Impact of orthologous gene replacement on the circuitry governing pilus gene transcription in streptococci

Abstract

Background: The evolutionary history of several genes of the bacterial pathogen Streptococcus pyogenes strongly suggests an origin in another species, acquired via replacement of the counterpart gene (ortholog) following a recombination event. An example of orthologous gene replacement is provided by the nra/rofA locus, which encodes a key regulator of pilus gene transcription. Of biological importance is the previous finding that the presence of the nra- and rofA-lineage alleles, which are approximately 35% divergent, correlates strongly with genetic markers for streptococcal infection at different tissue sites in the human host (skin, throat).

Methodology/principal findings: In this report, the impact of orthologous gene replacement targeting the nra/rofA locus is experimentally addressed. Replacement of the native nra-lineage allele with a rofA-lineage allele, plus their respective upstream regions, preserved the polarity of Nra effects on pilus gene transcription (i.e., activation) in the skin strain Alab49. Increased pilus gene transcription in the rofA chimera correlated with a higher rate of bacterial growth at the skin. The transcriptional regulator MsmR, which represses nra and pilus gene transcription in the Alab49 parent strain, has a slight activating effect on pilus gene expression in the rofA chimera construct.

Conclusions/significance: Data show that exchange of orthologous forms of a regulatory gene is stable and robust, and pathogenicity is preserved. Yet, new phenotypes may also be introduced by altering the circuitry within a complex transcriptional regulatory network. It is proposed that orthologous gene replacement via interspecies exchange is an important mechanism in the evolution of highly recombining bacteria such as S. pyogenes.

Figure 1. FCT-region maps of wt Alab49 and isogenic mutants.

A, Map of the FCT-region of wt Alab49; the direction of transcription for each gene is shown (arrows). The four genes encoding surface proteins are in green; also shown are genes whose products have a role in transcriptional regulation (blue) and pilus assembly (red). The 430 bp intergenic region between nra and cpa is indicated by an inverted triangle. Black arrows represent the highly conserved genes that form the boundary of the FCT-region; the hsp33gene lies downstream of nra, outside of the FCT-region. B, Maps of the modified portions of the FCT-region for the Alab49 Δnra mutant and nra::aad9 and rofA::aad9 constructs. The aad9 gene encoding spectinomycin resistance is inserted in the nra-hsp33 intergenic region, at a position of 97 nt downstream of nra/rofA and 33 nt upstream of hsp33. C, Nucleotide sequence of the 522 bp rofA-cpa intergenic region within the Alab49 rofA::aad9 chimeric construct, consisting of a 266 bp region upstream of rofA derived from strain D471 (M6), juxtaposed to the 256 bp region upstream of cpa derived from strain Alab49 (M53). Known and putative sequence motifs involved in DNA binding by RofA and MsmR (blue, underlined) are highlighted , , . Also indicated are 5′ end positions for forward oligonucleotide primers (UP-1F through UP-4F; red, underlined) used in PCR amplification. The transcriptional start sites for cpa transcripts in both wt Alab49 and the rofA::aad9 construct, as determined by RACE (see text), are depicted (black, underlined).

Figure 2. Increased quantity of pilus-like structures in the rofA::aad9 construct.

Immunoblots were treated with antiserum raised to rCpa (A) or rPrtF2 (B). Mutanolysin extracts subject to SDS-PAGE were prepared from wt Alab49 (lanes 1 and 2), Alab49 Δnra mutant (lanes 3 and 4), Alab49 nra::aad9 (lanes 5 and 6) and Alab49 rofA::aad9 (lanes 7 and 8). Extracts from cells grown to mid-logarithmic phase (4 h at 30°C) are shown in lanes 1, 3, 5 and 7; extracts from cells grown to stationary phase (16 h at 30°C) are shown in lanes 2, 4, 6 and 8. To account for the increase in bacterial cell mass at stationary phase, the loading volume of the samples obtained from mid log phase cultures was increased by 2.5-fold, a value which corresponds to the observed increase in OD₆₀₀ for the stationary phase cultures relative to mid log phase. Antiserum to rFctA and rFctB show staining patterns highly similar to those observed with anti-rCpa serum (supplement; Figure S1). Because PrtF2 is completely degraded by SpeB in stationary phase cultures , only extracts from mid log cultures are shown (panel B); also the protease inhibitor cocktail was not included during the extraction procedure. Molecular weight markers are indicated in kilodaltons.

Figure 3. Skin infection in the humanized mouse at 7 d post-inoculation.

Bacteria grown to either mid-logarithmic (filled symbols) or stationary (open symbols) phase in broth culture were used to inoculate scratched human skin engrafted on SCID mice. The inoculum dose (log₁₀ CFUs) is depicted on the x-axis. The net change (increase or decrease) in log₁₀ CFUs recovered from a graft at biopsy, relative to the inoculum dose, is shown on the y-axis. Each data point represents an inoculated skin graft. Bacterial inoculums are indicated as wt Alab49 (upper panel) and Alab49 rofA::aad9 (lower panel). Differences in inoculum doses tested, for wt Alab49 versus Alab49 rofA::aad9, at either the exponential or stationary growth phase, are not statistically significant according to either the parametric unpaired t-test or non-parametric Mann-Whitney U test (2-tailed).

Figure 4. Growth rate during the early stages of skin infection.

The number of bacterial population doublings (log₂ change in cfu) at the skin by 48 or 72 h post-inoculation is shown for mid-logarithmic phase broth cultures of wt Alab49 (circles) and the Alab49 rofA::aad9 construct (diamonds). Bars depict average mean values. The mean average inoculum dose and standard deviation is also indicated.

Figure 5. Start site for pilus gene transcription in the rofA::aad9 construct.

cDNA generated from RNA derived from wt Alab49 (A) or Alab49 rofA::aad9 (B) was used as a template for PCR amplification; amplicons were subject to agarose gel electrophoresis and ethidium bromide staining. All reactions are paired with reverse primer CPA-R in combination with UP-F1 (lane 1), UP-F2 (lane 2), UP-F3 (lane 3), UP-F4 (lane 4) and CPA-F (lane 5). Positions for primers UP-F1 through F4 are also depicted in Figure 1C. Molecular size markers are indicated.

Figure 6. Decrease in pilus gene transcript levels in rofA::aad9 ΔmsmR.

qRT-PCR data showing transcript abundance for rofA::aad9 and rofA::aad9 ΔmsmR relative to wt Alab49, for cpa (panel A) and fctA (panel B). RNA was prepared from three bacterial cultures - wt, rofA::aad9 and rofA::aad9 ΔmsmR - grown to the mid-logarithmic phase in three separate experiments; two independent cDNA preparations were generated from each RNA preparation. Matched pairs of strains from the six different cDNA preparations are plotted. Statistical significance is measured for transcript ratios of rofA::aad9 versus rofA::aad9 ΔmsmR by the paired t-test (2-tailed).

Figure 7. MsmR effects on transcripts corresponding to the 5- untranslated region of cpa.

qRT-PCR data showing transcript abundance for rofA::aad9 and rofA::aad9 ΔmsmR relative to wt Alab49, for reactions using UP-F3 (filled) and UP-F4 (open) primers targeting the 5′-untranslated region of cpa (Figure 1C). RNA was prepared from bacterial cultures grown to the mid-logarithmic phase in three separate experiments.

Figure 8. Effect of MsmR on extracted pilus and PrtF2 proteins.

Immunoblots were treated with antiserum raised to rCpa (A) or rPrtF2 (B). Mutanolysin extracts subject to SDS-PAGE were prepared from Alab49 rofA::aad9 (lanes 1) or Alab49 rofA::aad9 ΔmsmR (lanes 2). Extracts from cells grown to stationary phase (16 h at 30°C) are shown in panel A; extracts from cells grown to mid-logarithmic phase (4 h at 30°C) are shown in panel B. Molecular weight markers are indicated in kilodaltons.

Figure 9. Network motifs involving msmR, nra/rofA and cpa.

Pathways of the circuitry are shown for wt Alab49 and the Alab49 rofA::aad9 construct (this report). Positive and negative regulation is represented by arrows and bars, respectively.