Phage-Like Streptococcus pyogenes Chromosomal Islands (SpyCI) and Mutator Phenotypes: Control by Growth State and Rescue by a SpyCI-Encoded Promoter

Abstract

We recently showed that a prophage-like Streptococcus pyogenes chromosomal island (SpyCI) controls DNA mismatch repair and other repair functions in M1 genome strain SF370 by dynamic excision and reintegration into the 5' end of mutL in response to growth, causing the cell to alternate between a wild type and mutator phenotype. Nine of the 16 completed S. pyogenes genomes contain related SpyCI integrated into the identical attachment site in mutL, and in this study we examined a number of these strains to determine whether they also had a mutator phenotype as in SF370. With the exception of M5 genome strain Manfredo, all demonstrated a mutator phenotype as compared to SpyCI-free strain NZ131. The integrase gene (int) in the SpyCIM5 contains a deletion that rendered it inactive, and this deletion predicts that Manfredo would have a pronounced mutator phenotype. Remarkably, this was found not to be the case, but rather a cryptic promoter within the int ORF was identified that ensured constitutive expression of mutL and the downstream genes encoded on the same mRNA, providing a striking example of rescue of gene function following decay of a mobile genetic element. The frequent occurrence of SpyCI in the group A streptococci may facilitate bacterial survival by conferring an inducible mutator phenotype that promotes adaptation in the face of environmental challenges or host immunity.

Keywords: DNA mismatch repair; SpyCI; Streptococcus pyogenes; chromosomal islands; group A streptococcus; mutator phenotype; prophages.

Figure 1

MMR regulation by SpyCIM1 in strain SF370. Prophage-like chromosomal island SpyCIM1 integration separates mutL, lmrP, ruvA, and tag from the promoter upstream of mutS, preventing expression of these genes and leading to a mutator phenotype in strain SF370 (Scott et al., 2008). mutS is constitutively expressed from mRNA_A, which is truncated by the presence of the SpyCI. When cells enter early logarithmic growth phase, approximately at the time of initiation of DNA replication, the SpyCI excises from the host chromosome and restores transcription of the polycistronic message from mutS to tag (mRNA_B). The excised phage circularizes and replicates in the host cell as an episome. As cell densities increase, the phage genome integrates into attB located at the 5′ end of mutL, returning the cell to the mutator phenotype. Large, unfilled arrows indicate transcriptionally inactive genes of the MMR operon (mutS, mutL, lmrP, ruvA, and tag), while transcriptionally active ones are large arrows filled in black. Small, filled arrows indicate the SpyCI ORFs; except for int (Scott et al., 2008), the transcriptional patterns of the SpyCI genes are unknown at this time.

Figure 2

Mutation rate and UV sensitivity of S. pyogenes strains. The grouping of the S. pyogenes strains by mutation rate (μ) is shown as in Table 3. Next to the calculated mutation rate, the sensitivity to UV irradiation is shown for each strain. UV exposure time in seconds is indicated at the top and all strains were plated at the same dilution. All experiments were performed at least three times for each strain.

Figure 3

Alignment of the promoter and 5′ ORF of the integrase genes from SpyCIM1 and SpyCIM5. The sequence alignment of the promoters and 5′ portion of the int coding regions of SpyCIM1 and SpyCIM5 is shown (labeled M1 and M5, respectively). The predicted promoter for SpyCIM1 int is double underlined, and the single line indicates the probable beginning of the coding region. The SpyCIM5 promoter has significantly diverged from SpyCIM1 and the other SpyCI-like chromosomal islands. The alignment shows that 128 bp have been deleted from the SpyCIM5 int, resulting in not only a loss of genetic information but also the formation of a frameshift. Thus, computational analysis predicts that the Manfredo SpyCIM5 integrase is a non-functional pseudogene.

Figure 4

Expression of mutL in Manfredo is independent of SpyCIM5 excision. (A) Expression of both mutS and mutL in strain Manfredo occurs without SpyCIM5 excision. PCR was used to amplify cDNA from strain Manfredo logarithmically growing cells or after overnight (ON) incubation (the absorbance at A₆₀₀ when the cells were harvested is indicated above each sample). Both mutS and mutL were constitutively expressed while the SpyCIM5-associated sequences of attP and attB were never detected in Manfredo, indicating that SpyCIM5 excision does not normally occur in response to growth. (B) The SpyCIM1 integrase from SF370 was expressed in Manfredo by introduction of plasmid pWM448 containing the SpyCIM1 integrase ORF under control of the strong CAMP promoter. Gene int expression is absent from wild type Manfredo, and the target of the qRT-PCR amplification was the deleted region of the integrase ORF not present in Manfredo, confirming that expression was from the cloned SpyCIM1 gene. (C) The SpyCIM1 integrase mediates excision of SpyCIM5 in Manfredo. DNA was isolated from early logarithmic grown cells from Manfredo, SF370, or Manfredo (pWM448). PCR specific for attP, which is present only when the prophage-like element has excised from the mutS-mutL junction, produces no product in wild type Manfredo but appears after introduction of pWM448. Mitomycin C (MC), while stimulating the excision of the SpyCIM1 in strain SF370, inhibits the formation of attP in Manfredo (pWM448), suggesting inhibition of CAMP promoter activity.

Figure 5

Manfredo mutLmRNA is constitutively expressed under normal growth. Strains Manfredo and SF370 were grown to increasing cell densities [A₆₀₀ = 0.2–0.8 or overnight (ON)]. The mRNA was harvested from the cells, converted into cDNA, and the mutL region amplified using primers positioned as shown. For mitomycin C induction, cells were grown to A₆₀₀ = 0.2 before mitomycin C was added. In SF370, mutL mRNA was transcribed only early in logarithmic phase and then quickly diminished, following the pattern of SpyCIM1 excision and re-integration (Scott et al., 2008). Mitomycin C induction briefly increased the presence of this mRNA. Strain Manfredo, by contrast, expressed this mRNA constitutively throughout logarithmic growth, and small amounts were detectable still after ON incubation. Mitomycin C treatment inhibited expression of the mRNA during logarithmic growth but greatly enhanced its presence after ON incubation.

Figure 6

Mapping of the Manfredo mutL transcription to a promoter within the int pseudogene. Primer extension of the SpyCIM5 int cDNA revealed the presence of a cryptic promoter within the int pseudogene. The fluorescent primer (arrow labeled “F”) extended to an mRNA start position (35 nucleotides upstream) that exactly matched a predicted promoter (Reese, 2001) region (in capitals) and mRNA start (large open “C”) within the pseudogene ORF. The large arrow indicates the initiation and direction of the predicted mRNA. The encoded protein sequence of int is shown below for reference. No mRNA initiating from the region upstream of the integrase ORF could be detected. The X-axis of the figure corresponds to length of product in basepairs. The left Y-axis shows the relative fluorescence units of the sample peaks and right Y-axis shows the relative fluorescence units of the GeneScan 600 LIZ size standards. The small vertical arrow indicates the peak corresponding to the product of primer extension.

Figure 7

Transcription from the SpyCIM5 int pseudogene is dependent upon host background. Gene chimeras were made that fused increasing portions of the SpyCIM5 int pseudogene and its upstream promoter to the green fluorescent protein gene (gfp); the constructs are shown below the micrographs. The downstream region of int following the 128 bp deletion is hatched in the figure. An encircled “P” indicates the predicted promoters (Reese, 2001). P1 is immediately upstream of the beginning of the int ORF, while P2 is positioned within the pseudogene and maps to the beginning of transcription (Figure 6). The plasmids with these constructs were introduced into strains NZ131 and Manfredo (SpyCIM5+). In E. coli JM109, no GFP fluorescence could be detected for any plasmid (not shown).

Figure 8

Phylogenetic analysis of the repressor-operator regions of SpyCI. (A) The chromosomal DNA region of the SpyCI, containing the predicted repressors, operators, and antirepressors, were aligned using Geneious (Drummond et al., 2012), and a phylogenetic tree was constructed from the alignment by neighbor-network analysis (Huson, 1998). Below the tree is the sequence view of the alignment of the operator regions; variants from the consensus are colored by nucleotide. The SpyCI regions analyzed were from strains SF370 (M1), MGAS10270 (M2), MGAS10750 (M4), Manfredo (M5), MGAS10394 (M6), MGAS6180 (M28), Alab49 (M53), MGAS15252 (M59), and MGAS1882 (M59.1). (B) Shown is the identity matrix for the DNA alignment of the SpyCI repressor-operator regions.