Molecular Mechanisms of hsdS Inversions in the cod Locus of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae(pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferasehsdSgenes (hsdSA,hsdSB, andhsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all threehsdSgenes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression ofhsdSAbut nothsdSBandhsdSC, indicating thathsdSBandhsdSCdo not produce functional proteins and instead act as sources for altering the sequence ofhsdSAby DNA inversions. Consistent with our previous finding that thehsdSinversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion betweenhsdSAandhsdSB. Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in thepsrAmutant. Surprisingly, PsrA-independent inversions were also detected in thehsdSsequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed thatS. pneumoniaevaries the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in thecodlocus.IMPORTANCEStreptococcus pneumoniaeis a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferasehsdSgenes of thecodlocus. This work revealed that only thehsdSgene that is in the same orientation ashsdMis actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of thehsdSinversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed thatS. pneumoniaeswitches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.

Regulation of pneumococcal epigenetic and colony phases by multiple two-component regulatory systems

“…Our previous study has found that the reversible switch between O and T colony phenotypes is controlled by the PsrA-catalyzed inversions of the three invertible regions in the cod locus [5,9] (Fig 2A). Therefore, enzymatic inactive psrA Y247A mutant (with a point mutation in the catalytic residue tyrosine 247) is locked in either O or T colony phase [9].…”

“…Our previous study has found that the reversible switch between O and T colony phenotypes is controlled by the PsrA-catalyzed inversions of the three invertible regions in the cod locus [5,9] (Fig 2A). Therefore, enzymatic inactive psrA Y247A mutant (with a point mutation in the catalytic residue tyrosine 247) is locked in either O or T colony phase [9]. To determine whether the impact of RR06, RR08, RR09, RR11 and RR14 on phase variation requires PsrA-mediated inversions, we constructed deletion mutant in each of these genes in the O-locked psrA Y247A strain, which was previously shown to produce only the O colonies [5] ( Fig 2B; S2 Fig).…”

“…The cod locus, a type-I restriction-modification (RM) system, consists of the hsdR (restriction endonuclease), hsdM (DNA methyltransferase), psrA (invertase), and three homologous hsdS (hsdS A , hsdS B and hsdS C ) genes [5]. PsrA catalyzes extensive inversions between hsdS A and two downstream homologs (hsdS B and hsdS C ) [9,10]. As a typical sequence recognition or S subunit of the type-I RM DNA methyltransferase (MTase) [11], hsdS A encodes two target recognition domains (TRDs), each of which recognizes a half of the type-I RM methylation bipartite sequence [5].…”

“…As a typical sequence recognition or S subunit of the type-I RM DNA methyltransferase (MTase) [11], hsdS A encodes two target recognition domains (TRDs), each of which recognizes a half of the type-I RM methylation bipartite sequence [5]. Our recent study has shown that only hsdS A is actively transcribed and produces a functional S subunit for the MTase, whereas hsdS B and hsdS C merely serve as templates for replacement of one or two TRD domains in the HsdS A MTase by the PsrA-catalyzed inversions [5,9]. The inversions generate six different hsdS A alleles, each of which drives methylation of a unique DNA motif in the genome of S. pneumoniae [5,7].…”

Selective and non‐selective bottlenecks as drivers of the evolution of hypermutable bacterial loci

“…For example, sub‐components of the hsdS gene of the well‐characterised complex PV RM of Streptococcus pneumoniae , SpnIII, can exist in one of six possible orientations. Each of these unique TRD combinations is associated with a different DNA methylation pattern and some of them can impact on invasive disease potential in mice and the invasion‐associated opaque colony phenotype (Li et al, , ; Manso et al, ; Oliver, Roy, Kumar, Lefkowitz & Swords, ). The simplicity of these findings is challenged by variable experimental observations, suggesting that genetic background may influence links between methylation and colony opacity, and by the extensive hsdS allelic variation resulting in differing dominant methylation patterns among pneumococcal strains (De Ste Croix et al, ).…”