Novel indole-mediated potassium ion import system confers a survival advantage to the Xanthomonadaceae family

Abstract

Interspecific and intraspecific communication systems of microorganisms are involved in the regulation of various stress responses in microbial communities. Although the significance of signaling molecules in the ubiquitous family Xanthomonadaceae has been reported, the role bacterial communications play and their internal mechanisms are largely unknown. Here, we use Lysobacter enzymogenes, a member of Xanthomonadaceae, to identify a novel potassium ion import system, LeKdpXFABC. This import system participates in the indole-mediated interspecies signaling pathway and matters in environmental adaptation. Compared with the previously reported kdpFABC of Escherichia coli, LekdpXFABC contains a novel indispensable gene LekdpX and is directly regulated by the indole-related two-component system QseC/B. QseC autophosphorylation is involved in this process. The operon LekdpXFABC widely exists in Xanthomonadaceae. Moreover, indole promotes antimicrobial product production at the early exponential phase. Further analyses show that indole enhances potassium ion adsorption on the cell surface by upregulating the production of O-antigenic polysaccharides. Finally, we confirm that LeKdpXFABC mediation by indole is subject to the intraspecific signaling molecules DSFs, of which the biosynthesis genes always exist together with LekdpXFABC. Therefore, as a new idea, the signal collaborative strategy of indole and DSFs might ensure the persistent fitness advantage of Xanthomonadaceae in variable environments.

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

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.

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

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.

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

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.

Fig. 4. LekdpXFABC is regulated by QseB.

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 P_LekdpXFABC 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 P_LekdpXFABC is enough. Biotin can be detected with the help of streptavidin-horseradish peroxidase conjugates. Results of unrelated DNA control, BSA control and QseB_D51A 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.

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

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.

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

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.

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

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.