The bacterial strains and plasmids used in this study are listed in Table 1. Unless otherwise stated, L. capsici X2-3 and its derivative strains were grown at 28°C in nutrient broth (NB) medium or on NA (NB with 1.5% agar) medium. Transformants from the first crossover for the LC_GidA knockout were cultured on NBN (NB without 1% sucrose) or NAN (NBN with 1.5% agar) medium. Transformants bearing the second crossover were plated on NAS (NAN plus 10% sucrose) medium (Zou et al., 2011). All bacterial strains were incubated at 28°C. Escherichia coli strains were cultured in Luria-Bertani (LB) or LB plus 1.5% agar plates at 37°C. When necessary, the media were supplemented with the antibiotic ampicillin (Amp, 50 μg/ml), kanamycin (Km, 50 μg/ml), or gentamicin (Gm, 50 μg/ml), depending on the strains used.

The LC_GidA mutant was generated from the wild-type X2-3 strain by allelic homologous recombination. Briefly, two LC_GidA flanking regions were amplified by PCR using the primer pairs up F/R and down F/R (Table 1). The upstream and downstream PCR products were digested with BamHI and HindIII, respectively. The digested fragments were ligated into the suicide vector pKMS1 (Table 1) to obtain the recombinant plasmid pKMS1-AB (Zou et al., 2011). The plasmid was transformed into X2-3 by electroporation. The LC_GidA mutant MT16 was obtained after two recombination events and confirmed by PCR and sequencing of the PCR products.

The fragment harboring the intact LC_GidA gene, which was amplified by PCR using the primers gidAF and gidAR (Table 1), was cloned into the expression vector pBBR1-MCS5 (Table 1) at the EcoRI and BamHI site, resulting in the recombinant plasmid pBBR1-gidA, and then pBBR1-gidA was transformed into the mutant MT16 by electroporation (1.8 KV, 200 Ω, and 25 μF). The complemented mutant strain Com-16 was selected on NA plates with gentamycin (Kovach et al., 1994).

The X2-3, MT16, and Com-16 strains were grown for 24 h at 28°C in NA medium and then inoculated into NB medium to OD₆₀₀ = 1.0. The cultures were diluted 1:100 into NB medium. The strains were incubated at 28°C for 48 h with shaking at 180 rpm, and bacterial growth was examined every 4 h (Rehl et al., 2013).

The motility assay was performed as previously described (Rashid and Kornberg, 2000; Tomada et al., 2016). To test twitching motility, bacteria were grown for 24 h in NA medium at 28°C, and 3 μl of the bacterial cultures at a normalized OD₆₀₀ were added to NYGB medium (0.6% agar) plates. The diameters of the areas occupied by the bacterial cells were measured after 3 days.

The crystal violet technique was used to analyze the attachment of the different strains to an abiotic surface. The X2-3, MT16, and Com-16 strains were cultured in NB medium and adjusted to OD₆₀₀ = 1. The cultures were diluted 1:100 into a glass tube containing 10 ml of NB medium supplemented with 1% sucrose or glucose. Then, the glass tubes were incubated at 28°C for 3 days with shaking at 180 rpm. The growth medium was removed, and the tubes were washed three times with sterile distilled water. Then, the glass tubes were stained with a 0.2% crystal violet solution for 10 min. The unbound crystal violet was removed, and the tubes were washed three times with sterile distilled water. Crystal violet was extracted with absolute ethanol, and the absorbance was measured at 575 nm (Zhang et al., 2018).

All Lysobacter strains obtained throughout the study were tested for their ability to produce biofilms, which were visualized as floating pellicle at the air–broth interface that completely blocked the surface of the culture and could not be dispersed by shaking. The X2-3, MT16, and Com-16 strains were grown in glass test tubes containing NB medium (with 1% sucrose or 1% glucose) at 28°C for 5 days without shaking (Latasa et al., 2012).

Seven-day-old plants were collected, and the roots were cut into 1.5 cm segments. Fragments of uniform shape and size were placed into 96-well microtiter plate. Two hundred microliters of bacterial culture with an OD₆₀₀ = 1.0 was added to the wells, and the plates were incubated at 28°C for 3 days. After the incubation period, the roots were removed from the cultures, washed with sterile water, and then added to 1 ml sterile water. The bacteria on the root surface were removed and dispersed in sterile water by shaking. One hundred microliters of the dispersed preparation was plated on NA agar and counted after 5 days (Tariq et al., 2014).

The plasmid pBBR1-gfp was transformed into the X2-3, MT16, and Com-16 strains by electroporation, and the transformants were selected on NA plates with gentamycin. The treatment was the same as above. To view the colonization of L. capsici X2-3-gfp, MT16-gfp, and Com-16-gfp on the root surfaces, the roots were observed using a confocal laser scanning microscope system (Zeiss LSM 800, Carl Zeiss AG, Jena, Germany) with an excitation wavelength of 488 nm. Images of at least 12 roots were obtained for each treatment (Liu et al., 2020).

The bacterial strains were diluted 1:100 into NB medium, and experiments were conducted to test the OD₆₀₀ under five environmental stresses. Stress treatments were applied as follows: for UV radiation, the cells were exposed to shortwave UV radiation (254 nm in a biological safety cabinet) at a distance of 60 cm for 45 min. For salt stress, NaCl was added to the bacterial cultures at final concentrations of 0.15, 0.25, and 0.35 mol/L (Li et al., 2014). For temperature stress, the cultures were incubated at 37 and 42°C with shaking at 180 rpm. Resistance against H₂O₂ was determined as described previously with slight modifications (Liu et al., 2019). H₂O₂ at concentrations of 0.1, 0.01, and 0.001 mM was added to the bacterial cultures and, the samples were incubated at 28°C for 10 min with shaking. After serially diluting the bacteria five times (10⁻¹–10⁻⁵), 3 μl of each cell sample was dropped onto NA plates and incubated at 28°C for 3 days. The pH stress test was similar to the H₂O₂ test. The bacterium was serially diluted five times (10⁻¹–10⁻⁵), and then 3 μl of each cell sample was dropped onto NA plates with pH values ranging from 5.0 to 9.0.

The wild-type strain X2-3 and the mutant strain MT16 were cultivated until they reached an OD₆₀₀ = 1. Total RNA was extracted using AG RNAex Pro Reagent [Accurate Biotechnology (Hunan) Co., Ltd.], and cDNA was synthesized by reverse transcription. Nineteen genes related to DNA replication, cell division, motility, and biofilm formation were chosen for RT–qPCR (Table 2). RT–qPCR experiments were carried out as instructed by the manufacturer [Accurate Biotechnology (Hunan) Co., Ltd.]. The 16S rRNA gene was used as an internal control (Qian et al., 2013). The relative transcription levels were calculated using the 2^–ΔΔCT method (Livak and Schmittgen, 2001).

All data were reported as mean standard at least triplicate experiments. The data were analyzed using the statistical SPSS software (version 18.0) by one-way ANOVA, and the mean was compared by Duncan’s multiple range test (DMRT) at the 5% probability level.

Glucose-inhibited division protein as a tRNA modification enzyme is highly conserved in bacteria and plays an important role in bacterial growth, stress response, and virulence (Shippy and Fadl, 2014). We conducted a search of the L. capsici X2-3 genome annotation (GenBank accession No. LBMI00000000.1) and observed that a potential ORF of approximately 1,890 bp in size was predicted to encode GidA (Supplementary Figure S1), which was named LC_GidA in L. capsici. BLAST analyses showed that the LC_GidA gene shares 62.43% identity with the E. coli gidA gene (GenBank accession No. NC_011750.1). The putative LC_GidA protein showed 63.81% identity with the E. coli GidA protein (GenBank accession No. YP_002410220.1; Supplementary Figure S1), which is a tRNA modification enzyme responsible for the proper biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm5s2U) at position 5 of the wobble uridine (U34) of tRNAs.

To determine the function of the LC_GidA gene in L. capsici X2-3, a LC_GidA deletion mutant, termed MT16, was generated by integration of the pKMS1 plasmid (Supplementary Figure S2). The mutant was identified for the loss of 1,890 bp fragment coding region of the gidA gene by PCR with the primers gidAup-F and gidAdown-R (Supplementary Figure S3). Additionally, the complemented mutant Com-16 was generated by insertion of the full-length LC_GidA into pBBR1-MCS5 and transfer of the resultant plasmid into MT16. The growth of wild-type strain X2-3 and the LC_GidA gene deletion mutant MT16 was assayed by measuring OD₆₀₀ values from 4 to 48 h at 4 h intervals. As shown in Figure 1A, the cell density of MT16 was lower than that of X2-3 and Com-16, and the MT16 colony size was obviously smaller than that of X2-3 at the same timepoints. These results suggest that the loss of LC_GidA resulted in the attenuation of bacterial growth.

The twitching motility of X2-3 and the mutant MT16 were tested on 0.6% agar plates. After 3 days of incubation at 28°C, the diameter of the Com-16 complemented strain was 2.30 cm in NYGB media, very similar to the X2-3 wild-type strain (2.57 cm). In contrast, the MT16 LC_GidA mutant had decreased twitching motility significantly (Figure 1B). These results indicated that the LC_GidA gene is required for the motility of L. capsici X2-3.

To measure the difference in the biofilm biomass of the MT16 and X2-3 strains, they were cultured in NB medium supplemented with 1% sucrose or 1% glucose for 3 days. The samples were then stained with crystal violet, and the biofilm biomass was quantified by measuring their OD₅₇₅. Staining of bacterial cells with CV-staining showed that X2-3 and Com-16 produced much more biofilms of cell mass adhered to the glass surface than those produced by MT16 strain (Figure 2A). The biofilm biomass of MT16 was 17 and 30% lower than that of X2-3 in 1% sucrose and 1% glucose media, respectively. By contrast, the biofilm biomass of Com-16 was similar to that of the wild-type strain (Figure 2B). Furthermore, the pellicle, robust biofilm formed at the air–liquid interface of the culture, could be observed in 1% sucrose or 1% glucose NB medium after static culture for 5 days. The MT16 pellicle was much thinner than that of X2-3, both in 1% sucrose and 1% glucose NB medium, while pellicle formation was partially or fully restored in the Com-16 strain (Figure 2C). From these results we also determined that the rate at which X2-3 utilized different C sources varied, for example, the utilization rate of sucrose was higher than that of glucose; the utilization rate of glucose by the LC_GidA deletion strain was relatively low. These results indicated that deletion of the LC_GidA gene in MT16 decreased the biofilm biomass, while the Com-16 complemented strain recovered biofilm formation ability.

Considering that the LC_GidA gene plays a role in biofilm formation, a quantitative measurement of root colonization was performed. Wheat roots were cultured in X2-3, MT16, or Com-16 for 3 days, and then 100 μl of the bacterial suspensions were plated on NA agar and cultured for 3 days. The results are shown in Figure 3B. The ability of the MT16 mutant to colonize wheat roots was significantly lower than that of the wild-type X2-3 strain; wheat root colonization was recovered in the Com-16 complemented strain. Green fluorescent protein-labeled X2-3, MT16, and Com-16 (X2-3-gfp, MT16-gfp, and Com-16-gfp) were used to detect the root colonization of L. capsici X2-3 under a confocal laser scanning microscope (Zeiss LSM 800, Carl Zeiss AG, Jena, Germany). GFP fluorescence shows successful colonization of X2-3 in root tip cells of wheat, the difference of colonization was determined by observing the GFP fluorescence area. As can be seen from Figure 3A, that the fluorescence area of the wild type is significantly larger than that of the mutant. The images showed that more X2-3-gfp cells were bound to the roots than MT16-gfp cells (Figure 3A). These results indicated that the inactivation of LC_GidA may affect the colonization of wheat roots.

To assess the role of LC_GidA in stress tolerance, the growth yields of MT16, Com-16, and X2-3 were tested under different conditions, including temperature, salt, pH, and UV radiation. The growth of MT16 was significantly lower than that of X2-3 at 37 and 42°C, while Com-16 growth was basically restored to the level of the wild-type strain (Figure 4A). As shown in Figure 4A, the mutant had decreased survival at high osmotic pressure. When treated with UV radiation, there were no significant differences between the MT16 and X2-3 strains (Figure 4A). Compared with the wild-type strain, the growth of the mutant was inhibited at all concentrations of H₂O_2, and the growth of Com-16 was also slightly affected under the high and low H₂O₂ conditions (Figure 4B). The pH resistance of L. capsici was significantly affected by the deletion of LC_GidA (Figure 4C).

To assess the role of LC_GidA as a global regulatory factor and further show that the deletion of LC_GidA leads to a decrease in growth, motility, and biofilm formation, 19 genes related to DNA replication, repair, cell division, motility, and biofilm formation in X2-3 were chosen for RT–qPCR. The results showed that the expression of genes related to motility, replication, cell division, and biofilm formation was significantly downregulated. The genes radC, gyrA, recN, n6amt, dnaA, rmuC, ftsQ, ftsI, and ftsB, which are related to DNA replication, repair, and cell division, were markedly downregulated in the LC_GidA mutant (Figure 5A). Six genes related to motility, pilA, flgD, fliF, flhB, fliQ, and fliP, were significantly decreased in the mutant compared with wild-type X2-3 (Figure 5B). Among the biofilm formation genes, four genes, pgaA, pgaB, pgaC, and surA, were significantly repressed in the LC_GidA mutant (Figure 5C).