Phage-associated mutator phenotype in group A streptococcus

Abstract

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 downstream 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(-7) to 10(-8) mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10(-9) to 10(-10) mutation/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.

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.

FIG. 2.

Expression of mutL is growth dependent in GAS strain SF370. (A) cDNA from SF370 cells was synthesized from RNA isolated 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 dividing 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 chromosomal DNA.

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.

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 isolated 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.

FIG. 5.

Induction of prophage SF370.4 and expression of MutL in relation 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 analyzed by quantitative real-time PCR to determine the presence and quantities 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 were exactly coordinated, while attP was detectable after a short lag time. This 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 SF370 was monitored by determining the absorbance at 600 nm, and samples 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 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nylon membrane for hybridization to polyclonal anti-MutL or anti-MutS antibodies. The relative amounts of the 74.3-kDa MutL band were measured by densitometry and normalized to the sample harvested at an A₆₀₀ 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 similar analysis of MutS expression showed that there was constant expression 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.

FIG. 6.

Mutator phenotype associated with prophages integrated into mutL. (A) Calculated spontaneous rates of mutation (μ) (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 μW/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.

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.