretti. 1991. Electrotransformation of Streptococcus
pyogenes with plasmid and linear DNA. FEMS Microbiol. Lett. 82:219–224.
53. Smoot, J. C., K. D. Barbian, J. J. Van Gompel, L. M. Smoot, M. S. Chaussee,
G. L. Sylva, D. E. Sturdevant, S. M. Ricklefs, S. F. Porcella, L. D. Parkins,
S. B. Beres, D. S. Campbell, T. M. Smith, Q. Zhang, V. Kapur, J. A. Daly,
L. G. Veasy, and J. M. Musser. 2002. Genome sequence and comparative
microarray analysis of serotype M18 group A streptococcus strains associ-
ated with acute rheumatic fever outbreaks. Proc. Natl. Acad. Sci. USA
54. Stewart, F. M. 1994. Fluctuation tests: how reliable are the estimates of
mutation rates? Genetics 137:1139–1146.
55. Sumby, P., S. F. Porcella, A. G. Madrigal, K. D. Barbian, K. Virtaneva, S. M.
Ricklefs, D. E. Sturdevant, M. R. Graham, J. Vuopio-Varkila, N. P. Hoe, and
J. M. Musser. 2005. Evolutionary origin and emergence of a highly successful
clone of serotype M1 group A streptococcus involved in multiple horizontal
gene transfer events. J. Infect. Dis. 192:771–782.
56. Sutton, M. D., B. T. Smith, V. G. Godoy, and G. C. Walker. 2000. The SOS
response: recent insights into umuDC-dependent mutagenesis and DNA
damage tolerance. Annu. Rev. Genet. 34:479–497.
57. Tormo, M. A., M. D. Ferrer, E. Maiques, C. Ubeda, L. Selva, I. Lasa, J. J.
Calvete, R. P. Novick, and J. R. Penades. 2008. Staphylococcus aureus patho-
genicity island DNA is packaged in particles composed of phage proteins. J.
58. Varhimo, E., K. Savijoki, J. Jalava, O. P. Kuipers, and P. Varmanen. 2007.
Identification of a novel streptococcal gene cassette mediating SOS mu-
tagenesis in Streptococcus uberis. J. Bacteriol. 189:5210–5222.
59. Worth, L., Jr., S. Clark, M. Radman, and P. Modrich. 1994. Mismatch repair
proteins MutS and MutL inhibit RecA-catalyzed strand transfer between
diverged DNAs. Proc. Natl. Acad. Sci. USA 91:3238–3241.
60. Wyatt, M. D., J. M. Allan, A. Y. Lau, T. E. Ellenberger, and L. D. Samson.
1999. 3-Methyladenine DNA glycosylases: structure, function, and biological
importance. Bioessays 21:668–676.
VOL. 190, 2008HOST GENE CONTROL BY GAS PHAGES 6301