Development and Application of Two Inducible Expression Systems for Streptococcus suis

ABSTRACT Streptococcus suis is an important zoonotic bacterial pathogen posing a threat to the pig industry as well as public health, for which the mechanisms of growth and cell division remain largely unknown. Developing convenient genetic tools that can achieve strictly controlled gene expression is of great value for investigating these fundamental physiological processes of S. suis. In this study, we first identified three strong constitutive promoters, Pg, Pt, and Pe, in S. suis. Promoter Pg was used to drive the expression of repressor genes tetR and lacI, and the operator sequences were added within promoters Pt and Pe. By optimizing the insertion sites of the operator sequence, we successfully constructed an anhydrotetracycline (ATc)-inducible expression system and an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible expression system in S. suis. We showed that these two systems provided inducer-concentration- and induction-time-dependent expression of the reporter gene. By using these tools, we investigated the subcellular localization of a key cell division protein, FtsZ, which showed that it could be correctly localized to the midcell region. In addition, we constructed a conditional knockout strain for the glmS gene, which is an essential gene, and showed that our ATc-inducible promoter could provide strictly controlled expression of glmS in trans, suggesting that our inducible expression systems can be used for deletion of essential genes in S. suis. Therefore, for the first time we developed two inducible expression systems in S. suis and showed their applications in the study of an important cell division protein and an essential gene. These genetic tools will further facilitate the functional study of other important genes of S. suis. IMPORTANCE Streptococcus suis is an important zoonotic bacterial pathogen. Studying the mechanisms of cell growth and division is important for the identification of novel antimicrobial drug targets. Inducible expression systems can provide strictly controlled expression of the protein of interest and are useful tools to study the functions of physiologically important proteins. However, there is a lack of convenient genetic tools that can achieve inducible protein expression in S. suis. In this study, we developed two (ATc-inducible and IPTG-inducible) inducible expression systems and showed their applications in a subcellular localization study of a cell division protein and the construction of conditional knockout of essential genes in S. suis. These systems will be useful for functional studies of important proteins of S. suis.

To date, extensive studies have been carried out and advances have been made regarding the pathobiology of this important pathogen (4)(5)(6)(7). However, it needs to be pointed out that convenient genetic tools for manipulating the genome of S. suis are a prerequisite for these studies. The most commonly used genetic tools for S. suis have been developed by D. Takamatsu and colleagues, who established a set of genetic tool systems including the temperature-sensitive (Ts) suicide vectors pSET4s, pSET5s, and pSET6s for gene knockout and the replicating vectors pSET1, pSET2, and pSET3 for gene expression (8,9). However, there is still a lack of genetic tools that can provide inducible expression of proteins in S. suis.
Bacteria replicate through binary fission, which involves DNA replication and segregation, cell growth, and cell division (10). These are the most fundamental and important physiological processes, which are attracting antimicrobial drug targets (11)(12)(13). As an oval coccus, S. suis undertakes a usual pattern of elongation and division, the molecular mechanisms of which are not yet fully understood. There are several dozen proteins that participate in cell growth and division, and many of them are reported to be essential and need precise regulation (10,14). FtsZ is one of the most important proteins during bacterial cell division and serves as a scaffold to recruit other cell division proteins (15). Proper subcellular localization of FtsZ ensures the subsequent assembly of other cell division proteins (16). GlmS is a glutamine-fructose-6-phosphate aminotransferase involved in the conversion of fructose-6-phosphate (Fru-6P) to glucosamine-6-phosphate (GlcN-6P), which is an initial substrate for peptidoglycan synthesis (17,18). In Streptococcus mutans, glmS is essential when cells are cultured in tryptic soy broth (TSB), while a glmS deletion mutant could survive in the presence of N-acetylglucosamine (GlcNAc) (19). Considering the importance and essentiality of the genes related to cell growth and division, achieving regulated or induced expression and conditional gene knockout is critical to elucidating the functions of these genes.
Inducible expression systems usually act at the level of transcription initiation by repressor proteins responding to small molecules. TetR and LacI are the most commonly used repressor proteins, for which the repression effect can be relieved by the presence of anhydrotetracycline (ATc) and isopropyl-b-D-thiogalactopyranoside (IPTG), respectively (20,21). TetR can bind to the operator sequence tetO, which inhibits the expression of the tetracycline resistance gene in the absence of tetracycline. Once tetracycline is present, the binding affinity is significantly reduced, leading to the expression of the resistance gene (22,23). The IPTG-inducible expression system comes from the lactose operon of Escherichia coli. When the inducer is present, the lac repressor binds to the inducer and reduces the binding to the operator sequence (20).
Here, we identified three constitutive promoters, P g , P t , and P e , from the S. suis genome that can be expressed steadily under different culture conditions. Based on them, we successfully developed two inducible expression systems in which the expression can be induced by ATc or IPTG. We demonstrate that the systems are useful tools that can be used to study the subcellular localization of cell division proteins and to construct conditional knockout mutants of essential genes. For the first time, we provide these genetic tools, which will greatly facilitate studies concerning essential genes or genes whose expression needs fine regulation in such an important zoonotic bacterial pathogen.

RESULTS
Construction of a stable expression system. Constitutive promoters can be used to engineer efficient inducible promoters (24). In S. suis, we selected three promoters, P g , P e , and P t , from the genome that drive the expression of the housekeeping gene gapdH, which has been used as an internal reference for quantitative reverse transcription-PCR (RT-qPCR) (25), the protein enolase, which has been used as an internal reference for Western blotting, and the elongation factor Tu, which has been reported to be constitutively expressed in other bacteria (26). The gfp gene was cloned downstream of the promoters to test their activity, resulting in three plasmids, i.e., pSET2-P g -gfp, pSET2-P e -gfp, and pSET2-P t -gfp. The plasmids were introduced individually into S. suis, and the cells were cultured for different periods of time (optical density at 600 nm [OD 600 ] of 0.3 to 0.9), at a pH of 6 or 8, or at different temperatures (28°C or 42°C). Fluorescence intensity results indicated that the expression of promoters P g , P t , and P e was relatively stable under most conditions except for growth at 42°C or at pH 8 ( Fig. 1). Since previous studies showed that high temperatures and alkaline medium can interfere with the fluorescence intensity of green fluorescent protein (GFP) (26,27), we further verified the expression level by Western blotting, which showed that GFP was expressed at similar levels under different growth conditions (Fig. 1D). These data suggest that promoters P g , P t , and P e are constitutive promoters in S. suis.
Construction of ATc-and IPTG-inducible expression systems. An inducible expression system harbors the element for expression of a repressor protein and a promoter inserted with an operator sequence. To construct an ATc-inducible expression system, the repressor gene tetR was driven by promoter P g , and the operator sequence was inserted into promoters P t and P e . The putative 210 and 235 regions and the transcription start site of the promoters were analyzed by using Softberry BPROM software (28) (Fig. 2A). First, the operator sequence tetO was inserted at the 127 position of promoter P t and at the 12 position of promoter P e , which were subsequently cloned FIG 1 (A to C) Evaluation of the expression of the constitutive promoters. S. suis cells harboring pSET2-P g -gfp (A), pSET2-P e -gfp (B), and pSET2-P t -gfp (C) were grown under different conditions based on TSB or to different growth stages. The pH was adjusted using HCl or NaOH. The cells were harvested and washed with PBS, and the fluorescence intensity was measured using a spectrometer. Statistical analysis was performed using the unpaired Student's t test to compare the fluorescence intensity of the cells cultured under each indicated growth condition with that of the cells cultured in TSB. *, P , 0. 05; **, P , 0. 01; ***, P , 0.001; ****, P , 0.0001; ns, not significant. (D) Detection of GFP expression by Western blotting. S. suis cells harboring plasmid pSET2-P tgfp and grown under different culture conditions were collected, washed with PBS, and lysed, followed by immunoblot analysis using anti-GFP or an-HylA antibody. The HylA antibody was used as a control. and 235 regions and the transcription start site of the promoters were analyzed by using Softberry BPROM software. The expression of tetR was driven by P g , and that of gfp was driven by P e . The operator sequence was inserted into different positions of P e . (B) Detection of the expression of the ATc-inducible expression systems. S. suis cells harboring the indicated plasmid were grown to the mid-log phase and induced with or without 200 ng/mL ATc for 60 min at 37°C. The cells were harvested and washed with PBS, and the fluorescence intensity was measured using a spectrometer. 2 ATc, average expression level in the absence of inducer; 1 ATc, average expression level in the presence of inducer; fold, fold change with ATc, compared with the value without (Continued on next page) Inducible Expression Systems for S. suis Microbiology Spectrum into pSET2 vector, together with the repressor cassette, resulting in two plasmids, pSSTett1-gfp and pSSTete1-gfp, respectively ( Fig. 2A). Fluorescence intensity results showed that the cells harboring pSSTett1-gfp did not show GFP expression even in the presence of inducer ATc (Fig. 2B), suggesting that the insertion of the operator sequence disrupted the promoter P t . In contrast, the cells harboring pSSTete1-gfp displayed strong expression of GFP. However, the expression was observed even in the absence of ATc, suggesting uncontrolled expression. Therefore, we next inserted the operator sequence at different positions within promoter P e and also tried to insert two operator sequences, resulting in three plasmids, pSSTete2-gfp, pSSTete3-gfp, and pSSTete4-gfp (Fig. 2B). It is shown in Fig. 2B that plasmid pSSTete2-gfp had higher expression of GFP in the presence of ATc and the GFP expression was tightly repressed in the absence of inducer. Therefore, pSSTete2-gfp was successfully constructed as an ATc-inducible expression plasmid.
Using the same strategy to construct an IPTG-inducible system, promoter P g was used to drive the expression of the repressor LacI. The operator sequence lacO was inserted at the 12 or 11 position of promoter P e , resulting in two IPTG-inducible plasmids, pSSlace1-gfp and pSSlace2-gfp, respectively (Fig. 2C). Fluorescence intensity results showed that, compared with the cells cultured in the absence of IPTG, the presence of the inducer significantly enhanced the expression of GFP, indicating an effective inducing effect (Fig. 2D). We also observed that the expression of GFP by pSSlace1-gfp was significantly higher than that by pSSlace2-gfp in the presence of IPTG (Fig. 2D). However, it should be noted that basal expression was observed for both of the plasmids when no IPTG was added (Fig. 2D). Therefore, the IPTG-inducible expression plasmid pSSlace1-gfp was successfully constructed.
Regulatory capacities of the inducible expression systems. We used another reporter gene, lacZ, to quantify the regulatory capacities of the inducible expression systems; lacZ was cloned into plasmids pSSTete2-gfp and pSSlace1-gfp to replace gfp. The resulting plasmids were introduced into S. suis and the expression of lacZ was quantified by measuring the b-galactosidase activity in the presence of different concentrations of inducers or with different induction times. For pSSTete2-lacZ, it is shown in Fig. 3A that, in the absence of ATc, b-galactosidase activity was barely detected (Fig. 3A). With the increase of the concentration of ATc, the expression of lacZ increased significantly, and ATc at 150 ng/mL could already give very high expression of the reporter gene (Fig. 3A). The expression of lacZ at a given concentration of inducer (150 ng/mL) but with different induction times showed that the expression of lacZ increased with the increase in induction time (Fig. 3B). For pSSlace1-lacZ, we also observed increased expression of lacZ in an inducer concentration-dependent manner, and it was fully induced with the presence of 0.1 mM IPTG (Fig. 3C). Also, prolonged induction time significantly promoted the expression of the reporter. These results suggested that plasmids pSSTete2-lacZ and pSSlace1-lacZ were both effective inducible expression systems in which the ATc-inducible plasmid pSSTete2-lacZ showed less basal expression.
Determination of the subcellular localization of FtsZ in S. suis using the inducible expression system. Cell division is a very important process for bacteria. Proteins involved in this process need to be correctly localized to the destined subcellular locations to exert their functions, and it is important to be able to characterize the subcellular localization of these proteins. Therefore, we used the above-established inducible expression system to study the subcellular localization of an essential cell division protein, FtsZ, in ATc. Statistical analysis was performed using the unpaired Student's t test to compare the activity of promoters P eT2 , P eT3 , and P eT4 with or without 200 ng/ mL ATc. **, P , 0. 01; ***, P , 0.001; ****, P , 0.0001; ns, not significant. The data are presented as the mean 6 standard deviation. (C) Characterization of IPTG-inducible promoters. The expression of lacI was driven by P g , and that of gfp was driven by P e . The operator sequence was inserted into different positions of P e . (D) Detection of the expression of the IPTG-inducible expression systems. S. suis cells harboring the indicated plasmid were grown to the mid-log phase and induced with or without 0.2 mM IPTG for 60 min at 37°C. -IPTG, average expression level in the absence of inducer; 1 ITPG, average expression level in the presence of inducer; fold, fold change with IPTG, compared with the value without IPTG. Statistical analysis was performed using the unpaired Student's t test to compare the activity of promoters P eI1 and P eI2 with or without 0.2 mM IPTG.
S. suis. A ftsZ-gfp fusion was cloned into pSSTete2-gfp and pSSlace1-gfp to replace gfp, resulting in plasmids pSSTete2-ftsZ_gfp and pSSlace1-ftsZ_gfp, respectively. The plasmids were transformed into S. suis, and the cells were cultured in the presence of inducer, followed by examination by fluorescence microscopy. It is shown in Fig. 4 that, by using either pSSTete2-ftsZ_gfp or pSSlace1-ftsZ_gfp in the presence of inducer, a clear FtsZ-GFP signal that was localized at the middle of the cells was observed (Fig. 4), which was consistent with the localization pattern of FtsZ reported previously for S. suis (29,30). In addition, by using plasmid pSSlace1-ftsZ_gfp, a weak FtsZ-GFP signal was observed even without induction, indicating basal expression, which was consistent with the observations above. These results suggest that the inducible expression systems can be used for subcellular localization studies for cell division proteins.
Use of the inducible expression system for conditional gene knockout in S. suis. It is difficult to study the functions of essential genes because knockout mutants cannot be obtained. By using an inducible expression system, however, essential gene mutants can be constructed (31). In S. suis, there were so far no available tools to construct knockout mutants for essential genes. The glmS gene is an essential gene that encodes glutamine-fructose-6-phosphate aminotransferase, which is involved in the conversion of Fru-6P to GlcN-6P (17). A previous study reported that the glmS deletion mutant was viable in the presence of GlcNAc (19) but lethal under normal growth conditions. Therefore, we constructed an S. suis glmS gene deletion mutant (DglmS) in the presence of GlcNAc. It was shown that, consistent with the previous finding, the S. suis DglmS mutant could not

Inducible Expression Systems for S. suis
Microbiology Spectrum grow in the absence of GlcNAc (Fig. 5). We next constructed an inducible expression plasmid encoding glmS, pSSTete2-glmS, which was transformed into the S. suis DglmS strain. As shown in Fig. 5, the S. suis DglmS strain and the DglmS strain harboring pSSTete2-glmS but without ATc induction could not grow on tryptic soy agar (TSA) plates in the absence of GlcNAc but could grow when GlcNAc was supplemented (Fig. 5). However, it was seen that the DglmS strain harboring pSSTete2-glmS could grow in the presence of 150 ng/mL ATc on TSA plates without GlcNAc (Fig. 5). These results suggest that pSSTete2-glmS can provide strict control of expression that is inducer dependent. Therefore, this inducible plasmid system enables conditional deletion of essential genes.

DISCUSSION
Bacterial resistance to antibiotics has become a serious threat to public health, and it is urgent to develop new drugs to deal with antimicrobial resistance. Common antibiotic drug targets are the protein machineries of fundamental biological processes, such as DNA and RNA synthesis, cell division, bacterial metabolism, and protein synthesis (11)(12)(13). Most of the coding genes are essential and cannot be knocked out using the traditional gene deletion approaches. This makes it difficult to study the functions of these essential genes. Unlike model microorganisms, S. suis is an oval coccus in which the mechanism of cell growth and division is distinct from that of typical bacilli or cocci (32). Here, we successfully developed two (ATc-inducible and IPTG-inducible) inducible expression systems that can be used for regulated FIG 4 Determination of subcellular localization of FtsZ in S. suis using the inducible expression system. (A) Determination of subcellular localization of FtsZ using pSSTete2-ftsZ_gfp. S. suis cells harboring pSSTete2-ftsZ_gfp were grown to the mid-log phase, followed by another 15-min incubation with or without ATc induction at 75 ng/mL. The cells were harvested, washed, stained with AF647 dye, and imaged using the NIS-Elements microscope (Nikon). (B) Determination of subcellular localization of FtsZ using pSSlace1-ftsZ_gfp. S. suis cells harboring pSSlace1-ftsZ_gfp were grown to the mid-log phase, followed by another 15-min incubation with or without IPTG induction at 0.2 mM. The cells were harvested, washed, stained with AF647 dye, and imaged using the NIS-Elements microscope (Nikon).

Inducible Expression Systems for S. suis
Microbiology Spectrum expression and conditional knockout of essential genes in the important zoonotic pathogen S. suis. Bacterial gene expression is influenced by a variety of environmental stimuli, including nutrient availability, ions, and temperature, on the basis of which several inducible expression systems have been developed, for example, the IPTG-inducible system (21), the ATc-inducible system (20), the arabinose-inducible system (33), and the Zn-inducible system (31). An inducible expression system usually includes a repressor protein that is normally expressed and binds the operator sequence within the promoter region, leading to repression of the target gene. Once the inducer is present, the binding between the repressor protein and the operator sequence is relieved and the target gene starts to be expressed. Therefore, to construct inducible expression systems in S. suis, we first identified three strong constitutive promoters, P g , P t , and P e . To ensure efficient gene repression in the absence of inducer, the relatively stronger promoter P g was used to drive the expression of the repressor gene. However, when the operator sequence was inserted into P t , its activity was disrupted. Therefore, we then optimized the insertion site of the operator sequence with promoter P e , and two inducible expression plasmids, pSSTete2-gfp (ATc inducible) and pSSlace1-gfp (IPTG inducible), were successfully constructed in S. suis.
We next evaluated the regulatory capacity of these two inducible expression systems. Both of the plasmids showed inducer-concentration-dependent and inductiontime-dependent expression of the reporter gene, indicating successful construction of the expression systems. However, it was noted that, compared with pSSTete2-lacZ, pSSlace1-lacZ showed higher basal expression in the absence of inducer. This is consistent with previous findings that the lac promoter shows leaky expression even in the presence of the repressor LacI (34). This can be resolved by increasing the expression of the repressor or by inserting more operator sequences into the promoter.
S. suis is an important zoonotic pathogen posing a severe threat to public health. To date, there are still many unknowns regarding the fundamental biology and pathogenesis of this pathogen that deserve deep investigation. Unfortunately, very limited genetic tools have been developed and no inducible expression systems are available for S. suis. The easiest way to develop inducible expression systems is to directly clone an inducible expression system that has already been developed from other bacteria. For example, the tetracycline-inducible promoter P xyl/tet from Staphylococcus aureus has been applied to Streptococcus pneumoniae and Streptococcus agalactiae (35,36). However, further optimizations are still needed; otherwise, they may not be able to provide ideal expression levels (35,37). Another way to develop novel inducible expression systems is to screen the natural inducible expression systems in the bacterial genome by adding different inducers. By using this strategy, the ComS peptideregulated system (38), the zinc-inducible promoter P czcD (31), the maltose-inducible promoter P M (39), and the fucose-inducible promoter P fcsK in S. pneumoniae (40) were developed.
Bacterial cell division is one of the most fundamental physiological processes. Proteins involved in this process need precise temporal and spatial regulation. Examining the subcellular localizations of important cell division proteins is critical to studying the details of the cell division process. FtsZ plays a central role during cell division; it forms a ring structure and orchestrates cell division. Therefore, it is important to investigate its subcellular localization. However, it has been reported that overexpression of FtsZ in bacteria results in abnormal cell division (41,42). Therefore, care is needed when performing localization studies with FtsZ. Utilizing the inducible expression systems we established in this study, it is feasible to achieve fine control of FtsZ expression, and our results showed that FtsZ-GFP expressed from both of the plasmids was localized correctly to the midcell region, suggesting that our inducible expression systems provide powerful tools for functional and localization studies of cell division proteins in S. suis. Many important proteins involved in cell growth and division are essential, and null mutants cannot be obtained using normal genetic tools, which hinders the functional study of these proteins. Recently, however, a conditional knockout strategy that can be used to construct deletion mutants of essential genes has been developed (43). In this study, by using the inducible expression system, we showed that, in the absence of inducer, pSSTete2-glmS barely expressed glmS, indicating that the growth of S. suis DglmS needs GlcNAc. Once induced, expression from pSSTete2-glmS complemented the need of S. suis DglmS for GlcNAc. Therefore, our inducible expression systems can be exploited to construct gene deletion mutants of essential genes in S. suis.
In summary, we successfully constructed two inducible expression systems in S. suis. We showed their practical applications in the subcellular localization study of the critical cell division protein FtsZ, and we demonstrated their potential use in the construction of null mutants of essential genes. These systems provide useful tools that will further facilitate the functional study of important proteins of S. suis.

MATERIALS AND METHODS
Bacterial strains and growth conditions. The bacterial strains and plasmids used in this study are listed in Table 1. S. suis strains in this study are derivatives of S. suis SC19, a highly virulent serotype 2 S. suis expression vector; Spc r 9 pET28a Expression vector; Kan r Novagen pRAB11 E. coli/S. aureus shuttle vector, containing a tetracycline-inducible promoter; Chl r 37 pSET2-P g -gfp pSET2 with P g -gfp fusion cloned at BamHI site; Spc r This study pSET2-P t -gfp pSET2 with P t -gfp fusion cloned at BamHI site; Spc r This study pSET2-P e -gfp pSET2 with P e -gfp fusion cloned at BamHI site; Spc r This study pSSTett1-gfp pSET2 with P g -tetR fusion and P tT1 -gfp fusion; Spc r This study pSSTete1-gfp pSET2 with P g -tetR fusion and P eT1 -gfp fusion; Spc r This study pSSTete2-gfp pSET2 with P g -tetR fusion and P eT2 -gfp fusion; Spc r This study pSSTete3-gfp pSET2 with P g -tetR fusion and P eT3 -gfp fusion; Spc r This study pSSTete4-gfp pSET2 with P g -tetR fusion and P eT4 -gfp fusion; Spc r This study pSSlace1-gfp pSET2 with P g -tetR fusion and P eI1 -gfp fusion; Spc r This study pSSlace2-gfp pSET2 with P g -tetR fusion and P eI2 -gfp fusion; Spc r This study pSSTete2-lacZ pSET2 with P g -tetR fusion and P eT2 -lacZ fusion; Spc r This study pSSlace1-lacZ pSET2 with P g -tetR fusion and P eI1 -lacZ fusion; Spc r This study pSSTete2-ftsZ_gfp pSET2 with P g -tetR fusion and P eT2 -ftsZ_gfp fusion; Spc r This study pSSlace1-ftsZ_gfp pSET2 with P g -tetR fusion and P eI1 -ftsZ_gfp fusion; Spc r This study pSET4S-glmS Derived from pSET4s for deleting glmS in SC-19; Spc r This study pSSTete2-glmS pSET2 with P g -tetR fusion and P eT2 -glmS fusion; Spc r This study a Spc r , spectinomycin resistance; Kan r , kanamycin resistance; Chl r , chloramphenicol resistance.
strain that was isolated from a diseased pig during the S. suis outbreak in 2005 in the Sichuan Province of China; its genome sequence GenBank accession number is NZ_CP020863.1 (44). S. suis cells were grown at 37°C with shaking at 180 rpm in TSB or on TSA (Difco, France) plates containing 5% inactivated newborn bovine serum (Sijiqing Biotech, Hangzhou, China). Spectinomycin was used at a final concentration of 100 mg/mL when needed. E. coli strain MC1061 was used for plasmid propagation (9). E. coli was cultured in Luria-Bertani (LB) broth (Difco) or on LB agar plates at 37°C. Cloning procedures and transformation. The promoters P g , P t , and P e are the 160-bp fragment upstream of the gene gapdH, the 160-bp fragment upstream of the gene tufA, and the 118-bp fragment upstream of the gene eno (encoding enolase), respectively. These promoter fragments were amplified by PCR using P g -F/P g -R, P t -F/P t -R, and P e -F/P e -R, respectively ( Table 2). The GFP coding sequence (gfp) was amplified from pMIDG301, which was kindly donated by Paul Langford, Imperial College London (London, UK). Fragments containing each promoter followed by gfp were cloned into the pSET2 vector by using the ClonExpress MultiS one-step cloning kit (Vazyme, China), resulting in plasmids pSET2-P g -gfp, pSET2-P t -gfp, and pSET2-P e -gfp. The recombinant plasmids were transformed into S. suis by electroporation (2.5 kV, 25 mF, and 200 X). To construct ATc-and IPTG-inducible expression plasmids, the tetR gene and lacI gene were amplified from plasmids pRAB11 and pET-28a, respectively (37). The promoter fragments with the operator sequence inserted were synthesized by Sangon Biotech Company (Shanghai, China).
Inducible expression of GFP. S. suis cells cultured overnight were transferred to 4 mL of TSB and grown at 37°C to the mid-log phase (OD 600 of ;0.5); 200 ng/mL ATc or 0.2 mM IPTG was then added, followed by another 1 h of incubation to induce expression. The cells were then harvested, washed three times with phosphate-buffered saline (PBS), resuspended in PBS, and diluted to give an OD 600 of about 0.5. The suspension (200 mL) in four replicates was subjected to measurement of GFP fluorescence intensity in 96-well plates using a microplate reader (TECAN SPARK10M, Switzerland) (excitation at 485 nm and emission at 535 nm). S. suis strain SC19 was used as the negative control. The expression of GFP was further verified by Western blotting as follows. S. suis cells cultured overnight were transferred to 40 mL of TSB and grown under different culture conditions. The samples were collected by centrifugation, washed three times with PBS, and lysed with lysozyme treatment, followed by homogenization. The concentration of total protein was measured using a micro-bicinchoninic acid (BCA) protein assay kit (Cwbiotech, Beijing, China) and normalized to 15 mg for each sample. The samples were subjected to 12% SDS-PAGE analysis, followed by transfer to a polyvinylidene fluoride (PVDF) membrane by electrophoretic transfer. The GFP protein was probed with anti-GFP antibody (catalog number 50430-2-AP; Proteintech) (1:10,000 dilution) as the primary antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (catalog number SA00001-2; Proteintech) (1:10,000 dilution) as the secondary antibody. The HylA protein as a control was detected with mouse anti-HylA serum (developed by our laboratory) (1:5,000 dilution) as the primary antibody and HRP-conjugated goat anti-mouse IgG (catalog number SA00001-1; Proteintech) (1:10,000 dilution) as the secondary antibody. Chemiluminescent signals of the protein bands were detected using a Western enhanced chemiluminescence (ECL) substrate kit (catalog number 1705060; Bio-Rad) and the ChemiDoc Touch imaging system (Bio-Rad).
Determination of b-galactosidase activity. In order to determine the optimal induction conditions, lacZ was used as a reporter gene; it was amplified by PCR amplification from the chromosomal DNA of E. coli BTH101 (45) and cloned into the inducible plasmids. S. suis cells cultured overnight were transferred to 80 mL of TSB and grown at 37°C to the mid-log phase (OD 600 of ;0.5). The samples were then induced with the addition of ATc and IPTG, respectively, with different inducer concentrations and induction times. The samples were collected by centrifugation, washed three times with PBS, and resuspended with Z-buffer (46). The OD 600 was recorded. The cell suspension was lysed with lysozyme treatment, followed by homogenization. o-Nitrophenyl-b-D-galactopyranoside (ONPG) was added to the cell lysate, followed by incubation at 37°C for 15 min, and the reaction was terminated by the addition of Na 2 CO 3 . The absorbance was measured at the wavelength of 420 nm and 550 nm, respectively. The b-galactosidase activity was calculated as 1,000 Â (OD 420 -[1.75 Â OD 550 ])/(T Â V Â OD 600 ), in which T represents the reaction time and V represents the volume of the reaction (47).
Construction of a glmS depletion/complementation strain. In order to construct the glmS deletion strain of S. suis, the regions upstream and downstream of the glmS gene were amplified by PCR using primers GU-F/GU-R and GD-F/GD-R, respectively ( Table 2). The PCR products were cloned into the Ts plasmid pSET4s via the BamHI site by using ClonExpress MultiS one-step cloning kit (Vazyme). The resulting plasmid was introduced into S. suis SC-19 by electroporation (2.5 kV, 25 mF, and 200 X). The transformants were grown in TSB with 10 mM GlcNAc. The single-and double-exchanged strains were screened as described previously (8) using culture medium supplemented with GlcNAc. The glmS deletion strain was verified by PCR using primers Rglms-F and Glms-R. To construct the induced complementary strain, the glmS gene was amplified by PCR using primers Rglms-F and Glms-R and fused to an ATc-inducible promoter amplified by PCR from plasmid pSSTete2-gfp. The PCR products were cloned into plasmid pSET2 via the BamHI site, and the resulting plasmid was then transformed into the glmS deletion strain to acquire the induced complementary strain.
Fluorescence microscopy. The S. suis cells were grown in 5 mL of TSB at 37°C until the OD 600 reached 0.3. The cells were transferred into 5 mL of fresh TSB with a ratio of 50:1 and grown for 0.5 h. To induce protein expression, ATc was supplemented at 100 ng/mL, followed by incubation at 37°C for 15 min. The cells were collected by centrifugation, washed with PBS three times, and resuspended in 500 mL of PBS. Fluorescent dye AF647 (catalog number A20006; Thermo Fisher Scientific) was then added to a final concentration of 20 mg/mL, followed by incubation at 37°C for 45 min. Twenty microliters of the sample was spotted on a microscope cover glass and covered with a 1% agarose pad. Images were collected by using the NIS-Elements microscope (Nikon, Japan) and analyzed by using ImageJ software. For FtsZ-GFP imaging, a 100-ms exposure time, excitation at 488 nm, and emission at 500 to 545 nm were used. For AF647 imaging, a 20-ms exposure time, excitation at 647 nm, and emission at 663 to 738 nm were used.
Data availability. The sequences of plasmids pSSTete2-gfp and pSSlace1-gfp are available in the NCBI GenBank database with the accession numbers ON391044 and ON391045, respectively.