Characterization of a Four-Component Regulatory System Controlling Bacteriocin Production in Streptococcus gallolyticus

Abstract

Bacteriocins are natural antimicrobial peptides produced by bacteria to kill closely related competitors. The opportunistic pathogen Streptococcus gallolyticus subsp. gallolyticus was recently shown to outcompete commensal enterococci of the murine microbiota under tumoral conditions thanks to the production of a two-peptide bacteriocin named gallocin. Here, we identified four genes involved in the regulatory control of gallocin in S. gallolyticus subsp. gallolyticus UCN34 that encode a histidine kinase/response regulator two-component system (BlpH/BlpR), a secreted peptide (GSP [gallocin-stimulating peptide]), and a putative regulator of unknown function (BlpS). While BlpR is a typical 243-amino-acid (aa) response regulator possessing a phospho-receiver domain and a LytTR DNA-binding domain, BlpS is a 108-aa protein containing only a LytTR domain. Our results showed that the secreted peptide GSP activates the dedicated two-component system BlpH/BlpR to induce gallocin transcription. A genome-wide transcriptome analysis indicates that this regulatory system (GSP-BlpH/BlpR) is specific for bacteriocin production. Importantly, as opposed to BlpR, BlpS was shown to repress gallocin gene transcription. A conserved operator DNA sequence of 30 bp was found in all promoter regions regulated by BlpR and BlpS. Electrophoretic mobility shift assays (EMSA) and footprint assays showed direct and specific binding of BlpS and BlpR to various regulated promoter regions in a dose-dependent manner on this conserved sequence. Gallocin expression appears to be tightly controlled in S. gallolyticus subsp. gallolyticus by quorum sensing and antagonistic activity of 2 LytTR-containing proteins. Competition experiments in gut microbiota medium and 5% CO₂ to mimic intestinal conditions demonstrate that gallocin is functional under these in vivo-like conditions.IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus, formerly known as Streptococcus bovis biotype I, is an opportunistic pathogen causing septicemia and endocarditis in the elderly often associated with asymptomatic colonic neoplasia. Recent studies indicate that S. gallolyticus subsp. gallolyticus is both a driver and a passenger of colorectal cancer. We previously showed that S. gallolyticus subsp. gallolyticus produces a bacteriocin, termed gallocin, enabling colonization of the colon under tumoral conditions by outcompeting commensal members of the murine microbiota such as Enterococcus faecalis Here, we identified and extensively characterized a four-component system that regulates gallocin production. Gallocin gene transcription is activated by a secreted peptide pheromone (GSP) and a two-component signal transduction system composed of a transmembrane histidine kinase receptor (BlpH) and a cytosolic response regulator (BlpR). Finally, a DNA-binding protein (BlpS) was found to repress gallocin genes transcription, likely by antagonizing BlpR. Understanding gallocin regulation is crucial to prevent S. gallolyticus subsp. gallolyticus colon colonization under tumoral conditions.

Keywords: Streptococcus; bacteriocins; regulation of gene expression.

FIG 1

A three-component system activates gallocin transcription in S. gallolyticus subsp. gallolyticus UCN34. (A) Gallocin locus in strain UCN34 (12,986 bp) extending from gallo_rs10325 to gallo_rs10405 (UCN34 genome reference NC_013798.1; new annotation). Genes with a predicted function are in gray; hypothetical genes are in black. Gene names are those given in this work or referred to using the novel “gallo_rs” annotation (e.g., “10325” for gallo_rs10325). Arrowheads above the genes indicate the presence of a 30-bp conserved motif in the promoter regions. (B) Agar diffusion assay revealing the capacity of S. gallolyticus subsp. gallolyticus UCN34 Δgsp, ΔblpH, and ΔblpR to inhibit the growth of the gallocin-sensitive S. gallolyticus subsp. macedonicus strain. Mutants were cultured either in THY or in THY supplemented with 20 nM synthetic GSP (THY GSP). Activity of one counterpart that reverted back to WT (bWT) is also shown on the right. (C) Schematic representation of the reporter plasmid pTCVΩPgllA-gfp to monitor gallocin promoter activity. (D) PgllA activity (fluorescence divided by OD₆₀₀) in strain UCN34 WT, Δgsp, ΔblpH, and ΔblpR in presence or absence of 20 nM synthetic GSP. One representative curve of three independent experiments is shown here for each condition. (E) PgllA activity in S. gallolyticus subsp. gallolyticus UCN34 Δgsp containing the reporter plasmid in the presence of growing concentrations of synthetic GSP (curves from bottom to top were obtained in culture medium containing 0 to 20 nM GSP, respectively; the concentration increasing by 2 nM between each curve. The three upper curves were obtained with 16, 18, and 20 nM GSP. One representative curve of three independent experiments is shown for each condition.

FIG 2

The whole regulon controlled by GSP/BlpHR. (A) Heat map representing the log₂ fold change in mRNA abundance (determined by whole-transcriptome analysis) of all the genes along the UCN34 genome in Δgsp, ΔblpH, and ΔblpR mutants compared to the parental S. gallolyticus subsp. gallolyticus UCN34 WT. (B) Analysis similar to that in panel A for selected genes whose log₂ fold change in mRNA abundance was significantly greater than 2 or less than −2 in at least one mutant (see Materials and Methods). All the genes belonging to the gallocin locus are in red, along with the corresponding gene product. Gene product was determined either with genome annotation or by BLAST analysis. A question mark indicates that the result of the BLAST analysis was not found to be relevant. (C) qRT-PCR data showing the fold change in mRNA abundance in S. gallolyticus subsp. gallolyticus UCN34 Δgsp, ΔblpH, and ΔblpR compared to the WT. The identity of each mutant was confirmed by the absence of transcript (triangles). Results are means and standard deviations (SD) from three independent cultures in triplicate. Asterisks represent statistical differences relative to WT strain UCN34. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., no significant difference as assessed by using ANOVA in R package version 1.4.2.

FIG 3

BlpS inhibits gallocin gene transcription. (A) SMART domains identified in BlpR and BlpS proteins. REC, cheY-homologous receiver domain; LytTR, LytTR DNA-binding domain. The percent identity between the two LytTR domains was determined using the Geneious alignment tool. (B) Agar diffusion assay showing gallocin activity in the culture supernatant against S. gallolyticus subsp. macedonicus. The strains tested were S. gallolyticus subsp. gallolyticus UCN34 WT and ΔblpS (top) and the same strains containing pTCVΩPtetO-blpS with or without induction of blpS expression with 200 ng/ml anhydrotetracycline (bottom). (C) Promoter activity of PgllA (circles) and Pgsp (squares) during growth in S. gallolyticus subsp. gallolyticus WT and the ΔblpS mutant. One representative curve of three independent experiments is shown here for each condition. (D) qRT-PCR data showing the fold change in mRNA abundance between S. gallolyticus subsp. gallolyticus UCN34 pTCVΩPtetO-blpS (WT) and ΔblpS pTCVΩPtetO-blpS (ΔblpS). blpS+ indicates the induction of blpS transcription with 200 ng/ml anhydrotetracycline. Results are means and SD from three independent cultures carried out in triplicate. Statistical differences for each gene in the various groups were assessed using ANOVA in R package version 1.4.2. ***, P < 0.001. (E) Agar diffusion assay to test gallocin activity in the culture supernatant of S. gallolyticus subsp. gallolyticus UCN34 ΔblpS ΔblpR (deletion of blpS in ΔblpR), UCN34 ΔblpR ΔblpS (deletion of blpR in ΔblpS), and the respective bWT strains against S. gallolyticus subsp. macedonicus. (F) PgllA activity during growth in S. gallolyticus subsp. gallolyticus UCN34 WT, ΔblpS and ΔblpR ΔblpS. One representative curve of three independent experiments is shown for each condition.

FIG 4

Overexpression of gallocin in blpS mutant allows better killing of E. faecalis in a competition experiment under gut-like conditions. (A) E. faecalis V583 counts after a 5-h competition against different strains of S. gallolyticus subsp. gallolyticus. (B) E. coli pks⁺, a strain resistant to gallocin used as a control. S. gallolyticus subsp. gallolyticus Δblp is the mutant initially constructed (4), which has the three genes of the gallocin-encoding core operon deleted (ΔgllA1 ΔgllA2 Δgip). Statistical differences for each strain were assessed using ANOVA in R package version 1.4.2. ***, P < 0.001; n.s., no statistically significant difference found.

FIG 5

A conserved DNA motif is present upstream of all the genes regulated by GSP-BlpHR. (A) The 15-bp DNA motif obtained by alignment of the promoters of the regulatory system, the bacteriocin accessory protein gene, gallocin genes, the ABC transporter gene, and the Abi domain protein gene on https://weblogo.berkeley.edu/logo.cgi. (B) A 30-bp consensus sequence identified by MEME in the 12 putative promoters regulated by BlpHR. The initial 15-bp motif is located at the 3′ end of the larger consensus motif. (C) Mapping of the 30-bp consensus sequence on the S. gallolyticus subsp. gallolyticus chromosome (with a maximum of 6 mismatches). The consensus sequences are represented by arrowheads, and the name of the gene downstream of the consensus is given. (D) Determination of the transcription start site of gllA mRNA and localization of the conserved motif. Putative −10 (TAGACT) and −35 (CGTGCA) promoter boxes were assigned based on the location of the (+1) transcription start site according to the canonical procaryotic promoter sequence (TTGACA-X17-TATAAT).

FIG 6

Binding of BlpR and BlpS to three regulated promoters. EMSA demonstrating the binding of BlpS and BlpR to the promoter regions of gsp (Pgsp), blpA (PblpA), gllA (PgllA), and PgllA where the consensus sequence was randomly scrambled (PgllA scramble). PgyrA was used as a negative control. The full sequences of the various promoters are presented in Table 3. Serial 2-fold dilutions of the recombinant protein (from right to left) were incubated with purified radiolabeled promoters before migration. The leftmost band corresponds to migration of the promoter alone. All these experiments were carried out in the presence of 0.1 μg/μl poly(dI-dC) to prevent aspecific binding of proteins to DNA. Results of one representative EMSA of three independent experiments are shown for each condition.

FIG 7

The binding site of BlpR and BlpS, mapped precisely by DNase I footprint experiment. Analysis of BlpR and BlpS footprint on the gllA promoter. The gllA promoter was incubated with increasing concentrations of BlpR or BlpS (indicated on the left) and digested with DNase. The sites protected from DNase by BlpR or BlpS binding are indicated by black squares. The sequence was determined by G+A sequencing and mapped on the gllA promoter. (A) Footprint with BlpS protein. (B) Footprint with BlpR protein.

FIG 8

Genetic evidence demonstrating that non-phosphorylated BlpR cannot activate gallocin (gllA2) gene transcription. (A) Agar diffusion assay to assess gallocin activity in the culture supernatant against S. gallolyticus subsp. macedonicus. S. gallolyticus subsp. gallolyticus UCN34 ΔblpS Δgsp, ΔblpS bWT gsp, ΔblpS ΔblpH, and ΔblpS bWT blpH strains were tested. (B) qRT-PCR data showing the fold change in mRNA abundance of the gllA2 gene between S. gallolyticus subsp. gallolyticus UCN34 WT and S. gallolyticus subsp. gallolyticus mutants.

FIG 9

Hypothetical model of transcription regulation by BlpR and BlpS. At low cell density, i.e., in the absence of GSP or at low concentrations of GSP, BlpR is not phosphorylated by BlpH and hence has no affinity for the conserved binding motif located in the promoter of gallocin genes, which is occupied by the BlpS repressor. At high cell density, the GSP concentration is sufficient to induce BlpH-mediated phosphorylation of BlpR. Phosphorylated BlpR (BlpR-P) outcompetes BlpS, resulting in RNA polymerase recruitment and transcription of gallocin genes. The antagonistic effect of BlpS and BlpR-P controls the level of expression of the gallocin genes, which is shut down when GSP concentration decreases.