Competence for genetic transformation in Streptococcus pneumoniae: mutations in σA bypass the comW requirement

Abstract

Competence for genetic transformation in the genus Streptococcus depends on an alternative sigma factor, σ(X), for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of σ(X) is coordinated between cells by transcription factors linked to peptide pheromone signals. In Streptococcus pneumoniae, elevated σ(X) is insufficient for development of full competence without coexpression of a second competence-specific protein, ComW. ComW, shared by eight species in the Streptococcus mitis and Streptococcus anginosus groups, is regulated by the same pheromone circuit that controls σ(X), but its role in expression of the σ(X) regulon is unknown. Using the strong, but not absolute, dependence of transformation on comW as a selective tool, we collected 27 independent comW bypass mutations and mapped them to 10 single-base transitions, all within rpoD, encoding the primary sigma factor subunit of RNA polymerase, σ(A). Eight mapped to sites in rpoD region 4 that are implicated in interaction with the core β subunit, indicating that ComW may act to facilitate competition of the alternative sigma factor σ(X) for access to core polymerase.

FIG 1

Strategy for enrichment of comW bypass mutants. A single colony was picked and grown for 35 generations to create pool 0, a library of potential suppressor mutations. Then, 10⁸ cells of pool 0 were transformed with Nv^r DNA and plated, and 2 × 10⁴ Nv^r transformant colonies were collected to create pool I. Next, 10⁸ cells of pool I were transformed with Em^r DNA, and 2 × 10⁴ Nv^r Em^r transformant colonies were collected to create pool II. After 10⁸ cells of pool II were transformed with Tc^r DNA, individual Nv^r Em^r Tc^r transformant colonies were picked and transformed with Spc^r DNA to determine the transformation efficiency. The dots in tubes represent cells; the dots on plates represent colonies.

FIG 2

Serial enrichment of suppressor mutants from 14 independent libraries. Shown are counts of pools with transformation efficiencies in four ranges, for the initial mutant library (stage 0) and 3 successive cycles of enrichment. Values for enrichment stage III reflect averages (Avg) of subclone efficiencies (Table 3).

FIG 3

Locations of 68 amino acid changes identified by whole-genome and rpoD-targeted sequencing. (A) Mapping of base changes identified by WGS in 14 independent comW bypass mutants. Nonsynonymous substitutions are organized by relative genome positions, and gene designations are as annotated in accession number NC_003098.1. Black, unique gene hit; color, 2 or more hits in the same gene; black horizontal line, entire S. pneumoniae genome. The names of the bypass strains sequenced by WGS are on the right. (B) Map of predicted amino acid residue changes in RpoD (σ^A) among 27 suppressor mutants (Table 3). Boxes, four conserved regions of σ^A, 369 amino acids (aa), as assigned by Vassylyev et al. (32). The WT residues and positions are shown above the protein, and the suppressor residues and numbers of cases are shown below.

FIG 4

Linkage between rpoD* mutations and the comW bypass phenotype. (A) Backcross analysis of the bypass phenotypes of four rpoD* isolates. Shown is a comparison of the transformation efficiency of the suppressor isolate (line 3) to those of isolates with rpoD* in a WT background (line 4) and with rpoD* in a transformant ΔcomW WT background (line 5) and a suppressor isolate cured of the rpoD* mutation (line 6). Solid lines, WT genome; dashed lines, genome of isolate recovered from enrichment; ■, comW⁺; □, ΔcomW; ●, rpoD⁺; ○, rpoD* (the bypass mutant residues are indicated at the tops of the data columns). For mutations A171V, R316H, R355H, and L363F, the suppressor isolates used were ALT4, ILT1, FLT4, and NYT1, respectively; the strains with backcrossed rpoD* alleles in a ΔcomW WT background were CP2456, CP2458, CP2457, and CP2455; and the rpoD⁺ cured strains were CP2459, CP2461, CP2460, and CP2462. Standard deviation (SD) values were below 20%. (B) Growth of strains CP2451 to -54 containing rpoD* mutations over 17 generations. The arrows indicate 1:100 dilutions of the exponentially growing culture in fresh medium. (C) Allele-specific alternative primer pairs used for mismatch PCR genotyping of segregants after transformation with 5.5-kb donor rpoD amplicons. The SNP in the A171V mutant is underlined. The productive allele-specific primer for each allele is shown in red.

FIG 5

Alignment of σ^A homologs. S. pneumoniae σ^A amino acid residues 147 to 369 were aligned with σ^A sequences from accession numbers Q9EZJ8.1 (T. aquaticus), YP_491259.1 (E. coli), EIA15345.1 (S. aureus), NP_721232.1 (S. pneumoniae), and NP_358573.1 (S. mutans), using Clustal Omega with default parameters (33). Asterisks, identical residues; colons, conserved residues; periods, semiconserved residues; red, residues in S. pneumoniae σ^A replaced by bypass mutations.

FIG 6

Locations of DNA-contacting and protein interaction residues, comW bypass residues, and bypass and affinity-affecting residues in region 4 of σ^A in a holoenzyme. Shown is the crystal structure of a holoenzyme from E. coli (34), Protein Data Bank (PDB) ID 4LJZ. σ⁷⁰, space-filling blue; β-subunit, green ribbon; β′, gray ribbon. (A) Residues contacting DNA or regulatory proteins in region 4. Beige, DNA-binding residues; dark green, CAP-interacting residues; pink, FNR-interacting residues; yellow, λCI-interacting residues; purple, PhoB-interacting residues (all according to Campbell et al. [26]). (B) comW suppressor residues in region 4. Orange, residues corresponding to comW bypass mutations identified in this study. (C) Bypass and affinity residues from E. coli and T. thermophilus in region 4. Red, residues corresponding to bypass mutations that facilitate the activity of an alternative sigma factor according to Laurie et al. (29); magenta, mutations that reduce σ^A affinity for RNA according to Dove et al. (27) and Nickels et al. (28).

FIG 7

Model of potential ComW functions. Possible functions of ComW as a prosigma, anti-protease-adapter or as an anti-anti-σ. The prosigma function promoting competition with σ^A is in boldface because it is consistent with recovery of bypass mutations exclusively in rpoD. Anti-protease and anti-anti-σ functions are lighter because no bypass mutations were in protease subunits/adapters or in other proteins orthologous to anti-σ factors. Dashed lines, possible functions; pentagons, genes; orange half-circle, core RNA polymerase; blue circles, σ^X; purple squares, ComW; yellow, anti-σ^X; red triangles, protease.