Role of VicRKX and GlnR in pH-Dependent Regulation of the Streptococcus salivarius 57.I Urease Operon

Dental plaque rich in alkali-producing bacteria is less cariogenic, and thus, urease-producing Streptococcus salivarius has been considered as a therapeutic agent for dental caries control. Being one of the few ureolytic microbes in the oral cavity, S. salivarius strain 57.I promotes its competitiveness by mass-producing urease only at acidic growth pH. Here, we demonstrated that the downregulation of the transcription of the ure operon at neutral pH is controlled by a two-component system, VicRKX, whereas the upregulation at acidic pH is mediated by the global transcription regulator of nitrogen metabolism, GlnR. In the absence of VicR-mediated repression, the α subunit of RNA polymerase gains access to interact with the AT-rich sequence within the operator of VicR, leading to further activation of transcription. The overall regulation provides an advantage for S. salivarius to cope with the fluctuation of environmental pH, allowing it to persist in the mouth successfully.

acidic pH shifts the metabolism from glutamine synthesis to ATP generation to enhance acid tolerance (19).
The regulation by VicR and GlnR of p ureI expression under different growth conditions was investigated in this study. We found that the regulation by VicR and GlnR of p ureI is modulated by the growth pH. GlnR activates the expression of p ureI at acidic pH, whereas VicR represses p ureI activity. In the absence of VicR, the AT tract within the VicR box of p ureI acts as an UP element to further enhance p ureI expression.
the reaction mixture, no shifted band was detected. As above, the recombinant MalE protein failed to interact with both probes, confirming the binding specificity of GlnR (data not shown). Notably, the addition of glutamine and glutamine synthetase to the EMSA reaction mixture did not enhance the binding (data not shown), indicating that, unlike in B. subtilis, FBI-GS was not required for the binding activity of S. salivarius GlnR. Finally, the in vivo interaction between GlnR and p ureI was confirmed by the ChIP-PCR assay (Fig. 3C).
VicRKX represses the transcription of p ureI . A vicR-deficient strain is needed to investigate whether VicR is involved in the urease regulatory circuit. However, we failed to generate a vicR-null recombinant strain in S. salivarius 57.I. Several studies have shown that VicR is essential for the viability of several oral streptococci (16,23), and thus, it is possible that VicR plays a similar role in S. salivarius 57.I. To circumvent this difficulty, we first investigated the regulatory function of VicRKX in p ureI expression by using a promoter fusion with mutations in the predicted VicR box. A derivative of S. salivarius strain MC308 was constructed, strain MC308_mVicR_box, in which the sequence of Ϫ64 to Ϫ59 of p ureI (5=-TGTAAA) in the p ureI -cat fusion was mutated to 5=-GTCGAC. Notably, the CodY box in this fusion remains untouched. Both strain MC308 and strain MC308_mVicR_box were cultivated in brain heart infusion (BHI) at pH 7.5 and pH 5.5. A 4-fold increase in CAT activity was observed in strain MC308_mVicR_box compared to its activity in strain MC308 in cells grown at pH 7.5, whereas only a 2.1-fold increase was detected in cells grown at pH 5.5 (Fig. 4A), indicating that the putative VicR box was involved in the negative regulation of p ureI , and the repression was more evident at pH 7.5.
Since a vicRKX-null strain is available in S. gordonii CH1 (22), we examined p ureI expression in a vicRKX-null derivative of S. gordonii strain SL17 (strain SL17_ΔvicRKX) and its derivative that harbors the vicRKX operon of S. salivarius 57.I (strain SL17_CΔvicRKX). Notably, S. gordonii SL17 harbors a single copy of the p ureI -cat fusion on gtfG. In agreement with the cis element analysis, elevated CAT activity was detected in the vicRKX-null background at both pH 7.5 and pH 5.5. A wild-type level of CAT activity was observed in strain SL17_CΔvicRKX, confirming that VicR repressed the transcription of p ureI (Fig. 4B). It was also noted that the CAT activity of strain SL17_CΔvicRKX was consistently lower (approximately 30%) than that of strain SL17, suggesting that S. salivarius VicR represses p ureI expression more efficiently than S. gordonii VicR does.
To investigate the potential effects of growth pH and glucose concentration on the regulation of VicR, strains SL17_CΔvicRKX and SL17_ΔvicRKX were grown in a chemostat, a culture system that allows tight control of the growth pH and glucose concentration. The CAT activities in cells grown at pH 7 and pH 5.5 with 20 and 100 mM glucose were examined. At pH 7, a 4.7-fold increase in CAT activity was seen in SL17_ΔvicRKX compared to the activity in SL17_CΔvicRKX in the presence of 20 mM glucose, but comparable levels of CAT activity were detected between these two strains when cells were grown with 100 mM glucose (Fig. 4C). On the other hand, 1.8-and 1.5-fold increases in activity were seen in cells grown at pH 5.5 with 20 mM and 100 mM glucose, respectively (Fig. 4C). Collectively, the activity of VicR was modulated by both pH and carbohydrate concentration and VicRKX repressed p ureI most effectively at neutral pH under glucose limitation.
GlnR activates p ureI more strongly at pH 5.5. A glnR-deficient strain is essential to investigate how GlnR regulates urease expression. Unfortunately, multiple attempts failed to generate a glnR-null mutant strain in S. salivarius 57.I, indicating that mutations in glnR are also lethal in S. salivarius 57.I. Thus, we initiated the study by using p ureI -cat fusions with mutations in the putative GlnR boxes, since the interaction between GlnR and the putative GlnR boxes 5= to p ureI has been confirmed (Fig. 3). Previous promoter deletion analysis indicates that a 40-bp region 22 bases 5= to the Ϫ35 element of p ureI exhibits a positive effect on p ureI transcription (14). This region contains the 3= portion of GlnR box 2 and the entire GlnR box 1, suggesting that GlnR activates p ureI expression. As a positive effect would be more evident in a repressor-free host, i.e., strain S. salivarius ΔcodY, the activity of all promoter derivatives was examined in the codY-deficient background. In agreement with the hypothesis, mutations in the putative GlnR boxes reduced CAT activity in batch-grown cells at both pH 7.5 and pH 5.5 and the reduction is slightly more evident at pH 5.5 (Fig. 5A), suggesting that GlnR acts as an activator for p ureI expression and its regulatory activity on p ureI is modulated by the growth pH. As stated above, the nonureolytic S. gordonii strain seems to be a logical alternative host for studying p ureI regulation, and luckily, a glnR-null S. gordonii derivative could be obtained. Thus, the effect of GlnR on p ureI expression was examined in S. gordonii by using a similar approach as for analyzing VicR regulation. The CAT activities in the glnR-null S. gordonii SL17 (strain SL17_ΔglnR) and its derivative harboring the glnR gene of S. salivarius 57.I (strain SL17_CΔglnR) were examined in batch-grown cells at pH 7.5 and pH 5.5. In agreement with the cis element analysis, the CAT activity in strain SL17_ΔglnR was lower than the levels in strains SL17 and SL17_CΔglnR at both pH 7.5 and pH 5.5, indicating that GlnR activates p ureI expression (Fig. 5B). It was also noticed that the CAT activity of strain SL17_CΔglnR was consistently higher (approximately 40%) than that of strain SL17, suggesting that S. salivarius GlnR works more efficiently on p ureI than S. gordonii GlnR does.
To further investigate the effects of growth pH and carbohydrate concentration on the regulation of GlnR on p ureI , strains SL17_CΔglnR and SL17_ΔglnR were cultivated in a chemostat at pH 7 or pH 5.5 with 20 or 100 mM glucose. At pH 7, comparable expression levels were observed in strains SL17_CΔglnR and SL17_ΔglnR under glucose limitation (20 mM), whereas a 1.8-fold increase in CAT activity was detected in strain SL17_CΔglnR compared to that in strain SL17_ΔglnR under glucose excess (100 mM). At pH 5.5, 4-fold and 3.2-fold increases in CAT activity were seen in strain SL17_CΔglnR compared to the levels in strain SL17_ΔglnR under 20 mM and 100 mM glucose, respectively (Fig. 5C). These results indicated that the activation by GlnR of p ureI was modulated by both carbohydrate concentration and growth pH, and the activation was most pronounced at pH 5.5.
The VicR box acts as a UP element of p ureI . As the VicR box is rich in AT, it is hypothesized that this region could act as a UP element to enhance the activity of RNA polymerase in the absence of the cognate repressor. Thus, the VicR box in the 5= flanking region of p ureI was mutated in strain SL17_ΔvicRKX to verify the possibility. Notably, the CodY box remains intact in this mutant strain. Mutations in the VicR box downregulated the CAT activity in strain SL17_ΔvicRKX at both pH 7.5 and pH 5.5 (Fig. 6A), suggesting that this region exhibited a positive effect on p ureI expression in the absence of VicR. To further investigate whether this region could interact with the C-terminal domain (CTD) of the RNA polymerase ␣ subunit (␣-CTD), EMSA was carried out with a probe of 21 bp covering the entire VicR box. The result indicated that the MalE-tagged ␣-CTD recombinant protein interacted with the probe, and the shift was abolished when an unlabeled probe in 300-fold excess was included in the reaction mixture (Fig. 6B), confirming that the VicR box acted as a UP element to enhance p ureI expression.

DISCUSSION
Urease expression in both H. pylori and S. salivarius is upregulated during growth at acidic pH; however, H. pylori activates the expression at acidic pH by the activity of NikR and ArsR (27)(28)(29), which is different from the repression by CodY and VicR at neutral pH in S. salivarius. It seems most cost-effective for H. pylori to activate urease expression at acidic pH, as the pH of the stomach is generally below pH 5. On the other hand, the pH of oral mucosal pH is close to neutral normally (30), and therefore, S. salivarius gains the greatest advantage by repressing urease expression at neutral pH. The repression of S. salivarius p ureI by the VicRKX system was most evident in cells cultivated at pH 7 with 20 mM glucose and less evident at pH 5.5, but surprisingly, the repressive effect was absent when cells were cultivated at pH 7 with 100 mM glucose, raising the possibility that the VicRKX system is insensitive to environmental pH under glucose excess. An attempt was made using ChIP-quantitative PCR to verify the DNA binding activity of VicR under different growth pH conditions in batch-grown S. salivarius 57.I. More VicR binding was detected in cells grown at pH 7.5 than at pH 5.5 (data not shown), suggesting that VicR is more active at neutral pH. Thus, the absence of repression of p ureI by VicR at pH 7 under 100 mM glucose may result from the repression of an additional regulatory protein that exerts regulation mainly at neutral pH under glucose excess, and/or the repression by this regulatory protein is augmented in the absence of VicR under this growth condition. Our recent observations suggested that the catabolite control protein A (CcpA) also participates in the regulation of p ureI expression. Inactivation of ccpA led to upregulation of p ureI in cells grown at pH 7 with 100 mM glucose, but only marginal upregulation was seen in cells grown at pH 7 with 20 mM glucose (unpublished data). The regulation by CcpA of p ureI is not understood currently, as no CcpA binding consensus element is found in the flanking region of p ureI . However, the effect of CcpA in response to carbohydrate concentration at pH 7 may explain the absence of upregulation in SL17_ΔvicRKX grown at pH 7 with 100 mM glucose.
It is intriguing that p ureI was also repressed by the VicRKX system at pH 5.5 regardless of the glucose concentration, if neutral pH is required to activate the system. A study in S. gordonii indicates that inactivation of VicR reduces the tolerance of S. gordonii for oxidative stresses (22). Furthermore, studies in S. mutans have suggested that acidic growth pH could induce the oxidative stress response (31,32). Specifically, the expression levels of genes encoding enzymes metabolizing reactive oxygen species, e.g., sod, ahpC, and ahpF, are upregulated in chemostat-grown S. mutans at pH 5 compared to their expression levels in cells grown at pH 7 (31). Thus, the activity of VicR at pH 5.5 may be part of the oxidative stress response.
The results of site-directed mutagenesis of the GlnR boxes and p ureI expression in chemostat cultures (Fig. 5) suggest that growth pH modulates the activity of GlnR. Although it is not understood how the acidic pH activates the DNA binding activity of GlnR, the activation by acidic pH is not unique to S. salivarius. A recent study in S. mutans demonstrates that the repression of GlnR is activated at pH 5.5 (19). Thus, GlnR of oral streptococci is likely to be activated by both excess amounts of the nutrient nitrogen and acidic growth pH. Additionally, the presence of more than one GlnR box in the promoter region is also not unique to p ureI . For instance, the glnRA operon, the ureABC operon, and tnrA of B. subtilis all possess two GlnR boxes in the promoter regions (7,18,33), and cooperative binding of GlnR to the two GlnR boxes, 6 bp apart, has been demonstrated in the promoter of glnRA (33). As both GlnR box 1 and GlnR box 2 are required for GlnR-dependent activation (Fig. 5A), these two sites may participate in cooperative binding of GlnR. Furthermore, the positive regulation of GlnR in p ureI transcription is similar to the activity of TnrA in B. subtilis (26). As a tnrA homolog is absent in most streptococcal species with known genomes (8,(34)(35)(36) and studies in Lactococcus lactis and Streptococcus pneumoniae have shown that GlnR carries out some of the functions that are exerted by TnrA in B. subtilis (37,38), GlnR of S. salivarius is likely to function in the same way.
Studies in S. mutans have demonstrated that chemostat-grown cells under glucose limitation have an excess of amino acid nutrients at a high growth rate (D ϭ 0.4 Ϫ1 ) (39). Reduced expression of glnRA was observed in chemostat-grown S. salivarius 57.I supplemented with 20 mM glucose compared to that with 100 mM glucose (data not shown), suggesting that the nutrient nitrogen is likely to be in excess under glucose limitation. If this is true, it is expected that the activation of p ureI transcription by GlnR should be more pronounced under glucose limitation than under glucose excess. However, we did not observe downregulation of p ureI in strain SL17_ΔglnR cultivated at pH 7 under 20 mM glucose, suggesting that repressor(s) which are most active at pH 7 with limited sugar supply could mask the activation by GlnR. Based on what we have learned, the repression is likely to be governed by CodY (15) and VicR (Fig. 4B).
A working model for the regulatory network governing urease expression is proposed (Fig. 7). CodY and VicR inhibit urease expression via binding to the cognate operators in the 5= flanking region of p ureI at neutral pH to avoid overalkalinization of the oral cavity. GlnR activates p ureI transcription via binding to the GlnR boxes at acidic pH to enhance acid tolerance. In the absence of the cognate repressors, both the CodY and VicR boxes could act as UP elements to enhance p ureI expression. The complex regulation of the urease operon by these regulators links nitrogen metabolism and the acid stress response, which could ensure optimal fitness of S. salivarius against environmental stresses.

MATERIALS AND METHODS
Bacterial strains, growth conditions, and general genetic manipulations. The bacterial strains used in this study are listed in Table 1. S. salivarius 57.I, S. gordonii CH1, and their derivatives were grown routinely in BHI (Difco) at 37°C under 10% CO 2 atmosphere. When necessary, kanamycin (Km) was added to the culture medium at 1,000 and 250 g · ml Ϫ1 for recombinant S. salivarius and S. gordonii strains, respectively. When necessary, 750 g · ml Ϫ1 of spectinomycin (Sp) and 5 g · ml Ϫ1 of erythromycin (Em) were used for other recombinant streptococcal strains. Recombinant Escherichia coli strains were maintained in L broth supplemented, where indicated, with Sp at 50 g · ml Ϫ1 . To obtain batch cultures grown under neutral or acidic pH conditions, cells were cultivated to mid-exponential phase (optical density at 600 nm [OD 600 ] of Ϸ0.6) in BHI containing 50 mM KPO 4 at pH 7.5 and in BHI that was adjusted to pH 5.5 by the addition of 2 N HCl, respectively. For continuous-culture studies, recombinant S. gordonii strains were grown in a Biostat B plus bioreactor (Sartorius Stedim Biotech) at a dilution rate (D) of 0.3 h Ϫ1 (generation time, 2.3 h) in medium containing 3% tryptone and 0.5% yeast extract (TY) (13). Cultures were kept at a specific growth condition for at least 10 generations to reach steady state. Furthermore, when 20 mM glucose was included in the medium, glucose was undetectable in the culture supernatant, whereas approximately 50 mM glucose remained in the culture supernatant when 100 mM glucose was used. Thus, 20 and 100 mM glucose present glucose limitation and excess, respectively.
The oligonucleotides used in this study are listed in Table 2. Synthetic DNA oligonucleotides were purchased from Genomics BioSci & Tech (Taiwan) and Integrated DNA Technologies (Singapore). Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs (NEB). PCRs were performed with the high-fidelity DNA polymerase Blend Taq plus (Toyobo).
Purification of recombinant proteins and generation of polyclonal antisera. The coding region of VicR was PCR amplified from S. salivarius 57.I using primers VicR_BamHI_S and VicR_PstI_AS and cloned into pQE30 (Qiagen) in E. coli M15. The identity of the recombinant plasmid was confirmed by sequencing analysis. The recombinant His-tagged VicR (His-VicR) was induced and purified under denaturing conditions using the standard methods. The identity of the recombinant protein was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. The concentration of the purified protein was determined by Bio-Rad protein assay based on the method of Bradford (40). Using a similar approach, the coding region of GlnR was amplified from S. salivarius 57.I by using primers GlnR_PstI_S and GlnR_BamHI_AS, and a His-tagged GlnR protein (His-GlnR) was prepared. The purified His-VicR and His-GlnR were used to generate polyclonal antisera in rabbits by LTK BioLaboratories (Taiwan). The titers and specificities of all antisera were tested by immunoblotting.
To purify recombinant VicR and GlnR under the native condition, constructs to produce maltose binding protein-tagged VicR (MalE-VicR) and GlnR (MalE-GlnR) were generated by using pMAL-c2X (NEB). The recombinant proteins were purified by amylose affinity chromatography (NEB) and verified by MALDI-TOF analysis prior to performing the EMSA analysis.  mechanical disruption in the presence of an equal volume of glass beads (0.1 mm in diameter) by homogenization in a BeadBeater (BioSpec Products) for a total of 120 s at 4°C. Amounts of 1 mg and 500 g of the total lysate were incubated with 20 nM biotin-labeled DNA probes specific for the VicR box and for the GlnR boxes, respectively, in the DAPA binding buffer. The binding reaction was carried out under rotation at 4°C for 1 h. The DNA-protein complexes were captured by using 50 l of streptavidin MagneSphere paramagnetic particles (Promega). The mixture was incubated at 4°C for 1 h, followed by five washes with the binding buffer. Finally, the proteins of the DNA-protein complexes were eluted in electrophoresis sample buffer, separated on 12% SDS-PAGE, and then detected by immunoblotting.
Anti-VicR and anti-GlnR antisera were used at 1:2,000 and 1:20,000 dilution, respectively. The signals were detected by horseradish peroxidase-conjugated anti-rabbit IgG (GeneTex) and luminol-based reagents (Merck Millipore). EMSA and ChIP-PCR. Two biotin-labeled, complementary oligonucleotides containing the target site were annealed and used in EMSA. An amount of 0.01 pmol of the annealed probe was incubated with increasing amounts of recombinant MalE-VicR, MalE-GlnR, and MalE-␣-CTD in the EMSA binding buffer [50 mM Tris-HCl (pH 7.4), 50 mM KCl, 2 mM MgCl 2 , 100 ng poly(dI-dC), and 0.5 g bovine serum albumin (BSA)]. All reactions were carried out at 4°C for 30 min, and the products were resolved on 6% nondenaturing polyacrylamide gels. Specific competition was carried out by including the same probe without labeling in a 300-fold excess. The DNA-protein complex was electrotransferred to a piece of Hybond blotting membrane (Amersham), and the signal was detected by the chemiluminescent nucleic acid detection module kit (Pierce).
ChIP-PCR assay was performed as previously described (15). The PCR was carried out by using primers pureI_4870_S and pureI_5090_AS.
Construction of a p ureI -cat fusion and its derivatives in S. gordonii CH1 and S. salivarius 57.I. The p ureI -cat fusion was tagged with an Sp resistance gene (spe) (41) and cloned into the integration vector for S. gordonii CH1, pMJB6 (14), which allows the integration of the promoter fusion at gtfG. The resulting plasmid, pSL16, was introduced into S. gordonii CH1 by transformation (42), and the double-crossover recombination was verified by colony PCR. The resulting strain was designated S. gordonii SL17.
The putative VicR box in the p ureI -cat fusion was mutated by site-directed mutagenesis. Briefly, the primer pairs pureI_VicR_box_SalI_S plus pureI_VicR_box_SalI_AS and pureI_320_SalI_S plus pureI_320_SmaI_AS were used in an inverse PCR using pMC300 (15) (for S. salivarius) and pSL16 (for S. gordonii) as the template, respectively. The PCR products were digested, ligated, and established in E. coli. The resulting plasmids were confirmed by sequencing analysis, and the correct constructs were introduced into S. salivarius 57.I by electroporation (12) and into S. gordonii ΔvicRKX by transformation (42). The double-crossover recombination was verified by colony PCR, and the resulting strains are designated S. salivarius MC308_mVicR_box and S. gordonii ΔvicRKX_mVicR_box, respectively.
The GlnR boxes in the p ureI -cat fusion were mutated by a similar approach. Briefly, the primer pairs GlnR_box 1_SalI_S plus GlnR_box 1_SalI_AS and GlnR_box 2_NsiI_S plus GlnR_box 2_NsiI_AS were used in PCR to introduce mutations into GlnR box 1 and 2, respectively.
Construction of the S. gordonii vicRKX-deficient strain and its derivative. All recombinant S. gordonii strains were generated by using PCR ligation mutagenesis (43). To inactivate vicRKX in S. gordonii SL17, the 5= and 3= flanking fragments of vicR were generated from S. gordonii CH1 by using the primer pairs CH1_VicR_S plus CH1_VicR_XhoI_AS and CH1_VicR_BamHI_S plus CH1_VicR_AS, respectively. The PCR products were digested and then ligated to the 5= and 3= ends of an ⍀kan (44) fragment. The ligation mixture was used to transform S. gordonii SL17, with selection for Km resistance. The double-crossover recombination was verified by colony PCR, and the resulting strain was designated SL17_ΔvicRKX. The region encoding the 41st to 92nd amino acids of VicR in strain SL17_ΔvicRKX was replaced by ⍀kan.
To generate a vicRKX recombinant strain, two fragments for integrating the vicRKX gene of S. salivarius at gtfG were generated from SL17_ΔvicRKX by primer pairs CH1_GtfG_S plus CH1_GtfG_BamHI_AS and CH1_GtfG_XhoI_S plus CH1_Spec_AS, respectively. A DNA fragment containing the vicRKX operon was amplified from S. salivarius 57.I by PCR using primers 57I_VicR_XhoI_S and 57.I_VicX_SphI_AS. A DNA fragment containing the nonpolar Em resistance gene (erm) from Tn916ΔE (45), which does not possess a promoter or a transcription terminator, was also prepared by PCR. All PCR products were digested and used in a ligation reaction. The ligation mixture was used to transform strain SL17_ΔvicRKX, and the allelic exchange event in the Em-resistant transformants was verified by colony PCR. The resulting strain, SL17_CΔvicRKX, harbors a copy of erm-tagged vicRKX on gtfG.
Construction of the S. gordonii glnR-deficient and derivative strains. The glnR gene of S. gordonii CH1 was inactivated by the method described above. Briefly, the 5= and 3= flanking fragments of glnR were generated from S. gordonii CH1 by primer pairs CH1_SGO_0212_S plus CH1_GlnR_XhoI_AS and CH1_GlnR_SphI_S plus CH1_GlnA1_AS, respectively. These two fragments were ligated to the 5= and 3= ends of a nonpolar erm fragment, and the ligation mixture was used to transform S. gordonii SL17. The double-crossover recombination was verified by colony PCR, and the resulting strain was designed strain SL17_ΔglnR. The region encoding the 12th to 76th amino acids of GlnR was replaced by a nonpolar erm in this strain.
An S. gordonii glnR-derived strain was generated as described above. Briefly, the 5= and 3= flanking fragments were generated from SL17_ΔglnR by PCR with the primer pairs CH1_GlnR_S plus CH1_GlnR_NcoI_AS and CH1_GlnA_XbaI_S plus CH1_GlnA2_AS, respectively. A DNA fragment containing glnR was generated from S. salivarius 57.I by PCR using primers 57I_GlnR_NcoI_S and 57.I_GlnR_Bam-HI_AS. A ligation mixture of all three fragments and a DNA fragment containing a nonpolar kan cassette (44) that lacks a promoter and a transcription terminator was prepared and used to transform strain SL17_ΔglnR. The ligation was prepared to favor the formation of a construct comprising the 5= flanking fragment followed by glnR of S. salivarius, the kan fragment, and the 3= flanking fragment. The correct allelic exchange event in the Km-resistant transformants was verified by colony PCR, and the resulting strain was designed SL17_CΔglnR.
CAT assay. Total protein lysates from the concentrated cell suspensions were subjected to mechanical disruption as described previously (14). The CAT activities were determined by the method of Shaw (46), and the specific activities were calculated as nmol of Cm acetylated min Ϫ1 mg Ϫ1 .
Statistical analysis. Statistical analysis was performed using one-way analysis of variance (ANOVA) with GraphPad Prism (version 5) software.