A Markerless Gene Deletion System in Streptococcus suis by Using the Copper-Inducible Vibrio parahaemolyticus YoeB Toxin as a Counterselectable Marker

Streptococcus suis is an important zoonotic pathogen causing severe infections in swine and humans. Induction of the Vibrio parahaemolyticus YoeB toxin in Escherichia coli resulted in cell death, leading to the speculation that YoeBVp can be a counterselectable marker. Herein, the counterselection potential of YoeBVp was assessed in S. suis. The yoeBVp gene was placed under the copper-induced promoter PcopA. The PcopA-yoeBVp construct was cloned into the S. suis-E. coli shuttle vector pSET2 and introduced into S. suis to assess the effect of YoeBVp expression on S. suis growth. Reverse transcription quantitative PCR showed that copper induced yoeBVp expression. Growth curve analyses and spot dilution assays showed that YoeBVp expression inhibited S. suis growth both in liquid media and on agar plates, revealing that YoeBVp has the potential to be a counterselectable marker for S. suis. A SCIY cassette comprising the spectinomycin-resistance gene and copper-induced yoeBVp was constructed. Using the SCIY cassette and peptide-induced competence, a novel two-step markerless gene deletion method was established for S. suis. Moreover, using the ΔperR mutant generated by this method, we demonstrated that PmtA, a ferrous iron and cobalt efflux pump in S. suis, was negatively regulated by the PerR regulator.


Introduction
Streptococcus suis is a Gram-positive, facultative anaerobe that threatens the swine industry and public health worldwide [1]. It is responsible for various swine diseases, including meningitis, septicemia, pneumonia, endocarditis, and arthritis [2]. Generally, S. suis is considered one of the most important bacterial pathogens that lead to significant economic losses to the swine industry [3]. Indeed, a recent survey revealed that its isolation rate was 16.9%, ranking first among the bacterial pathogens isolated from Chinese pig farms from 2013 to 2017 [4]. More seriously, S. suis can be transmitted to humans by minor skin injuries or the gastrointestinal tract, causing meningitis, streptococcal toxic shock-like syndrome, and some other clinical symptoms [5]. In 1968, the first human case of S. suis infection was described in Denmark; since then, over 1600 human cases have been reported worldwide by the end of 2013, some of which were fatal [6]. Remarkably, two great outbreaks of human S. suis infections occurred in China in 1998 and 2005, resulting in 25 cases with 14 deaths and 215 cases with 39 deaths, respectively [7,8]. In recent years, S. suis still frequently caused sporadic human cases worldwide [9][10][11][12][13][14].
Over the past few decades, significant progress has been made toward understanding the physiology and pathogenesis of S. suis. A number of virulence-related factors have been described in S. suis [15,16]. Recently, in vivo transcriptomes and transposon mutant libraries have been applied to identify genes involved in the virulence traits of S. suis [17][18][19]. Usually, studies related to the physiology and pathogenesis of S. suis rely on gene deletion mutants. In S. suis, the most frequently used gene deletion system is the pSET4s thermosensitive suicide vector [20]. For gene deletion using pSET4s, a knockout vector is constructed and introduced into the wild-type (WT) S. suis strain by electroporation; subsequently, the mutant is selected after two steps of allelic exchange. As this system contains no counterselectable marker, the mutant must be picked out from many potential colonies. In addition, electrotransformation does not work well for certain S. suis isolates [21]. Except for allelic exchange using the pSET4s vector, a cloning-independent method employing peptide-induced competence has been established in S. suis [22]. This method allows high-throughput mutation; however, the mutant carries a spectinomycin resistance gene, limiting its vaccine potential. Only recently, Zhu et al. developed a markerless gene deletion method in S. suis Chz serotype with the utilization of the ComRS system and sucrose sensitivity [21].
Toxin-antitoxin (TA) systems are small genetic modules widely distributed in the plasmids or chromosomes of bacteria and archaea [23]. Typically, they are composed of a gene encoding a stable toxin and a gene encoding an unstable antitoxin [24,25]. Under stress conditions, toxins are released from the TA complex and target various cellular functions to inhibit cell growth, making them valuable for counterselection [26][27][28][29][30]. In a previous study, we identified a chromosomal type II toxin-antitoxin system, YefM-YoeB, in Vibrio parahaemolyticus; induction of YoeB Vp in Escherichia coli resulted in cell death [31]. This result has led us to speculate that YoeB Vp can be a counterselectable marker for S. suis.
In this study, the YoeB Vp toxin was tested for the counterselection potential in S. suis. Using YoeB Vp as a counterselectable marker, we successfully established a novel two-step markerless gene deletion method for S. suis. Finally, using the ∆perR mutant generated by this method, we demonstrated that pmtA, a gene encoding a ferrous iron and cobalt efflux pump in S. suis [32] was negatively regulated by the PerR regulator.

Bacterial Strains, Plasmids, Primers, and Culture Conditions
Bacterial strains and plasmids used in this study are listed in Table 1. All primers are listed in Table S1. Unless otherwise specified, S. suis strains were cultured at 37 • C in Tryptic Soy Broth (TSB) or on Tryptic Soy Agar (TSA; Becton, Dickinson and Company, Suzhou, China) supplemented with 5% (vol/vol) newborn bovine serum (Sijiqing, Hangzhou, China). E. coli strains were grown in Luria-Bertani (LB) broth or on LB agar. When required, spectinomycin was added to the medium at 50 µg/mL for E. coli and 100 µg/mL for S. suis.

Preparationof Synthetic Peptide and Natural Transformation Experiment
The peptide (GNWGTWVEE) was synthesized by Sangon Biotech (Shanghai, China) at 90-95% purity. It was dissolved in deionized water at a final concentration of 5 mM, divided into aliquots of 50 µL, and stored at −80 • C.
A natural transformation experiment was performed as previously described [22], with slight modifications. Overnight culture of S. suis was diluted 1:100 in fresh medium and grown to an OD 600 of 0.035-0.05 (about 1-2 h). Next, a 100 µL sample was removed from the culture; 5 µL of the peptide and 1.2 µg of DNA (plasmid or PCR products) were added to the sample. Following 2 h of incubation, the sample was plated on agar plates containing spectinomycin or diluted in fresh media containing 0.5 mM CuSO 4 .

Construction of a S. suis Strain Expressing the Copper-Inducible YoeB Vp Toxin
The promoter PcopA was amplified from the S. suis SC19 genome using primers PcopA-F/PcopA-R. The DNA fragment containing yoeB Vp and its terminator was amplified from V. parahaemolyticus RIMD 2210633 genome using primers yoeB Vp -F/yoeB Vp -R. The two DNA fragments were fused into a fragment using overlap PCR with primers PcopA-F/yoeB Vp -R. Following digestion with the BamH I and EcoR I enzymes, the fused DNA fragment was cloned into the pSET2 vector, to generate pSET2-PcopA-yoeB Vp . Next, the vector was introduced into the S. suis SC19 strain by natural transformation. The resultant strain, SC19/pSET2-PcopA-yoeB Vp was confirmed by PCR, DNA sequencing, and reverse transcription quantitative PCR (RT-qPCR).

RNA Extraction
The SC19/pSET2-PcopA-yoeB Vp strain was grown to an OD 600 of 0.6-0.8 and divided into four aliquots of 1 mL, which were supplemented with deionized water or CuSO 4 at final concentrations of 0.1 mM, 0.2 mM, or 0.5 mM. After 15 min of incubation, bacterial cells were collected and subjected to RNA extraction using an Eastep Super Total RNA Isolation Kit (Promega, Shanghai, China). RNA was evaluated for integrity by gel electrophoresis and determined for concentration using a Nanodrop 200.
In another assay, the WT and ∆perR strains were grown to an OD 600 of 0.6-0.8; each strain was then divided into four aliquots of 1 mL. Three of the aliquots were supplemented with 1 mM FeSO 4 , 1 mM CoSO 4 , and 1 mM NiSO 4 , respectively; the remaining aliquot was supplemented with deionized water. After 15 min of incubation, bacterial cells were collected for RNA extraction.

RT-qPCR Analysis
cDNA was generated from approximately 0.2 µg of RNA using the NovoScript Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge) (novoprotein, Shanghai, China). Quantitative PCR was performed using NovoStart SYBR qPCR SuperMix Plus (novoprotein, Shanghai, China) and the specific primers listed in Table S1. The reaction mixture was as follows: 2×NovoStart SYBR qPCR SuperMix Plus 10 µL, each primer 0.5 µM, 10-fold diluted cDNA 1 µL, ROX 0.4 µL, and finally RNase-free water added to 20 µL. Quantitative PCR was conducted on the StepOnePlus Real-Time PCR System (Applied Biosystems). The procedure was 95 • C for 1 min, followed by 40 cycles of 95 • C Microorganisms 2021, 9, 1095 4 of 12 for 20 s, and 60 • C for 1 min. A melting curve analysis (starting from 60 • C and continuing to 95 • C, with 0.3 • C increments for 5 s each) was performed to verify the specificity of the products. The amplification efficiency of each primer pair was determined using serially diluted genomic DNA as the template. The gene expression level was calculated using the 2 −∆∆CT method [35], with 16S rRNA as the reference gene.

Growth Curves Analyses
Overnight cultures of the SC19/pSET2-PcopA-yoeB Vp and SC19/pSET2 strains were diluted in fresh medium and grown to an OD 600 of approximately 0.3. Next, each culture was divided into five aliquots (1 mL per aliquot), to which CuSO 4 was added at final concentrations of 0, 0.05, 0.1, 0.2, and 0.5 mM, respectively. Each aliquot was sub-packed in triplicate in 96-well plates (200 µL/well) and cultured at 37 • C for 6 h. The OD 595 values were measured hourly using the CMax Plus plate reader (Molecular Devices, Shanghai, China).

Spot Dilution Assays
Overnight cultures of the SC19/pSET2-PcopA-yoeB Vp and SC19/pSET2 strains were diluted in fresh medium and grown to an OD 600 of approximately 0.6. Next, each culture was serially diluted 10-fold up to 10 −5 dilution, and 5 µl of each dilution was spotted onto the plates supplemented with varying concentrations of CuSO 4 (0, 0.1, 0.2, and 0.5 mM). The plates were photographically documented following 18 h of incubation at 37 • C.

Construction of the SCIY Positive-Negative Selectable Cassette
The spectinomycin-resistance gene was amplified from pSET2 using primers spc-F/spc-R. The PcopA-yoeB Vp construct was amplified from pSET2-PcopA-yoeB Vp using primers PcopA-yoeB Vp -F/PcopA-yoeB Vp -R. The two DNA fragments were fused into a fragment using overlap PCR with primers spc-F/PcopA-yoeB Vp -R. The fused DNA fragment was confirmed by DNA sequencing, and designated SCIY.

Construction of Markerless Gene Deletion Mutants Using the SCIY Cassette
The ∆pmtA mutant was constructed using the SCIY cassette via a two-step procedure. For the first step, the left and right arms of pmtA were amplified from S. suis SC19 genome using primer pairs pmtA-LA-F/pmtA-Fir-LA-R and pmtA-Fir-RA-F/pmtA-RA-R, respectively. The SCIY cassette was amplified using primers pmtA-SCIY-F/pmtA-SCIY-R. The three DNA fragments were fused into a fragment using overlap PCR with primers pmtA-LA-F/pmtA-RA-R. The fused DNA fragment was transformed into S. suis SC19 by natural transformation. The spectinomycin-resistant colonies were selected, confirmed by PCR, and designated the intermediate strain. For the second step, the left and right arms of pmtA were amplified from the S. suis SC19 genome using primer pairs pmtA-LA-F/pmtA-Sec-LA-R and pmtA-Sec-RA-F/pmtA-RA-R, respectively. The two DNA fragments were fused into a fragment using overlap PCR with primers pmtA-LA-F/pmtA-RA-R. The fused DNA fragment was transformed into the intermediate strain by natural transformation. Following 2 h of incubation, the sample was diluted 1:100 in fresh medium containing 0.5 mM CuSO 4 and cultured at 37 • C for another 12 h. In total, the culture was repeatedly diluted three to five times for enrichment of the mutant. After each incubation, the culture was diluted and plated on agar plates. One hundred colonies were tested for spectinomycinsensitivity. Spectinomycin-sensitive colonies were selected, and the absence of pmtA was confirmed by PCR using primer pairs pmtA-in-F/pmtA-in-R and pmtA-out-F/pmtA-out-R. The efficiency of the SCIY cassette for counterselection was evaluated as the proportion of spectinomycin-sensitive colonies. The ∆perR and ∆lysR mutants were constructed using the same procedure to verify the method.

Identification of the S. suis Strain Expressing the Copper-Inducible YoeB Vp Toxin
To evaluate the effect of YoeB Vp induction on S. suis growth, we constructed a S. suis strain expressing the copper-inducible YoeB Vp toxin using the PcopA promoter and pSET2 vector [34,36]. The strain, termed SC19/pSET2-PcopA-yoeB Vp , was identified by PCR ( Figure 1A) and DNA sequencing (data not shown). RT-qPCR analysis was also performed to detect whether copper can induce yoeB Vp expression. As shown in Figure 1B, the expression of yoeB Vp was significantly induced by copper, and the inductive effects increased with increasing copper concentrations. structed using the same procedure to verify the method.

Identification of the S. suis Strain Expressing the Copper-Inducible YoeB
To evaluate the effect of YoeBVp induction on S. suis growth, we co strain expressing the copper-inducible YoeBVp toxin using the PcopA pr vector [34,36]. The strain, termed SC19/pSET2-PcopA-yoeBVp, was ident ure 1A) and DNA sequencing (data not shown). RT-qPCR analysis was detect whether copper can induce yoeBVp expression. As shown in Figu sion of yoeBVp was significantly induced by copper, and the inductiv with increasing copper concentrations.

YoeBVp Expression Results in Growth Defect in S. suis
The SC19/pSET2-PcopA-yoeB Vp and SC19/pSET2 strains were grown in fresh media containing various concentrations of CuSO 4 , and their growth curves were measured. As shown in Figure 2A, the two strains exhibited similar growth in the absence of CuSO 4 . However, when supplemented with CuSO 4 , the SC19/pSET2-PcopA-yoeB Vp strain displayed a remarkable growth defect compared with the SC19/pSET2 strain ( Figure 2B-D).
The effect of YoeBVp expression on S. suis growth was also detected on agar plates. In the absence of CuSO4, the two strains formed colonies of equal sizes (Figure 3). However, in the presence of CuSO4, the SC19/pSET2-PcopA-yoeBVp strain formed colonies of smaller sizes than the SC19/pSET2 strain (Figure 3).
Taken together, YoeBVp expression in S. suis led to growth inhibition both in liquid media and on agar plates. Thus, YoeBVp has the potential to be a counterselectable marker for S. suis.  The effect of YoeB Vp expression on S. suis growth was also detected on agar plates. In the absence of CuSO 4 , the two strains formed colonies of equal sizes (Figure 3). However, in the presence of CuSO 4 , the SC19/pSET2-PcopA-yoeB Vp strain formed colonies of smaller sizes than the SC19/pSET2 strain (Figure 3).

Establishment of the Cloning-Independent and Counterselectable Markerless Gene Deletion System in S. suis
The spectinomycin-resistance gene and PcopA-yoeBVp construct were combined to generate the SCIY cassette, which was further used for markerless gene deletion in S. suis. The strategy for markerless gene deletion in S. suis using the SCIY cassette is shown in Figure 4. In the first step, an intermediate strain was generated, in which the SCIY cassette Taken together, YoeB Vp expression in S. suis led to growth inhibition both in liquid media and on agar plates. Thus, YoeB Vp has the potential to be a counterselectable marker for S. suis.

Establishment of the Cloning-Independent and Counterselectable Markerless Gene Deletion System in S. suis
The spectinomycin-resistance gene and PcopA-yoeB Vp construct were combined to generate the SCIY cassette, which was further used for markerless gene deletion in S. suis. The strategy for markerless gene deletion in S. suis using the SCIY cassette is shown in Figure 4. In the first step, an intermediate strain was generated, in which the SCIY cassette replaced the target gene. As the SCIY cassette contains the spectinomycin-resistance gene, the intermediate strain could be selected with spectinomycin. In the second step, the markerless gene deletion mutant was generated. The intermediate strain contains the PcopA-yoeB Vp construct; thus, its growth was inhibited in the presence of copper. However, the mutant could grow well in the presence of copper. After three to five dilutions in media supplemented with copper, the mutant was enriched to be easily isolated.

Markerless Deletion of the pmtA, perR, and lysR Genes in S. suis
To assess whether the strategy is effective, we firstly constructed a markerless deletion mutant of the pmtA gene. As seen in Figure 5A, PCR amplification of the ΔpmtA mutant using primers pmtA-in-F/pmtA-in-R generated no products, whereas amplification of the WT strain generated products with expected sizes (755 bp). Furthermore, PCR amplification of ΔpmtA and the WT strain using primers pmtA-out-F/pmtA-out-R generated products with expected sizes for ΔpmtA (2472 bp) and the WT strain (4199 bp), respectively ( Figure 5A). DNA sequencing confirmed that the pmtA gene was successfully deleted in the ΔpmtA mutant. To further verify the strategy, markerless deletion mutants of

Markerless Deletion of the pmtA, perR, and lysR Genes in S. suis
To assess whether the strategy is effective, we firstly constructed a markerless deletion mutant of the pmtA gene. As seen in Figure 5A, PCR amplification of the ∆pmtA mutant using primers pmtA-in-F/pmtA-in-R generated no products, whereas amplification of the WT strain generated products with expected sizes (755 bp). Furthermore, PCR amplification of ∆pmtA and the WT strain using primers pmtA-out-F/pmtA-out-R generated products with expected sizes for ∆pmtA (2472 bp) and the WT strain (4199 bp), respectively ( Figure 5A). DNA sequencing confirmed that the pmtA gene was successfully deleted in the ∆pmtA mutant. To further verify the strategy, markerless deletion mutants of the perR ( Figure 5B) and lysR genes ( Figure 5C) were also constructed. Overall, the two-step strategy applying the SCIY cassette is effective in markerless gene deletion in S. suis.

Markerless Deletion of the pmtA, perR, and lysR Genes in S. suis
To assess whether the strategy is effective, we firstly constructed a markerless de tion mutant of the pmtA gene. As seen in Figure 5A, PCR amplification of the ΔpmtA m tant using primers pmtA-in-F/pmtA-in-R generated no products, whereas amplification the WT strain generated products with expected sizes (755 bp). Furthermore, PCR amp fication of ΔpmtA and the WT strain using primers pmtA-out-F/pmtA-out-R generat products with expected sizes for ΔpmtA (2472 bp) and the WT strain (4199 bp), resp tively ( Figure 5A). DNA sequencing confirmed that the pmtA gene was successfully d leted in the ΔpmtA mutant. To further verify the strategy, markerless deletion mutants the perR ( Figure 5B) and lysR genes ( Figure 5C) were also constructed. Overall, the tw step strategy applying the SCIY cassette is effective in markerless gene deletion in S. su DNA markers, respectively. Lanes 3 and 5 indicate PCR amplification of the mutants using the primer pairs in-F/in-R and out-F/out-R, respectively. Lanes 4 and 6 indicate PCR amplification of the WT strain using the primer pairs in-F/in-R and out-F/out-R, respectively.

The SCIY Cassette Is Highly Efficient for Counterselection in S. suis
The proportion of the mutant after each subculture was evaluated to determine SCIY counterselection efficiency in S. suis. As shown in Table 2, approximately 95% of the colonies were the ∆pmtA mutant after subculturing three times. For the perR and lysR genes, approximately half or greater than 80% of the colonies were the mutant strain after five times of subculture ( Table 2). The results indicate that the SCIY cassette is highly efficient as a counterselectable marker for S. suis. The percentage of spectinomycin-sensitive colonies (mutants) was determined by analysis of 100 colonies. The experiments were performed three times for deletion of each gene, and the results for each repetition are shown.

PerR Is a Transcriptional Repressor of the Ferrous Iron and Cobalt Efflux Pump in S. suis
In a previous study, we demonstrated that the pmtA gene encodes a ferrous iron and cobalt efflux pump in S. suis; its expression was significantly induced by ferrous iron, cobalt, and nickel [32]. Upstream of the pmtA gene is a gene encoding the PerR regulator. RT-qPCR analysis was performed to determine whether the pmtA gene is under the control of PerR. As shown in Figure 6, pmtA expression in the WT strain was upregulated following treatment with ferrous iron, cobalt, and nickel. However, pmtA expression in the ∆perR mutant was upregulated without metal supplementation ( Figure 6). The results reveal that deletion of perR led to derepression of the pmtA gene; thus, pmtA expression in ∆perR was upregulated without treatment with ferrous iron, cobalt, or nickel.
In a previous study, we demonstrated that the pmtA gene encodes a ferrous ir cobalt efflux pump in S. suis; its expression was significantly induced by ferrous ir balt, and nickel [32]. Upstream of the pmtA gene is a gene encoding the PerR reg RT-qPCR analysis was performed to determine whether the pmtA gene is under th trol of PerR. As shown in Figure 6, pmtA expression in the WT strain was upreg following treatment with ferrous iron, cobalt, and nickel. However, pmtA expres the ΔperR mutant was upregulated without metal supplementation ( Figure 6). The reveal that deletion of perR led to derepression of the pmtA gene; thus, pmtA expres ΔperR was upregulated without treatment with ferrous iron, cobalt, or nickel.

Discussion
S. suis is an important zoonotic pathogen that causes severe infections in swine and humans. Research on the physiology and pathogenesis of S. suis usually relies on gene deletion mutants. In the present study, we describe a novel two-step method for markerless gene deletion in S. suis. This method is established based on natural transformation in S. suis [22] and the utilization of V. parahaemolyticus YoeB toxin as a counterselectable marker.
TA systems are widely prevalent in bacteria and archaea [23]. Some toxin genes have been developed as counterselectable markers for genetic manipulation based on toxins' antibacterial activity [26][27][28][29][30]. In a previous study, induction of V. parahaemolyticus YoeB toxin in E. coli was found to cause cell death [31]. This finding led to the speculation that YoeB Vp could be an ideal counterselectable marker. YoeB Vp expression should be precisely controlled to be an available counterselectable marker. In S. suis, the copA gene, which encodes a copper efflux system, could be specifically induced by copper [36]. The promoter PcopA might be reliable to control YoeB Vp expression in S. suis. Herein, a S. suis strain expressing the copper-inducible YoeB Vp toxin was constructed to test the counterselection potential of YoeB Vp . As expected, the addition of copper to the culture induced yoeB Vp expression and inhibited S. suis growth. It should be noted that a homologous TA system of YefM-YoeB is present in S. suis [37]. We also evaluated the counterselection potential of YoeB Ss . Induction of YoeB Ss resulted in drastic growth inhibition in E. coli [37], whereas no growth defect was observed when YoeB Ss was induced in S. suis ( Figure S1). We speculate that the endogenous YefM Ss antitoxin counteracted the toxicity of YoeB Ss . While YoeB Vp shares 63% identity with YoeB Ss at the amino acid level, YefM Vp shares only 29% identity with YefM Ss . Therefore, it is not surprising that the toxicity of YoeB Vp was not counteracted by YefM Ss .
A SCIY cassette composed of the spectinomycin-resistance gene and PcopA-yoeB Vp construct was generated to explore its application for markerless gene deletion in S. suis. The first step, by which the SCIY cassette replaced the target gene, was adopted from a previously described method [22]. The intermediate strain was easily selected from plates containing spectinomycin. Since YoeB Vp toxin exerts a bacteriostatic effect rather than a bactericidal effect on S. suis, the mutant generated from the second step should not be selected directly from plates containing copper. Instead, several dilutions in media containing copper were performed for the enrichment of the mutant. Our results showed that after subculturing three to five times, the mutant was easy to isolate. However, the mutant's proportion after each subculture should be correlated with the efficiency of natural transformation and homologous recombination.
In a previous study, a cassette containing a kanamycin resistance gene and a gene encoding the ParE toxin has been used to introduce a single mutation in Salmonella Typhimurium [30]. Similarly, the SCIY cassette could be applied in site-directed mutagenesis or deletion of a few bases in the genome of S. suis, which is an outstanding advantage of the two-step method. The conventional method using pSET4s generates the mutant and WT genotype simultaneously, which were preliminarily identified by PCR. It would be difficult to distinguish the mutant and WT strains by PCR when only a few bases were deleted. If using the two-step method, the SCIY cassette in the intermediate strain could be easily replaced by the target gene with desired site-directed mutagenesis or a few bases deletion in the second step. The two-step method would facilitate research of the role of a single amino acid or protein domain in S. suis.
Although the two-step method is highly efficient in markerless gene deletion in S. suis, it does not mean that it could not be further improved. Next, the effect of other toxins on S. suis growth will be evaluated. If a toxin is found to exert bactericidal activity against S. suis, the yoeB Vp gene in the SCIY cassette will be replaced by this gene. Then, the intermediate strain is expected to be killed in the presence of copper, so that the mutant can be easily isolated in the second step without enrichment. In addition, some undesired mutations might be introduced into the genome during construction of the mutant. Therefore, it would be better to generate a complementation strain for the mutant when performing a functional study of a gene.
BlastP analysis also revealed that YefM Vp and YoeB Vp share 30% and 63% amino acid sequence identity with the homologous antitoxin and toxin from Streptococcus pneumoniae, respectively [38]. It is likely that the YoeB Vp toxin could exert a toxic effect against S. pneumoniae, which might not be counteracted by the endogenous YefM Sp antitoxin. Therefore, further studies could be performed to detect the counterselection potential of the YoeB Vp toxin in other species such as S. pneumoniae. Yet, a suitable promoter should be selected to control yoeB Vp expression in the corresponding species.
In a previous study, we demonstrated that the pmtA gene is involved in ferrous iron and cobalt efflux in S. suis [32]. One of the remaining questions is which regulator modulates pmtA expression. In Streptococcus pyogenes, the PmtA homolog is regulated by PerR [39,40]. In S. suis, the perR gene is located upstream of the pmtA gene. Using the ∆perR mutant generated by the novel two-step method, we demonstrated that in the absence of metal supplementation, pmtA expression in the ∆perR mutant was significantly upregulated compared to that in the WT strain. This result is consistent with the observations in S. pyogenes [39,40]. Thus, PerR is a transcriptional repressor of pmtA in S. suis.
In conclusion, a novel two-step markerless gene deletion method was established for S. suis. This method is cloning-independent and can also be used for site-directed mutagenesis or deletion of a few bases in the genome of S. suis. Moreover, we demonstrate that PerR is a transcriptional repressor of ferrous iron and cobalt efflux pump (PmtA) in S. suis.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/microorganisms9051095/s1, Table S1: Primers used in this study. Figure S1: YoeB Ss expression had no significant effect on S. suis growth in liquid media. Text S1: DNA sequence of the SCIY cassette.