Table 1.
The ratios of T and O colonies formed by the rr mutants.
Fig 1.
Colony phenotypes of the rr mutants.
A. Representative colonies formed by selected rr mutants. ST606 (WT) and isogenic rr mutants were separately spread on catalase-TSA plates, incubated for 17 hrs before the colonies were photographed under a dissection microscope. Genotype (top) and identification (bottom) of each strain are marked. Representative colonies are indicated by red (opaque) and light blue (transparent) arrowheads, respectively. B. Ratio between opaque (O) and transparent (T) colonies of selected rr mutants and their revertants. O and T colonies on each plate were prepared as in Fig 1A to quantify O and T colonies formed by ST606 (WT), isogenic rr mutants and their revertants. The mean ± SEM of 3 values (from 3 plates) for the O (filled) and T (open) colonies of each strain is presented in a single bar. C. Ratio between O and T colonies derived from single O seeding colonies of the six selected rr mutants. Three well-separated O colonies were spread on three catalase-TSA plates to assess the relative ratio between O and T colonies for each strain as described in Fig 1B. D. Same as Fig 1C except for using T colonies as the seeding bacteria.
Fig 2.
Characteristics of the colonies produced by the psrAY247A-rr double mutants.
A. Schematic illustration of the gene organization in the cod locus. The genes encoding the restriction enzyme (hsdR), DNA methyltransferase (hsdM), sequence recognition proteins (hsdSA, hsdSB and hsdSC) and invertase (psrA) are depicted as thin arrows at the top. Three pairs of inverted repeats (IR1, IR2 and IR3) flanking the invertible regions that mediate DNA inversions are indicated as small arrowheads in the first row of the lower panel which depicts the six major hsdSA allelic configurations generated by PsrA-catalyzed inversions. B. Ratio between O and T colonies produced by the five selected rr mutants that were generated in the psrAY247A background. The colonies were prepared and processed as in Fig 1B.
Fig 3.
Relative methylation rate of the DNA motifs recognized by four HsdSA allelic variants in the rr mutants.
Relative methylation rate of each DNA motif recognized by HsdSA1 (5’-CRAm6AN8CTT-3’/3’-GYTTN8Gm6AA-5’, 2,058 loci), HsdSA2 (5’-CRA m6AN9TTC-3’/3’-GYTTN9m6AAG-5’, 2,054 loci), HsdSA3 (5’-CRAm6AN8CTG-3’/3’-GYTTN8G m6AC-5’, 1,468 loci) and HsdSA4 (5’-C m6ACN7CTG-3’/3’-GTGN7G m6AC-5’, 888 loci) was calculated in each strain by dividing the number of methylated chromosomal loci for each motif with the total loci of the motif in the ST556 genome. Only the values for the parental strain ST606 (A) and isogenic mutants of rr10 (B), rr06 (C), rr08 (D), rr09 (E), rr11 (F) and rr14 (G) are presented. The results for the other rr mutants are described in Table 2.
Table 2.
Methylation sequences specified by the cod MTases1.
Fig 4.
The colony characteristics of the rr06, rr08, rr09 and rr11 mutants in the P384 and ST877 strain backgrounds.
A. Representative colonies formed by P384 (6A) and its isogenic rr mutants. The colonies were generated, photographed and marked as in Fig 1A. The ratio between the O and T colonies (bottom) is displayed as in Fig 1B except for different strains. B. Representative colonies produced by ST877 (35B) and its isogenic rr mutants. Same as in (A) with the exception of different strains.
Fig 5.
Colony opacity characteristics of the tcs11 mutants.
A. Colonies characteristics of the rr11 mutants. The colony phenotypes of ST606 derivatives lacking rr11 or carrying the D53A, D53E or wildtype rr11 allele were characterized and presented essentially as in Fig 1. B. Colonies characteristics of the hk11 mutants. Same as in A except for using ST606 derivatives with various hk11 alleles. C. Colonies characteristics of the tcs11 mutants. Same as in A except for using ST606 derivative lacking both the rr11 and hk11 of the 11th two-component system (Δtcs11) and tcs11 revertant.
Fig 6.
Impact of the rr11 mutations on the hsdSA1 allelic configuration.
A. Relative abundance of the hsdSA1 mRNA in the rr11 mutants. The mRNA levels of the hsdSA1 allele in the clonal populations of ST606 (WT), isogenic Δrr11 and psrAY247A mutants were detected by qRT-PCR. The transcripts of the 5’ non-invertible segment of hsdSA were similarly detected in all strains as a reference to calculate the relative CT values. The relative CT values of the ST606 and Δrr11 strains were then normalized to those of the psrAY247A strain that has the locked hsdSA1 allele (100%). The data are shown as mean ± SEM of a representative experiment. Each experiment was replicated at least twice. B. Colony opacity of the psrAY247A-rr11 double mutants. ST606 (WT) and its derivatives with either the psrAY247A allele alone or additional rr11 allelic modifications were processed for colony enumeration, photographing and data presentation as in Fig 1B. C. Relative abundance of the hsdSA1 mRNA in the psrAY247A-rr11 double mutants. Same as in Fig 6A except for different strains.
Table 3.
Differentially expressed genes in the rr11 mutant.
Fig 7.
Impact of the RR11-regulated genes on the colony phases.
A. Colony opacity characteristics of the RR11-regulated gene mutants. The O and T colonies were prepared and photographed as in Fig 1A. B. Relative composition of the O and T colony types in each strain were enumerated and presented as in Fig 1B. C. Relative abundance of the hsdSA1 mRNA in the populations of the RR11-regulated gene mutants. The mRNA levels of the hsdSA1 allele in ST606 (WT) and its mutant lacking RR11-regulated genes were detected, analyzed and presented in the same manner as in Fig 6A.
Fig 8.
Working model depicting modulation of pneumococcal colony phases by the two-component systems.
In response to unspecified environmental cue(s) sensed by HK11, the 53rd conserved aspartic acid residue of RR11 is phosphorylated by HK11 or an alternative donor(s) of phosphoryl group, which in turn activates the comW and hyaluronate utilization locus. By the undefined mechanism(s), the RR11-activated genes act to drive the directions of the hsdS inversions toward the hsdSA1 allelic configuration and eventually the O colony phase. Likewise, RR06, RR08 and RR09 promote the hsdSA1 allelic configuration and O colony phase by the uncharacterized mechanisms. RR14 modulates colony phases in a non-epigenetic manner.