Introduction

Microbial networks in natural environments have important significance [1,2,3]. These networks are involved in biochemical cycles, host cell life processes and environmental adaptation adjustment [4,5,6,7,8]. Communication among bacteria is one of the core ways to constitute microbial networks. To respond to environmental changes and stress pressure, by sensing specific signaling molecules, bacteria adjust their physiological states and group behaviors, including the production and release of virulence factors, the production of secondary metabolites and biofilm formation [9,10,11,12]. The Xanthomonadaceae family is a typical class of environmental microorganisms. Members of Xanthomonadaceae are widely distributed in natural environments. A large number of species in this family are plant pathogens [13, 14]. In addition, some human pathogen members can inhabit human-made niches, such as Stenotrophomonas maltophilia, which plays an important role in both plant interactions and human infectious diseases [15]. As a ubiquitous member of Xanthomonadaceae, Lysobacter has unique environmental adaptability [16,17,18,19,20,21]. Lysobacter can produce a variety of natural bioactive products, such as the broad-spectrum antifungal compound the heat-stable antifungal factor (HSAF) dihydromaltophilin and WAP-8294A2, a compound against Gram-positive bacteria [22,23,24,25,26,27,28,29]. In recent studies, indole has been reported as an extracellular signaling molecule that is sensed and responded to by Lysobacter [30]. This small-molecule compound is also involved in the regulation of antibiotic resistance and twitching activity [31, 32].

In microbial networks, indole is a ubiquitous interspecific signaling molecule [33, 34]. Indole induces bacteria to sense environmental changes and activates complex responses. For example, indole regulates the persister cell formation of Escherichia coli (E. coli) through different mechanisms [33, 35, 36]. The biofilm formation of E. coli is also regulated by indole in a dose-dependent manner [33, 37,38,39]. For non-indole-producing bacteria, such as Pseudomonas aeruginosa (P. aeruginosa), indole promotes the metabolic pathway of degrading anthranilate and thus stimulates biofilm formation [40, 41]. In terms of bacterial resistance, indole can not only enhance the antibiotic tolerance of its producer [35], but also strengthen the antibiotic tolerance of other bacterial species in the same environment [42, 43]. In some cases, however, the intrinsic resistance of bacteria can be reversed by indole [30, 31]. In addition, indole plays an important role in bacterial community competition [44, 45]. Upon some stress conditions leading to growth arrest, including heat stress and removal of nutrition, indole secretion is promoted in E. coli [33, 46]. Moreover, indole affects the survival ability of E. coli under conditions of acid and heat stress, and these effects are complex [47,48,49,50]. Therefore, although many studies have been reported, the interaction and ecological functions of indole signaling among different bacterial species are still largely unknown.

Here, we show the connection between Lysobacter enzymogenes YC36 (LeYC36) and indole signaling produced by E. coli. We identified a novel transport system, LeKdpXFABC, and confirmed its internal mechanism and universality. After it was sensed by LeYC36, indole enhanced potassium ion uptake and activated a higher level of antimicrobial substance production at the early exponential phase, thus strengthening the resistance to high osmotic pressure and the survival ability in complex habitats.

Materials and methods

Bacterial strains, plasmids and general methods

Lysobacter strains were grown in 40% strength tryptic soy broth (TSB) medium. Indole (0.5 mM) was added in the indole treatment. Contact-independent bacterial culture devices (Transwell permeable supports, 6.5 mm diameter inserts, 0.4 μm pore size, Corning, USA) were used to establish a co-culture system of LeYC36 and E. coli K12. Secondary metabolites and bacterial viability were detected at different time points. The concentration of LeDSF3 in some experiments was 0.5 μM. E. coli strains S17-1, BL21 and DH5α were used for DNA manipulation. Among these strains, the DH5α strain was used to amplify plasmids. The S17-1 strain was used for conjugation assays. The BL21 strain was used for protein purification. The strains and plasmids used in this study are described in Supplementary Table S1. The extractions of plasmids and DNA fragments were performed with the Plasmid Mini Kit I and Gel Extraction Kit (OMEGA, USA). All the molecular manipulations in this study were performed according to previously described methods [30, 51]. Reagents related to molecular biology were purchased from Takara (TaKaRa Bio Group, Japan). The PCR primers used in this study were synthesized by Tsingke Biological Technology Company, China. The primers used in this study are listed in Supplementary Table S2.

Generation of in-frame deletion and complementary mutants

To construct vectors for gene in-frame deletion in LeYC36, two homologous arms (upstream and downstream fragments) were amplified from genomic DNA. Two homologous arms of each gene were linked with pEX18 to generate pEX18-Target. The resulting vectors were transferred into LeYC36 cells according to the protocol described previously [52]. For the gene complementation assay on ∆LekdpX, a DNA fragment containing the upstream arm, the gene to be supplemented and the downstream homologous arm was amplified from LeYC36 genomic DNA with the primers LekdpX-C-up and LekdpX-C-down and then connected to the pEX18 vector. Then, the recombinant vector pEX18-LekdpX-C was transferred to the gene knockout strain ∆LekdpX, and the complementary strain ∆LekdpX::LekdpX was obtained. The complementary strain and mutants were verified by PCR and sequence analyses.

Gene transcription detection

LeYC36 and related mutants (∆rpfC and ∆rpfF) were cultured under different conditions until the cell density was the specific value (OD600 = 0.1 as low cell density or OD600 = 1.0 as high cell density). Then, RNA was extracted at various time points by using RNA extraction kits (OMEGA, USA). The RNA samples were reverse-transcribed to cDNA, and then real-time PCR was performed in a total reaction volume of 20 μL (250 nM primers, 0.5 μL of 10-fold-diluted cDNA template, 10 μL of Eva Green 2× qPCR Master Mix and 8.5 μL of RNase-free water), and 16 S rRNA was used as the reference gene. This program was designed as described previously [53]. Real-time PCR was carried out with a StepOne Real-time PCR System (Applied Biosystems, USA).

Transcriptional analysis

Analysis of the transcriptome of LeYC36 under indole-treated and control conditions was performed at Biozeron Company in Shanghai, China (PRJNA508225). Bioanalyzer 2100 (Agilent, USA) and NanoDrop 2000 (Thermo Scientific, USA) instruments were used to determine and quantify RNA quality. The RNA preparation kit (Illumina, USA) was used to construct RNAseq transcriptional libraries, and the sequencing was performed on the HiSeq platform (Illumina, USA). Then, the raw paired-end reads were handled with tools including SeqPrep, Sickle and Rockhopper. The results were used for differential gene expression analysis with the help of EdgeR [54]. Finally, Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Changes in the abundance of genes greater than 2-fold with p value < 0.005 were regarded as significant differences.

Bioinformatics analyses

Based on the NCBI and UniProt databases, BLAST analysis was performed to annotate the specific gene sequences so that the highest protein homology results of annotated key genes and nonannotated key genes were obtained. To verify the universality of this newly identified operon, sequence alignment was carried out by using DNAMAN and MEGA-X software, and then the evolutionary tree was constructed. By using ClustalX and ESPript 3, each sequence of the key proteins was compared with its homologous sequences. Then, collaborative analyses between the comparison results and the secondary structures of the homologous proteins reviewed in the UniProt database were performed. The secondary structure information came from the RCSB PDB database. Based on SoftBerry, the promoter of LekdpXFABC was predicted. To verify the universality of LekdpX, the IMG/M database was used to search for other strains that contain similar genes from different environments. Based on the standard criteria e value < e−10 and identity >50%, the results were selected (Supplementary Fig. S1 and Supplementary data). For the LeKdpX family proteins, based on the standard criteria e value < e−10 and identity >30%, the results from the UniProt database were selected (Supplementary Fig. S2).

QseB purification

The method described by Hughes et al. [55] was applied with some modifications. By using the primers Pu-qseB-up and Pu-qseB-down, qseB with both BamH I and EcoR I sites was amplified from the genome of LeYC36. Then, this DNA product was integrated into the plasmid pET28a (+). This recombinant plasmid qseB-pET28a (+) was transformed into E. coli BL21. Then, the heterologously expressed strain qseB-E. coli-BL21 was separated from E. coli BL21 without qseB-pET28a (+) in the culture with kanamycin (100 μg/mL). The resistance genes of qseB-pET28a (+) to kanamycin made qseB-E. coli-BL21 survival in the culture with kanamycin. After culturing at 37 °C for 6 h (100 μg/mL kanamycin, OD600 = 0.6), isopropyl β-D-1-thiogalactopyranoside (IPTG, 0.5 mM) was added. Then, this expression system was cultured at 16 °C for 12 h. After centrifugation (12,000 × g, 10 min), these cells were collected and then resuspended in binding buffer (20 mM Tris-Cl, 0.5 M NaCl, pH 8.0). By using an ultrasonic homogenizer (Scientz, China), the ultrasonic crushing was performed for 20 min at 35% power for three times. After centrifugation (12,000 × g, 10 min), the supernatant (30 mL) was collected. Then, 3 mL of Ni-NTA Agarose (QIAGEN, Germany) was equilibrated with binding buffer (10 mL) after it was transferred to a column. The supernatant was transferred to the nickel column at least three times. A series of eluted buffers (20 mM Tris-Cl, 0.5 M NaCl, 10/20/50/75/100 mM imidazole, pH 8.0) were used to isolate other proteins. The final eluted buffer (20 mM Tris-Cl, 0.5 M NaCl, 250/500 mM imidazole, pH 8.0) was used to purify QseB. The purity degree of QseB was tested by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Supplementary Fig. S3).

Protein site-directed mutation

The method described by Kostakioti et al. [56] was applied with some modifications. The mutant plasmid D51A-qseB-pET28a (+) was amplified from the previously obtained recombinant plasmid qseB-pET28a (+) with the primers D51A-qseB-up and D51A-qseB-down. Then, based on the method of QseB purification, the strain D51A-qseB-E. coli-BL21 was constructed, which was then used to purify the target mutant protein QseBD51A.

EMSA

Based on the method described by Si et al. [57], some steps were modified. We used the biotin 5′-end-labeled promoter probe Bio-PLekdpXFABC to perform electrophoretic mobility shift assay (EMSA). Following the instructions of the Chemiluminescent EMSA Kit (Beyotime Biotechnology, China), each reaction solution was prepared. For the EMSA containing QseC, the reaction solutions (25 μL) contained 1× binding buffer, 5 mM MgCl2, 2.5% glycerol, 50 ng of poly(dI-dC), 0.05% Nonidet P-40, 1 mM ATP, 10 μg of QseC, 0.5 mM indole, 10 ng of biotin-DNA Bio-PLekdpXFABC, 10 ng of unlabeled DNA PLekdpXFABC which was amplified from LeYC36 genomic DNA with primers PLekdpXFABC-up and PLekdpXFABC-down as competitors, 10 ng of unrelated DNA as control and different concentrations of QseB. In a control group, indole was not added. For the EMSA containing acetyl phosphate, the reaction solutions (10 μL) contained 1× binding buffer, 5 mM MgCl2, 2.5% glycerol, 0.1 M acetyl phosphate, 50 ng of poly(dI-dC), 0.05% Nonidet P-40, 10 ng of biotin-DNA Bio-PLekdpXFABC, 10 ng of unlabeled DNA PLekdpXFABC which was amplified from LeYC36 genomic DNA with primers PLekdpXFABC-up and PLekdpXFABC-down as competitors, 10 ng of unrelated DNA as control, 2 μg of BSA as the control, 2 μg of QseBD51A as the protein control and different concentrations of QseB. These compounds were incubated for 30 min at room temperature, and their mixtures were separated in a 6.5% polyacrylamide native gel. Then, the compounds were transferred to a nylon membrane (Millipore, USA). Streptavidin-horseradish peroxidase conjugates were used to detect the migration of biotin-labeled probes.

Adversity viability analysis

LeYC36 and its mutant strains including ∆qseC, ∆kdpA, ∆LekdpX and ∆LekdpX::LekdpX were cultured in 40% TSB medium (with or without indole) for 12 h. Then, each culture was adjusted to the same value (OD600 = 0.1). One milliliter of bacterial suspension was centrifuged, and the cells were collected. Next, the cells were resuspended in the corresponding medium. The viability test under high osmotic pressure was carried out under high NaCl concentration (final concentration 5%). These bacterial suspensions were incubated at 28 °C for 6 h. Then, the numbers of colonies on plates were counted and compared.

Atomic force microscopy and dynamic imaging analysis

LeYC36 cells were cultured overnight, washed with 40% TSB medium for three times and diluted to 106 cells/mL. These samples were coated on freshly dissociated mica sheets and observed under an atomic force microscope (Agilent 5400, tapping mode, USA). The scanning speed was 0.4 lines/second, and the resolution was 512 × 512. In the experiment of dynamically observing the bacterial division rate, an FCS2 flow cytometry system (Bioptechs, USA) was used to record real-time images. The cells were cultured for 12 h, diluted, and washed with 40% TSB medium for three times. Subsequently, the cells were imaged on a gel pad containing 2% low-temperature agarose. Cell division was continuously observed at 30 °C under a microscope and 0.5 mM indole was added to the medium at the same time.

Determination of the intracellular potassium ion content

LeYC36 and its mutant strains including ΔqseB, ΔkdpA, Δwzm, ΔrpfC, ΔrpfF, ΔLekdpX and ΔLekdpX::LekdpX were cultured overnight. After centrifugation (4000 × g, 10 min) was performed on the diluted bacterial suspension (OD600 = 1.0, 50 μL), the same number of bacterial cells were collected, and they were washed twice with normal saline (0.85%) through resuspension. By using an ultrasonic homogenizer (Scientz, China), the ultrasonic crushing was performed for 30 min at 35% power, and the supernatant was collected. Each sample was diluted 100-fold in 2% molecular nitric acid, and the total volume was 5 mL. These samples were analyzed by the inductively coupled plasma–mass spectrometry (ICP-MS) (Shimadzu ICPMS-2030, Japan), and the results were corrected with the appropriate buffer as a reference and by dilution factors. Each strain was cultured three times in a single experiment, and the experiment was repeated at least three times.

Determination of the ability of LPSs to adsorb potassium ions

LeYC36 was cultured under different conditions (with or without indole). After centrifugation (12,000 × g, 10 min), bacterial cells in 100 mL culture medium (OD600 = 0.1) were collected and then resuspended in 30 mL of distilled water. A 3-min water bath at 70 °C and a later 3-min ice bath were used. These samples were frozen and thawed 4 times. Lipopolysaccharides (LPSs) were prepared through the hot phenol water method. The bacterial lysate was treated with 30 mL of 45% aqueous phenol solution for 5 min, and then 30 mL of 95% aqueous phenol was added. This solution was stirred at 68 °C for 20–30 min. After this solution was cooled, it was centrifuged at 5000 × g/min for 30 min. The upper layer was taken for dialysis with distilled water at 4 °C for 48 h. Then, polyethylene glycol (40 mg/mL), DNase I (0.5 mg) and RNase A (0.5 mg) were added. Enzymatic hydrolysis was performed for 4 h. After centrifugation (3800 × g, 10 min), 60 mL of acetone was added to the precipitate and resuspended it to purify LPSs. After centrifugation (3800 × g, 10 min), purified LPSs were obtained. LPSs were incubated with potassium ions, and 10 mL of this reaction mixture contained 100 mg of purified LPSs, distilled water and 10 mg/mL potassium ions. The mixture was shaken at 28 °C and 150 r/min for 30 min and then centrifuged at 5000 × g/min. The content of residual potassium ions in the supernatant was detected. The content of potassium ions accumulated in LPSs was obtained by calculation.

Determination of rhamnose in LPSs

The purified LPSs were hydrolyzed by trifluoroacetic acid, and then the mixture was blown dry by N2. The sample residue was dissolved with 50% acetonitrile, and the HPLC-MS analysis was performed. The HPLC (Agilent 1260, USA) conditions were as follows: column, Waters Acquity BEH Amide column (2.1 mm×150 mm, 1.7 μm); mobile phase A, acetonitrile (containing 0.1% ammonia); mobile phase B, water (containing 0.1% ammonia); gradient program, the concentration of B was changer from 20% to 80% in 10 min; flow rate, 0.15 mL/mine; and column temperature, 35 °C. Mass spectrometry (Agilent 6120 quadrupole LC/MS, USA) was performed with the following conditions: The MS was operated in negative mode, and m/z = 163.1 was chosen as the ion of interest in selected ion monitoring (SIM) mode. The relative content of rhamnose was obtained by comparing the peak area with that of the reference substance.

HSAF yield determination

Indole (0.5 mM) was added to 15 mL of 40% TSB medium inoculated with LeYC36. The solution was incubated at 28 °C and 200 rpm for 48 h. The supernatant was collected, evaporated and concentrated. Then, the product was dissolved in ~2 mL of methanol, evaporated and concentrated again. The residue was dissolved in 0.5 mL of methanol and 1 mL of DMSO. HSAF and its analogs were determined by HPLC. The indole-free group was the control. The relative yield of the compound was obtained by determining the semiquantitative peak areas of HSAF and its analogs. Mobile phase A was water containing 0.1% formic acid, mobile phase B was acetonitrile containing 0.1% formic acid, and the flow rate was 1 mL/min. The Pack Pro C18 columns (250 mm × 4.6 mm, S-5 μm, 12 nm, YMC, Japan) were used.

Time-lapse recording of bacterial growth under a microscope

Based on the method we previously described [31], some steps were modified. LeYC36 was cultured in 40% TSB overnight. After centrifugation (4000 × g, 15 min), the cells were collected and were diluted to a suitable OD value (OD600 = 0.1). Then, cells in 1 mL of bacterial suspension were washed 3 times with 1 mL of 10% TSB through resuspension. After centrifugation (4000 × g, 15 min), these cells were resuspended in 10% TSB (with or without indole). The cells from different groups (with or without indole) were imaged on a gel pad (2% low-melting-temperature agarose) and were observed under a microscope at 30 °C.

Bacterial viability analysis in a nutrient-poor environment

LeYC36 and its mutant strains, including ∆rpfC and ∆rpfF, were cultured until their cell densities reached the same value (OD600 = 1.0). Then, 2 μL of bacterial suspensions of different culture mixtures were transferred onto the surface of 10% TSB solid media (with or without indole). The suspensions were cultured at 28 °C for 36 h. The state of the bacterial patch reflected the viability of bacteria in the environment with poor nutrition.

QseC purification

The method described by Tsang et al. [58] was applied with slight modifications. By using the primers Pu-qseC-up and Pu-qseC-down, qseC with both Nde I and BamH I sites was amplified from the genome of LeYC36. Then, this amplicon was integrated into the plasmid pET19b. The recombinant plasmid qseC-pET19b was obtained and transformed into E. coli BL21. The heterologously expressed strain qseC-E. coli-BL21 was screened in LB culture with ampicillin (100 μg/mL). After culturing at 37 °C for 6 h (100 μg/mL ampicillin, OD600 = 0.6), isopropyl β-D-1-thiogalactopyranoside (IPTG, 1 mM) and rhamnose (0.1% final concentration) were added. Then, this expression system was cultured at 16 °C for 16 h. After centrifugation (12,000 × g, 10 min), these cells were collected and then resuspended in binding buffer (50 mM phosphate buffer, pH 8.0, 500 mM NaCl, 1% Triton X-100, 10% glycerol, 10 mM imidazole). By using a low-temperature ultra-high pressure cell disruptor (JNBIO, China), the pressure crushing was performed at 100,000 kPa. After centrifugation (6000 × g, 15 min), the supernatant was collected, and Ni-NTA Agarose (QIAGEN, Germany) was used to purify QseC. Eluted buffers (50 mM phosphate buffer, pH 8.0, 500 mM NaCl, 1% Triton X-100, 10% glycerol, 25/100 mM imidazole) were used to isolate other proteins. The final eluted buffer (50 mM phosphate buffer, pH 8.0, 500 mM NaCl, 1% Triton X-100, 10% glycerol, 250 mM imidazole) was used to purify QseC.

In vitro phosphorylation assay of QseC

Based on the method described by Clarke et al. [59], some steps were modified. Ten microliters of QseC were added to the reaction buffer (50 mM Tris-HCl, pH 7.5, 1 mM DTT, 10 mM MgCl2). ATP (1 mM) was added to each reaction solution. Indole was not added to the control group. In other reaction solutions, indole (0.5 mM) was added. All the reaction systems were 25 μL in volume, and the gradient reaction mixtures were incubated for 0.5, 1, 2, 4 and 8 min at 25 °C. The reaction time of the control group was 8 min. Then, 10 μL of each reaction mixture was subjected to SDS-PAGE with Phos-tags. The Phos-tag gel (Wako, Japan) was used to detect phosphorylated QseC. In this SDS-PAGE experiment, phosphorylated QseC was separated from the unphosphorylated QseC.

Statistical analysis

Statistical analyses were performed by using Origin software. Significant differences and other analysis data of bacterial viability and other physiological indicators were obtained in this process.

Results

Co-culture of different bacterial species causes stress responses of LeYC36

The interspecies communication of bacteria endows them with a variety of physiological and biochemical behaviors [9, 10]. In this study, we used a contact-independent bacterial culture device to establish a co-culture system between LeYC36 and E. coli K12. This device avoided contact between these two species of bacteria, but the small-molecule compounds released by the cells could penetrate the membrane (Fig. 1a). In the co-culture system, the production of antifungal and antibacterial products of LeYC36 was activated at the early exponential phase (Fig. 1b). When LeYC36 was cultured alone, this production was activated at the late stage of the exponential phase, indicating a time-scale promotion of antimicrobial compound production. At the early stage of bacterial culture (low density), the relatively high expression of antimicrobial product genes of LeYC36 in the co-culture system was consistent with this phenomenon. For example, co-culture enabled LeYC36 to initiate high expression of the antifungal factor HSAF at the 12th h of culture, while LeYC36 alone initiated high expression at the 48th h (late exponential phase) (Fig. 1c). For the anti-Gram-positive bacterial compound WAP-8294A2, co-culture induced its high expression at the 12th h, while LeYC36 alone induced its high expression at the 36th h (Fig. 1d). In addition, unknown substances released by E. coli markedly enhanced the high osmotic pressure adaptability of LeYC36 (Fig. 1e). We speculated that E. coli in the co-culture system produced a class of permeable substances, which could help LeYC36 sense survival pressure from the environment and respond to it from the sides of both “attack” (antimicrobial substance production) and “defense” (the ability to resist adversity) in an unknown way.

Fig. 1: Stress responses of LeYC36 are activated in the co-culture system between LeYC36 and E. coli K12.
figure 1

a The device allows penetrable material produced from E. coli K12 to be sensed by LeYC36 without direct contact between these two bacteria. b In the co-culture system between LeYC36 and E. coli K12, the high-level expression of antifungal and anti-Gram-positive bacterial secondary metabolites of LeYC36 was activated ahead of time. At the 12th h, they were produced in the co-culture system, while anti-Gram-positive bacterial secondary metabolites were produced by LeYC36 alone at the 36th h. c The real-time PCR result. The transcription of the antifungal secondary metabolite HSAF genes was increased significantly at the early exponential phase in the co-culture system. In the LeYC36-alone culture system, a similar level of transcription was achieved at 48 h. d The real-time PCR result. In the co-culture system, the transcription of anti-Gram-positive bacterial secondary metabolite WAP genes was increased significantly at the early exponential phase. In the LeYC36-alone culture system, a similar level of transcription was achieved at the 36th h, the end of exponential phase. e Survival rate of LeYC36 in high osmotic pressure for 6 h. After indole (0.5 mM) was added, the viability of LeYC36 cells under high osmotic pressure was enhanced. Error bars show the standard deviation of three replicates. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All data are mean ± s.e.m.

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The interspecific signaling molecule indole enhances the survival ability of LeYC36

Bacterial stress responses are usually induced by some environmental signals [55]. To explore the main functional components of these permeable substances, transcription analysis of two-component systems (TCSs) contained in LeYC36 was performed. The results showed that qseC and qseB were significantly upregulated in the co-culture system (Fig. 2a). In our previous study, we showed that QseC was the sensor of L. enzymogenes, which directly sensed indole signaling [30]. QseB is regulated by QseC and plays a key role in indole regulation [30,31,32]. In this study, the new evidence showed that indole could induce the autophosphorylation of QseC (Fig. 2b). It further proved the function of QseC in sensing indole directly. Considering that indole is an important interspecific signaling molecule in microbial communities [33, 34], indole might be the main functional permeable substance that induces the stress response in the co-culture system.

Fig. 2: Indole produced from E. coli activates different stress responses of LeYC36.
figure 2

a The real-time PCR result. In the co-culture system of LeYC36 and E. coli K12, the transcription of qseC and qseB was increased significantly at low cell density (OD600 = 0.1). b The result of Phos-tag SDS-PAGE on QseC. The phosphorylated QseC (P-QseC) was separated from the unphosphorylated QseC. The exogenous indole induced the formation of P-QseC. With the increase of the reaction time, the content of P-QseC in the reaction increased. c Survival rate of LeYC36 in high osmotic pressure for 6 h. Viability changes under high osmotic pressure were avoided in the co-culture of LeYC36 and E. coli-ΔtnaA. E. coli-ΔtnaA is unable to produce indole. d Survival rate of LeYC36 in high osmotic pressure for 6 h under the indole treatment condition. When indole (0.5 mM) was added in the co-culture of LeYC36 and E. coli-ΔtnaA, the bacterial viability under high osmotic pressure was enhanced. The concentration (0.5 mM) of indole was used in all the indole-related experiments of this study. e Survival rate of LeYC36-ΔqseC in high osmotic pressure for 6 h. Viability changes under high osmotic pressure were avoided in the co-culture of LeYC36-ΔqseC and E. coli K12. LeYC36-ΔqseC is unable to sense indole. f Survival rate of LeYC36 in high osmotic pressure for 6 h. When indole was added, the viability of LeYC36 under high osmotic pressure was greatly enhanced. g Indole can maintain the integrity of LeYC36 cells in a high osmotic pressure environment. Compared with those in the indole-free group (green and yellow cells), LeYC36 cells in the indole-supplemented group were plump, and their integrity was maintained (red cells). The flagella are marked with green arrows and white boxes. h At the 6th and 12th h, the production of HSAF under the indole-supplemented condition (red) was higher than that under the indole-free condition (blue). The yellow part is the purified HSAF as the control. The arrows with different colors (red, blue and yellow) point out the main peak corresponding to HSAF. i Indole reduced the division speed of LeYC36 cells in the environment with poor nutrition (10% TSB). Under the indole-free condition (yellow time axis), 8 cells existed at the 5th h. In the indole-supplemented culture system (green time axis), 8 cells existed at the 8th h. Split points are indicated by black arrows and colorful dots. Error bars show the standard deviation of three replicates. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All data are mean ± s.e.m.

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To ascertain whether the possible effect of indole was involved in the stress response in the co-culture system, the indole-deficient strain E. coli-ΔtnaA was used. The bacterial resistance of LeYC36 in a new co-culture between E. coli-ΔtnaA and LeYC36 to high osmotic pressure was the same level as that in the LeYC36-only culture (Fig. 2c). However, when indole (0.5 mM) was added, this resistance was enhanced in the co-culture (Fig. 2d). These two results showed that the lack of indole in the co-culture led to the same level of high osmotic pressure resistance as that in the LeYC36-alone culture. Furthermore, the enhancement of this resistance was avoided in the co-culture between the qseC deletion mutant LeYC36-ΔqseC and E. coli (Fig. 2e). The lack of indole-sensing ability prevented the high osmotic pressure resistance of LeYC36 from being enhanced in the co-culture. In further studies, exogenously added indole (0.5 mM) enhanced the high osmotic pressure adaptability of LeYC36 (Fig. 2f). Furthermore, indole could maintain the integrity of LeYC36 cells in a high osmotic pressure environment and promote HSAF expression at the early exponential phase (Fig. 2g, h). These phenomena were consistent with the results of the co-culture system between LeYC36 and E. coli. In the environment with poor nutrition, indole reduced the division speed of LeYC36 cells (Fig. 2i). Conclusively, all these results showed that indole was the functional signaling molecule in the co-culture and enhanced the environmental adaptability and survival advantage of LeYC36.

The novel potassium ion transport system operon (LekdpXFABC) is involved in the indole-mediated pathway

There was no doubt that indole enhanced the bacterial resistance of LeYC36 to high osmotic pressure. To search for the key genes involved in this indole-regulated pathway, transcription analysis of osmolality-related genes under different conditions (with or without indole) was performed. The results showed that the kdp gene cluster in LeYC36 was stimulated and significantly upregulated by indole (Fig. 3a, b). The kdp operon encodes Kdp-ATPase, a potassium ion pump, to increase potassium ion uptake [60,61,62]. The inductively coupled plasma–mass spectrometry (ICP–MS) assay showed that the intracellular potassium ion content was greatly increased with the addition of indole (Fig. 3c). This observation was consistent with the results of the transcription test. Additionally, the bioinformatics analysis of this kdp cluster was performed. Instead of the canonical potassium ion pump operon kdpFABC, we identified a novel operon composed of five genes (LekdpX-LekdpF-kdpA-kdpB-kdpC) in LeYC36 (Fig. 3d–I). Among these genes, three of them were annotated as kdpA, kdpB and kdpC, and their highest protein homology results included KdpA of Pseudomonas putida (homology 44.2%), KdpB of Xanthomonas campestris (homology 82.4%) and KdpC of Xanthomonas oryzae (homology 56.9%) (Supplementary Figs. S4–S6). The other two genes upstream of kdpA were both annotated to encode putative proteins. One was a novel type of KdpF for its highest homology with KdpF from Halobacterium salinarum (homology 29.03%) (Supplementary Fig. S7). We named this gene product LeKdpF. Unexpectedly, the other gene product was completely unknown and was named LeKdpX (Supplementary Fig. S8). Further bioinformatics analysis showed that the LekdpXFABC operon widely existed in the main members of the family Xanthomonadaceae, including Lysobacter, Luteimonas, Pseudoxanthomonas, Stenotrophomonas, Xanthomonas, Thermomonas and Vulcaniibacterium (green background in Fig. 3d-II), while the canonical operon kdpFABC was the only form in other common environmental bacteria (yellow background in Fig. 3d-II), such as E. coli.

Fig. 3: A novel kdp operon, LekdpXFABC, is involved in indole-related pathways.
figure 3

a The transcriptome analysis result. b The real-time PCR result. Expressions of LekdpX, LekdpF, kdpA, kdpB and kdpC were upregulated upon indole treatment at low cell density (OD600 = 0.1). c The intracellular potassium ion content was enhanced when indole was added. d In d-I, the novel operon LekdpXFABC consists of LekdpX, LekdpF, kdpA, kdpB and kdpC. In d-II, the operon LekdpXFABC mainly exists in the family Xanthomonadaceae, including Lysobacter, Luteimonas, Pseudoxanthomonas, Stenotrophomonas, Xanthomonas, Thermomonas and Vulcaniibacterium (green background). The canonical operon kdpFABC exists in other common environmental bacteria (yellow background). * is the strain which possesses a special type of LekdpXFABC. In this type, a gap sequence space between LekdpX and kdpFABC exists. LekdpXFABC strains belong to DSFs strains (green square on the right) while kdpFABC strains belong to DSFs-lack strains (yellow square on the right). For the description of the DSFs-related result, see below discussion. e, f Survival rate of LeYC36 in high osmotic pressure for 6 h. Intracellular potassium ion content of LeYC36. Compared with that in the wild-type strain, the viability in a high osmotic pressure environment and intracellular potassium ion content were reduced in ΔkdpA. g, h Survival rate of ΔkdpA in high osmotic pressure for 6 h. Intracellular potassium ion content of ΔkdpA. Indole could not regulate the viability in a high osmotic pressure environment and intracellular potassium ion content of ΔkdpA. i, j Survival rate of LeYC36 in high osmotic pressure for 6 h. Intracellular potassium ion content of LeYC36. Viability in a high osmotic pressure environment and intracellular potassium ion content were significantly reduced in ΔLekdpX. After LekdpX was complemented, they were enhanced greatly. Error bars show the standard deviation of three replicates. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All data are mean ± s.e.m.

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To verify the function of this novel operon and its role in the indole-regulated pathway, gene knockouts of the function-known gene kdpA and the novel function-unknown gene LekdpX were performed (Supplementary Figs. S9 and S10). The inactivation of kdpA reduced the viability of ΔkdpA under osmotic stress and significantly decreased its intracellular potassium ion content, suggesting that this operon was responsible for potassium ion import and related to the osmotic pressure response (Fig. 3e, f). Moreover, unlike that for the wild-type strain, indole could not produce a positive effect on ΔkdpA, nor could it increase the intracellular potassium ion content (Fig. 3g, h). Thus, indole enhanced the high osmotic pressure resistance by upregulating the expression of the kdp operon, and the potassium ion uptake regulated by the kdp operon was involved in the formation of high osmotic pressure resistance. In addition, LekdpX was the key point. For the ΔLekdpX mutant, its intracellular potassium ion content and high-osmotic stress viability were significantly decreased, but they were restored after LekdpX was complemented (Fig. 3i, j and Supplementary Fig. S11). As a newly identified gene of the LekdpXFABC operon, LekdpX could be regarded as one of the key genes in the potassium ion transport system of LeYC36.

In the indole-mediated pathway, LekdpXFABC is directly regulated by QseB

Our previous studies [30,31,32] and the above results have shown that QseC/QseB is involved in the indole-induced regulation. Under indole treatment, the inactivation of qseB significantly reduced the intracellular accumulation of potassium ions (Fig. 4a). Moreover, the transcription levels of qseB and qseC were significantly upregulated in the indole-treated environment (Fig. 4b, c). It is possible that QseB, the regulator of QseC/QseB, could be related to the LekdpXFABC regulation. Through further bioinformatics analysis, the potential promoter of LekdpXFABC was forecasted (Supplementary Fig. S12). Then, an EMSA between phosphorylated QseB of LeYC36 and PLekdpXFABC, a 200 bp length specific DNA sequence that included the promoter area, was performed. The in vitro binding of them showed that the operon LekdpXFABC was directly regulated by phosphorylated QseB and this regulation depended on the phosphorylated QseC and the presence of indole (Fig. 4d). For QseB, D51 has been reported as its phosphorylation site [56]. The result of sequence alignment on QseBs of different bacterial species showed D51 as the conserved site (Fig. 4e). Then, QseBD51A, the mutant protein that could not be phosphorylated, was obtained in the method of site mutation. The result of the new EMSA showed that QseBD51A could not combine with PLekdpXFABC (Fig. 4f). In this EMSA, QseB was phosphorylated by acetyl phosphate in vitro. As the further evidence (Fig. 4f), the result of this EMSA showed that the QseB-LekdpXFABC regulation depended on the phosphorylation state of QseB. Thus, in the indole-supplemented environment, after sensing indole, QseC of LeYC36 phosphorylated QseB. Then, the phosphorylated QseB and the LekdpXFABC promoter were involved in the formation of a promoter-protein complex so that the expression of the LekdpXFABC operon was turned on (Fig. 4g).

Fig. 4: LekdpXFABC is regulated by QseB.
figure 4

a The lack of qseB reduced the intracellular potassium ion content under indole treatment. b The transcriptome analysis result. c The real-time PCR result. When indole was added, the transcription level of qseC and qseB increased. d Binding of QseB phosphorylated by phosphorylated QseC to the LekdpXFABC promoter. In the reaction systems, 10 ng of PLekdpXFABC is enough, and 10 μg QseC is enough. Biotin can be detected with the help of streptavidin-horseradish peroxidase conjugates. The result of unrelated DNA control verified the binding specificity. The result of the indole-free group verified the QseB-promoter binding depended on the presence of indole. e The sequence alignment among QseB of different bacterial species. Conserved sites were marked with red and yellow. D51 was proved as the conserved site. f Binding of QseB phosphorylated by acetyl phosphate in vitro to the LekdpXFABC promoter. In the control group, 10 ng of PLekdpXFABC is enough. Biotin can be detected with the help of streptavidin-horseradish peroxidase conjugates. Results of unrelated DNA control, BSA control and QseBD51A control verified the binding specificity. g After QseC senses indole, QseB is phosphorylated and combines with the promoter of LekdpXFABC to enhance the expression of this operon. Error bars show the standard deviation of three replicates. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All data are mean ± s.e.m.

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Indole enhances the potassium ion uptake with O-antigenic polysaccharides

To transport potassium ions into cells, especially in a potassium-poor environment, potassium ions need to be adsorbed more efficiently around LeYC36 cells. For the reason that LPSs can adsorb cations [63], the potassium ion contents of LPSs under different conditions (with or without indole) were tested. The results showed that the accumulation capacity of potassium ions in LPS after indole treatment was twice that of the untreated group (Fig. 5a). Transcriptome analysis and real-time PCR results showed that multiple O-antigenic polysaccharide transport genes were highly expressed in the indole-treated environment (Fig. 5b, c). A differentially expressed gene cluster was identified that contained three genes, namely, wxcA (glycosyltransferase family), wzm (O-antigenic polysaccharide transfer system, ATP binding protein) and wzt (O-antigenic polysaccharide transfer system, ABC transporter transmembrane domain) (Fig. 5d). The function of this gene cluster was predicted to help O-antigenic polysaccharide be transported to the periplasmic space after biosynthesis. Then, O-antigenic polysaccharide was integrated into the core polysaccharide chain to form complete LPS with an O-specific chain [64]. In LPS, O-antigenic is the only part containing rhamnose [65]. To some extent, the content of rhamnose in LPSs reflects the content of O-antigenic in LPSs. When indole was added, the content of rhamnose in LPS highly increased, which meant that indole enhanced the O-antigenic production (Fig. 5e). Therefore, we speculated that the O-antigenic polysaccharide in LPS was the key point for LPS to absorb potassium ions under indole treatment. Further wzm knockout experiments showed that the intracellular potassium ion content of the Δwzm mutant strain (O-antigenic polysaccharide could not be integrated into the core polysaccharide chain) was significantly reduced compared with that of the wild-type strain (Fig. 5f and Supplementary Fig. S13). These results showed that the accumulation of potassium ions by O-antigenic polysaccharides was beneficial to the potassium ion uptake of LeYC36.

Fig. 5: O-antigenic polysaccharide is involved in the indole-related pathway by helping bacteria collect potassium ions in the environment.
figure 5

a In the indole-treated group, the accumulation capacity of potassium ions in LPS was enhanced. b The transcriptome analysis result. c The real-time PCR result. In the indole treatment, the transcription of wxcA, wzm and wzt was increased significantly. d The products of wxcA, wzm and wzt compose the O-antigenic polysaccharide transfer system which transports O-antigenic polysaccharides into the periplasmic space. The extension of the O-antigenic polysaccharide chain occurs in glycosyltransferases (GTs). e Compared with that of the untreated strain, the rhamnose content in LPSs of LeYC36 under indole treatment greatly increased. The content of rhamnose was used to reflect the content of O-antigenic polysaccharides in LPSs. f Compared with that of the wild-type strain, the intracellular potassium ion content of Δwzm was greatly reduced. Error bars show the standard deviation of three replicates. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All data are mean ± s.e.m.

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Indole-mediated potassium ion absorption is coordinated by intraspecific signaling molecules DSFs

As the population density of LeYC36 increased, the way that LeYC36 coordinated both intraspecies and interspecies communication was worth studying. Subsequent studies showed that indole-mediated potassium ion transport in LeYC36 was strictly dependent on its population density. When its cell density reached a certain threshold (from the end of the exponential phase), indole could no longer regulate the expression of the LekdpXFABC operon (Fig. 6a). Then, we focused on Lysobacter enzymogenes diffusible signaling factors (LeDSFs), the only intraspecies quorum sensing molecules identified in Lysobacter thus far (Fig. 6b). LeDSFs are members of the DSF family. At the high cell density (OD600 = 1.0), exogenous indole (0.5 mM) applications enhanced the transcription level of LekdpXFABC in ΔrpfC (the mutant strain lacks DSFs-sensing ability) and stimulated the potassium ion uptake, while the transcription level of LekdpXFABC in the wild type was not affected by indole application (Fig. 6c, d). Furthermore, for ΔrpfF (the mutant strain lacks DSFs-producing ability), indole also upregulated the transcription level of LekdpXFABC and enhanced the intracellular potassium ion content (Fig. 6e, f). These results indicated that the lack of DSFs sensing or the inactivation of DSFs biosynthesis could restore the ability of high-density cells to sense indole. In addition, further results showed that indole greatly inhibited the viability of ΔrpfC and ΔrpfF cells at high cell density while the viability of the wild type was not affected by indole treatment at high cell density (Fig. 6g). Therefore, we speculated that LeDSFs could inhibit indole-induced potassium ion transport to promote economical nutrient usage and ultimately allow its population to survive in the environment with poor nutrition under the stress of intraspecies competition.

Fig. 6: Indole-mediated potassium ion absorption is coordinated by DSFs.
figure 6

a The real-time PCR result. The addition of indole could not relate the expression of LekdpXFABC at the end of the exponential phase, stationary phase and decline phase. b The structure of LeDSF3. c The real-time PCR result at high cell density (OD600 = 1.0). Compared with that for the wild-type strain, indole greatly increased the transcription of LekdpXFABC in ΔrpfC at a high cell density. In the wild-type strain, the effect of indole was avoided. d Under the high cell density condition, indole greatly enhanced the intracellular potassium ion content of ΔrpfC. e The result of real-time PCR on LekdpXFABC in ΔrpfF under different conditions (with or without indole) at high cell density (OD600 = 1.0). Indole significantly enhanced the transcription level of LekdpXFABC in ΔrpfF. f Indole significantly increased the content of intracellular potassium ions in ΔrpfF. g Analysis of adversity viability at high cell density (OD600 = 1.0). The bacterial patch of the wild type, ΔrpfC and ΔrpfF on the 10%TSB solid medium with poor nutrition under different conditions (with or without indole). For the wild type, indole did not affect the viability. For ΔrpfC and ΔrpfF, indole decreased the viability. Error bars show the standard deviation of three replicates. NS not significant; *p < 0.05; **p < 0.01; ***p < 0.001. All data are mean ± s.e.m.

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Discussion

Bacterial communication systems are closely related to physiological behaviors. Bacteria can sense environmental changes and stress pressure through signal communication and optimize survival strategies by regulating the expression of key genes to better adapt to the external environment. As an interspecific signaling molecule, indole plays a key role in information transmission among bacteria. The family Xanthomonadaceae is widely present in natural environments, and Lysobacter is a typical non-indole-producing member of this family. Previous studies have shown that Lysobacter can sense indole, and its antibiotic resistance can be reversed in an indole-mediated manner [31]. However, the mechanism by which Lysobacter responds to indole signaling and makes adaptive adjustments in bacterial ecosystems is largely unknown.

In this study, we used LeYC36 and the indole-producing bacterial strain E. coli K12 to simulate their communication and responses in a noncontact way. We found that LeYC36 underwent a series of physiological adjustments after sensing indole signaling produced from E. coli K12. Indole promoted LeYC36 to produce more anti-Gram-positive bacterial and antifungal metabolites at the early stage of the exponential phase. When LeYC36 was cultured alone, the yield of these metabolites reached the same level at the late stage of the exponential phase (after 48–60 h of cultivation). This phenomenon was speculated to be related to the intraspecies quorum sensing effect of LeYC36. Lysobacter can produce a variety of novel natural products, but their production is relatively low. Moreover, the biosynthesis genes are nearly not expressed in the first half of the culture process. The conclusion that indole can enable LeYC36 to activate the expression of these genes at the early growth stage provides a new idea for improving the production of natural products.

Furthermore, the presence of indole enhances the adsorption and transport capacity of potassium ions and improves the viability of LeYC36 (Fig. 7). Our study identifies a novel potassium ion transport system LeKdpXFABC in LeYC36 and a novel gene LekdpX, which has not been studied before. In previous studies, the function of the canonical operon kdpFABC has been studied extensively [61, 62], but the gene cluster pattern of LekdpXFABC has not been described. Here, we identify LekdpX as a necessary gene for the LeKdpXFABC potassium ion transport system, for the first time. Moreover, the novel pathway that LekdpXFABC is directly regulated by QseB was identified. Previously, KdpD/KdpE was reported as the main TCS which regulates kdpFABC, and some studies have reported that QseC can phosphorylate KdpE [57, 60, 62, 66]. However, our study shows that QseC regulates LekdpXFABC through the phosphorylation of QseB and that QseC/QseB plays a key role in this indole-regulated potassium ion transport system. For KdpD/KdpE, although the transcription levels of kdpD and kdpE were not upregulated by indole (Supplementary Table S3 and Supplementary Fig. S14), the possibility of its regulatory effect could not be ruled out. But it is clear that QseC/QseB is involved in this regulation and plays the key role, which has not been reported before. The universality of this mechanism deserves further studies and analyses. In addition, we found that the indole treatment induced high expression of the O-antigenic polysaccharide which enabled LeYC36 to more efficiently collect potassium ions around cells. Previous studies have shown that negatively charged LPSs have the function of cation adsorption [63], while the function of O-antigenic polysaccharides toward cation recruitment is still unknown. We speculated that the O-antigen plays a core role in the indole-related potassium ion recruitment of LeYC36.

Fig. 7: Indole-mediated potassium ion uptake signaling pathway.
figure 7

After being produced by E. coli, indole is sensed by LeYC36. Then, indole is involved in the QseC/QseB-LekdpXFABC regulatory pathway to enhance the potassium ion import. The productions of O-antigenic polysaccharides and antimicrobial compounds are promoted by indole. Moreover, the signal collaboration of indole and DSFs existed. DSFs can inhibit the effect of indole.

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Our above results show that indole promotes the potassium ion uptake of LeYC36 and enhances its resistance to high osmotic pressure to confer a survival advantage. Our study speculates that indole can help LeYC36 sense the presence of other bacteria and adjust its survival strategy which might be beneficial to E. coli. For the reason that L. enzymogenes tends to produce various extracellular lytic enzymes [67], it is possible for L. enzymogenes to degrade more extracellular organic matter and provide E. coli with a nutritious environment. Considering that the production of the anti-Gram-positive bacterial metabolite WAP and antifungal metabolite HSAF is promoted in the co-culture between LeYC36 and E. coli, inhibiting other competitors by inducing indole regulation of LeYC36 might be a possible survival strategy of E. coli. Possibly, the communication between E. coli and LeYC36 might be beneficial to both of these two species and promote their cooperation to survive in complex environments.

The cooperation between intraspecies and interspecies communication of bacteria in complex natural environments has always been an important topic, but many questions still need to be answered. Here, we provide a possible strategy for the signal collaboration of indole signaling and DSFs in LeYC36 (Fig. 7): When other bacteria are present in the environment, LeYC36 might initiate a stress response to gain a survival advantage, even under the low self-population density condition. With the increase in its self-population, intraspecies signal regulation might begin to be initiated. At this point, bacterial cells sense an increase in self-population and then invest more resources in intraspecies competition. To maintain the survival of the whole population in a resource-limited environment, the uptake of nutrients including potassium ions might be reduced, and metabolic activities might be downregulated. In other words, when the population of LeYC36 is high enough, DSFs might take precedence over indole signaling. Conversely, the lack of DSFs sensing in ΔrpfC cells or the lack of DSFs biosynthesis in ΔrpfF cells might lead to the constant indole responses at high cell density. These responses might compete for energy and nutrients. The mode of bacterial environmental adaptation might be regulated strictly by indole and DSFs.

The gene LekdpX exists widely in a large number of bacterial species from various environments, such as seawater, river, soil, plant hosts and human hosts (Supplementary Fig. S1). The genes of LeKdpX protein family are contained in members of 59 bacterial families at least (Supplementary Fig. S2). Considering such a wide distribution, LeKdpX and other members of this protein family might play a key role. However, the operon LekdpXFABC is identified in the main members of the family Xanthomonadaceae. In other bacteria, the operon kdpFABC is widely contained. All these identified LekdpXFABC members possess DSFs biosynthesis genes while those kdpFABC strains lack them (Fig. 3d-II). Based on these results, we speculated that the signal collaborative strategy of indole signaling and DSFs might provide a corresponding state under the specific microbial conditions so that the maximum fitness advantage is conferred. The family Xanthomonadaceae plays an important role in ecosystems. Many pathogens of it cause various diseases of plants and human beings [13,14,15,16]. For example, Stenotrophomonas maltophilia is hard to be eliminated totally and is harmful to public health [16]. Therefore, studies on LekdpXFABC could provide a new idea to develop new tools on crop disease control and treatment of human diseases. This study reveals the wide distribution of LekdpXFABC-mediated survival strategy and explains a mode of stress resistance formation. It broadens the understanding of bacterial communications.

In this study, we show that indole can enhance the survival ability of LeYC36 under adverse conditions. And a possible connection between indole and terminal flagella production (green arrows and white boxes in Fig. 2g) in the high osmotic pressure environment is indicated. Flagella existed in the indole-free high osmotic pressure system and indole-added high osmotic pressure system. However, they seemed to be different. In the untreated culture system, LeYC36 cells had no flagella (Fig. 2g). In addition, in the environment with poor nutrition, indole slowed the cell division of LeYC36 (Fig. 2i). Based on this result, we speculate that indole might activate a possible state of LeYC36 and that this state might enhance the resistance to adverse circumstances with the slow speed of cell division. This phenomenon might be related to the contradictions of indole functions described previously [33]. Therefore, we speculate that different methods will yield seemingly contradictory results, but their underlying mechanisms may be highly similar. This inference is worth investigating so that the function of indole, a mysterious signaling molecule, will be better understood.