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Article

Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae

by
Weiwei Yan
1,2,
Yiming Wei
1,
Susu Fan
3,
Chao Yu
1,
Fang Tian
1,
Qi Wang
2,
Fenghuan Yang
1,* and
Huamin Chen
1
1
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
2
The MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
3
Shandong Provincial Key Laboratory of Applied Microbiology, Ecology Institute, Shandong Academy of Sciences, Jinan 250014, China
*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(3), 495; https://doi.org/10.3390/microorganisms9030495
Submission received: 28 January 2021 / Revised: 19 February 2021 / Accepted: 23 February 2021 / Published: 26 February 2021
(This article belongs to the Section Molecular Microbiology and Immunology)
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Abstract

:
Cyclic diguanylate monophosphate (c-di-GMP) is a secondary messenger present in bacteria. The GGDEF-domain proteins can participate in the synthesis of c-di-GMP as diguanylate cyclase (DGC) or bind with c-di-GMP to function as a c-di-GMP receptor. In the genome of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, there are 11 genes that encode single GGDEF domain proteins. The GGDEF domain protein, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo 6]) was characterized in the present study. Firstly, the DGC and c-di-GMP binding activity of GdpX6 was confirmed in vitro. Mutation of the crucial residues D403 residue of the I site in GGDEF motif and E411 residue of A site in GGDEF motif of GdpX6 abolished c-di-GMP binding activity and DGC activity of GdpX6, respectively. Additionally, deletion of gdpX6 significantly increased the virulence, swimming motility, and decreased sliding motility and biofilm formation. In contrast, overexpression of GdpX6 in wild-type PXO99A strain decreased the virulence and swimming motility, and increased sliding motility and biofilm formation. Mutation of the E411 residue but not D403 residue of the GGDEF domain in GdpX6 abolished its biological functions, indicating the DGC activity to be imperative for its biological functions. Furthermore, GdpX6 exhibited multiple subcellular localization in bacterial cells, and D403 or E411 did not contribute to the localization of GdpX6. Thus, we concluded that GdpX6 exhibits DGC activity to control the virulence, swimming and sliding motility, and biofilm formation in Xoo.

1. Introduction

The phytopathogen Xanthomonas oryzae pv. oryzae (Xoo) causes the bacterial leaf blight in rice, one of the most devastating bacterial diseases of rice [1]. To infect a plant, Xoo mainly invades the rice through the wound or water pores, colonizes the xylem vessels of rice leaves [1]. The bacterial leaf blight disease causes different degrees of reduction rice yield with 70% yield reduction in the most severe cases [2,3]. The successful Xoo infection of a rice plant depends on several protein secretion systems, such as the type II secretion system that plays important roles in the degradation of plant cell walls and the type III secretion system which transfers the effectors into the plant cells [4,5,6]. In addition, multiple virulence factors like exopolysaccharide (EPS), biofilm, extracellular enzymes, and adhesins contribute to the virulence of Xoo [4]. These virulence factors have been found to be regulated by several signaling systems, including the two-component systems, diffusible signal factor signaling pathway, and cyclic diguanylate monophosphate (c-di-GMP) signaling pathway in Xoo [7,8,9,10,11].
c-di-GMP has been recognized as an essential secondary messenger that regulates biofilm formation, motility, cell differentiation, and virulence of pathogenic bacteria [12,13,14,15]. Diguanylate cyclase (DGC) with a GGDEF domain and phosphodiesterase (PDE) with an EAL or HD-GYP domain are responsible for the synthesis and degradation of c-di-GMP, respectively [16,17]. In general, high levels of c-di-GMP in bacteria can promote biofilm formation and switching of bacterial lifestyle from motile to sessile. However, low concentrations of c-di-GMP stimulate the motility and the expression of virulence factors [16,17]. Identification of multiple receptors can help to explain how c-di-GMP elicits specific responses. Several studies have revealed that c-di-GMP exerts its multiple regulatory roles by binding to RNA, various effectors, and regulatory proteins, including PilZ domain proteins, transcriptional regulators, degenerate GGDEF or EAL domain proteins, polynucleotide phosphorylase, riboswitches, and kinases, to name a few [18].
Many bacterial genomes encode dozens of GGDEF-domain proteins, and some of these are characterized by biochemical characteristics, molecular structure, and physiological functions. It has been reported that an active site (A-site) of GGDEF domain is crucial for the GGDEF-domain proteins to catalyze the condensation of two GTP molecules to form a molecule of c-di-GMP [16,17]. Additionally, the RXXD motif of the allosteric inhibition site (I site), which is located before the A site in GGDEF domain participates in c-di-GMP binding [19]. Several proteins contain a conserved A site in GGDEF domain function as DGCs, such as PleD of Caulobacter crescentus, WspR of Pseudomonas aeruginosa and the GGDEF-domain protein YeaP of Escherichia coli [20,21,22,23]. There is a general regulatory mechanism of some active DGCs including PleD and WspR, which allows them to bind c-di-GMP via the I site to feedback inhibit its DGC activity. Nonetheless, there are some exceptions, for instance, GGDEF-domain proteins with degenerated GGDEF motif including VCA0965 of Vibrio cholerae and ECA3270 of Pectobacterium atrosepticum catalyze the synthesis of c-di-GMP [24,25]. Besides the role of the I-site in DGC feedback inhibition, the non-catalytic GGDEF domain with degenerate A-site motifs including PopA in C. crescentus and SgmT in Myxococcus xanthusmmi can exert their regulatory roles by conserved c-di-GMP binding via the I site and represents an important class of c-di-GMP effector proteins [26,27].
In Xoo, c-di-GMP signaling plays important roles in regulating the expression of virulence factors [7,8,9,10,11]. The genome of Xoo PXO99A codes for eleven proteins with single GGDEF domain [28]. So far, only two have been characterized: GdpX1, which is involved in the regulation of virulence, EPS production, and swimming motility, and DgcA, an active DGC which negatively regulates the pathogenicity of Xoo on rice, EPS production, motility, and auto-aggregation by production of c-di-GMP [10,29]. In the present study, PXO_02019 (here GdpX6 [GGDEF-domain protein of Xoo 6]), which contains an extracellular domain Cache_1, a transmembrane domain (TM domain) and a GGDEF domain was characterized. In vitro analysis revealed that GdpX6 is not only an active DGC, but also binds to c-di-GMP with low affinity. Furthermore, the present study provides evidence that GdpX6 controls the virulence, swimming and sliding motility, and biofilm formation in Xoo. Subcellular localization analysis identified that GdpX6 protein has multisite distribution in the cell. These findings demonstrate that GdpX6 functions as a DGC to negatively regulate the virulence in Xoo.

2. Materials and Methods

2.1. Bacterial Strains Plasmids and Growth Condition

The bacterial strains and plasmids used in this study are list in Table 1. E. coli strains were cultured in Luria–Bertani (LB) medium at 37 °C. Xoo wild-type strain PXO99A, derived mutants and complementary strains were cultured on peptone sucrose agar (PSA) medium or M210 liquid medium with appropriate antibiotics at 28 °C [30]. Relevant antibiotics were used at the following concentrations: gentamycin, 20 µg/mL; kanamycin (Km), 50 µg/mL; and ampicillin (Ap), 100 µg/mL [31].

2.2. Bioinformatics Analysis of GdpX6

Nucleotide and amino acid sequences were downloaded from National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/pmc/) (accessed on 10 January 2021). The domain structures of GdpX6 were analyzed by the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/) (accessed on 10 January 2021). Multiple-sequence alignments were performed using the DNAMAN software (version 5.2.2, Lynnon BioSoft, Sanramon, CA, USA).

2.3. Protein Expression and Purification

The truncated DNA fragment GdpX6GGDEF encoding the intact GGDEF domain of GdpX6 was amplified by using the GdpX6PF1/GdpX6PR1 primer pairs. Point mutations of GdpX6 were generated by Bridge PCR, as described previously [8]. For point mutation of D403 in the GGDEF domain of GdpX6, the upstream and downstream fragments were amplified using primers GdpX6PF1/D403R, and D403F/GdpX6PR1. Subsequently, the fragments GdpX6GGDEF-D403A were amplified using two fragments as the templates by the GdpX6PF1/GdpX6PR1 primers. Point mutation for E411 was constructed using the methods as that of GdpX6GGDEF-D403A. The primers used in the study are listed in Table S1. The fragments GdpX6GGDEF, GdpX6GGDEF-D403A, and GdpX6GGDEF-E411A were subjected to treatment with corresponding restriction endonuclease, followed by their insertion into the pColdSUMO plasmid, resulting in pCGdpX6GGDEF, pCGdpX6GGDEF-D403A, pCGdpX6GGDEF-E411A constructs. The presence of the correct fragments GdpX6GGDEF, GdpX6GGDEF-D403A and GdpX6GGDEF-E411A was confirmed by sequencing. The plasmids were transferred into E. coli strains BL21(DE3) for protein expression. Expression of target proteins was induced by addition of isopropyl β- d-1-thiogalactopyranoside (IPTG) at the final concentration of 0.5 mM. Then, the bacterial cultures were incubated for 16 h at 16 °C. And the target proteins were purified as described previously [8,36]. At the same time, purified SUMO-His6 protein was purified as a negative control.

2.4. DGC Activity Assay

DGC activity of GdpX6 was firstly verified by using the riboswitch-based dual-fluorescence reporter system as described previously by Zhou et al. [34]. The constructs pCGdpX6GGDEF, pCGdpX6GGDEF-E411A and the empty vector pColdSUMO were transformed to E. coli BL21(DE3) strain containing a triple-tandem riboswitch BC3-5 RNA. The E. coli strains containing pETPleD/BC3-5 RNA and pET28b/BC3-5 RNA were used as positive control and its corresponding negative control, respectively [34]. All bacterial strains were grown in LB medium at 28 °C until OD600 reached approximately 0.8. Thereafter 1 mM IPTG was added to the bacterial culture for 20 h for induction of the protein expression. The culture was then maintained at 4 °C for subsequent experiments. Samples were diluted to an OD600 of 0.1 with water, and the ratio of fluorescence intensity (RFI) was detected by Flex station 3 (Moleculer Devices, Sunnyvale, CA, USA).
The DGC activity of the proteins was further confirmed by using GTP as the substrate [37]. Around 100 µg target purified proteins were added into a reaction buffer (75 mM Tris-HCl (pH 7.8), 250 mM NaCl, 25 mM KCl, 10 mM MgCl2) containing 100 μM GTP. The mixture was incubated at 37 °C for 12 h. The reaction was terminated by heating the reaction mixture at 95 °C for 5 min, followed by centrifugation at 12000 rpm for 5 min. The c-di-GMP production in supernatant was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

2.5. ITC Isothermal Titration Calorimetry (ITC) Assay

The binding of the GdpX6 with c-di-GMP was performed on an ITC200 calorimeter (MicroCal, Northampton, MA). Briefly, the proteins (30 μM SUMOHis6, 10 μM SUMOHis6-GdpX6GGDEF, 10 μM SUMOHis6-GdpX6GGDEF-D403A) were syringed into the cell pool and 2 μL c-di-GMP solution (300 μM, 1 mM, 1 mM) as ligand was titrated with the sample at 120 sec intervals with stirring speed of 1000 rpm at 25 °C. The heat changes accompanying these additions were recorded. The data of the titration experiment was calibrated with a buffer control. The dissociation constant (Kd) was analyzed through the single-site model using the MicroCal ORIGIN version 7.0 software [35].

2.6. Construction of Gene Deletion Mutant, Complementation and Overexpression Strains

The ΔgdpX6 mutant was generated using the suicide vector pKMS1 by homologous recombination, as described previously [38]. The left and right arms of gdpX6 were amplified by PCR using PXO99A genomic DNA as template with specific primers (Table S1). The fragments were digested and subsequently ligated into the pKMS1vector to generate the pKgdpX6 construct. The plasmid was transformed into PXO99A by electroporation and subsequently screened on NAS plate with 10% sucrose. The single colonies that were resistant to 10% sucrose but sensitive to Km were further confirmed as mutant by PCR analysis.
The coding region of gdpX6 with ribosome-binding site but without the termination codon was amplified with gdpX6CF/gdpX6CR primers and inserted into broad-host-range pBBRMCS1 plasmid containing a gfp gene which encoded a green fluorescent protein (GFP), resulting in pBgdpX6gfp plasmid. Point mutation of D403 and E411 of GdpX6 were constructed using the method described above, resulting in the plasmids pBgdpX6D403Agfp and pBgdpX6E411Agfp. pBgdpX6gfp, pBgdpX6D403Agfp and pBgdpX6E411Agfp. These plasmids were then transformed into ∆gdpX6 as well as PXO99A by electroporation and screened on PSA plate containing 100 μg/mL ampicillin antibiotic to acquire complementation and gdpX6-overexpressing strain, respectively. The expression of GFP-fusion proteins was confirmed by western blotting. The primers used in the study are listed in Table S1.

2.7. Western Blotting Analysis

For analysis of the expression of GFP-fusion proteins in PXO99A, Xoo strain was cultured in M210 medium at 28 °C to OD600 of 1.0. Bacteria cells were collected by centrifugation at 12,000 rpm for 5 min. The cells were resuspended in phosphate-buffered saline (PBS) and ultrasonicated for 2 min. The samples were boiled, separated on 12% SDS-PAGE and subsequently transformed onto polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany) for immunoblotting using anti-GFP primary antibodies (Huaxingbio, Beijing, China). Goat anti-mouse secondary antibody conjugated with HRP (horseradish peroxidase) were used to recognize the primary antibodies (TransGen Biotech, Beijing, China). Enhanced HRP-DAB Chromogenic Kit (TransGen Biotech, Beijing, China) was used to check the target proteins on the membrane according to the manufacturer’s guidelines.

2.8. Virulence Assay

The pathogenicity assay on susceptible rice cultivar (Oryza sativa L. cv. Nipponbare) was used to assess the virulence of wild-type PXO99A and its derived strains [8]. Firstly, all strains were cultured in M210 medium at 28 °C to OD600 of 0.8–1.0. Bacterial cells were harvested by centrifugation and subsequently resuspended in the same volume of sterilized ddH2O. The bacterial cells were inoculated onto the rice leaves by the leaf clipping method. At least ten leaves were inoculated for each strain in each experiment. After 14 days, the lesion length was measured, and the leaves were photographed. The experiment was repeated three times.

2.9. Motility Assay

Xoo strains were grown in M210 at 28 °C until OD600 reached 0.8. Thereafter, bacteria cells were harvested and resuspended in ddH2O. A total of 2 μL of bacterial suspension was stabled into semi-solid plates containing 0.25% agar for testing swimming assay or SB medium plates containing 0.6% agar for analyzing sliding motility, respectively [39,40]. Pictures were taken after bacterial growth at 28 °C for 4 days. The diameters of the swimming or sliding zones were measured. The experiment was repeated thrice in triplicate.

2.10. Biofilm Formation Assay

Xoo cells were grown in M210 and diluted in M210 to an OD600 of 0.5. 200 μL of the cultures were transferred to a 96-well polystyrene microplate and incubated at 28 °C for 4 days. The medium was discarded, and biofilm was washed with distilled water. The biofilm was subjected to staining with 0.1% crystal violet for 15 min [41]. The biofilm was washed twice with distilled water and photographed. For quantification, the biofilm was dissolved in ethanol, and the absorbance was detected at 490 nm with Flex station 3 (Molecule Devices, Sunnyvale, CA, USA). All experiments were performed thrice in triplicate.

2.11. EPS Production Assay

The ethanol precipitation method was used to quantify the EPS production of Xoo strains [42]. The Xoo strain was cultured in M210 medium until the OD600 reached 2.5. The supernatants were collected by centrifugation at 6,000 rpm for 10 min. Supernatants were subsequently incubated at −20 °C overnight following the addition of two volumes of absolute ethanol to the supernatants. The EPS molecules were collected by centrifugation at 10,000 rpm for 20 min and then dried overnight at 55 °C. The weights of EPS molecules were determined. At the same time, 2 μL of bacterial supernatant was stabbed onto a PSA plate and incubated at 28 °C for 3–4 days and then photographed. All experiments were performed thrice in triplicate.

2.12. Extracellular Enzymatic Activities Assay

Extracellular enzymatic activities of Xoo strains were tested as described previously [43]. Bacterial cells were cultured in M210 at 28 °C until an OD600 of 0.8 was reached. Thereafter, 2 μL cultures were stabbed onto PSA plate with 0.2% RBB-xylan. Xylanase activity was detected by the appearance of white clear zones against a blue background on the plate after incubation at 28 °C for 2 days. On the other hand, 2 μL cultures were stabbed onto PSA plate with 0.5% carboxymethyl cellulose. Plates were cultured at 28 °C for 2 days. The plates were subsequently stained with 1% Congo red for 30 min. Thereafter, the plates were washed twice with 1 M NaCl solution for 20 min. Cellulase activity was observed by the appearance of the transparent circle under the red background. All experiments were performed thrice in triplicate.

2.13. Fluorescence Microscopy

The PXO99A(pBgdpX6gfp) and PXO99A(pBgdpX6D403Agfp), PXO99A(pBgdpX6E411Agfp) strains were grown in M210 medium to an OD600 of 1.0. The cell suspension was dripped on a glass slide and covered with a coverslip. The subcellular location signals of samples were observed and recorded using an Olympus BX61 microscope (Olympus, Tokyo, Japan).

2.14. Statistical Analysis

All analysis was performed using Microsoft Excel 2010. The means and standard deviations of experimental results were calculated using average function and STDEV function, respectively. And t test was used to determine significant differences between samples.

3. Results

3.1. GdpX6 Contains a Conserved GGDEF Domain

GdpX6, one of eleven genes encoding single GGDEF domain proteins in the genome of Xoo PXO99A, contains an extracellular Cache_1 protein domain (41 to 275 amino acids [aa]) that is predicted to have a role in small-molecule recognition [44], a TM domain (294 to 311 aa), and a GGDEF domain (318 to 495 aa) (Figure 1a). Blast searches revealed that GdpX6 has homologous proteins in several sequenced Xanthomonas species genomes (Figure S1a). GdpX6 was found to be homolog of XCC2731 in X. campestris pv. campestris (Xcc), XOC1515 in X. oryzae pv. oryzicola (Xoc) and XAC2897 in X. citri pv. citri (Xac), with the amino acid sequence similarities of 78.93%, 92.47%, and 65.01%, respectively. (Figure S1b). Previous studies have revealed the capacity of GGDEF domain to synthesize c-di-GMP or bind to c-di-GMP depends on the presence of A-site and I-site in GGDEF domain, respectively [16,17]. The amino acid sequence alignment with the reported active DGCs indicated that GGE411EF residues of A-site, RXXD403 residues of I-site, Mg2+, and GTP binding sites were conserved in GdpX6 (Figure 1b). Thus, GdpX6 might function as an active DGC and bind with c-di-GMP.

3.2. GdpX6 Demonstrates DGC Activity In Vitro

To evaluate whether GdpX6 possesses the DGC activity, the protein GdpX6GGDEF containing the GGDEF motif of GdpX6 and the mutagenized protein GdpX6GGDEF-E411A having mutation in important residue E411 of the GGDE411F motif for the catalysis of diguanylate cyclase, were expressed, respectively. Firstly, the DGC activity of GdpX6 was measured using the dual-fluorescence reporter system by visible fluorescence color changes and the RFI values in E. coli BL21(DE3) [34]. The results showed that the color of bacterial cells containing pCGdpX6GGDEF expression vector changed from green to red and the relative RFI values significantly increased about 13 folds as compared to the bacterial cells containing the negative vector pColdSUMO after 20 h IPTG induction (p < 0.05) (Figure 2a). These results were similar to those of the bacterial cells containing PleD from C. crescentus, which exhibited strong DGC activity compared to its negative control pET28b [34] (Figure 2a). The bacterial cells containing point mutant pCGdpX6GGDEF-E411A expression vectors showed the same color as that of the negative pColdSUMO control. However, the value of RFI decreased about 81% as compared to GdpX6GGDEF (Figure 2a), suggestive of the lower DGC activity of GdpX6GGDEF-E411A as compared to GdpX6GGDEF. Subsequently, the enzyme activity of purified SUMOHis6-GdpX6GGDEF, SUMOHis6-GdpX6GGDEF-E411A and SUMOHis6 proteins was tested using GTP as substrate and the concentration of c-di-GMP in the reaction was detected by LC-MS/MS. As shown in Figure 2b, approximately 600 ng/mL c-di-GMP was synthesized by the protein SUMOHis6-GdpX6GGDEF, while no c-di-GMP synthesis was detected in the negative SUMOHis6 control. SUMOHis6-GdpX6GGDEF-E411A did not exhibit the wild-type DGC activity (Figure 2b). These results suggest that GdpX6 is an active DGC.

3.3. GdpX6 Binds to c-di-GMP via the I Site of GGDEF Motif

The bioinformatic analysis demonstrated that GdpX6 contains a conserved I-site that can bind to c-di-GMP [45]. To identify whether GdpX6 binds to c-di-GMP, the c-di-GMP binding affinity of the proteins GdpX6, GdpX6GGDEF-D403A and SUMOHis6 was determined by ITC. The results showed that GdpX6GGDEF bound c-di-GMP with the dissociation constants (Kd) of 9 ± 2.98 µM (Figure 3b), while no interaction was detected between the control protein SUMOHis6 and c-di-GMP (Figure 3a). To further characterize the importance of I site of GdpX6, the D403 residue of I site within the protein was mutated, and the resultant mutant protein was evaluated for its c-di-GMP binding ability. It was found that the mutated protein GdpX6GGDEF-D403A failed to bind to c-di-GMP (Figure 3c). These results indicate that GdpX6 binds c-di-GMP through I-site.

3.4. GdpX6 Contributes to the Virulence of Xoo on Rice

Several GGDEF-domain proteins have been shown to be involved in the regulation of bacterial virulence in Xanthomonas species [10,29,46]. To investigate whether GdpX6 affects the virulence of Xoo, a gdpX6-deleted strain, complement strains of ∆gdpX6 and gdpX6-overexpressing strains were constructed. The expressions of GdpX6-GFP, GdpX6D403A-GFP and GdpX6E411A-GFP in wild-type PXO99A and ∆gdpX6 were confirmed at expected sizes by western blotting analysis (see Figure S2). The bacterial cells were inoculated onto the leaves of susceptible rice plants by leaf-clipping method. The disease symptoms of rice were measured and recorded after 14 days of inoculation. As shown in Figure 4a,b, the mutant ∆gdpX6 strain caused more severe disease symptoms and the lesion lengths were increased by about 20% as compared to wildtype strain PXO99A. In trans expression of the full length gdpX6-gfp in ∆gdpX6 restored the phenotype to near-wild-type levels. The results showed that expression of point mutation protein GdpX6D403A-GFP in ∆gdpX6 restored the virulence to near-wild-type levels, while expression of GdpX6E411A-GFP in ∆gdpX6 showed similar virulence to that of the mutant ∆gdpX6 strain (Figure 4a,b). These results demonstrate that the GFP fusion proteins of GdpX6 retained their function. Moreover, overexpression of GdpX6-GFP or GdpX6D403A-GFP in PXO99A significantly decreased the virulence (by about 25%) in comparison to PXO99A (p < 0.05), while as overexpression of GdpX6E411A-GFP in PXO99A failed to inhibit the virulence of Xoo. These results suggest that GdpX6 negatively regulates the bacterial virulence in Xoo and the GGDEF domain of GdpX6 plays a primary role in regulation of virulence in rice.

3.5. GdpX6 Is Involved in the Regulation of Swimming and Sliding Motility of Xoo

To investigate whether gdpX6 influences the motility of Xoo, the wild-type strain PXO99A, the mutant ∆gdpX6 strain and the complement strains of ∆gdpX6 and gdpX6-overexpressing strains were cultured on semi-solid plates containing 0.25% agar for flagellum-dependent swimming motility or SB medium containing 0.6% agar for type IV pilis-dependent sliding motility as described in previous studies [47]. As shown in Figure 5a, ∆gdpX6 displayed larger swimming zones, and the swimming diameters increased to about 3 mm than PXO99A. The complement strains ΔgdpX6 (pBgdpX6gfp) and ΔgdpX6(pBgdpX6D403Agfp) recovered the swimming motility near to that of wild-type, while ΔgdpX6(pBgdpX6E411Agfp) showed similar swimming motility to the ΔgdpX6 strain (Figure 5a). Overexpression of gdpX6 in PXO99A led to a significant decrease in the swimming motility of Xoo (p < 0.05) (Figure 5a). When the E411 or D403 were mutated in GdpX6, the influence of overexpression of gdpX6 in PXO99A on the swimming motility disappeared (Figure 5a). Moreover, results from the sliding motility assays showed that the sliding zones of ΔgdpX6 mutant were smaller than that of PXO99A, while complement strains ΔgdpX6(pBgdpX6gfp) restored it to that of the wild-type (Figure 5b). The complement ΔgdpX6(pBgdpX6E411Agfp) strain showed a similar sliding zone as that of the ΔgdpX6 mutant, while the ΔgdpX6(pBgdpX6D403Agfp) strain displayed similar sliding motility as that of the ΔgdpX6(pBgdpX6gfp) strain (Figure 5b). Overexpression of GdpX6 or GdpX6D403A in PXO99A resulted in the enlarged sliding motility as compared to the wild type, while expression in trans of GdpX6E411A in the PXO99A failed to enhance its sliding motility (Figure 5b). These results indicate that GdpX6 regulates swimming motility negatively but sliding motility positively in Xoo.

3.6. GdpX6 Promotes Biofilm Formation of Xoo

It has been reported that c-di-GMP affects the adhesion of bacteria to host cells via regulation of the biofilm formation in bacteria [48]. Therefore, we examined whether deletion and overexpression of gdpX6 influence biofilm formation of Xoo. Results from biofilm formation assays showed that biofilm formation of ΔgdpX6 mutant decreased by approximately 21 % relative to PXO99A, while complement strains ΔgdpX6 (pBgdpX6gfp) restored the phenotype near to PXO99A (Figure 6). The complement strain ΔgdpX6 (pBgdpX6D403Agfp) restored biofilm formation similar to that of the ΔgdpX6 (pBgdpX6gfp) strain, while ΔgdpX6 (pBgdpX6E411Agfp) strain displayed a biofilm formation level similar to that of the ΔgdpX6 mutant (Figure 6). In contrast, biofilm formation increased approximately 33% and 32% GdpX6 or GdpX6D403A under overexpression in PXO99A in comparison to PXO99A, while as there were no significant differences in terms of biofilm formation under GdpX6E411A overexpression in PXO99A (Figure 6). These results suggest that gdpX6 promotes biofilm formation in Xoo, where the residues E411 are indispensable for the regulation of biofilm formation by GdpX6.

3.7. GdpX6 Does Not Control EPS Production and Extracellular Enzymatic Activities of Xoo

The regulation of GGDEF-domain proteins on EPS production has been reported in several bacteria [29,49,50]. Deletion of PDEs or DGCs encoding genes in Xoo has a significant impact on EPS production [8,11,29], so we compared the EPS production levels between wild-type strain, mutant ∆gdpX6 strain, and gdpX6-overexpressing strain. Results from colony examining and EPS quantification showed that there were no differences between PXO99A, the mutant ∆gdpX6 strain and gdpX6-overexpressing strain in terms of EPS production (see Figure S3). These results indicate that GdpX6 is not involved in the EPS production of Xoo.
The cellulase and xylanase activities contribute to the virulence of Xoo [43]. We analyzed whether GdpX6 affected the cellulase and xylanase activities of Xoo. No significant differences in cellulase and xylanase activities of PXO99A, the mutant ∆gdpX6 strain and gdpX6-overexpressing strain were found (see Figure S4). These results indicate that GdpX6 does not regulate the extracellular enzymatic activities of Xoo.

3.8. Subcellular Localization of GdpX6 in Xoo

The subcellular localization of a protein is considered essential for the execution of its biological function in bacteria [51,52]. Therefore, we analyzed the subcellular localization of GdpX6-GFP, GdpX6D403A-GFP and GdpX6E411A-GFP proteins in PXO99A. In all bacterial cells tested, GdpX6-GFP, GdpX6D403A-GFP and GdpX6E411A-GFP proteins displayed multisite distributions of 96%, 96.0% and 93.4%, respectively, while less than 7% of the cells showed other subcellular location types including bipolar and unipolar (Figure 7). These results demonstrate that GdpX6 mainly exhibits multisite localization in the Xoo cells, and point mutants in the A or I site residues of the GGDEF domain do not influence the subcellular localization of GdpX6.

4. Discussion

GGDEF-domain proteins are widely distributed in bacterial genomes, many of these function as DGCs or receptors to regulate a diversity of biological phenotypes [53,54]. Previous studies have shown that GGDEF-domain proteins, including GdpX1 and DgcA, play crucial roles and participate in regulation of biological functions via c-di-GMP signaling in Xoo [10,29]. In this study, we functionally characterized the GGDEF-domain protein GdpX6, which was demonstrated to be an active DGC that negatively regulates the virulence and swimming motility, and positively regulates sliding motility and biofilm formation of Xoo. Therefore, GdpX6 is a novel DGC in Xoo and modulates multiple virulence-related phenotypes.
The homologs of GdpX6 exist in Xanthomonas species including Xcc, Xoc, and Xac (Figure S1). GdpX6 shows protein sequence similarity of 78.93% to XCC2731 from Xcc. XCC2731 regulates the aggregation of cells, motility, extracellular enzymes, and EPS production in Xcc, while the transcript level of XCC2731 is regulated by Clp [46]. The present study did not find any role of gdpX6 in EPS production and extracellular enzymes as reported for XCC2731. However, it was found that GdpX6 not only regulates motility and virulence but also controls biofilm formation and sliding motility in Xoo. It indicates that the homologs of GdpX6 from different species with high sequence similarity exhibit differences in the regulation of biological functions. Moreover, we confirmed that GdpX6 functions as novel DGC in Xoo. Based on the studies carried on the homologs of GdpX6 [46], our findings provide further information about special regulatory roles of GdpX6 in Xoo and new evidence on the mechanism of GdpX6 in the regulation of expression of virulence factors might be an DGC to affect the concentration of c-di-GMP in bacterial cells.
Structural analyses of GGDEF domains have revealed diverse mechanism for c-di-GMP synthesis and c-di-GMP binding [55]. DGCs have a conservative GG(D/E)EF motif in their A-site, which is crucial for the synthesis of c-di-GMP [16,17]. The RXXD403 residues of I-site in GGDEF motif are necessary for the c-di-GMP binding activity [19]. It is considered that some DGCs including PleD and WspR with conserved I-site are mainly involved in c-di-GMP synthesis. Moreover, the binding of DGC to c-di-GMP through I-site is to inhibit its DGC activity to maintain the balance of c-di-GMP level in vivo [20,21,22]. Bioinformatic analysis revealed that GdpX6 possesses both conserved A and I sites in GGDEF motif. We firstly confirmed that GdpX6 not only can synthetize the c-di-GMP but also binds to c-di-GMP in vitro. Point mutation in A or I site results in the loss of the activity of GdpX6. It suggests that GdpX6 might function as the reported DGCs with conserved I site like PleD or WspR [20,21,22]. Moreover, in vivo functional analysis demonstrated that E411 residue in A site but not D403 residue in I site of GdpX6 is essential for biological functions of GdpX6. These results of the present study suggest that GdpX6 mainly exerts its biological functions as a DGC. It is possible that the binding affinity of GdpX6 with c-di-GMP is not enough to affect its function in bacterial cells. However, the DGC activity of GdpX6 might be affected by binding to c-di-GMP in certain conditions as in the case of the PleD or WspR.
It is well established that DGCs play distinct roles in processes associated with motility, attachment, and biofilm formation [12,13]. Opposite regulation of swimming motility and biofilm formation by DGCs have been demonstrated. For example, deletion of DGCs EdcC and EdcE in Erwinia. amylovora increased swimming motility but decreased biofilm formation [56]. Moreover, flagellum-dependent biofilm regulatory response can be induced through the elimination of flagellum, which can improve the level of c-di-GMP and enhances the biofilm formation, and this response requires at least three specific DGCs in the V. cholerae [57]. In this study, we showed that deletion or overexpression of gdpX6 in Xoo resulted in the opposite effects on biofilm formation and swimming motility that is consistent with the above active DGCs [56,57]. These findings revealed the role of DGCs in regulation of motile and static lifestyle in bacteria. Although EPS as a main component of biofilm is not regulated by GdpX6, it is possible that GdpX6 affects biofilm formation mainly by affecting flagellum-dependent motility of Xoo. The regulatory network that connects c-di-GMP signaling, motility, and biofilm formation in Xoo needs further investigation.
Type IV pili (T4P) are surface filamentous bacterial organelle involved in many phenotypes including adhesion, twitching or gliding motility, biofilm formation, and virulence in Gram-negative bacteria [58]. The c-di-GMP signaling and specific diverse c-di-GMP receptors have been demonstrated to be involved in the regulation of T4P. In V. cholerae, c-di-GMP could bind to ATPase MshE associated with mannose-sensitive haemagglutinin T4P formation, thus regulates pilus extension and retraction dynamics [59]. The c-di-GMP receptor FimXxcc and its interactor PilZxcc from Xcc, as well as its homologue Filp and PilZX3 from Xoo, regulate the bacterial T4P-depending sliding motility via direct interaction with or by affecting the expression of the pilus-related proteins, respectively [36,60,61,62]. On the other hand, T4P reversely regulates c-di-GMP signaling. PilR, a regulatory protein of pilus synthesis, controls the intracellular levels of c-di-GMP to inhibit production of the antifungal antibiotic HSAF in the soil bacterium Lysobacter enzymogenes [63]. In the present study, GdpX6 was found to act as a positive regulator of sliding motility of Xoo. This indicates that GdpX6 might be involved in the regulation of TP4. Previous studies have shown that the concentration of c-di-GMP plays an essential role in assembly of TP4. For example, the assembly of TP4 requires FimX with both c-di-GMP binding and PDE activity at low c-di-GMP concentrations, but this dependence disappeared at high c-di-GMP concentrations in P. aeruginosa [64,65]. Moreover, high intracellular c-di-GMP concentration increased the transcript levels of T4P genes as a result of its binding to an upstream transcriptionally active riboswitch in Clostridium difficile [66]. Therefore, we propose that GdpX6 might participate in regulation of sliding motility by affecting TP4 biogenesis or assembly as a DGC.
In the present study we observed that GdpX6-GFP displayed a multisite subcellular localization in Xoo, and both GdpX6D403A-GFP and GdpX6E411A-GFP exhibit similar multisite subcellular localization as that of GdpX6-GFP. The D403 is important for the c-di-GMP binding activity of GdpX6, and the E411 of GdpX6 is crucial for the DGC activity and the biological functions of GdpX6. It suggests that the DGC activity or c-di-GMP binding activity is not important for subcellular localization of GdpX6. These phenotypes are in agreement with other observations on subcellular localization of DGC or PDEs. For example, mutation of the residue G368 in the GGDEF motif of PleD, E153, and E176 in the EAL domain of EdpX1 abolish the DGC or PDE activity but do not affect the localization in the bacterial cell [11,52]. Moreover, the signal transduction domain of the protein can sense the specific signals and regulate the activity of DGC or PDE, thus affecting the subcellular localization of the protein. In P. aeruginosa, mutation of the phosphor-accepting residue D70 in the REC domain of WspR, which is essential for its DGC activity, alters the subcellular location of WspR from clustering to dispersion [67,68]. However, the TM domain which does not influence the function of the proteins has been shown to pay important roles in the subcellular localization of the proteins [11,69]. Besides the GGDEF domain, GdpX6 contains an N-terminal extracellular protein domain Cache_1 domain and a TM domain. Further identifying whether these domains are involved in the regulation of functions of GdpX6, and influence the subcellular location of GdpX6 in the cell will provide more information about the functional characteristics and localization patterns of GdpX6.

5. Conclusions

This study showed that the GGDEF-domain protein GdpX6 is an active DGC with c-di-GMP binding activity. Moreover, GdpX6 negatively regulated virulence and swimming motility, and positively regulated sliding motility and biofilm formation of Xoo. The A site of GGDEF domain is necessary for its regulatory functions in Xoo. Thus, we concluded that GdpX6 exists as a DGC to control the virulence expression in Xoo.

Supplementary Materials

The following are available online at https://www.mdpi.com/2076-2607/9/3/495/s1, Figure S1: Sequence analysis of the homologs of GdpX6 from Xanthomonas species. (a) The neighbor-joining tree reconstructed based on the sequences of homologs of GdpX6 from Xanthomonas species by MEGA (version 7.0.21). (b) Sequence alignment of GdpX6 with the proteins XCC2731, XOC1515 and XAC2897 by using the software DNAMAN. Black and gray highlighting show amino acid residues with100% and ≥75% homology, respectively; Figure S2: Western blot analyses showing expression of GFP fusion proteins with GdpX6, GdpX6GGDEF-D403A, GdpX6GGEDF-E411A in Xoo strains. A total of 10 μg proteins were loaded in each lane and analyzed by immunoblotting with anti-GFP antibodies. All individual blots were repeated three times using samples from independent cultures. Figure S3: GdpX6 does not affect the EPS production of Xoo. The EPS production of all Xoo strains were determined by ethanol precipitation method. No significant difference was observed between PXO99A and ∆gdpX6, ΔgdpX6(pBgdpX6gfp), PXO99A (pBgdpX6gfp) in terms of EPS production by quantification. All experiments were performed thrice in triplicate. The error bar represents standard deviations; Figure S4: GdpX6 does not regulate xylanase and cellulase activities of Xoo. The xylanase and cellulase activities were assayed on the PSA plates with 0.2% RBB-xylan and 0.5% carboxymethyl cellulose, respectively. No significant difference in cellulase and xylanase activities of PXO99A and ∆gdpX6, ΔgdpX6(pBgdpX6gfp), PXO99A(pBgdpX6gfp) was found. All experiments were performed thrice in triplicate. Table S1: Primers used in this study.

Author Contributions

F.Y. designed the experiments; W.Y. performed the experiments; W.Y., Y.W., S.F., C.Y., F.T., Q.W. and H.C. analyzed the data; F.Y. and W.Y. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the grants from Natural Science Foundation of China (31671990 and 31400117), National Key R&D Program of China (2016YFD0300701).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank C.Y. He for giving us important suggestions. We thank J. He for providing us with strains and plasmids of riboswitch-based dual-fluorescence reporter system.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Domain organization and sequence alignment of GdpX6. (a) Schematic representation of the domain structures of GdpX6 (GenBank accession no. WP_012445692.1). The numbers represent the start and end amino acid of the predicted domains based on National Center for Biotechnology Information’s (NCBI’s) conserved domain database and SMART database. The Cache_1 domain (gray), transmembrane domain (blue) and GGDEF domain (red) are shown. (b) The amino sequence alignment of the GGDEF domain of GdpX6 with active DGCs, including PleD (GenBank accession no. AAA87378.1) from C. crescentus CB15, WspR (GenBank accession no. NP_252391.1) from P. aeruginosa PAO1, GcpA (GenBank accession no. NP_460938.1a) from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Dcsbis (GenBank accession no. NP_251461.1) from P. aeruginosa PAO1, Vc0395_0300 (GenBank accession no. ABQ19213.1) from V. cholerae O395, DgcA (GenBank accession no. AAW77242.1) from Xoo KACC10331, GdpX1 (GenBank accession no. WP_011259000.1) from Xoo PXO99A, by using the software DNAMAN. represents the putative residue confirmed by isothermal titration calorimetry assay involved in c-di-GMP binding and represents the putative residue confirmed by DGC activity assays involved in the synthesis of c-di-GMP. The amino acids highlighted in black and gray represent a homology level of 100% and ≥ 75%, respectively.
Figure 1. Domain organization and sequence alignment of GdpX6. (a) Schematic representation of the domain structures of GdpX6 (GenBank accession no. WP_012445692.1). The numbers represent the start and end amino acid of the predicted domains based on National Center for Biotechnology Information’s (NCBI’s) conserved domain database and SMART database. The Cache_1 domain (gray), transmembrane domain (blue) and GGDEF domain (red) are shown. (b) The amino sequence alignment of the GGDEF domain of GdpX6 with active DGCs, including PleD (GenBank accession no. AAA87378.1) from C. crescentus CB15, WspR (GenBank accession no. NP_252391.1) from P. aeruginosa PAO1, GcpA (GenBank accession no. NP_460938.1a) from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Dcsbis (GenBank accession no. NP_251461.1) from P. aeruginosa PAO1, Vc0395_0300 (GenBank accession no. ABQ19213.1) from V. cholerae O395, DgcA (GenBank accession no. AAW77242.1) from Xoo KACC10331, GdpX1 (GenBank accession no. WP_011259000.1) from Xoo PXO99A, by using the software DNAMAN. represents the putative residue confirmed by isothermal titration calorimetry assay involved in c-di-GMP binding and represents the putative residue confirmed by DGC activity assays involved in the synthesis of c-di-GMP. The amino acids highlighted in black and gray represent a homology level of 100% and ≥ 75%, respectively.
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Figure 2. Analysis of diguanylate cyclase (DGC) activity of GdpX6. (a) Verification of the DGC activity of GdpX6 in E. coli BL21(DE3) using riboswitch (Bc3-5 RNA) based dual-fluorescence system. All bacteria strains were induced with 1 mM IPTG at 28 °C for 20 h. Visible fluorescence of bacterial suspensions was photographed. The relative fluorescence intensity (RFI) is calculated as the ratio of fluorescence intensity at 489 nm to fluorescence intensity at 547 nm. (b) The DGC activity of GdpX6 was detected by LC-MS-MS. Purified proteins were incubated with 100 μM of GTP overnight. HPLC was carried out to analyze the products. Three independent experiments were carried out with similar results. * indicates p < 0.05 as determined by t-test.
Figure 2. Analysis of diguanylate cyclase (DGC) activity of GdpX6. (a) Verification of the DGC activity of GdpX6 in E. coli BL21(DE3) using riboswitch (Bc3-5 RNA) based dual-fluorescence system. All bacteria strains were induced with 1 mM IPTG at 28 °C for 20 h. Visible fluorescence of bacterial suspensions was photographed. The relative fluorescence intensity (RFI) is calculated as the ratio of fluorescence intensity at 489 nm to fluorescence intensity at 547 nm. (b) The DGC activity of GdpX6 was detected by LC-MS-MS. Purified proteins were incubated with 100 μM of GTP overnight. HPLC was carried out to analyze the products. Three independent experiments were carried out with similar results. * indicates p < 0.05 as determined by t-test.
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Figure 3. The binding activity of the GdpX6 with c-di-GMP were analyzed using isothermal titration calorimetry assays. The titration calorimetry of 30 μM SUMOHis6, 10 μM SUMOHis6-GdpX6GGDEF, and 10 μM SUMOHis6-GdpX6GGDEF-D403A were syringed into the cell pool and 2 μL aliquots of 300 μM, 1 mM, or 1 mM c-di-GMP at 25 °C. The c-di-GMP binding to the control protein SUMOHis6 (a), the recombinant proteins SUMOHis6-GdpX6GGDEF (b) and SUMOHis6-GdpX6GGDEF-D403A (c).
Figure 3. The binding activity of the GdpX6 with c-di-GMP were analyzed using isothermal titration calorimetry assays. The titration calorimetry of 30 μM SUMOHis6, 10 μM SUMOHis6-GdpX6GGDEF, and 10 μM SUMOHis6-GdpX6GGDEF-D403A were syringed into the cell pool and 2 μL aliquots of 300 μM, 1 mM, or 1 mM c-di-GMP at 25 °C. The c-di-GMP binding to the control protein SUMOHis6 (a), the recombinant proteins SUMOHis6-GdpX6GGDEF (b) and SUMOHis6-GdpX6GGDEF-D403A (c).
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Figure 4. GdpX6 inhibited the virulence of Xoo on rice. (a) The bacterial cells were tested on rice by the leaf-clipping method. The disease symptoms were observed at 14 days post-inoculation. (b) The lesion lengths were recorded. The error bars represented standard deviations of the lesion lengths from ten leaves. Three independent experiments were performed with similar results. * indicates p < 0.05 as determined by t-test.
Figure 4. GdpX6 inhibited the virulence of Xoo on rice. (a) The bacterial cells were tested on rice by the leaf-clipping method. The disease symptoms were observed at 14 days post-inoculation. (b) The lesion lengths were recorded. The error bars represented standard deviations of the lesion lengths from ten leaves. Three independent experiments were performed with similar results. * indicates p < 0.05 as determined by t-test.
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Figure 5. GdpX6 regulated swimming motility and sliding motility of Xoo. Xoo strains were inoculated on (a) semi-solid plates containing 0.25% agar for testing the swimming motility and (b) Super Broth (SB) plates containing 0.6% agar for testing the sliding motility at 28 °C for 4 days. The values are the means ± standard deviations from three replicates of three independent experiments. * indicates p < 0.05 as determined by t-test.
Figure 5. GdpX6 regulated swimming motility and sliding motility of Xoo. Xoo strains were inoculated on (a) semi-solid plates containing 0.25% agar for testing the swimming motility and (b) Super Broth (SB) plates containing 0.6% agar for testing the sliding motility at 28 °C for 4 days. The values are the means ± standard deviations from three replicates of three independent experiments. * indicates p < 0.05 as determined by t-test.
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Figure 6. GdpX6 promoted biofilm formation of Xoo. Biofilm formation of all Xoo strains were tested by crystal violet staining method in polystyrene 96-well microplates. After suspension in ethanol, biofilm was quantified by measuring the optical density at 490 nm. Error bars represent standard deviations from three biological repeats. * indicates p < 0.05 as determined by t-test.
Figure 6. GdpX6 promoted biofilm formation of Xoo. Biofilm formation of all Xoo strains were tested by crystal violet staining method in polystyrene 96-well microplates. After suspension in ethanol, biofilm was quantified by measuring the optical density at 490 nm. Error bars represent standard deviations from three biological repeats. * indicates p < 0.05 as determined by t-test.
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Figure 7. Subcellular localization of GdpX6 in Xoo PXO99A. PXO99A (pBgfp), PXO99A (pBgdpX6gfp), PXO99A (pBgdpX6D403Agfp) and PXO99A (pBgdpX6E411Agfp) were grown in M210 to an OD600 of 1.0. Photograph of the GFP fusion protein in PXO99A detected using a fluorescence microscope (Olympus BX61). Different subcellular locations of proteins GdpX6-GFP, GdpX6D403A-GFP, and GdpX6E411A-GFP including nonpolar, bipolar, multisite were calculated the proportion were counted. ± indicates the standard deviations from three biology repeats. NA, not available.
Figure 7. Subcellular localization of GdpX6 in Xoo PXO99A. PXO99A (pBgfp), PXO99A (pBgdpX6gfp), PXO99A (pBgdpX6D403Agfp) and PXO99A (pBgdpX6E411Agfp) were grown in M210 to an OD600 of 1.0. Photograph of the GFP fusion protein in PXO99A detected using a fluorescence microscope (Olympus BX61). Different subcellular locations of proteins GdpX6-GFP, GdpX6D403A-GFP, and GdpX6E411A-GFP including nonpolar, bipolar, multisite were calculated the proportion were counted. ± indicates the standard deviations from three biology repeats. NA, not available.
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Table
Table 1. The bacterial strains and plasmids used in this study.
Table 1. The bacterial strains and plasmids used in this study.
Strain or PlasmidRelevant Characteristics aSource or Reference
Strains
 Escherichia coli
  DH5αF-φ80(lacZ)ΔlacX74hsdR(rk−, mk+)ΔrecA1398endA1tonATransGene Biotech, Beijing, China
  BL21F- omp T hsdS (rB− mB−)gal dcm (DE3)TransGene Biotech, Beijing, China
 Xanthomonas. oryzae pv. oryzae
  PXO99AWild-type strain, Philippine race 6, Cpr[32]
  ∆gdpX6gdpX6 gene deletion mutant derived from PXO99A, CprThis study
Plasmid
  pKMS1Suicidal vector carrying sacB gene for mutagenesis, Kmr[33]
  pKgdpX6pKMS1 with gdpX6, KmThis study
  pColdSUMOProtein expression vector with N-terminal SUMO-His6-tag, AprHaigene Biotech, Harbin, China
  pCGdpX6GGDEFpColdSUMO carrying the coding sequence of the GGDEF domain (318 to 495 aa) of GdpX6, AprThis study
  pCGdpX6GGDEF-E411ApColdSUMO carrying the coding sequence for the point mutation of E411 in GGDEF domain of gdpX6, AprThis study
  pCGdpX6GGDEF-D403ApColdSUMO carrying the coding sequence for the point mutation of D403 in GGDEF domain of gdpX6, AprThis study
  pETPleDpET28b containing the PleD coding sequence, Kmr[34]
  pET28bpET28b protein expression vector, KmrLaboratory collection
  pBgfpBroad-host range expression vector pBBR1MCS-4 carrying gfp, Apr[35]
  pBgdpX6gfppBgfp carrying full-length gdpX6, AprThis study
  pBgdpX6E411AgfppBgfp carrying full-length gdpX6 with E411 point mutation, AprThis study
  pBgdpX6D403AgfppBgfp carrying full-length gdpX6 with D403 point mutation, AprThis study
a Kmr, Apr and Cpr indicate resistance to kanamycin, ampicillin, and gentamicin, respectively.
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Yan, W.; Wei, Y.; Fan, S.; Yu, C.; Tian, F.; Wang, Q.; Yang, F.; Chen, H. Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae. Microorganisms 2021, 9, 495. https://doi.org/10.3390/microorganisms9030495

AMA Style

Yan W, Wei Y, Fan S, Yu C, Tian F, Wang Q, Yang F, Chen H. Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae. Microorganisms. 2021; 9(3):495. https://doi.org/10.3390/microorganisms9030495

Chicago/Turabian Style

Yan, Weiwei, Yiming Wei, Susu Fan, Chao Yu, Fang Tian, Qi Wang, Fenghuan Yang, and Huamin Chen. 2021. "Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae" Microorganisms 9, no. 3: 495. https://doi.org/10.3390/microorganisms9030495

APA Style

Yan, W., Wei, Y., Fan, S., Yu, C., Tian, F., Wang, Q., Yang, F., & Chen, H. (2021). Diguanylate Cyclase GdpX6 with c-di-GMP Binding Activity Involved in the Regulation of Virulence Expression in Xanthomonas oryzae pv. oryzae. Microorganisms, 9(3), 495. https://doi.org/10.3390/microorganisms9030495

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