Fig 1.
Genetic arrangement in the Spn556II locus.
A. The three functional DNA methylation motifs recognized by the three R-M systems in strain ST556 according to our previous study [35]. The methylated bases are highlighted with red characters. B. The gene order and other features in the Spn556 locus of three pneumococcal strains. The orientations of the coding sequences are indicated by arrowheads. Each hsdS segment with identical or nearly identical sequences between the two of three strains (ST556, TIGR4 and D39) is indicated with a dashed line. Drawing is not to scale.
Fig 2.
Detection of DNA rearrangements in the Spn556II locus by PCR.
A. Positions of the primers used for PCR amplification in the Spn556II locus of ST556. The predicted rho-independent transcription terminator is indicated by a hairpin. The primers used in (B) and (C) are indicated by small arrows. The JC-replaced region in TH6501 is marked with dashed lines. B. Amplification of the Spn556II locus in ST556 and isogenic mutant TH5792 lacking the entire Spn556II locus with primers P1 and P11. The PCR mixtures were processed by DNA electrophoresis and stained by the Goldview dye (Yeasen, Beijing, China). The PCR products that were absent in the mutant strains are marked with asterisks (*). The sizes of the DNA markers are indicated in kilobases. C. Detection of DNA rearrangements in the hsdS regions of the Spn556II locus. PCR reactions were performed with the genomic DNA of ST556 using the same set of primer pairs indicated at the top of each lane, and marked as in (B). D. Same as in (C) except for using the genomic DNA from the ST556 derivative lacking hsdSA strain (TH6501).
Fig 3.
DNA configurations generated by inversions and excisions in the hsdS genes of the Spn556II locus.
DNA configurations derived by three inversions from form S1 (A), S3 (B), or S4 (C). Each gene and its orientation are indicated with a large arrow. The inverted repeats (IRs) in the hsdS genes are represented by yellow (IR1), black (IR2), and white (IR3) arrowheads. The inversion sites are indicated by dashed lines. Each DNA configuration is assigned with an S number. DNA configurations generated by excisions between hsdSA and hsdSC demarcated by direct repeat sets 1 (DR1, red arrows) and (DR2, purple arrows) (D). Excisions mediated by the DR1 and DR2 yields hsdSA variant S9 and S7. Further inversion in S7 generates S8. S7 may also generate variant S9 by further DNA excision between DR1.1 and DR1.2.
Fig 4.
The pneumococcal genomic DNA motifs methylated by the hsdSA allelic variants.
The DNA methylation sequences in the genomes of strain ST556 (WT) or its derivatives (A1-A9) each possessing one of the 9 hsdSA alleles were detected by SMRT sequencing. The methylation motifs recognized by Spn556I and Spn556III were also detected in all of the strains but are not shown here for the sake of space; methylated m6A bases are indicated with red characters. R = A or G, Y = T or C.
Fig 5.
Requirement of psrA for the DNA inversion between the inverted repeats (IR1.1 and IR1.2).
Amplification of the Spn556II hsdS region in ST556 (upper panel)(A), isogenic mutant lacking psrA (TH6012, middle panel)(B), or complemented TH6012 with the wild type psrA gene (TH6659, lower panel)(C) with primer pairs indicated at the top of each lane as in Fig 2C. The major band absent in TH6012 is marked with an asterisk (*).
Fig 6.
Colony morphology of six S. pneumoniae strains and their derivatives each carrying an invariable hsdSA allele.
Pneumococcal strains ST556 (19F), P384 (6A), TH2901 (6B), TH2835 (14), TH2886 (23F), and ST877 (35B) were grown on TSA plates supplemented with catalase; the colonies photographed under a dissection microscope as described in reference [33]. The Spn556II hsdSA genotype and corresponding profile of chromosomal methylation in each strain are marked at the top of each column. Strain designation is indicated at the bottom of each photograph. The representative colonies with opaque and transparent appearance in the wild types are highlighted with blue and red arrowheads, respectively.
Fig 7.
Colony morphology of strain D39 and its derivatives each carrying one of the 6 invariable hsdSA alleles.
The hsdSA allele-locked derivatives of strain D39 (WT, type 2) and isogenic capsule switch variant producing a type-19F capsule (D39cps19F) were grown for 16 or 24 hours and processed as described in Fig 6. The name of each strain is listed at the bottom of each photograph; the hsdSA allele genotype marked at the bottom and left side of each row. The representative colonies with opaque and transparent appearance in the parental strains are indicated with blue and red arrowheads, respectively.
Fig 8.
Epigenetic-driven phase variation in unencapsulated pneumococci.
The unencapsulated mutants of ST556 (parental strain TH8160) and D39 (parental strain TH7901) were used to generate single hsdSA allele-locked strains by counter selection as described in Fig 6. Colonies of each strain were prepared and photographed as in Fig 6. The strains in each row shared the same hsdSA allele (A1, A2, or A3). The strains in each column were constructed from the same unencapsulated parental strain (top of each column). The name of each strain is listed at the bottom of each photograph.
Fig 9.
Essential roles of the DNA methyltransferase activity in defining pneumococcal colony opacity.
A. Necessity and sufficiency of hsdM and hsdSA in defining pneumococcal colony opacity. Isogenic mutants each with an unmarked deletion in the coding region of hsdR, hsdSA, hsdRM or Spn556II were constructed in the Spn556II locus of ST556. Colonies of each strain were prepared, photographed, and marked as in Fig 6. B. Requirement of the DNA methyltransferase catalytic activity in defining pneumococcal colony opacity. Strain TH6113 lacking the entire coding region of hsdR (MYY572) and hsdM (MYY571) (producing transparent colonies) was complemented with either the wild type hsdM gene (MYY571) or its catalytically inactive mutant with an E228A or N255A point mutation. Colonies are presented as in (A).
Fig 10.
Allele A1 of hsdSA dictates the opaque colony phenotype of S. pneumoniae.
Chromosomal co-expression of hsdSA alleles A1-A3 in the bgaA locus of the ST556 derivatives carrying the locked hsdSA allele A2 (A), A3 (B) or A1 (C). A modified Janus cassette (JC1) was used to replace partially the coding sequence of bgaA in each parent strain. JC1 in the resulting strains were subsequently replaced by the fusion PCR product of the A1, A2, or A3 allele of hsdSA by counter selection, which consisted of the hsdRMS promoter and full coding sequence of each allele. Colonies of each strain were prepared, photographed, and marked as in Fig 6. The genotype and name of each strain are marked at the top and bottom of each photograph. All of the ST556 derivatives carrying allele A1 produced uniformly opaque colonies. In (D), the relationship of the hsdSA allelic variations by DNA inversions and the resulting epigenetic and phenotypic switch is diagrammatically illustrated; the methylated and unmethylated adenine nucleotides in the DNA motif by the HsdA1-associated methyltransferase is highlighted with red and blue characters, respectively. R = A or G, Y = T or C.
Fig 11.
Significant impact of epigenetic-mediated phase variation on pneumococcal adhesion to host epithelial cells.
The hsdSA1, hsdSA2, or hsdSA3 allele-carrying derivatives of strains ST556 (panels A and B), P384 (panels C and D), and ST877 (panels E and F) were cultured on the TSA plates supplemented with catalase as represented in Fig 6, and used to determine adhesion to human lung (A549 line) and nasopharyngeal (Detroit 562 line) cells in 24-well plates by counting CFU of adhering bacteria after extensive washing of the cell monolayers. The pneumococci carrying the hsdSA1 (A1) (producing opaque colonies) are significantly less adherent than those carrying the hsdSA2 (A2) or hsdSA3 (A3).
Fig 12.
Significant impact of epigenetic-mediated phase variation on nasopharyngeal colonization of the pneumococci in the mouse co-carriage model.
The pneumococcal derivatives of strains ST556 (A), P384 (B), and ST877 (C) each carrying the hsdSA1, hsdSA2, or hsdSA3 allele were grown on the TSA plates supplemented with catalase as represented in Fig 6. Two of the three unique hsdSA allelic derivatives (A1, A2, and A3) from each strain background were mixed at a 1:1 ratio before being used to inoculate intranasally C57BL/6 mice. The colonizing pneumococci were recovered from each mouse by washing the nasal cavity 7 days post inoculation. The output ratio of the two the hsdSA allele-specific variants co-infecting the same mouse was determined with the nasal lavage sample by PCR with the hsdSA allele-specific primers. The hsdSA1-specific variant derived from each of three different strain backgrounds (A1) (forming opaque colonies) was less fit than the counterpart carrying hsdSA2 (A2) or hsdSA3 (A3) (forming transparent colonies) in the nasopharynx.