Environmental Microbiology

Two-Component Systems Involved in Susceptibility to Nisin A in Streptococcus pyogenes

Miki Kawada-Matsuo, Ichiro Tatsuno, Kaoru Arii, Takeshi Zendo, Yuichi Oogai, Kazuyuki Noguchi, Tadao Hasegawa, Kenji Sonomoto, Hitoshi Komatsuzawa
C. M. Dozois, Editor
DOI: 10.1128/AEM.01897-16

ABSTRACT

Two-component systems (TCSs) are regulatory systems in bacteria that play important roles in sensing and adapting to the environment. In this study, we systematically evaluated the roles of TCSs in the susceptibility of the group A Streptococcus (GAS; Streptococcus pyogenes) SF370 strain to several types of lantibiotics. Using individual TCS deletion mutants, we found that the deletion of srtRK (spy_1081–spy_1082) in SF370 increased the susceptibility to nisin A, which is produced by Lactococcus lactis ATCC 11454, but susceptibility to other types of lantibiotics (nukacin ISK-1, produced by Staphylococcus warneri, and staphylococcin C55, produced by Staphylococcus aureus) was not altered in the TCS mutants tested. The expression of srtFEG (spy_1085 to spy_1087), which is located downstream of srtRK and is homologous to ABC transporters, was increased in response to nisin A. However, srtEFG expression was not induced by nisin A in the srtRK mutant. The inactivation of srtFEG increased the susceptibility to nisin A. These results suggest that SrtRK controls SrtFEG expression to alter the susceptibility to nisin A. Further experiments showed that SrtRK is required for coexistence with L. lactis ATCC 11454, which produces nisin A. Our results elucidate the important roles of S. pyogenes TCSs in the interactions between different bacterial species, including bacteriocin-producing bacteria.

IMPORTANCE In this study, we focused on the association of TCSs with susceptibility to bacteriocins in S. pyogenes SF370, which has no ability to produce bacteriocins, and reported two major new findings. We demonstrated that the SrtRK TCS is related to susceptibility to nisin A by controlling the ABC transporter SrtFEG. We also showed that S. pyogenes SrtRK is important for survival when the bacteria are cocultured with nisin A-producing Lactococcus lactis. This report highlights the roles of TCSs in the colocalization of bacteriocin-producing bacteria and non-bacteriocin-producing bacteria. Our findings provide new insights into the function of TCSs in S. pyogenes.

INTRODUCTION

Group A Streptococcus (GAS; Streptococcus pyogenes), a human-restricted pathogen, can cause invasive infections, including streptococcal toxic shock and necrotizing fasciitis, which are associated with high mortality rates (1). These common human pathogens colonize the epithelial cells of the throat and the epidermal layer of the skin and cause an array of mild to severe diseases, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome (2, 3). Serious sequelae of GAS infections include poststreptococcal glomerulonephritis and rheumatic fever. GAS encounters various bacteria in the upper respiratory tract and in the oral cavity during the early stages of infection and is exposed to antimicrobial agents produced by these bacteria (3, 4). Adapting to these antimicrobial agents is required to successfully infect a human host. From this perspective, overcoming antimicrobial agents derived from commensal bacteria is critical for GAS.

Many bacteria produce antimicrobial agents called bacteriocins (5, 6, 7). Bacteriocins in Gram-positive bacteria are primarily classified into class I and II bacteriocins (8). Class I bacteriocins (peptides of <5 kDa) are called “lantibiotics” and contain unusual amino acids, such as dehydrated amino acids, lanthionine, and 3-methyllanthionine, which form a ring structure (8, 9). Class II bacteriocins are composed of unmodified amino acids (10). Lantibiotics are subdivided into A (linear peptide) and B (globular peptide) types (8, 9, 10). Type A lantibiotics are further classified into two subtypes based on their structure. Type AI includes nisin, subtilin, and epidermin, and type AII includes lacticin 481 and nukacin ISK-1 (11). In addition, two-component lantibiotics, such as lacticin 3147 and staphylococcin C55, have been identified (11). The antimicrobial action of these lantibiotics occurs via disturbance of the bacterial membrane and inhibition of cell wall biosynthesis, which can occur via transglycosylation. Class II bacteriocins are non-lanthionine-containing bacteriocins and are classified into four subclasses: IIa (pediocin-like), IIb (two peptides), IIc (circular), and IId (non-pediocin-like linear single peptide) (9). Bacteriocins promote the survival of these bacteria in this community (5, 6). Bacteriocins effectively function as biological weapons that eliminate other bacteria for the purpose of ensuring their own survival. However, there are also bacteria that can coexist with bacteriocin-producing bacteria because they are resistant to specific bacteriocins.

Recently, two-component systems (TCSs) have been shown to be associated with susceptibility to bacteriocins in several bacterial species. Staphylococcus aureus utilizes BraRS to decrease susceptibility to several types of lantibiotics (12). Bacillus subtilis utilizes a SpaRK system both for the synthesis of the lantibiotic subtilin and for self-immunity to subtilin (13). In S. mutans, NsrRS and LcrRS are associated with susceptibility to lantibiotics (14). TCSs are bacterium-specific signal transduction systems that are composed of a sensory histidine kinase (HK) and a cognate response regulator (RR) (15). When the HK is stimulated by an environmental signal, it undergoes autophosphorylation of a histidine residue and relays the phosphate group to an aspartic acid residue on the cognate RR (16, 17). The phosphorylated RR controls the expression of target genes by binding to target DNA elements. Thus, bacteria can quickly adapt to the external environment by regulating gene expression. TCS-based mechanisms for altering the susceptibility to bacteriocins have been identified in several bacterial species.

GAS, including the SF370 strain, has 13 sets of TCSs (18), but no systematic analysis of TCS-mediated sensitivity to a range of lantibiotics has been performed. In this study, we evaluated the roles of S. pyogenes TCSs in the susceptibility to several types of bacteriocins. We determined that SrtRK is important in the susceptibility to nisin A, specifically via control of the ABC transporter SrtFEG.

MATERIALS AND METHODS

Bacterial strains and growth conditions.The bacterial strains used in this study are listed in Table 1. S. pyogenes was grown in brain heart infusion broth with 0.3% yeast extract broth (BHIYE) (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) at 37°C with 5% CO2. Lactococcus lactis was grown in BHI broth at 37°C with 5% CO2. Escherichia coli XL-II was grown in Luria-Bertani (LB) broth at 37°C. S. aureus TY4 and S. warneri were grown in Trypticase soy broth (TSB; Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) at 37°C. When necessary, spectinomycin (Spc; 100 μg/ml) and/or kanamycin (Km; 100 μg/ml) were used for S. pyogenes, and ampicillin (Amp; 100 μg/ml) was used for E. coli.

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TABLE 1

Strains used in this study

Evaluation of bacteriocin susceptibility.Two methods were used to evaluate bacteriocin susceptibility. In a direct method modified from a previously described protocol (19), an overnight culture of each bacteriocin-producing strain was stab-inoculated onto a BHI agar plate and cultivated overnight at 37°C with 5% CO2. After confirming that the diameter of the growth zone of the bacteriocin-producing strain was uniformly 2 mm, 5 ml of prewarmed half-strength BHIYE soft agar (0.8%) containing wild-type or mutant S. pyogenes (106 cells/ml) was poured over the BHI agar plate. The plates were incubated for 20 h at 37°C with 5% CO2. The diameters of the clearing zones surrounding the bacteriocin-producing strains were measured in three directions. Three independent experiments were performed, and the average diameter was calculated.

In the MIC method, the MICs of nisin A were determined by microdilution, as described previously (14). Nisin A was purified as described previously (20). The wild-type S. pyogenes strain SF370 and its mutants (105 cells) were inoculated into 100 μl of BHIYE containing serial 2-fold dilutions of nisin A. MICs were determined after incubation for 16 h at 37°C with 5% CO2.

Construction of deletion mutants and complemented strains.Gene deletion mutants of S. pyogenes SF370 were constructed using a previously described method (21). Primers for the construction of 13 sets of TCS mutants in SF370 were the same as those used previously (22). Nonpolar inactivated mutants of the srtR, srtRK, and srtFEG genes were constructed through double-crossover allelic replacement in the chromosome of S. pyogenes SF370 and 1529. Primers used in this study are listed in Table S1 in the supplemental material.

For the construction of the srtR knockout plasmid, a DNA fragment of srtR was amplified with oligonucleotide primers 1081-n9SmaI and 1081-c7 (fragment 1). Fragment 1 was subcloned into the SmaI site of the pFW12 vector (21) (plasmid pFW1081-1). A second round of PCR was performed using primers 1081-n8NheI and 1081-c6 (fragment 2). Both the NheI-digested fragment 2 and the spc3 DNA fragment containing aad9 (promoterless Spc resistance gene) obtained from an SmaI-digested fragment of pSL60-3 (21) were cloned into the NheI-SmaI site of plasmid pFW1081-1 (named plasmid pFWsrtR-ko). For the construction of the plasmid for the srtRK knockout, a DNA fragment of srtK was amplified with 1082-n6Sma and 1082-c2 (fragment 3). Fragment 3 was digested with SmaI and RsaI and was subcloned into the SmaI site of pFW12 (plasmid pFW1082-1). The NheI-digested fragment 2 and the spc1 DNA fragment containing aad9, obtained from an SmaI-digested fragment of pSL60-1, were cloned into the NheI-SmaI site of plasmid pFW1082-1 (named plasmid pFWsrtRK-ko). For the construction of the srtFEG knockout plasmid, a DNA fragment of srtG was amplified with primers srt-n2Sma and srt-c2 (fragment 4). Fragment 4 was subcloned into the SmaI site of pFW12 (plasmid pFWsrt-1). A second round of PCR was performed using primers srt-n1NheI and srt-c1 (fragment 5). The NheI-digested fragment 5 and the spc3 DNA fragment were cloned into the NheI-SmaI site of plasmid pFWsrt-1 (named plasmid pFWsrtFEG-ko). All plasmids for knockouts were suicide vectors for S. pyogenes. For preparation of competent cells, strain SF370 or 1529 was harvested at the early to mid-exponential phase (optical density at 660 nm [OD660] of 0.4 to 0.5) and washed twice with 0.5 M sucrose buffer. The constructed suicide vector was transformed into the strain by electroporation. The electroporation conditions were 1.25 kV mm−1, 25 μF capacitance, and 200 Ω resistance using a Gene Pulser II (Bio-Rad, Hercules, CA). After incubation at 37°C for 3 h, competent cells were spread onto BHIYE agar plates containing Spc (final concentration, 100 μg/ml). Selected colonies on the plates were cultured. Cultured bacteria were washed once with saline, resuspended in 10 mM Tris–1 mM EDTA, and boiled for 10 min. Genomic DNA was obtained from the supernatant of boiled bacteria. Double-crossover replacement with genomic DNA was analyzed by PCR. Successful double-crossover replacement was further confirmed by DNA sequencing.

For the construction of a plasmid for complementation, a DNA fragment of srtRK was amplified with oligonucleotide primers 1081-n1 and 1082-c1 with PrimeSTAR HS DNA polymerase (TaKaRa, Ohtsu, Japan). The fragment was treated with T4 polynucleotide kinase and was then inserted into the SmaI site of the pLZ12-Km2 plasmid (23) with ligase. For srtFEG complementation, the DNA fragment of srtFEG was amplified with primers srt-n1NheI and srt-c2 and inserted into the SmaI site of pLZ12Km2. The protocol for transformation was the same as that described above, except that the competent cells were spread onto BHIYE agar plates containing Km (at a final concentration of 200 μg/ml).

Analysis of gene expression by quantitative PCR.Overnight cultures of S. pyogenes (108 cells) were inoculated into 10 ml of fresh BHIYE and were then grown at 37°C with 5% CO2. When the optical density at 660 nm reached 0.5, nisin A was added to the medium. After 15 min of incubation, bacterial cells were harvested. Total RNA was extracted as described previously (14). Briefly, 1 μg of total RNA was reverse transcribed to cDNA using a first-strand cDNA synthesis kit (Roche, Tokyo, Japan). Using cDNA as the template DNA, quantitative PCR was performed using a LightCycler Nano system (Roche, Tokyo, Japan). Prior to cDNA synthesis, we also analyzed total RNA samples using quantitative PCR to ensure that no DNA remained. The results were normalized to the housekeeping gene gyrA (12). The primers used in this study are shown in Table S1 in the supplemental material.

Transcriptional analysis of the srt locus.Reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) were performed to identify the mRNA from the srt locus. For RT-PCR experiments, primers were constructed to amplify the intergenic region between two genes. Primers used in this experiment are listed in Table S1 in the supplemental material. RT-PCR was performed using the method described above. The transcriptional start sites of srtA, srtF, and srtR were determined by RACE experiments. RACE was performed with a 5′-full RACE core set (TaKaRa Bio Inc., Shiga, Japan) according to the manufacturer's protocol. The primers used are listed in Table S1.

Antiserum production and immunoblotting.To produce antiserum against SrtF, we first constructed the recombinant protein tagged with 6× histidine. The DNA fragment encoding srtF (Spy_1085) was amplified using the specific primers listed in Table S1 in the supplemental material and was then cloned into pQE30 (Qiagen, Tokyo, Japan). The plasmid was then transformed into E. coli M15 (pREP4). The recombinant SrtF (rSrtF) protein was purified according to the manufacturer's instructions. Antiserum against rSrtF was obtained by immunizing mice as described previously (14). For immunoblotting, exponential-phase wild-type and mutant S. pyogenes was harvested from a 10-ml culture, and the cells were washed with phosphate-buffered saline (PBS). The cells were then resuspended in 200 μl of 5% sodium dodecyl sulfate (SDS) and disrupted by ultrasonication three times at 10-s intervals. The cells were then heated at 100°C for 10 min. After centrifugation, the supernatant was obtained as a whole-cell lysate. The protein concentration in each whole-cell lysate was quantified with a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Lysate proteins mixed with equal volumes of sample loading buffers were resolved using 12.5% SDS-PAGE. The proteins next were transferred to a nitrocellulose membrane. We confirmed that equivalent quantities of proteins were loaded for each sample using Coomassie brilliant blue staining of a duplicate SDS-PAGE gel. After blocking with 2% skim milk in Tris-buffered saline (TBS; 20 mM Tris, 137 mM NaCl [pH 8.0]) containing 0.05% Tween 20 (TBS-T), the membrane was incubated with rSrtF-specific antiserum (diluted 1:1,000 in 1% skim milk in TBS-T) for 1 h at 37°C. The membrane was washed with TBS-T and was incubated with horseradish peroxidase-conjugated anti-mouse IgG (diluted 1:1,000 in TBS-T) (Promega, Madison, WI, USA) for 1 h at 37°C. The membrane was then washed 5 times with TBS-T. The reaction of the protein band to the antiserum was detected using a chemiluminescence detection system (PerkinElmer, Waltham, MA, USA).

Coculture of S. pyogenes with L. lactis.Coculture assays were performed using previously described methods (14). Briefly, aliquots (105) of overnight cultures of S. pyogenes C-1 (Kmr), S. pyogenes ΔsrtR (Spcr), ΔsrtRK (Spcr), ΔsrtR-comp (Spcr/Kmr), and ΔsrtRK-comp (Spcr/Kmr) strains and L. lactis ATCC 11454 and L. lactis NZ9000 were inoculated into BHIYE and grown to an OD660 of 0.5. For coculture assays, appropriate numbers of S. pyogenes (105) and L. lactis (ATCC 11454 or NZ9000; 105) cells were spotted on BHIYE agar plates and grown at 37°C with 5% CO2 for 8 h. Optimal ratios for the coculture assays were determined with preliminary experiments that investigated the effects of various ratios of L. lactis on the growth of S. pyogenes. Appropriate dilutions were plated on BHIYE agar plates with or without 100 μg/ml of Spc or Km for the selection of S. pyogenes. After 2 days, the numbers of CFU on BHIYE agar plates with and without Spc or Km were determined, and the percentage of the population represented by the S. pyogenes strain was calculated.

RESULTS

Susceptibility of TCS-inactivated mutants to bacteriocins.We first examined each of the 13 S. pyogenes TCS mutants for susceptibility to the class I lantibiotics nisin A, nukacin ISK-1 (20), and staphylococcin C55 bacteriocin (24) using the direct method (Table 2). The srtK (Spy_1082) mutant (ΔTCS6) exhibited increased susceptibility to nisin A compared with that of the wild-type SF370 strain, but the susceptibility of the srtK (Spy_1082) mutant to the other two bacteriocins was not altered, similar to the other TCS mutants. We also determined the MICs of nisin A for the mutants with the ΔsrtR or ΔsrtRK alteration in SF370 and obtained results similar to those for mutants with ΔsrtK (Fig. 1AB and Table 2). The MICs of nisin A against the wild-type and ΔsrtK strains were 2 and 0.5 μg/ml, respectively. To determine whether this effect applies to other strains, we constructed the srtK mutant in S. pyogenes 1529 strain (ΔsrtK-1529) and found that this mutant also exhibited increased susceptibility to nisin A (Table 3). Furthermore, we constructed complemented strains in each mutant and found that the MICs of the complemented strains were similar to that of the wild type (Fig. 1A and B and Table 3).

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TABLE 2

Susceptibility to lantibiotics in S. pyogenes mutants

FIG 1
FIG 1

Susceptibility of S. pyogenes strains to nisin A. (A) Image of the direct assay. The susceptibility of the S. pyogenes SF370 strain and mutant strains to nisin A produced by L. lactis ATCC 11454 was evaluated using a direct method, as described in Materials and Methods. Overnight cultures of L. lactis ATCC 11454 were stabbed on BHI agar plates. After 16 h of incubation at 37°C with 5% CO2, prewarmed BHIYE soft agar (0.8%) containing individual S. pyogenes strains was poured over the surface of the BHI agar plates. The plates were then incubated for 20 h at 37°C with 5% CO2. White bars represent 3 mm. (B) Diameter of direct assays. Three independent experiments were performed, and the data are shown as the means ± standard errors of the means (SEM) from triplicate determinations. Statistical significance versus the corresponding SF370 or 1529 strain was assessed by Dunnett's post hoc test or Student's t test. *, P < 0.05 (relative to SF370); #, P < 0.05 (relative to 1529).

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TABLE 3

Susceptibility to nisin A in S. pyogenes mutants

Effects of nisin A on the expression of srtRK and its neighboring genes.Because the srtRK mutant exhibited increased susceptibility to nisin A, we investigated the effects of nisin A on the expression of open reading frames (ORFs) neighboring srtRK. Treatment with nisin A did not affect the expression of srtI (Fig. 2), whereas srtA, srtT, and srtFEG expression was increased with 1/8 MIC (0.25 μg/ml) of nisin A (Fig. 2). Since srtF was homologous to nisF, which has been reported to be one of the ABC transporter clusters of nisFEG and plays a role in self-immunity against nisin A-producing L. lactis (25), we searched for other genes homologous to NisF. Although seven genes, including srtF, were identified in the search for homologous genes in the SF370 genome database (see Table S2 in the supplemental material), the addition of nisin A did not increase the expression of any of these genes, except srtF (see Fig. S1 in the supplemental material). We next investigated whether srtF expression was induced at various concentrations of nisin A. srtF expression was induced by nisin A concentrations greater than 1/32 MIC (Fig. 3).

FIG 2
FIG 2

Expression of the srt genes with or without nisin A. Aliquots of cells (105 cells) were inoculated into fresh BHIYE medium and incubated at 37°C with 5% CO2 until the early exponential phase, and then 1/8 MIC (0.25 μg/ml) nisin A was added. After 15 min of incubation, bacterial cells were collected for mRNA extraction. Using cDNA, mRNA expression levels of srt genes were evaluated using quantitative PCR as described in Materials and Methods. Three independent experiments were performed, and the data are shown as the means ± SEM from triplicate determinations. Simple comparisons of the means and SEM of individual gene expression with or without the addition of nisin A to the SF370 wild type were performed using Student's t test. *, P < 0.005.

FIG 3
FIG 3

Dose-dependent induction of srtF expression by nisin A. Various concentrations of nisin A were added to the SF370 strain during the early exponential phase, and the samples were incubated for 15 min at 37°C with 5% CO2. After harvesting the bacterial cells, mRNA was extracted and cDNA was synthesized as described in Materials and Methods. Three independent experiments were performed, and the data are shown as the means ± SEM from triplicate determinations. Statistical significance versus the corresponding data without the addition of nisin A was assessed by Dunnett's post hoc test. *, P < 0.05; **, P < 0.01.

We also analyzed the transcription of the srt locus. From the results of RT-PCR, three transcripts (srtI, srtRK, and srtA-srtG) were identified (Fig. 4). We then further investigated the transcriptional start sites of srtR, srtA, and srtF. Although srtA to srtG formed one transcript by RT-PCR analysis, we examined the transcriptional start site of srtF because we found an 81-bp intergenic region between srtT′ and srtF. The transcriptional start sites of srtR and srtA were 132 bp and 43 bp upstream of the coding region, respectively. However, we found no transcriptional start site for srtF in the consensus region (Fig. 4).

FIG 4
FIG 4

RT-PCR analysis of srt region. RT-PCR analysis was performed using SF370 wild-type strain with 0.1 μg/ml nisin A. Lanes: M, size marker; 1, srtI-R; 2, srtR-K; 3, srtK-A; 4, srtA-T; 5, srtT-F; 6, srtF-E; 7, srtE-G. RT(+), with transcriptase; RT(−), no transcriptase. The lines of individual genes indicate the PCR amplification regions by RT-PCR. Gray arrows indicate the transcriptional start site (−132 bp or −43 bp from the coding region of srtR or srtA, respectively) by RACE.

Susceptibility of srtFEG deletion mutants to nisin A.To identify the srtFEG genes, which encode an ABC transporter, that are directly involved in the susceptibility to nisin A, we constructed an srtFEG deletion (ΔsrtFEG) mutant and its complemented (ΔsrtFEG-comp) strain. We then investigated the susceptibility of these mutants to nisin A and observed that the ΔsrtFEG mutant exhibited increased susceptibility, and its complement strain recovered to the same level of susceptibility as that of the wild type (Fig. 1A and B and Table 3).

Regulation of the expression of srtEFG by SrtRK.Because nisin A susceptibility was altered by inactivation of the ABC transporter srtFEG, we employed quantitative PCR and immunoblot analysis to further investigate SrtRK-regulated expression of the srtFEG genes following nisin A treatment. Quantitative PCR analysis revealed that nisin A induced a greater than 10-fold upregulation of srtF expression in wild-type SF370 cells, but this induction was not observed in ΔsrtR and ΔsrtRK cells. However, the induction was restored in the ΔsrtR-comp and ΔsrtRK-comp strains (Fig. 5A). These results were also confirmed by immunoblot analysis of the SrtF protein in wild-type SF370 and its mutants (Fig. 5B). The data showed that nisin A induced srtF expression via the SrtRK TCS in the wild-type strain.

FIG 5
FIG 5

Expression of SrtF. Protein and mRNA expression levels of SrtF were evaluated by quantitative PCR and immunoblotting, as described in Materials and Methods. (A) Quantitative analysis of srtF expression in SF370, ΔsrtR, ΔsrtRK, ΔsrtR-comp, and ΔsrtRK-comp strains following treatment with 1/8 MIC (0.25 μg/ml) of nisin A. Three independent experiments were performed, and the data are shown as the means ± SEM from triplicate determinations. Statistical significance was determined versus the corresponding data from wild-type strain SF370 using Dunnett's post hoc test. *, P < 0.05; **, P < 0.005. (B) Immunoblot analysis of SrtF expression following treatment of SF370, ΔsrtR, ΔsrtRK, ΔsrtR-comp, and ΔsrtRK-comp cells with 1/8 MIC (0.25 μg/ml) of nisin A. The ΔsrtFEG strain was used as a negative control.

Coculture of S. pyogenes with L. lactis.To determine whether bacteriocins had a function in survival in mixed live bacteria, coculture assays were performed. First, we investigated the effect of nisin A on the proportion of S. pyogenes SF370 by coculturing two bacterial strains at different ratios. Wild-type S. pyogenes SF370 with a Km resistance gene (C-1) was cocultured with nisin A-producing L. lactis ATCC 11454 or nonproducing L. lactis NZ9000. As shown in Fig. 6A, the total population of S. pyogenes SF370 cocultured with L. lactis ATCC 11454 (L. lactis/S. pyogenes = 100, 10, 1, or 0.1) was higher than that with L. lactis NZ9000. When wild-type S. pyogenes SF370 cells were cocultured with various ratios of L. lactis ATCC 11454, S. pyogenes comprised 0.5% (L. lactis/S. pyogenes = 100), 8.7% (L. lactis/S. pyogenes = 10), 38.4% (L. lactis/S. pyogenes = 1), 72.6% (L. lactis/S. pyogenes = 0.1), and 99.1% (L. lactis/S. pyogenes = 0.01) of the total population (Fig. 6A, left). However, when wild-type S. pyogenes cells were cocultured with L. lactis NZ9000, a strain that does not produce nisin A, S. pyogenes comprised 5.2% (L. lactis/S. pyogenes = 100), 34.4% (L. lactis/S. pyogenes = 10), 56.7% (L. lactis/S. pyogenes = 1), 100% (L. lactis/S. pyogenes = 0.1), and 100% (L. lactis/S. pyogenes = 0.01) of the total cell population (Fig. 6A, right).

FIG 6
FIG 6

Coculture of S. pyogenes with L. lactis. The coculture assay is described in Materials and Methods. (A) Percentage of the total population represented by S. pyogenes when various densities of S. pyogenes SF370 (C-1) cells were mixed with L. lactis ATCC 11454 cells (left) and L. lactis NZ9000 cells that did not produce nisin A (right). Three independent experiments were performed, and data shown are the means ± SEM from triplicate determinations. (B) Populations of S. pyogenes SF370 (C-1), ΔsrtR, ΔsrtRK, ΔsrtR-comp, and ΔsrtRK-comp strains were determined after L. lactis cells were cocultured. Three independent experiments were performed, and data are shown as the means ± SEM from triplicate determinations. P < 0.01 (*) and P < 0.001 (**) as determined by Tukey's honestly significant difference for the percentage of the S. pyogenes population.

We then investigated the effect of SrtRK on the survival of S. pyogenes colocalized with nisin A-producing strains using coculture assays with various srtRK mutants. The left part of Fig. 6B shows the ratios of S. pyogenes populations when 5 × 106 S. pyogenes SF370 (C-1) and the mutant cells were cocultured with 5 × 106 L. lactis cells (nisin A-producing strain). The ΔsrtR (S. pyogenes proportion, 7.8%) and the ΔsrtRK (14.3%) strains exhibited dramatically decreased population ratios than the wild type (55.4%) when cocultured with the nisin A-producing strain (L. lactis ATCC 11454). In contrast, when these mutants were cocultured with the L. lactis strain that did not produce nisin A, the proportion of each mutant increased (Fig. 6B, right). The proportion of the srtRK or srtR complemented strain was the same as that of the wild type when the mutants were cocultured with the nisin A-producing strain (L. lactis ATCC 11454).

DISCUSSION

In a comprehensive analysis of the involvement of TCSs in susceptibility to a range of lantibiotics, we found that SrtRK was involved in susceptibility to nisin A in the S. pyogenes SF370 strain. Additionally, we found that srtEFG, which is downstream of srtRK, was directly involved in susceptibility to nisin A, and its expression was controlled by SrtRK. Figure 7 shows a map of the genes involved in lantibiotic synthesis and immunity; these genes are homologous to srtRK and srtEFG in 3 bacterial species. In L. lactis, biosynthesis and immunity proteins of nisin A are encoded by nisABTCP and nisIFEG, respectively (Fig. 7) (25, 26). The protein encoded by nisA is involved in the nisin A peptide, whereas the other lantibiotic biosynthetic proteins, encoded by the nisBC and the nisTP genes, are involved in prelantibiotic modification reactions and in the translocation process of lantibiotics, respectively (25). The proteins encoded by nisI and nisFEG have been implicated in resistance against lantibiotics by extracellular processing of a fully matured precursor nisin A or efflux of nisin A (25, 27). NisRK is a TCS that regulates the gene expression of biosynthesis and self-immunity and consists of nis clusters (25, 28). Similar to the nis clusters, the biosynthesis, immunity, and regulatory systems of lantibiotics are widely present among type AI lantibiotic-producing bacteria, including Bacillus subtilis (subtilin) and S. pyogenes (streptin) (Fig. 7; see also Table S3 in the supplemental material) (29, 30). Furthermore, HKs are classified into three major groups based on the architecture of the N-terminal region (31). SrtK is the prototypical periplasmic-sensing HK, which is composed of two transmembrane regions with an intervening extracytoplasmic domain that detects external signals (see Fig. S3A and C). NisK and SpaK also belong to this group (see Fig. S3A and C). Two other groups are HKs with more than 2 transmembrane regions that lack an apparent sensory domain and HKs that have a cytoplasmic sensory domain. SrtR is also homologous to NisR and SpaR, possessing the 2 most conserved segments (see Fig. S3B and C). Interestingly, the neighboring region of srtRK in the S. pyogenes SF370 strain is similar to that of the S. pyogenes BL-T strain, although the SF370 strain lacks srtC and possesses partial srtT and srtB genes and also has srtT′ (Fig. 7). SrtB and SrtC are dehydratases (LanB) and cyclases (LanC), respectively, and SrtT is involved in the translocation of lantibiotics (29). We confirmed that no bacteriocins were produced by the S. pyogenes SF370 strain by direct analysis using the bacteriocin-susceptible Bacillus coagulans (data not shown). This may be because the SF370 strain lacks the function of SrtTBC and is believed to be unable to produce lantibiotics. Therefore, we assumed that gene deletion of the srt gene cluster in the SF370 strain promoted a loss of bacteriocin-producing ability; however, genes encoding TCSs and immunity factors against lantibiotics are still maintained.

FIG 7
FIG 7

ORFs related to streptin in S. pyogenes and synteny of streptin loci in L. lactis and Bacillus subtilis. The organization of genes around the individual TCSs was deduced from the complete genome sequences retrieved from NCBI. Diagonal pattern, TCS; black, export and processing of lantibiotics and ABC transporter (putative immunity); white, lantibiotics; gray, others; dotted-line border, gene that does not exist on the SF370 genome. The base numbers of srtT and srtB refer to the S. pyogenes BL-T strain. The numbers of srtT′ indicate the base numbers of the SF370 strain.

In this study, we demonstrated the contribution of SrtEFG to the susceptibility to nisin A. This finding suggests that the bacteriocin immunity system functions independently of the export and processing system, which is systematically regulated by the TCS in SF370. In this study, we also evaluated the susceptibility of S. pyogenes 1529 to nisin A. The 1529 strain does not produce bacteriocins (data not shown). From the results of genomic PCR analysis, the 1529 strain has the same srt gene cluster as the SF370 strain (data not shown). Thus, the 1529 strain may lack the function of SrtTBC but still possesses resistance to nisin A due to SrtFEG (Fig. 1B and Table 3). We previously reported that the non-lantibiotic-producing S. mutans UA159 strain and the S. aureus MW2 strain are resistant to nisin A produced by L. lactis via TCSs (12, 14). Both bacteria also have resistance proteins, which are similar to the lantibiotic self-immunity proteins of type AI lantibiotic-producing bacteria that are regulated by TCSs. However, there are no genes involved in lantibiotic synthesis neighboring the TCS and immunity genes in S. mutans UA159 and S. aureus MW2.

SrtRK induced srtFEG expression with nisin A in this study. srtFEG expression was gradually increased in a dose-dependent manner, and the SrtRK TCS was active at low concentrations. This phenomenon indicates that coexistence between bacteriocin-producing and nonproducer bacteria is possible. Based on evaluations of srt gene expression using wild-type and srtRK knockout strains, SrtRK regulates srtA (encoding a prepeptide), srtT (encoding the ABC transporter), and srtFEG, but srtI expression was not regulated by SrtRK (Fig. 5; see also Fig. S2 in the supplemental material). Previously, NisI was shown to contribute to self-immunity by cooperating with NisFEG in L. lactis (32, 33). However, srtI expression was not regulated by srtRK, suggesting that srtI was not involved in nisin susceptibility via srtRK in S. pyogenes (see Fig. S2). Based on the RT-PCR and the RACE results, srtATFEG was cotranscribed (Fig. 4 and 5). Because the nisin-responsive promoter elements, such as nisA, nisF, spaB, spaI, and spaS, contain a pentanucleotide direct repeat (PDR; TCTGA or TTGAT) separated by 6 nucleotides (34), we looked for the promoter elements in srtA to strG and found the PDR sequences (TCTGA) upstream of srtA (Fig. 5 and 8). We also found PDR sequences upstream of srtF with one mismatch, but the PDR in srtF (TTGAT) was not identical to that of srtA, and the distance between PDRs is 7 nucleotides. These results suggested that SrtRK regulates the expression of srtA to srtG together.

FIG 8
FIG 8

Sequence alignment of srtA, nisA, nisF, spaB, spaI, and spaF promoter regions. Previously determined transcription start positions (34) are indicated by an arrow and boldfaced letter, and transcriptional start sites of srtA are indicated by an asterisk and a boldfaced letter. The −35 and −10 regions are underlined. The PDR sequences are indicated by a gray background.

In the coculture assays, nisin A produced by L. lactis affected the survival ratio of S. pyogenes, whereas SrtRK of S. pyogenes maintains the proportion of S. pyogenes when S. pyogenes coexists with nisin-producing strains. Several bacteria have been reported to produce type AI lantibiotics similar to nisin A. These bacteria are present in the pharynx and oral cavity (35). Therefore, S. pyogenes may utilize SrtRK for coexistence with commensal bacteria in the pharynx or oral cavity. In conclusion, we found that one of the TCSs (SrtRK) regulates the ABC transporter (SrtFEG) involved in susceptibility to nisin A in S. pyogenes SF370. These results highlight the important roles of TCSs in the interactions between different bacterial species that produce bacteriocins.

ACKNOWLEDGMENT

This study was supported in part by a grant-in-aid (15K11017) from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan.

FOOTNOTES

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