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. 2025 Jan 6;372:fnae117. doi: 10.1093/femsle/fnae117

Distribution and functional analysis of two types of quorum sensing gene pairs, glaI1/glaR1 and glaI2/glaR2, in Burkholderia gladioli

Kazumi Takita 1, Nobutaka Someya 2, Tomohiro Morohoshi 3,✉
PMCID: PMC11753530  PMID: 39762131

Abstract

Burkholderia gladioli produces a yellow-pigmented toxin called toxoflavin, and causes disease on a variety of plants. Previous studies have suggested that the pathogenicity of B. gladioli is regulated by an N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing (QS) system. In this study, complete genome sequencing revealed that B. gladioli pv. gladioli MAFF 302385 possesses two types of AHL synthase and AHL receptor gene pairs: glaI1/glaR1 and glaI2/glaR2. Disruption of QS genes revealed that the glaI1/glaR1 QS system regulated swarming motility, biofilm formation, and colony formation via N-octanoyl-l-homoserine lactone. Although Escherichia coli harboring glaI2 produced N-(3-hydroxyoctanoyl)-l-homoserine lactone and N-(3-hydroxydecanoyl)-l-homoserine lactone, the expression of glaI2 was not confirmed in MAFF 302385 cells. We also found that toxoflavin production was regulated by the glaI1/glaR1 QS system in liquid medium, but not on agar medium. When pathogenicity tests were performed on gladiolus leaves, the wild-type and QS mutants showed a similar level of disease. Our results demonstrated that only the glaI1/glaR1-mediated QS system is active in MAFF 302385, but major virulence factors, especially toxoflavin, are not completely dependent on the QS system.

Keywords: quorum sensing, Burkholderia gladioli, acylhomoserine lactone, toxoflavin, plant pathogen

This study investigated the plant pathogen Burkholderia gladioli, which possesses two cell–cell communication systems, revealing that one regulates its virulence factors and the other is silent in the genome.

Introduction

Burkholderia gladioli is a Gram-negative environmental bacterium. It has been reported that B. gladioli is a well-known pathogenic bacterium and isolated from various putrid vegetables, flowers, and even from patients with cystic fibrosis (Graves et al. 1997, Stoyanova et al. 2014). Burkholderia gladioli is classified into four types of pathovars that can infect a wide range of plant hosts. Burkholderia gladioli pv. gladioli was initially isolated from putrid gladiolus bulbs (Severini 1913). Burkholderia gladioli pv. cocovenenans, which was isolated from contaminated tempeh, causes food poisoning due to the production of toxic secondary metabolites (Ross et al. 2014). Burkholderia gladioli pv. agaricicola causes soft rot in mushrooms, and B. gladioli pv. allicola causes onion scale rot (Young et al. 1978, Gill 1995). In addition, B. gladioli strains have been isolated from rice, iris, sweet potato, pineapple, and sugarcane (Kato et al. 1992, Wrobel et al. 1992, Seynos-García et al. 2019, Cui et al. 2020, Zhang et al. 2020). Burkholderia gladioli produces a yellow-pigmented toxin called toxoflavin, which acts as an electron carrier by bypassing the cytochrome system (Latuasan and Berends 1961). Hydroperoxide, the final product of toxoflavin synthesis, is highly toxic to plants, fungi, animals, and bacteria (Latuasan and Berends 1961, Fenwick et al. 2011). Despite their toxoflavin-producing activities, some avirulent B. gladioli strains are expected to serve as novel biocontrol agents against fungi and bacteria (Jung et al. 2013, Seynos-García et al. 2019, Cui et al. 2020, Kim et al. 2021).

Quorum sensing (QS) is a cell-to-cell communication mechanism that regulates the expression of specific genes in response to increased cell density (Miller and Bassler 2001). N-acyl-l-homoserine lactone (AHL) is a well-known QS signaling molecule found in Gram-negative bacteria (Miller and Bassler 2001). The luxI/luxR QS system is used by many Gram-negative bacteria, including plant pathogens. Burkholderia glumae BGR1, isolated from rice, uses a tofIMR-QS system that regulates swarming motility, toxoflavin production, protection against visible light, lipase secretion, and oxalate acid biosynthesis (Kim et al. 2004, Devescovi et al. 2007, Kim et al. 2007, Chun et al. 2009, Goo et al. 2012). Burkholderia gladioli BSR3, isolated from rice, also possesses tofIMR-QS system and regulates phenotypes, such as toxoflavin production, swarming motility, polyketide synthesis, inositol catabolism, and the glyoxylate cycle (Kim et al. 2014). The mechanisms of toxoflavin production regulated by the tofIMR-QS system are very similar between B. glumae BGR1 and B. gladioli BSR3 (Kim et al. 2004, 2014, Lee et al. 2016).

In the present study, we designated the tofI and tofR homologs present in B. gladioli strains as glaI1 and glaR1, respectively. Previous studies have shown that most B. gladioli strains possess the glaI1/glaR1-QS system, which regulates various phenotypes. In contrast, some B. gladioli strains, such as ATCC 10248T and KACC 11889, possess toxoflavin biosynthesis genes but lack the glaI1/glaR1-QS system (Lee et al. 2016, Cui et al. 2020). Instead of the glaI1/glaR1-QS system, both strains possessed another novel luxI/luxR-QS gene pair, identified by BLAST analysis (Altschul et al. 1990). We designated these novel luxI and luxR homologs as glaI2 and glaR2, respectively. To date, AHL production and phenotypes regulated by the glaI2/glaR2-QS system have not been elucidated. In this study, we investigated the distribution of the glaI1/glaR1 and glaI2/glaR2-QS systems among B. gladioli strains deposited in the Japanese Culture Collection and whole-genome sequences deposited in the genome database of the National Center for Biotechnology Information (NCBI). In addition, the unique B. gladioli strain MAFF 302385, which has both glaI1/glaR1 and glaI2/glaR2 gene pairs (Fig. 1), was selected as a model pathogenic strain and investigated for phenotypic regulatory mechanisms, such as toxoflavin production, swarming motility, colony morphology, and virulence, against the leaves of the gladiolus, which are under the control of two QS systems.

Figure 1.

Figure 1.

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Arrangement of glaI1/glaR1 and glaI2/glaR2 homolog genes on chromosome 2 (accession no. CP002600) and plasmid bgla_1p (CP002601) from the genome of B. gladioli BSR3, chromosomes 1 (AP024337) and 2 (AP024338) from the B. gladioli MAFF 302385, and chromosome 1 (CP009323) from the B. gladioli ATCC 10248T. The scale represents 1 kb of nucleotides.

Materials and methods

Bacterial strains, compounds, and growth conditions

The bacterial strains and plasmids used in this study are listed in Table S1. All strains of B. gladioli were grown in trypticase soy broth (TSB; Becton, Dickinson and Co., Sparks, MD, USA) at 30°C. Luria-Bertani (LB) medium was used to extract toxoflavin from B. gladioli. Escherichia coli DH5α was grown in LB medium at 37°C. Solid bacterial agar plates were prepared by adding agar to LB medium at a final concentration of 1.5%. All the bacterial liquid media were incubated with shaking at 200 rpm. Antibiotics such as ampicillin, kanamycin, and gentamycin were added at final concentrations of 100, 50, and 50 µg/ml, respectively. The AHLs used in this study, N-octanoyl-l-homoserine lactone (C8-HSL), N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL), N-(3-hydroxyoctanoyl)-l-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxydecanoyl)-l-homoserine lactone (3-OH-C10-HSL), were synthesized using a previously described method (Chhabra et al. 2003).

Identification of glaI1 and glaI2 genes in B. gladioli strains

To identify QS genes in 80 strains of B. gladioli obtained from the National Agriculture and Food Research Organization (NARO) GeneBank (Tsukuba, Ibaraki, Japan), glaI1 and glaI2 were amplified using the KOD One PCR Master Mix (TOYOBO, Osaka, Japan) with specific primers (Table S2). The polymerase chain reaction (PCR) cycling conditions were as follows: 30 cycles of 98°C for 10 s, 60°C for 5 s, and 68°C for 10 s. Agarose gel electrophoresis was performed to confirm amplification of each gene.

Genome sequencing of B. gladioli MAFF 302385

The genomic DNA of MAFF 302385 was extracted using a NucleoSpin Tissue DNA extraction kit (Takara Bio, Shiga, Japan). Genome sequencing was performed on the PacBio RSII platform (Pacific Biosciences, Menlo Park, CA) using libraries prepared using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) by Macrogen Japan Corp. (Kyoto, Japan). Sequencing reads were assembled using Canu version 1.6 software (Koren et al. 2017). The assemblies were annotated using the DDBJ Fast Annotation and Submission Tool (DFAST) version 1.2.18, which is a bacterial genome annotation pipeline (Tanizawa et al. 2018). Briefly, coding sequences were predicted using Prodigal 2.6.3 (Hyatt et al. 2010). Genes coding for tRNA and rRNA were identified using Aragorn 1.2.38 (Laslett and Canback 2004) and Barrnap 0.8 (https://github.com/tseemann/barrnap), respectively.

Construction of QS gene mutants of B. gladioli MAFF 302385

The QS genes in MAFF 302385 were amplified using the KOD One PCR Master Mix with the specific primers listed in Table S2. An A-overhang was added to the 3′ ends of the PCR products using Blend Taq DNA polymerase (TOYOBO). The PCR products were cloned into the pGEM-T easy cloning vector (Promega, Madison, WI, USA). To remove the internal regions of the target genes, the sequences upstream and downstream of the target genes were amplified using the pGEM-T easy vector containing QS genes as a template. PCR was performed using KOD FX Neo DNA polymerase (TOYOBO). The specific primers used for gene deletion are listed in Table S2. The PCR cycling conditions were as follows: 30 cycles of 98°C for 10 s, 60°C for 30 s, and 68°C for 2 min. The amplified PCR fragments were excised by digesting with BamHI and then self-ligated. The gene-coding region with a deletion in the internal sequence was excised using EcoRI and inserted into the suicide vector pK18mobsacB (Schafer et al. 1994) to create plasmids for gene deletion. The gene deletion plasmids were transformed into MAFF 302385 cells by electroporation. Recombinants generated by a single crossover event were selected using TSB agar plates containing kanamycin. The single-crossover mutants were streaked onto a TSB agar plate containing 10 wt% sucrose to select the second homologous recombinants by removing the sacB gene, which is sensitive to sucrose, from pK18mobsacB. The presence of the expected internal gene deletion was confirmed using PCR and agarose gel electrophoresis.

Cloning and expression of AHL synthases in E. coli

Each glaI1 and glaI2 coding region was amplified by PCR from the MAFF 302385 genome using the KOD One PCR Master Mix with specific primers (Table S2). The PCR cycling conditions were as follows: 30 cycles of 98°C for 10 s, 60°C for 5 s, and 68°C for 10 s. The PCR products were excised using EcoRI and BamHI (for glaI1), SalI and PstI (for glaI2), and inserted into the same restriction sites in the broad-host-range vector pBBR1MCS5 (Kovach et al. 1995). The constructed plasmids were transferred into E. coli DH5α and used for the experiments described below.

Detection of AHL production

To determine AHL production by B. gladioli strains, cross-streaking assays were performed by co-inoculation with Chromobacterium violaceum CV026 and VIR07 as AHL reporter strains, which were used to detect AHLs with short (C4–C8) and long (C8–C18) acyl chains, respectively (McClean et al. 1997, Morohoshi et al. 2008). Burkholderia gladioli strains were inoculated on TSB agar medium prepared in 24-well plates. CV026 and VIR07 were inoculated on the plates close to the inoculation sites of the MAFF strains. After incubation at 30°C for 2 days, AHL production was confirmed by the secretion of purple-pigmented violacein in response to AHLs.

Identification, extraction, and quantification of AHL molecules

MAFF 302385 and its QS gene mutants were inoculated in 4 ml of TSB medium. After overnight cultivation, 40 µl of the subculture was transferred to 4 ml of fresh TSB medium and incubated for 18 h. After centrifugation, 700 µl of the supernatant was mixed with an equal volume of ethyl acetate and vortexed for 30 min. Next, 600 µl of the ethyl acetate layer was transferred to a new microtube, evaporated to dry the solvent, and dissolved in 50 µl of DMSO. AHL molecules were identified and quantified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) as previously described (Morohoshi et al. 2017).

Swarming motility assay

The overnight cultures of MAFF 302385 and its QS gene mutants were diluted at OD600 = 0.01. One microliter of the cell suspension was dropped onto a TSB medium containing 0.5% agar, and the plate was incubated at 30°C for 32 h. Swarming motility was confirmed by dendritic pattern formation in each colony.

Toxoflavin production

To extract toxoflavin from the liquid medium, the overnight cultures of MAFF 302385 and its QS gene mutants were diluted 100 times with LB medium and incubated for 24 h. If necessary, 1 µM C8-HSL was added to the medium. After centrifugation, 700 µl of the supernatant was mixed with an equal volume of chloroform and vortexed for 30 min. For the extraction of toxoflavin from solid medium, bacterial strains were streaked on LB agar plates and incubated at 30°C for 4 days. The bacterial colonies were removed from the agar plates, and 0.15 g of agar medium was cut and placed into a microtube with 700 µl of chloroform and vortexed for 30 min. Next, 600 µl of the chloroform layer was transferred to a new microtube, evaporated for drying, and dissolved in 300 µl of an HPLC mobile phase (50% water, 50% acetonitrile, and 0.1% acetic acid). The samples (20 µl) were chromatographed on an HPLC system (Jasco, Tokyo, Japan) connected to a UV/VIS detector set at 380 nm using a Mightysil RP-18GP column (250 mm × 4.6 mm, 5 µm particle diameter; Kanto Kagaku, Tokyo, Japan) at a flow rate of 2 ml/min.

Pathogenicity assay of gladiolus leaves

The overnight cultures of MAFF 302385 and its QS gene mutants were diluted at OD600 = 1.0 with sterile distilled water. Gladiolus leaves were pierced using the tip of a chip, and 1 µl of cell suspension or sterile distilled water as a negative control was inoculated into the punctured sites, and the leaves were incubated at 30°C. After incubation for 3 days, the lesion areas formed on the gladiolus leaves were observed and measured. At least 15 leaves per strain were used for the pathogenicity assay.

Biofilm assay

The overnight cultures of MAFF 302385 and its QS gene mutants were diluted 100 times with TSB medium. If needed, 1 µM C8-HSL was added to the medium. Subsequently, 100 µl of the cell suspension was transferred to a 96-well polystyrene plate (AsOne, Osaka, Japan), and the plate was incubated for 3 days. Subsequently, 25 µl of 1% crystal violet solution was added to each well. After 15 min of standing, the plates were rinsed twice with distilled water. Crystal violet was dissolved in 100 µl of 99.5% ethanol, and biofilm formation was analyzed at 570 nm using the Infinite M200 microplate reader (Tecan Japan, Kanagawa, Japan).

Nucleotide sequence accession number

The complete genome of B. gladioli MAFF 302385 has been deposited in the DDBJ/ENA/GenBank database under the accession numbers AP024337 (chromosome 1), AP024338 (chromosome 2), AP024339 (plasmid pBGLAD1), and AP024340 (plasmid pBGLAD2).

Results

The dominant QS system in B. gladioli is glaI1/glaR1 and rarely possesses glaI2/glaR2

Burkholderia gladioli BSR3 possesses the glaI1/glaR1 gene pair as its sole QS system, whereas the B. gladioli type strain ATCC 10248T has only the glaI2/glaR2 pair (Fig. 1). To investigate the distribution of these genes, PCR was performed using 80 B. gladioli strains isolated in Japan. All the strains possessed the glaI1/glaR1 genes, and none had glaI2/glaR2 as their sole QS system (Table S3). Notably, only one strain, MAFF 302385, harbored both the gene pairs. Further, BLAST analysis of 258 B. gladioli strains in the NCBI genome database (https://www.ncbi.nlm.nih.gov/genome, accessed on 20 August 2022) revealed that 246 strains, including BSR3, had only glaI1/glaR1, whereas four strains, including ATCC 10248T, had only glaI2/glaR2. Three strains contained both QS gene pairs, whereas any QS gene pairs have not been confirmed in five strains (Tables 1 and S3). These findings indicate that most B. gladioli strains possess only glaI1/glaR1, with glaI2/glaR2 being primarily found in strains isolated from clinical or gladiolus samples. Interestingly, six of the eight strains containing glaI2/glaR2 were isolated from the gladiolus, including MAFF 302385.

Table 1.

Distribution of two types of QS gene pair in B. gladioli strains.

QS gene type Number of strains
  MAFF strains NCBI genome database Total
glaI1/glaR1 79 246 325
glaI2/glaR2 0 4 4
Both 1 3 4
None 0 5 5
Total 80 258 338
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C8-HSL is a dominant AHL synthesized by glaI1/glaR1-QS system in MAFF 302385

To identify the positions of QS genes on the chromosome, we obtained a complete genome sequence of MAFF 302385 using the PacBio RSII platform. We obtained 127 371 reads with an average length of 10 678 bp which corresponded to sequences of approximately 1.36 Gbp. After assembly, the MAFF 302385 genome included two circular chromosomes, 1 and 2; and two endogenous plasmids, pBGLAD1 and pBGLAD2; which were 4 563 288, 3 800 749, 180 710, and 120 226 bp in size, respectively. Gene annotation analysis revealed that glaI1/glaR1 is located on chromosome 2, and glaI2/glaR2 is located on chromosome 1 (Fig. 1).

QS gene deletion mutants were constructed to evaluate AHL-producing activities of the glaI1/glaR1 and glaI2/glaR2-QS systems. The AHLs produced by MAFF 302385 and its QS gene mutants were extracted from the culture supernatant and analyzed using LC–MS/MS. The spectra of the AHLs extracted from MAFF 302385 are shown in Fig. S1. C8-HSL was the dominant AHL produced by MAFF 302385. In addition to C8-HSL, small amounts of 3-OH-C8-HSL and 3-oxo-C6-HSL were detected. C8-HSL is a well-known AHL molecule synthesized by TofI from B. glumae BGR1, B. glumae 336gr-1, and B. gladioli BSR3 (Kim et al. 2004, 2014, Chen et al. 2012, Choudhary et al. 2013, Lee et al. 2016). Results of the quantified C8-HSL are shown in Fig. 2A. The production of C8-HSL completely disappeared in supernatants extracted from the glaI1 deletion mutants 2385ΔI1 and 2385ΔI1ΔI2. Regarding the glaR1 deletion mutant, production of C8-HSL was drastically reduced. It has been reported that the production of C8-HSL is under a positive feedback loop, as it is often the case with these QS systems in B. glumae BGR1 (Kim et al. 2004). Thus, it was assumed that the mechanism of C8-HSL production in MAFF 302385 was similar to that of B. glumae BGR1. In addition, since there was no difference in the AHL production between MAFF 302385 and 2385ΔI2, it was assumed that glaI2 was a nonfunctional gene or silent in MAFF 302385 cells under our experimental condition.

Figure 2.

Figure 2.

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(A) Quantification of C8-HSL produced by MAFF 302385 and its QS mutants using LC–MS/MS. The average production of C8-HSL by wild-type MAFF 302385 was defined as 100%. The experiments were repeated at least three times, and the error bars represent standard deviations. (B) Identification of AHLs produced by E. coli DH5α harboring glaI1- or glaI2-expressing plasmids. E. coli DH5α harboring pBBR1MCS5 was used as a negative control. The strains were incubated in 4 ml LB medium at 30°C for 18 h. AHLs were extracted from the culture supernatant and visualized as purple pigments produced by the AHL reporter strain, C. violaceum VIR07. C8-HSL stock solution was used as a positive control.

Both GlaI1 and GlaI2 are functional for AHL biosynthesis

To investigate the AHL-producing activities of GlaI1 and GlaI2 in detail, glaI1 and glaI2 were cloned into pBBR1MCS5, resulting in plasmids pBBR-I1 and pBBR-I2, respectively. AHLs extracted from the culture supernatants of DH5α harboring pBBR-I1 or pBBR-I2 were detected using AHL reporter strains. Interestingly, although glaI2 deletion in MAFF 302385 did not affect AHL production, E. coli DH5α harboring pBBR-I2 showed AHL production (Fig. 2B). The structures of the AHLs produced by DH5α harboring pBBR-I1 or pBBR-I2 were determined by LC–MS/MS analysis. Similar to that observed in MAFF 302385, C8-HSL was the dominant AHL produced by DH5α harboring pBBR-I1; however, the minor AHLs, 3-OH-C8-HSL and 3-oxo-C6-HSL, did not reach detectable levels (Fig. S2). The cell-free supernatant of E. coli DH5α harboring pBBR-I2 contained two AHLs: 3-OH-C8-HSL and 3-OH-C10-HSL (Fig. S3). To investigate whether GlaI2 functions as an AHL synthase in MAFF 302385 cells, AHLs extracted from 2385ΔI1ΔI2 harboring pBBR-I2 were subjected to LC–MS/MS. The results showed that 2385ΔI1ΔI2 harboring glaI2 mainly produced 3-OH-C8-HSL and less amounts of 3-OH-C10-HSL and C8-HSL (Fig. S4). These results suggest that GlaI2 has the ability to synthesize 3-OH-C8-HSL and 3-OH-C10-HSL, but its expression level might be low in MAFF 302385 cells.

The glaI1/glaR1-QS system regulates swarming motility, colony morphology, and biofilm formation in MAFF 302385

Because swarming motility is a typical phenotype regulated by the QS system, the swarming motility of MAFF 302385 and its QS mutants was evaluated. MAFF 302385 exhibited motility on a TSB plate containing 0.5% agar. Likewise, the glaI2 and glaR2 mutants showed motility similar to that of the MAFF 302385 wild-type strain (Fig. 3A). In contrast, deletion of glaI1 or glaR1 resulted in deficient swarming motility. Addition of 1 µM C8-HSL restored the swarming motility of the glaI1 deletion mutants but not that of the glaR1 deletion mutants. Next, we examined whether the glaI1/glaR1-QS system affected colony morphology. Although the MAFF 302385 wild-type strain showed wrinkled and slightly dry colonies, the glaI1 and glaR1 deletion mutants displayed smooth and wet colonies (Fig. 3B). The colony morphology of the glaI1 mutant changed to that of the MAFF 302385 wild-type strain upon the addition of 1 µM C8-HSL, whereas that of the glaR1 mutant did not. We also examined whether the QS system affected biofilm formation of MAFF 302385. The results demonstrated that deletion of glaI1 or glaR1 resulted in a loss of biofilm formation ability; however, addition of 1 µM C8-HSL resulted in the formation of biofilm to the same extent as that of the MAFF 302385 wild-type strain (Fig. S5). These results demonstrated that swarming motility, colony morphology, and biofilm formation were regulated by the glaI1/glaR1-QS system, but the glaI2/glaR2-QS system did not affect these phenotypes in MAFF 302385.

Figure 3.

Figure 3.

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(A) Swarming motility of MAFF 302385 and its QS mutants. Overnight cultures were diluted to OD600 = 0.01. Each cell suspension was spotted on the center of TSB plates containing 0.5% agar with or without 1 µM of C8-HSL. After 32 h incubation at 30°C, the presence (+) or absence (−) of dendritic pattern was evaluated. (B) Colony morphology of MAFF 302385 and its QS mutants. Bacterial colonies were transferred onto the center of TSB plates with or without 1 µM C8-HSL. After incubation for 3 days at 30°C, colony morphology of each strain was observed. The presence (+) or absence (−) of the wrinkled surface was evaluated.

Toxoflavin production is regulated by glaI1/glaR1-QS system in liquid medium but not on solid medium

To investigate the regulation of toxoflavin production by the QS system, toxoflavin was first extracted from culture supernatants of MAFF 302385 and its QS mutants and analyzed using HPLC (Fig. 4A). MAFF 302385, 2385ΔI2, and 2385ΔR2 mutants produced toxoflavin, but 2385ΔI1 and 2385ΔR1 did not. Toxoflavin produced by 2385ΔI1 could be recovered by the addition of 1 µM C8-HSL, but that of 2385ΔR1 could not. These results demonstrate that the glaI1/glaR1-QS system regulates toxoflavin biosynthesis in liquid LB medium. Interestingly, when MAFF 302385 and its mutants were grown on LB agar medium, the production of a yellow pigment, thought to be toxoflavin, was observed around all colonies. Therefore, we attempted to extract toxoflavin from the LB agar medium after culturing MAFF 302385 and its mutants. HPLC analysis revealed that the yellow pigment extracted from the LB agar medium corresponded to toxoflavin (Fig. 4B). Previous studies have shown that mutation of the AHL synthase gene in B. gladioli BSR3 results in defective toxoflavin biosynthesis on LB agar plates (Kim et al. 2014). However, our findings regarding MAFF 302385 differ significantly from those of previous experiments with BSR3.

Figure 4.

Figure 4.

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Toxoflavin production of MAFF 302385 and its QS mutants cultivated in LB liquid medium (A) and on LB agar medium (B). The samples were incubated with/without adding 1 µM C8-HSL in LB broth. Toxoflavin was extracted from the medium using chloroform and quantified by HPLC. All results expressed as a percentage relative to the MAFF 302385 wild type. The results were reproduced in three experiments, and the error bars indicate standard deviations. Different lowercase letters indicate significant differences as determined by the Tukey's HSD test (P < 0.05).

QS does not affect pathogenicity on gladiolus leaves in MAFF 302385

Because MAFF 302385 has been isolated from rotten gladiolus in Japan, we investigated QS regulation of the pathogenicity of MAFF 302385 and its QS mutants in gladiolus leaves (Fig. 5). When MAFF 302385 was inoculated onto gladiolus leaves, lesions were clearly observed around the inoculation sites. In all the QS mutants, the lesions were observed to be at a similar level as those in MAFF 302385 wild type. These results indicate that pathogenicity against gladiolus leaves is not regulated by any QS system in the MAFF 302385 strain. In addition, these results were different from B. gladioli BSR3 and B. glumae BGR1, in which the QS-mutant lost the pathogenicity against its host plant, rice (Chun et al. 2009, Lee et al. 2016). Table S4 shows the pathogenic relationships of B. gladioli and B. glumae strains against their plant hosts, including some phenotypes.

Figure 5.

Figure 5.

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Pathogenicity assay of MAFF 302385 and its QS mutants using gladiolus leaves. Bacterial strains were inoculated into the center of each gladiolus leaf, and the leaves were incubated for 3 days. After incubation, lesions formed on the leaves (A) were observed and their areas (B) were measured. Sterilized distilled water was used as a negative control. The results were reproduced in 25 pieces of gladiolus leaves, and the error bars indicate standard deviations. Different lowercase letters indicate significant differences, as determined by the Tukey's HSD test (P < 0.05).

Discussion

Burkholderia gladioli is a known plant pathogen with several pathovars and infects a variety of host plants. Both B. gladioli and B. glumae infect rice and cause bacterial panicle blight by producing toxoflavin, which is regulated by a tofIMR-mediated QS system. Previous studies have shown that B. glumae BGR1, B. glumae 336gr-1, B. gladioli BSR3, and B. gladioli CGB10 possess a tofIMR-type QS system that is activated by C8-HSL (Kim et al. 2004, 2014, Chen et al. 2012, Choudhary et al. 2013, Lee et al. 2016, Cui et al. 2020). In the present study, we identified a unique B. gladioli strain, MAFF 302385, which possesses two QS gene pairs (glaI1/glaR1 and glaI2/glaR2) in its genome. However, the remaining 79 MAFF strains did not possess the glaI2/glaR2-QS system (Table 1). Analysis of 258 B. gladioli genomes deposited in the NCBI database revealed that only seven strains possessed glaI2/glaR2 (Table 1). It has been reported that B. gladioli KACC11889, which lacks glaI1/glaR1, does not produce toxoflavin and is nonvirulent in rice (Lee et al. 2016). Thus, it can be inferred that the glaI1/glaR1-QS system is more strongly associated with pathogenic expression rather than glaI2/glaR2-QS. In this study, we revealed that the glaI1/glaR1-QS system regulated several virulence factors, but the glaI2/glaR2-QS system did not play an important role in MAFF 302385. GlaI2 functions in the biosynthesis of 3-OH-C8-HSL and 3-OH-C10-HSL in E. coli DH5α, but may not be expressed or suppressed by any other regulation system in MAFF 302385 cells. The two QS systems of Pseudomonas fuscovaginae UPB0736 are inactive in a standard laboratory setting, and their expression is restored when exogenous AHLs are added (Špacapan et al. 2024). This suggests the possibility that glaI2 in MAFF 302385 strain might also be expressed in natura by other triggers. It has been reported that Burkholderia pseudomallei, which is known as an etiologic agent of melioidosis in humans, and Burkholderia thailandensis, which is known as a non-pathogenic soil bacterium, secrete several types of AHLs including 3-OH-C8-HSL and 3-OH-C10-HSL (Ulrich et al. 2004, Le Guillouzer et al. 2017). The fact that the habitat of B. gladioli is the same as that of these bacteria suggests that they create a molecular chemical network and QS gene expression mechanism for 3-OH-C8-HSL and 3-OH-C10-HSL between different Burkholderia species. A BLAST search revealed that glaI2 and glaR2 homologs were present in the genome sequences of some strains belonging to the family Burkholderiaceae, Paraburkholderia tropica IAC135 (GenBank assembly accession number GCF_014171495.1), Paraburkholderia xenovorans LB400 (GCF_000013645.1), and Caballeronia sp. SBC1 (GCF_011493005.1) and SBC2 (GCF_011039955.1). Multiple insertions in the genome, called genome islands, between Burkholderia species account for more than 10% of the genome, and the structure and content vary among different species (Mannaa et al. 2018). Thus, the unexpressed glaI2 and glaR2 in the MAFF 302385 genome may have been acquired by horizontal insertion from other closely related species during evolution.

The QS system mediated by tofIMR has been well studied in B. gladioli BSR3 and B. glumae BGR1. Previous studies have shown that the lack of tofIMR in these strains resulted in the absence of toxoflavin production on agar media (Kim et al. 2004, 2007, 2014, Chun et al. 2009). However, in our study, MAFF 302385 mutants lacking glaI1 or glaR1 did not produce toxoflavin in liquid medium but did on LB agar medium, unlike BGR1 and BSR3. This suggests an alternative mechanism regulates toxoflavin biosynthesis in MAFF 302385 independent of the glaI1/glaR1-QS system. The tofI and tofR mutants of B. glumae MAFF 302934 and B. glumae 336gr-1 also produced toxoflavin on agar plates (Kato et al. 2014). The QS mutant of B. glumae 336gr-1 switches on/off for toxoflavin production using different inoculation methods. This indicates that toxoflavin production is regulated under certain growth conditions, regardless of the QS system. However, no report has explained the detailed mechanism of toxoflavin production switching in liquid and solid media. The 2385ΔI1 mutant strain produced more toxoflavin than the wild-type strain on LB agar (Fig. 4B). In B. glumae 336gr-1, toxoflavin production increased in the tofI mutant with the addition of 1 µl C8-HSL to the LB medium (Chen et al. 2012). Unlike 336gr-1, the 2385ΔI1 mutant did not exhibit overproduction of toxoflavin, which remained comparable to that of the wild-type strain when supplemented with C8-HSL in LB medium. These findings indicate that toxoflavin production in MAFF 302385 is not regulated solely by the QS system and that overproduction is suppressed by C8-HSL on LB agar (Fig. S6). Thus, it is clear that C8-HSL-mediated QS affects the regulatory mechanism of toxoflavin production on LB agar medium; however, the detailed mechanism for toxoflavin production on agar medium is not clear. Further research is needed to elucidate the complex system governing toxoflavin production.

A genomic functional study revealed distinct functionalities in certain B. gladioli strains compared to other strains. Specifically, strains ATCC 10248T, KACC 11889, and ATCC 25417 lack specific metabolic pathways, including nitrogen reduction and nitrite oxidation. Additionally, some strains exhibit differences in their secretory systems (Waterworth et al. 2020). Several strains of B. gladioli, including ATCC 10248T and KACC 11889, have been reported to be non-QS-regulating strains (Seo et al. 2015, Lee et al. 2016). Intriguingly, all these strains were isolated from the gladiolus and possessed the glaI2/glaR2 gene pair (Table S3). Based on differences in QS genes and metabolic pathways compared with those of other strains, these strains are thought to have unique gene functions. This indicates that the gene structures and functions of B. gladioli possibly differ according to their hosts, and the strains infecting the gladiolus tend to have specific genes, including the glaI2/glaR2-QS system.

In conclusion, our study revealed that MAFF 302385 possesses unique QS and pathogenic gene functions distinct from those of other B. gladioli strains. However, virulence factors, including the toxoflavin system, remain largely unexplored and their characteristics vary depending on the strain and host. Given the ability of B. gladioli to infect a wide range of hosts, it poses a significant threat to important crops. Therefore, it is imperative to elucidate the pathogenic mechanisms of each strain. We hope that future research will focus on the functions of genes related to pathogenic factors, which may ultimately lead to the development of effective methods for controlling bacterial and plant infections.

Supplementary Material

fnae117_Supplemental_File
fnae117_supplemental_file.docx (577.9KB, docx)

Acknowledgements

We thank Prof. Xiaonan Xie from Utsunomiya University for technical assistance with the LC–MS/MS analysis.

Contributor Information

Kazumi Takita, Department of Innovation Systems Engineering, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya 321-8585, Japan.

Nobutaka Someya, Institute for Plant Protection, National Agriculture and Food Research Organization (NARO), 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8666, Japan.

Tomohiro Morohoshi, Department of Innovation Systems Engineering, Graduate School of Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya 321-8585, Japan.

Conflict of interest

None declared.

Funding

None declared.

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