Ala-Gln

Characterization of a transcriptional regulator PtxS from Pseudomonas plecoglossicida for regulating 2-ketogluconic acid metabolism

Lei Sun 1, Daming Wang 2, Wenjing Sun 3, Xiaofei Zhang 2, Fengjie Cui 4, Chang Su 5, Xiaomei Zhang 5, Guoqiang Xu 1, Jinsong Shi 5, Zhenghong Xu 6

Abstract

Glucose negatively affected the molecular binding of PpPtxS and 2KGA, and gluconic acid inhibited the PpPtxS2KGA binding reaction. PpPtxS in water solution mainly existed as a dimer and bound to two molecules of 2KGA. The effector 2KGA mainly bound to the region close to the C-terminal of PpPtxS by interacting with the 299th to the 301st amino acids (Ala, Gln, Pro, Thr, Glu and Arg). PpPtxS specifically recognized and bound to a 14-bp palindrome sequence (5′-TGAAACCGGTTTCA-3′) due to its conserved HTH motif at the N-terminal. The characterization of PpPtxS in this study would provide a theoretical guidance for the industrial production of 2KGA. Homologs of PtxS are ubiquitous transcriptional regulators controlling the expression of the glucose dehydrogenase and kgu operon to globally regulate the 2-ketogluconic acid (2KGA) metabolism in Pseudomonas. In the present study, a PtxS from a 2KGA industrial producer Pseudomonas plecoglossicida JUIM01 (PpPtxS) was heterologously expressed in E. coli BL21(DE3), then structurally and functionally characterized. The obtained results showed that PpPtxS was a 36.65-kDa LacI-family transcriptional regulator. 2KGA was the sole effector of PpPtxS.

Keywords:
PtxS
Pseudomonas plecoglossicida
2-ketogluconic acid (2KGA)
LacI-family transcriptional regulator
Transcriptional regulation

1. Introduction

2-Ketogluconic acid (2KGA) is an organic acid with a wide range of applications in cosmetic, pharmaceutical and environmental industries. It is currently mainly used as a precursor for the synthesis of food antioxidant D-erythorbic acid and its salts [1,2]. The industrial 2KGA production presently prefers the microbial fermentation method using Pseudomonas strains due to high 2KGA concentration, yield and productivity [3–11]. Metabolic flux analysis also showed glucose in Pseudomonas aeruginosa and Pseudomonas putida was mainly converted to gluconic acid rather than directly phosphorylated, and the 2KGA bypass plays a significant role in gluconic acid metabolism [12–15]. In Pseudomonas, glucose is firstly oxidized to gluconic acid by glucose dehydrogenase [16], and then oxidized to 2KGA by gluconate dehydrogenase in the periplasmic space [17]. Glucose, gluconic acid and 2KGA can be transported into the cytoplasm through energy-dependent processes mediated by the GntP and KguT transporters, and further catabolized, respectively. 2KGA intracellular metabolism includes 2KGA phosphorylation to 2-keto-6-phosphogluconic acid by 2ketogluconate kinase (KguK), 2-keto-6-phosphogluconic acid reduction to 6-phosphogluconic acid by 2-keto-6-phosphogluconate reductase (KguD), and 6-phosphogluconic acid metabolization within the Entner-Doudoroff pathway (Fig. 1) [8,18,12–14].
The genes such as gad operon (encoding gluconate 2-dehydrogenase for 2KGA synthesis) and kgu operon (encoding 2KGA transporter, 2ketogluconate kinase and 2-keto-6-phosphogluconate reductase for 2KGA utilization) in the model species P. putida KT2440 were proved to responsible for the reactions converting gluconic acid to 2KGA, and 2KGA to 6-phosphogluconic acid, respectively. PtxS is a member of the LacI-family transcriptional regulators with some of the characteristic features of the GalR-LacI repressors. PtxS modulates expression of gad and kgu operon and is induced in cells growing with glucose, gluconic acid, or 2KGA [19]. PtxS is also identified and clustered as a transcriptional repressor to globally regulate the 2KGA metabolism in the conditional pathogenic species P. aeruginosa [20–25]. Although homologs of the PtxS are ubiquitous in Pseudomonas [20], almost only those in P. putida and P. aeruginosa are reported so far. The function of PtxS in 2KGA production of other Pseudomonas species, especially industrial 2KGA producers, still requires a thorough understanding because its potential binding to target promoters to affect 2KGA periplasmic and intracellular metabolization.
Pseudomonas plecoglossicida JUIM01 is an industrial producer with 2KGA yield of over 90% (of theoretical value) [16,17,8–11]. Our previous study has cloned a 1023-bp ptxS gene (GenBank accession No.: KU168041.1) from P. plecoglossicida JUIM01 encoding a protein composed of 340 amino acids, and sharing approximate 80% identity with the ptxS genes from P. aeruginosa and P. putida [26]. In the present study, the ptxS gene from P. plecoglossicida JUIM01 was cloned and heterologously expressed in Escherichia coli BL21(DE3). Structural characteristics and functions of the purified recombinant P. plecoglossicida PtxS (PpPtxS) were elucidated using circular dichroism far-ultraviolet scanning, isothermal titration calorimetry, SEC-MALS, molecular sieve filtration chromatography and molecular docking simulation. Our findings would benefit for understanding the potential mechanism of ptxS gene binding models to regulate 2KGA production and metabolism.

2. Materials and methods

2.1. Microorganism and media

P. plecoglossicida JUIM01, an industrial 2KGA producing strain, was screened and stored in our laboratory [8–11,16–18]. Stock culture was prepared by inoculating the activated cells to a slant stock medium containing: 10 g/L peptone, 5 g/L beef extract, 5 g/L NaCl and 20 g/L agar at pH 7.0. The culture medium consisted of 20 g/L glucose, 10 g/L corn syrup powder, 2 g/L KH2PO4, 0.5 g/L MgSO4·7H2O and 5 g/L CaCO3 at pH 7.0. P. plecoglossicida cells were cultivated at 30 °C and 265 rpm for 20 h to collect cells for extracting P. plecoglossicida JUIM01 genome DNA.

2.2. Construction of the recombinant plasmid and strains

The ptxS gene was amplified by PCR using P. plecoglossicida JUIM01 genome DNA as the template and the pairs of primers ptxS-P1 (5′AGCCATATGGTGGACAAGACGCTTTCCCA-3′) and ptxS-P2 (5′-ATGCTC GAGTCAATTGCGTATCGGGGTGG-3′). The purified PCR product and the vector pET-28a(+) were double-digested by the restriction enzymes Nde I and Xho I at 37 °C, and ligated by T4 DNA ligase. The ligated product was transformed into E. coli DH5α competent cells, and cultured overnight at 37 °C on LB plates supplemented with 50 μg/mL of kanamycin (Kan). The constructed plasmid was extracted, sequenced and named as pET-28a-ptxS. The pET-28a-ptxS and the empty vector pET28a(+) were transformed into E. coli BL21(DE3) to construct PtxSheterologous-expression strain E. coli BL21(DE3)/pET-28a-ptxS for PpPtxS expression and its negative strain BL21(DE3)/pET-28a(+) for control, respectively.

2.3. Expression and purification of the recombinant PpPtxS

Recombinant E. coli strains were activated by inoculating into 10 mL LB medium (containing 5 g/L yeast extract, 10 g/L tryptone and 10 g/L NaCl, and 50 μg/mL kanamycin) and cultivating at 37 °C and 220 rpm overnight. The activated E. coli BL21(DE3)/pET-28a-ptxS and BL21 (DE3)/pET-28a(+) were inoculated into 50 mL LB(Kan) with inoculum size of 1% (v/v), respectively, and cultured at 37 °C and 220 rpm until OD600 nm = 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.4 mM for induction at 18 °C and 220 rpm for 16 h. After induction, the recombinant E. coli cells were collected by centrifugation at 4 °C and 4000 rpm for 10 min and crushed to collect cell-lysate supernatants at 4 °C and 13,000 rpm for 30 min. The expression of PpPtxS was analyzed by SDS-PAGE and Western Blot (WB).
Buffers used for purification of recombinant PpPtxS are listed in Table 1. The above collected cell-lysate supernatant was loaded to a HisTrap HP (5 mL) affinity column (GE Healthcare Life sciences, Pittsburgh, PA, USA) for the first-round purification, using Ni-A buffer as the loading buffer, then mixing Ni-B buffer in different proportions (6%, 10%, 16%, 20%, and 60%) for gradient elution. The eluted samples were collected and detected by SDS-PAGE. The eligible eluted samples were mixed, diluted by 5 times, and loaded to a Hitrap Q HP (5 mL) ion-exchange column (GE Healthcare Life sciences, Pittsburgh, PA, USA) for the second-round purification. The sample was loaded with Q-A buffer and eluted with 50% Q-A and 50% Q-B mixture. The eluate was ultrafiltrated and concentrated with a 15 mL/10 kD ultrafiltration tube (Millipore China, Shanghai) by centrifuging at 4 °C and 6000 rpm. The concentrated sample was transferred to an MD44 dialysis bag (Beijing Solarbio Science﹠Technology Co., Ltd., Beijing, China), dialyzed overnight to replace the buffer. The molecular sieve buffer was used for dialysis, and changed every 3–4 h for 3 times.

2.4. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS)

The target recombinant PpPtxS band on the SDS-PAGE gel was cut and treated according to the methods developed by Kumarathasan et al. [27]. Then the sample was identified by MALDI-TOF-MS on AXIMA resonance MALDI-TOF-MS (Shimadzu Corp., Kyoto, Japan) equipped with a 337 nm nitrogen laser in positive ion detection. Spectra were accumulated until a satisfactory S/N had been obtained. Parent mass speaks with the range from 400 to 1600 m/z were picked out for MS/MS analysis. The peptide mass fingerprinting of the recombinant PpPtxS and MS/MS data was acquired and processed using Mass-Lynx V4.1 software (Micromass) and were converted to PKL files by the Protein-Lynx 2.2.5 software (Waters). The PKL files were analyzed using the MASCOT search engine (http://www.matrixscience.com/). The search parameters were defined as follows: database, Swiss-Prot; taxonomy, Eucaryotes; enzyme, trypsin; and allowance of one missed cleavage. The resulting peptide information was uploaded to National Center of Biotechnology Information (NCBI) for alignment and analysis. The results are credible with scores >53 (p < 0.05). 2.5. Circular dichroism (CD) far-ultraviolet scanning analysis A Chirascan™ Plus CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK) was used to analyze the secondary structure (far-UV CD spectra) of PpPtxS and PpPtxS-2KGA. The far-UV CD spectra were obtained in the intervals of 190–260 nm, in quartz cuvettes with a path length of 0.1 cm, at room temperature. The spectra were collected by continuous scanning at 0.1 nm intervals, a scanning speed of 100 nm/ min, a response time of 1 s, a bandwidth of 1 nm, and 15 accumulations. The baseline was corrected in all experiments using a protein-free buffer as control. Data processing including subtract baseline and smoothing was performed with the Pro-Data Viewer software (Applied Photophysics Ltd., Leatherhead, UK). 2.6. Isothermal titration calorimetry (ITC) A GE MicroCal iTC200 isothermal titration calorimeter (GE Healthcare Life Science, Pittsburgh, PA, USA) was used to detect the affinity between PpPtxS and various small molecules including calcium 2ketogluconate (2KG-Ca), glucose, gluconic acid, glucose-6-phosphate, pyruvic acid, 2-keto-L-gulonic acid and synthesized 14-bp palindrome DNA (5′-TGAAACCGGTTTCA-3′). The PpPtxS sample was desalted with a Zeba™ Spin Desalting Column (7K MWCO, 0.5 mL, Thermo Fisher Scientific, Shanghai, China) by replacing the buffer with ultrapure water, and adjusted to the concentration of 40 μM as the titrate. The aqueous solutions of 2KG-Ca, glucose, gluconic acid, glucose-6phosphate, pyruvic acid and 2-keto-L-gulonic acid were prepared to the concentration of 1 mM, and the DNA was diluted to around 100 mM. The experiment was carried out at 25 °C, and pure water was used as the blank titrate. Data were processed with MicroCal Concat ITC software (Malvern Panalytical Ltd., Malvern, UK). 2.7. Oligomerization state analysis of PpPtxS in solution The oligomerization state of PpPtxS in solution was analyzed by Size exclusion chromatography combining Multi-angle laser light scattering (SEC-MALS) and molecular sieve filtration chromatography. The SEC-MALS equipped with a DAWN HELEOS-II multi-angle laser light scattering detector (Wyatt Technology, Pittsburgh, PA, USA), an Optilab (U) TrEX differential refractive index detector (Wyatt Technology, Pittsburgh, PA, USA) and a TSK G3000SWXL high-performance-liquidchromatography (HPLC) SEC column (7.8 mm × 30 cm, 5 μm, Tosoh Corporation, Tokyo, Japan) was used to analyze the molecular weight (MW) of the recombinant PpPtxS. The purified PpPtxS was separated by HPLC with a mobile phase containing 25 mM PBS buffer (pH 7.0) and 150 mM NaCl. The conditions were set with a column temperature of 30 °C, a flow rate of 0.5 mL/min, 40-min equi-gradient elution, and a UV detection wavelength of 280 nm. Then the eluate entered the MALS detector to analyze the molecular sizes, and finally entered the refractive index detector to detect the concentration. The data was processed and analyzed by the Astra software (Wyatt Technology, Pittsburgh, PA, USA). The molecular sieve filtration chromatography was performed with a GE ÄKTA™ Pure protein purification system (GE Healthcare Life sciences, Pittsburgh, PA, USA) and a molecular sieve HiPrep 16/60 Sephacryl S-200 HR column (GE Healthcare Life sciences, Pittsburgh, PA, USA). The Gel Filtration Calibration Kit (High Molecular Weight) including ovalbumin (43 kDa), conalbumin (75 kDa) and aldolase (158 kDa) (GE Healthcare Life sciences, Pittsburgh, PA, USA) was used as the standards. One milligram of standard or filtered protein was loaded into the ÄKTA™ Pure system and then separated by the molecular sieve column using 40 mM PBS containing 150 mM NaCl (pH 7.4) as the mobile phase, with a column temperature of 4 °C, a flow rate of 0.3 mL/min, and a UV detection wavelength of 280 nm. The separated peaks were collected and further analyzed by SDS-PAGE. 2.8. Homologous modelling and molecular docking simulation The 340-amino-acid sequence of PpPtxS from P. plecoglossicida JUIM01 was uploaded to the fully automated protein structure homology-modelling server SWISS-MODEL (https://swissmodel.expasy. org/) for alignment. The result with the highest sequence identity was selected as the template for the tertiary-structure modelling of PpPtxS in both forms of dimer and its monomer. The three-dimensional structure of the ligand 2KGA was searched on the PubChem database (https:// pubchem.ncbi.nlm.nih.gov/). The molecular docking simulation between PpPtxS and 2KGA was carried out by the AutoDock software [28]. The simulation output results were sorted according to binding energy, and the highest-ranking results were selected. 3. Results and discussion 3.1. Heterologous expression and purification of PpPtxS PpPtxS was heterologously expressed by constructing the recombinant strain E. coli BL21(DE3)/pET-28a-ptxS. After 16-h induction by IPTG, the heterologously expressed PpPtxS in E. coli BL21(DE3)/pET28a-ptxS was analyzed by SDS-PAGE. As shown in Fig. S1A, the negative control BL21(DE3)/pET-28a(+) had the absence of the 36.65-kDa protein. A high-expression band in whole-cell lysate of E. coli BL21(DE3)/ pET-28a-ptxS was shown with a molecular weight (MW) between 29.0 and 44.3 kDa, which was consistent with the predicted PpPtxS MW of 36.65 kDa. Similarly, a PtxS from Pseudomonas aeruginosa is overexpressed in E. coli T7 system with a MW of 37.4 kDa, and shows an interference with the effect of a positive regulatory gene ptxR of the exotoxin A [23]. WB results further proved the presence of recombinant PpPtxS in E. coli BL21(DE3)/pET-28a-ptxS (Fig. S1B). Recombinant PpPtxS was firstly purified by using gradient eluted Nicolumn affinity chromatography and polyethyleneimine (PEI) precipitation. Purities of the eluted samples were verified with SDS-PAGE. As shown in Fig. 2A, PEI precipitation had no positive effect on PpPtxS purification. With the increase of Ni-B buffer concentration to over 20%, purity of PpPtxS significantly increased with a single band (Lane 8 # and 9 #). The eluates with 20% and 60% Ni-B buffers were further loaded onto a Hitrap Q HP chromatography column, and also showed a single band with high-purity (Fig. 2B). A recombinant PpPtxS yield of 0.5 mg/g wet cell weight was obtained after Ni-column affinity chromatography, Hitrap Q HP chromatography, ultrafiltration and dialysis. The potential PpPtxS band on the SDS-PAGE was identified using MALDI-TOF-MS and its peptide mass fingerprinting (PMF) (Fig. S2). NCBI BLAST results showed that PpPtxS sequence shared high identities (305 of scores and 14% of coverage rates) with two proteins including a transcriptional regulator from Pseudomonas (ID gi|497308403, consisting of 337 amino-acid residues) and a transcriptional regulator from Pseudomonas sp. M1 (ID gi|568070625, consisting of 340 aminoacid residues). The above results indicated that the recombinant protein, PpPtxS, could be classified as a transcriptional regulator with the potential regulation on 2KGA metabolism. 3.2. Secondary-structure analysis of PpPtxS The secondary structure of purified PpPtxS was analyzed by CD scanning and shown in Fig. S3. The ratios of α-helix, β-sheet (antiparallel and parallel), β-turn and random coils were calculated in the MilliDegress mode with CDNN software and summarized in Table 2. The proportions of secondary structure of PpPtxS showed a similar trend within 5 wavelength ranges of 190–260 nm, 195–260 nm, 200–260 nm, 205–260 nm, and 210–260 nm. α-Helix occupied highest proportion of 46.0–52.1%, followed by random coils accounting for about 25.2–31.2%. β-Sheet (including antiparallel and parallel) accounted for the lowest proportion of only 8.7%–11.9%. 3.3. The effector of PpPtxS Some small molecules work as effectors during transcription process by binding to the transcriptional repressors and inhibiting their transcriptional repressive activities, such as Lac repressor and its effector galactose [29]. In order to determine the effector(s) of PpPtxS, a variety of small-molecular substances related to glucose metabolism in Pseudomonas, including glucose, gluconic acid, 2KGA, glucose-6phosphate and pyruvate, were used as the titrants with concentrations of 1 mM, and pure water was used as the blank control. A significant exotherm was detected when using 1 mM 2KGA to titrate 40 μM PpPtxS, which meant that PtxS bound tightly to 2KGA. An ITC curve was fitted as shown in Fig. 3A, revealing the specific binding between 2KGA and PpPtxS. Binding was characterized with values of binding ratio N, constant Ka, and dissociation constant KD of 0.813 ± 0.0371 Sites, 4.91E5 ± 2.01E5 M−1, and 2.04E-6 M, respectively. However, the ITC curves could not be fitted when using 1 mM of glucose, gluconic acid, glucose-6-phosphate or pyruvate to titrate 40 μM PpPtxS (Fig. 3B–E), indicating that the above small-molecular substances had no molecular interaction with PpPtxS. The results were consistent with previous results by Daddaoua et al. that PtxS of P. putida KT2440 only binds to 2KGA among the candidate ligands of glucose, gluconic acid, 2KGA, pyruvate and 6-phosphogluconic acid [19]. Analogues of natural effectors in cells usually show similar molecular interaction with transcriptional repressors. For example, IPTG, a structural analogue of the natural effector, can also bind to Lac repressor to induce the initiation of transcription [29]. In order to explore other potential effectors of PpPtxS, the binding effect of an optical isomer of 2KGA, 2-keto-L-gulonic acid (Fig. S4), to PpPtxS was tested using ITC. The ITC curve was not successfully fitted, proving that no molecular binding of PpPtxS-2-keto-L-gulonic acid existed (Fig. 3F). During 2KGA production process, glucose (as substrate), gluconic acid (as intermediate) and 2KGA (as final product) usually co-exist in P. plecoglossicida JUIM01 cells and broth [30]. Hence, glucose-2KGA, gluconic acid-2KGA and glucose-gluconic acid-2KGA were mixed to find their mutual interactions when titrating. Glucose-2KGA mixture gave a fitted binding-curve with lower binding parameters N = 0.294 ± 0.201 Sites, Ka = 1.59E5 ± 1.20E5 M−1, and KD = 6.29E-6 M, which indicated that glucose negatively influenced the interaction between 2KGA and PpPtxS (Fig. 4A). Interestingly, mixing 2KGA with gluconic acid could not fit a binding curve (Fig. 4B). Glucose-gluconic acid-2KGA mixture also had no molecular binding reaction with PpPtxS, shown by overlapped blots with blank control (Fig. 4C). The above results could be concluded that 2KGA was solely effector of PpPtxS, glucose existence negatively affected the molecular binding of PpPtxS2KGA, and gluconic acid inhibited the PpPtxS-2KGA binding reaction. The effector-binding usually leads to the structural changes of the proteins [31,32]. Fig. 5 presents CD scanning results under the wavelength of 190–250 nm of PpPtxS and 2KGA-bound PpPtxS to find the secondary structure changes of PpPtxS before and after 2KGA addition. The positive peaks at 201 nm and 206 nm in the far-UV region disappeared while a stronger negative-peak signal appeared at 195 nm. Further analysis showed that with the addition of 2KGA, the proportion of random coils in the secondary structure of the protein increased significantly from 31.2% to 60.4%. The proportion of β-sheet and β-turn also increased slightly to 14.9% and 19.3%, respectively. Accordingly, the proportion of α-helix significantly reduced from 46.0% to 5.4%. Hence, it could be concluded that the secondary structure of PpPtxS significantly changed after binding 2KGA. 3.4. Oligomerization state of PpPtxS in solution Oligomerization state of PpPtxS in solution directly affects its physical stability and binding form. MALS combined with SEC was used to detect the status of PpPtxS in solution. As shown in Fig. 6A, the PpPtxS sample presented mainly three peaks. Peak 1# and 2# could be excluded from proteins due to no absorption in UV. MW of Peak 3# was calculated to about 74.6 kDa, which was close to twice of the theoretical MW of PpPtxS, indicating that the protein was in the dimeric form. A molecular sieve filtration chromatography was performed to further confirm a dimeric state of PpPtxS in water solution with ovalbumin (43 kDa), conalbumin (75 kDa) and aldolase (158 kDa) as MW standards. As shown in Fig. 6B, PpPtxS also showed mainly three peaks. Among them, Peak 3# was proven to be PpPtxS by SDS-PAGE (Fig. S5) while Peak 1# and 2# were excluded from proteins. As Peak 3# had the similar retention with the standard conalbumin (75 kDa), confirming that PpPtxS was dimeric in solution. The obtained results were in agreement with the previous report by Daddaoua et al. that PtxS from P. putida tended to be dimeric [19]. 3.5. Molecular docking of PpPtxS and its effector 2KGA The amino-acid sequence of the PtxS from P. plecoglossicida JUIM01 was uploaded to the SWISS-MODEL for alignment. The result with the highest identity (protein code: 5ysz.1.A) was selected as the template for modelling. The template is described as a LacI-family transcriptional repressor, with a sequence identity of 25.85% with PpPtxS. The predicted tertiary structures of PpPtxS in both forms of dimer and monomers are shown in Fig. S6. The three-dimensional structure of 2KGA was obtained from the PubChem database (Fig. 7A), and molecular docking simulation of the PpPtxS and its effector 2KGA was performed by the AutoDock software. The simulation output results were sorted according to the binding energy, and the highest-score result was selected, with binding energy = −3.16 and ligand efficiency = −0.24. As shown in Fig. 7, the effector 2KGA mainly bound to the region close to the C-terminal of PpPtxS. The 299th to the 301st amino acids (alanine, glutamine, proline, threonine, glutamic acid and arginine, respectively) of PpPtxS were predicted to be involved in the interaction of PpPtxS and 2KGA. Additionally, this region is predicted to be a putative protein kinase C phosphorylation P. aeruginosa [19,20,33]. The tertiary-structure prediction of PpPtxS and its molecular docking simulation with the effector 2KGA provided a theoretical basis for the future modification of the protein. However, structural information obtained by SWISS-MODEL still has some limits including incomplete sequence information and lower identity to the known proteins. The analysis of the tertiary and quaternary structures is still a challenge in the current study on the Pseudomonas PtxS proteins. Only Pineda-Molina et al. successfully crystallized the PtxS-DNA complex from P. putida by the counter-diffusion technique, but failed to crystallize the PtxS alone. The high sensitivity of crystallization also hinders the researchers from further investigation on its structure and function [33]. The amino-acid sequence identity between the reported protein (accession number: Q88HH7) and PpPtxS reaches 72.57%, but unfortunately the tertiary-structure model of the P. putida PtxS has not been reported. 3.6. PpPtxS recognized and bound to a 14-bp palindrome 5′-TGAAACCGGTT TCA-3′ Previously, PtxS from P. aeruginosa and P. putida were reported to specifically recognize a 14-bp palindrome sequence (5′-TGAAACCGG TTTCA-3′) in the promoter region of the regulated gene, which overlaps the RNA polymerase binding site. Whereby, these PtxS are able to regulate the transcription of the corresponding gene by binding to the region where the palindrome is located [19,21,22,24,34]. Both of the above two PtxS contain the typical helix-turn-helix (HTH) motif of the LacI-family regulators at the N-terminal, which is likely to be the DNA binding region of the PtxS [19,23]. Our previous bioinformatics analysis revealed that PpPtxS also has the conserved helix-turn-helix motif [26]. The ability of PpPtxS to recognize and bind to the 14-bp palindrome sequence (5′-TGAAACCGGTTTCA-3′) in vitro was verified by titrating PpPtxS with the DNA. ITC results showed that PpPtxS showed a similar ability of 14-bp-palindrome recognition and binding to the P. aeruginosa and P. putida PtxS (Fig. 8), which possibly owed to the conserved HTH sequence at the N-terminal of these PtxS. Our previous study also proved the existence of the 14-bp palindrome in the promoter region of the kgu operon in the P. plecoglossicida JUIM01 genome, which plays an important role in 2KGA metabolism [35]. Hence, PpPtxS should regulate 2KGA metabolism by specifically bind to the region where the 14-bp palindrome is located in P. plecoglossicida. 4. Conclusion In this study, a transcriptional regulator PpPtxS with a MW of 36.65 kDa from P. plecoglossicida JUIM01 was heterologously expressed, then structurally and functionally characterized. PpPtxS had a sole effector of 2KGA. Glucose existence negatively affected the molecular binding of PpPtxS and 2KGA, and gluconic acid completely inhibited the PpPtxS-2KGA binding reaction. PpPtxS in water solution mainly existed as a dimer and bound to two molecules of 2KGA. The effector 2KGA mainly bound to the region close to the C-terminal of PpPtxS by interacting with the 299th to the 301st amino acids (Ala, Gln, Pro, Thr, Glu and Arg). 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