Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

Eunhye Goo; Ingyu Hwang

doi:10.1371/journal.pone.0257257

. 2021 Sep 15;16(9):e0257257. doi: 10.1371/journal.pone.0257257

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

Eunhye Goo ^1,^2,^*, Ingyu Hwang ^1,²

Editor: Ya-Wen He³

PMCID: PMC8443046 PMID: 34525127

Abstract

The highly conserved ATP-dependent Lon protease plays important roles in diverse biological processes. The lon gene is usually nonessential for viability; however, lon mutants of several bacterial species, although viable, exhibit cellular defects. Here, we show that a lack of Lon protease causes pleiotropic effects in the rice pathogen Burkholderia glumae. The null mutation of lon produced three colony types, big (BLONB), normal (BLONN), and small (BLONS), in Luria–Bertani (LB) medium. Colonies of the BLONB and BLONN types were re-segregated upon subculture, while those of the BLONS type were too small to manipulate. The BLONN type was chosen for further studies, as only this type was fully genetically complemented. BLONN-type cells did not reach the maximum growth capacity, and their population decreased drastically after the stationary phase in LB medium. BLONN-type cells were defective in the biosynthesis of quorum sensing (QS) signals and exhibited reduced oxalate biosynthetic activity, causing environmental alkaline toxicity and population collapse. Addition of excessive N-octanoyl-homoserine lactone (C8-HSL) to BLONN-type cell cultures did not fully restore oxalate biosynthesis, suggesting that the decrease in oxalate biosynthesis in BLONN-type cells was not due to insufficient C8-HSL. Co-expression of lon and tofR in Escherichia coli suggested that Lon negatively affects the TofR level in a C8-HSL-dependent manner. Lon protease interacted with the oxalate biosynthetic enzymes, ObcA and ObcB, indicating potential roles for the oxalate biosynthetic activity. These results suggest that Lon protease influences colony morphology, growth, QS system, and oxalate biosynthesis in B. glumae.

Introduction

The ATP-dependent protease Lon is highly conserved in prokaryotes and eukaryotes and is involved in diverse biological processes [1]. Lon was first identified in Escherichia coli and derived its name from the long, non-septate filamentation phenotype of the lon mutant [2]. Since its discovery, studies have shown that Lon plays an important role in the degradation of abnormal or unstable regulatory proteins as a quality control protease [1]. In E. coli, the short-lived regulatory proteins selectively degraded by Lon include bacteriophage λ N protein, the cell division regulator SulA, the positive regulator of capsule synthesis, RcsA, and the F factor plasmid antidote protein CcdA [3–5]. The lon mutant of E. coli exhibited increased sensitivity to UV irradiation, filamentation, and overproduction of capsular polysaccharides [3–5]. In Caulobacter crescentus, Lon protease is involved in regulating the methylation of chromosomal DNA and cellular differentiation via degradation of cell-cycle-regulated DNA methyltransferase (CcrM) [6]. In pathogenic bacteria such as Brucella abortus and Salmonella typhimurium, Lon-mediated proteolysis of transcription factors controlling for the expression of virulence genes is necessary for host invasion [7, 8]. Lon protease influenced the biofilm formation, motility, and survival of Pseudomonas aeruginosa in a rat model of chronic infection [9].

The lon gene is generally nonessential for viability. However, LonV is essential for viability, while LonD plays an important role in sporulation in Mycobacterium xanthus [10, 11]. A recent study showed that Lon protease is essential for growth under stressful conditions in pathogenic bacteria such as Acinetobacter baumannii and Dickeya solani [12, 13]. Lon protease affects cell morphology in Agrobacterium tumefaciens [14]. A lon mutant in A. tumefaciens formed both large and small colonies, of which the large colonies formed homogeneously sized colonies after subculture, while the small colonies re-differentiated into small and large colonies after subculture [14].

Lon protease has been reported to play a negative regulatory role in the quorum sensing (QS) system of P. aeruginosa and P. putida, but the underlying molecular mechanisms have not yet been determined [15, 16]. We aimed to identify the roles of Lon protease in QS-dependent phenotypes including phytotoxin production, motility, protein secretion, and oxalate biosynthesis in Burkholderia glumae, the causal agent of rice panicle blight [17–20]. We generated a lon null mutant of B. glumae to assess its roles in QS and found that the mutant exhibited continuous segregation of cell morphology in the mutant upon subculture. Such segregation was an obstacle in deciphering the functional roles of Lon protease. Nonetheless, we selected one genetically complementary colony type from among the three different colony types present and analyzed the effect of Lon protease on the QS system and QS-dependent phenotypes. We found that defects in the Lon protease resulted in pleiotropic phenotypes, including difficulty reaching maximum growth capacity, failure to generate QS signals, and alkalization of the growth environment, in addition to giving colony size variations. Our findings provide insights into the critical functions of Lon protease in B. glumae, including maintenance of cellular integrity and roles in the QS system and the QS-dependent phenotypes.

Materials and methods

Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are listed in S1 Table. Strains of B. glumae and E. coli were grown in Luria-Bertani (LB) broth (Affymetrix) (0.1% tryptone, 0.5% yeast extract, and 0.5% NaCl [all wt/vol]) at 37°C, M9 minimal medium (Na₂HPO₄, KH₂PO₄, NaCl, and NH₄Cl) with 0.2% glucose, nutrient broth (NB) medium (BD) (0.1% beef extract, 0.2% yeast extract, 0.5% peptone and 0.5% NaCl [all wt/vol]), and King’s B medium (BD) (2% peptone, 0.15% K₂HPO₄, 0.15% MgSO₄ [all wt/vol]) with the following antibiotics: gentamycin, 20 μg/mL; rifampicin, 100 μg/mL; tetracycline, 10 μg/mL; ampicillin, 50 μg/mL; trimethoprim; 75 μg/mL; kanamycin, 50 μg/mL.

Cell viability and extracellular pH assay

Cells were inoculated in 2 mL LB broth with appropriate antibiotics and grown at 37°C at 250 rpm for 18 h. Overnight cultures were washed twice with fresh LB broth, and turbidity was adjusted to an optical density (OD) of 0.05 at 600 nm using a BioSpectrometer (Eppendorf) followed by 2 mL subculture in glass test tubes (PYREX) for all assays. To compare initial growth rate of wild type and mutant, an OD₆₀₀ of 0.05 was diluted 20 times followed by subculture (Figs 2A and S1A). Aliquots of 100 μL from each sample were serially diluted and 10 μL each of three repeats was spotted on LB agar medium to monitor colony-forming units (CFUs) at the designated time point. LB agar plates were incubated at 37°C for 24 h to allow colonies to grow. CFUs were counted under a dissecting microscope and multiplied by the appropriate dilution factor to calculate CFU/mL. In an extracellular pH assay, the culture supernatant was sampled from each vial at the designated time point and the pH was measured using a pH meter (Lab 860, SCOTT Instruments).

Fig 2 — (A) B. *glumae* strains were subcultured in LB medium with 20× dilution after adjusting samples to an O.D₆₀₀ of 0.05. CFUs were determined from serial dilution of 100 μL aliquots of each sample at the designated time points. The *lon* mutant had a significantly lower initial growth rate than that of the wild type. (B) B. *glumae* strains were subcultured in LB medium following adjustment of samples to an O.D₆₀₀ of 0.05. The population density of the *lon* mutant decreased gradually after 12 h, leading to a population crash. (C) The extracellular pH of the strains was measured at each time point using a pH meter.

Construction of the B. glumae lon::Gm, lon::Gm/lon, tofI::Sp/lon::Gm double mutant, and obcA::Tp

The lon (bglu_1g13520) gene was deleted via insertion of the gentamycin cassette ScaI and marker exchange as described previously [21]. The pLa2 is a 9.0-kb EcoRI-HindIII fragment cloned into pLAFR3, including the lon gene from cosmid clone pLa1. A gentamycin cassette was generated by PCR using ScaI-Gm-F and Gm-ScaI-R primers, cloned into pBluescript II SK(+), and termed “pBS_Gm4”. A 0.8-kb DNA fragment obtained from pBS_Gm4 via digestion performed with ScaI was ligated into pLa2 to produce pLa2_Gm. pLa2_Gm was mobilized from DH5α into B. glumae BGR1 or BGS2 by conjugation using pRK2013 as a helper plasmid and marker exchange, as described previously [21]. For genetic complementation of the lon mutant, a 2.876-kb DNA fragment containing the lon gene and its own promoter was generated by PCR using HindIII-La and La-EcoRI primers, cloned into pBluescript II SK(+), and termed “pLa3”. A 2.876-kb DNA fragment obtained by pLa3 by digestion performed with HindIII and EcoRI was ligated into pLAFR3 to produce pLa4. pLa4 was mobilized from DH5α into B. glumae BLONN through conjugation using pRK2013 as a helper plasmid. The obcA (bglu_2g18790) gene was deleted via insertion of the trimethoprim resistance gene KpnI-XhoI and marker exchange as described previously [21]. A 2.716-kb DNA fragment including the obcA and obcB genes and their promoter was generated via PCR using BamHI-obcA and obcB-EcoRI primers, cloned into pLAFR3, and termed “pOBC4”. pTP2 contained a trimethoprim-resistant gene produced by PCR using the Tp(BamHI)-F and Tp-R primers and then cloned into pBluescript II SK(+). A 0.887-kb KpnI-XhoI fragment from pTP2 was cloned into pOBC4 to produce pOBC4::Tp. The BOBCA strain was generated via triparental mating of B. glumae BGR1, DH5α (pRK2013), and DH5α (pOBC4::Tp). Southern blot analysis was performed to confirm the BLONN, S2LON, and BOBCA strains, and the BLONC was assessed via plasmid extraction.

Measurements of oxalate levels and oxalate biosynthetic activity assay

Oxalate levels and oxalate biosynthetic activity were measured as described previously [20, 22]. Oxalate was measured using an oxalate assay kit (Trinity Biotech) according to the manufacturer’s instructions. Briefly, oxalate was converted into carbon dioxide and hydrogen peroxide using oxalate oxidase, and the production of hydrogen peroxide was measured via reaction with 3-(dimethylamino) benzoic acid, during which it formed a blue compound catalyzed by peroxidase. Absorbance at 590 nm was measured using a microplate reader (PerkinElmer); 0.5 mM oxalate was used as a standard. The absorbance of the sample was divided by the absorbance of standard and multiplied by the appropriate dilution factor. The reaction buffer used in the assay measuring oxalate biosynthetic activity consisted of 200 mM Tris-Cl (pH 8.0), 10 mM EDTA, 20 mM CoCl₂, 2 mM acetyl-CoA, and 200 mM oxaloacetate. The total cell lysate was added to the reaction mixture, followed by incubation at 37°C for 10 min. The level of biosynthesized oxalate from the reaction was measured using an oxalate assay kit. Units were calculated as the concentration of biosynthesized oxalate divided by the amount of total protein and reaction time.

RNA extraction and quantitative reverse-transcription PCR (qRT-PCR)

Total RNAs were extracted from B. glumae BGR1, BLONN (BGR1 lon::Gm), and BLONC (BGR1 lon::Gm/lon), grown in LB medium at 37°C for 10 h after subculture, were extracted using RNeasy mini kits (Qiagen) following the manufacturer’s protocols. The extracted total RNA was treated with RNase-free DNase I (Ambion) to remove DNA. Total RNA (1 μg) was reverse-transcribed into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and incubated for 1 h at 42°C. The primer pairs used for qRT-PCR are listed in S2 Table. The 16S rRNA gene was used as the positive control. Transcription levels were determined using SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA) and the CFX96 Real-Time PCR System (Bio-Rad). The thermal cycling parameters were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 5 s. All reactions were performed in triplicate, and all data were normalized to the expression levels of the 16S rRNA gene expression using Bio-Rad CFX Manager software.

Overexpression of Lon and TofR and co-expression of Lon and TofR

The lon and tofR genes of B. glumae were amplified using the primers listed in S2 Table. The amplification products were cloned into the NheI and HindIII restriction sites of the pET-28b expression vector and into the NdeI and XhoI restriction sites of the pACYC-Duet expression vector (Novagen), resulting in pLa8 and pTOFR5, respectively. His-Lon-His and TofR-His were overexpressed in E. coli BL21(DE3), which was induced by addition of 1 mM isopropyl β-o-thiogalactopyranoside (IPTG), followed by additional growth for 1 h at 37°C. To test for protease resistance against Lon protease, 4 μM N-octanoyl-homoserine lactone (C8-HSL) was added to the BL21(DE3) cultures. The BL21(DE3) carrying both pLa8 and pTOFR5 was used for co-expression of Lon and TofR.

Immunoblotting using anti-ObcA, anti-ObcB, anti-TofR, anti-HA, and anti-His antibodies

Total lysates from the B. glumae and E. coli strains were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked using 4% skim milk and incubated with anti-ObcA (1:4000) plus anti-ObcB (1:3000), anti-TofR (1:3000), anti-HA (Invitrogen, 1:2500), and anti-His (Invitrogen, 1:3000) antibodies. Proteins were detected using a secondary anti-rabbit horseradish peroxidase-conjugated IgG antibody (Cell Signaling), a secondary anti-mouse horseradish peroxidase-conjugated IgG antibody (Cell Signaling), and a chemiluminescent substrate (Bio-Rad). Antibodies against B. glumae ObcA, ObcB, Lon, and TofR were generated in rabbits immunized with ObcA-6xHis, a peptide of ObcB (N-GNIEFYADQRRPQYLRELVRVTR-C), a peptide of Lon (N-GLPWRKKSKVNNDLSNAE-C), and TofR-6xHis. The density (pixels/inch) of each band was measured using ImageJ version 1.53a software (NIH). All experiments were performed using three independent replicates.

Coimmunoprecipitation (Co-IP) and western blot analysis

According to the manufacturer’s instructions, Co-IP was performed using the Pierce Direct IP kit (Thermo Fisher Scientific). The anti-ObcA (10 μg), anti-ObcB (10 μg), and anti-Lon (10 μg) antibodies were coupled to AminoLink Plus Coupling Resin (Thermo Fisher Scientific). Cell lysates from B. glumae BGR1 and S2HA containing 1 mg of protein were added to the antibody-coupled resin in a spin column and mixed at 4°C for 24 h. The immunoprecipitates were eluted, followed by the addition of a 5× non-reducing lane marker sample buffer. Samples were boiled for 5 min, cooled to room temperature, and separated via SDS-PAGE. The samples were then transferred to PVDF membranes. Anti-Lon, anti-ObcA, anti-ObcB, and anti-HA (Invitrogen) antibodies were used to detect interactions of ObcA with ObcB and Lon, of ObcB with Lon, and of TofI-HA with Lon, respectively. The immunoreactive bands were detected using electrochemiluminescence (ECL) reagents (Bio-Rad) and captured using the ChemiDoc™ XRS+ imager (Bio-Rad). All experiments were performed using at least three independent replicates.

Autoinducer assay

An autoinducer assay was performed as described previously [18] with the following modifications; B. glumae BGR1, BLONN (BGR1 lon::Gm), and BLONC (BGR1 lon::Gm/lon) were grown in LB supplemented with HEPES (pH 7.0) for 24 h. Autoinducer was extracted from the 500 μL culture-free supernatant using the same amount of ethyl acetate and dried. The dried pellet was dissolved in 10 μL dimethyl sulfoxide. Autoinducer was developed via the thin layer chromatography (TLC), using 70% methanol in the TLC tank. LB agar with biosensor strain Chromobacterium violaceum CV026 overlaid was used to develop the TLC plate to visualize C8-HSL and C6-HSL.

Statistical analysis

Statistical analyses were performed using SPSS version 23 (IBM Corp.). Comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc analysis (set at 5% significance level).

Results

Continuous segregation of the lon mutant into morphologically distinct colonies

To explore the role of Lon protease in B. glumae BGR1, we constructed a lon null mutant by inserting a gentamicin resistance cassette into the lon gene via double-crossover homologous recombination. The genome of B. glumae BGR1 contains three homologs of ATP-dependent Lon protease: bglu_1g13520, bglu_1g31260, and bglu_1g33380. The bglu_1g13520 homolog had typical domains of Lon protease, including the Lon substrate binding domain (accession number_pfam02190), P-loop_NTPase domain (accession number_ cl38936), and C-terminal proteolytic domain (accession number_pfam05362) (S1 Fig). On the other hand, bglu_1g31260 and bglu_1g33380 contained only N-terminal domain of Lon protease (accession number_cl19481) and P-loop_NTPase domain, respectively (S1 Fig). The bglu_1g13520 was selected for investigation of its biological roles in B. glumae BGR1. The marker-exchanged lon null mutant was segregated into three morphologically distinct colony types: big (BLONB), normal (BLONN), and small (BLONS) (Fig 1A). The BLONS-type cells grew very slowly and were too small to manipulate. The largest-sized colonies, called BLONB, grew faster than the other two types but segregated into BLONB and BLONS only, not BLONN, after subculture in LB medium (Fig 1B). BLONB was bigger than BLONN in all tested media, and was darker, than BLONN under a dissecting microscope (Fig 1A, 1B and 1E–1H). The occurrence frequency of BLONB and BLONS segregated from BLONB was 67.5% and 32.5%, respectively (Fig 1C). When a colony of the BLONN type was subcultured in LB medium, cells were segregated into the BLONB, BLONN, and BLONS types at frequencies of 5.35%, 84.52%, and 10.13%, respectively (Fig 1D). Such segregation of the BLONB and BLONN types did not change in M9 minimal medium supplemented with 0.2% glucose (Fig 1E), nutrient broth medium (Fig 1F), King’s B medium (Fig 1G), and LB supplemented with 100 mM HEPES (pH 7.0) (Fig 1H). We selected the BLONN type for further analysis, as this colony type exhibited fully complemented phenotypes with pLa4 carrying a full length of lon gene along with its own promoter in pLAFR3 (Fig 2A).

Fig 1 — (A) The *lon* mutant was segregated into three morphologically distinct colony types, big (BLONB), normal (BLONN), and small (BLONS). Colonies were grown in LB medium and observed under a dissecting microscope at ×30 magnification. (B) The BLONB segregated into BLONB and BLONS after subculture in LB medium. Colonies were observed under a dissecting microscope at ×30 magnification. (C) The BLONB and BLONS that had segregated from BLONB subcultured in LB medium accounted for 67.5% and 32.5% of the total population, respectively. (D) When the BLONN was subcultured in LB medium, 5.35%, 84.52%, and 10.13% of cells segregated into BLONB, BLONN, and BLONS types, respectively. The colony morphology of BLONN subcultured in (E) M9 minimal medium supplemented with 0.2% glucose, (F) nutrient broth medium, (G) King’s B medium, (H) LB supplemented with 100 mM HEPES (pH 7.0).

Impaired growth of the lon mutant

To determine the biological significance of Lon protease, we measured the growth and extracellular pH of the lon mutant. The population of the wild type BGR1 reached a maximum capacity at 3.20 × 10⁹ colony forming unit (CFU)/mL after 12 hours of subculture in LB medium (Fig 2B). On the other hand, the lon mutant increased up to 2.26 × 10⁸ CFU/ml after the stationary phase and decreased gradually for 1 to 3 days after subculture, followed by complete population death (Fig 2B). During the initial growth stage, the lon mutant had a significantly lower growth rate than that of the wild type (Fig 2A). The lon mutant showed alkalization of the LB culture medium, whereas the pH of wild-type BGR1 slightly increased to 7.33 and then decreased to 4.97, and finally returned to 6.67 (Fig 2C). Environmental pH increased during growth of the lon mutant, which resulted in a population crash (Fig 2). Addition of 100 mM HEPES buffer to the growth medium spared the lon mutant from cell death after the stationary phase but did not affect its initial growth rate (S2 Fig).

Failure of QS signal biosynthesis in the lon mutant

To determine whether impaired growth of the lon mutant affects QS systems, we performed an autoinducer (AI) assay of the wild type and the lon mutant. We performed AI assays with wild type BGR1 at two different population densities, approximately at 1.28 × 10⁸ and 1.33 × 10⁹ CFU/mL, as the lon mutant did not reach the maximum population density. AI signals were detected at 1.28 × 10⁸ CFU/mL but much higher levels were observed at 1.33 × 10⁹ CFU/mL of wild type BGR1 (Fig 3A). However, the lon mutant produced no detectable QS signals, and the genetic complementation resulted in recovery of QS signal biosynthesis to the wild type level (Fig 3A). As the failure of the lon mutant to reach maximum growth capacity might be one reason that the mutant did not produce detectable amounts of QS signals, we harvested cells from 10 culture tubes, each containing 3.57 × 10⁸ CFU/mL, and pooled them to reach approximately 3.57 × 10⁹ CFU/mL. Pooled samples were cultured for an additional 24 hours in the LB supplemented with 100mM HEPES (pH 7.0) prior to QS signal assays (S3 Fig). No QS signal was detected at 3.57 × 10⁹ CFU/mL of the lon mutant (S3 Fig). To determine whether the lack of QS signals in the lon mutant was due to low expression of QS signal synthase gene, tofI, we assessed tofI expression at the transcription level during the mid-log phase using qRT-PCR. Expression of tofI was lower in the lon mutant than in the wild type at the transcription level (Fig 3B). To detect the level of TofI-HA in the wild type and lon mutant via western blot using an anti-HA antibody, stationary-phase strains carrying the TofI-HA clone, pTOFI6, were used. The level of TofI-HA in the lon mutant was approximately 71.1% of the wild type level (Fig 3C).

Fig 3 — (A) A significantly larger amount of QS signal was produced by wild-type cells of 1.33 × 10⁹ CFU/mL than the wild-type cells of 1.28 × 10⁸ CFU/mL. The labels ×10⁸ and ×10⁹ denote the wild-type cell densities of 1.28 × 10⁸ CFU/mL and 1.33 × 10⁹ CFU/mL. TLC analysis of acyl-HSLs extracted from the wild type (3.48 × 10⁹ CFU/mL), the *lon* mutant (7.08 × 10⁸ CFU/mL), and the complemented strain (3.03 × 10⁹ CFU/mL) grown for 24 h in LB supplemented with 100 mM HEPES (pH 7.0). The C6-HSL and C8-HSL were visualized using the acyl-HSL sensor strain CV026. C6, C8, W, M, and C denote C6-HSL, C8-HSL, the wild type, *lon* mutant, and complemented strain, respectively. (B) Expression of the *tofI* gene in each strain was determined during mid-log phase by qRT-PCR. W, M, and C denote the wild type, *lon* mutant, and complemented strains, respectively. The letters (a, b, and c) above each mean indicate significant differences based on a one-way ANOVA, followed by Tukey’s post-hoc analysis. A value of p < 0.05 indicates significant differences among the strains. (C) The amount of TofI-HA in the wild type and *lon* mutant during early stationary phase was determined via western blot analysis using an anti-HA antibody. The densities (pixels/inch) of the TofI-HA bands are presented alongside the blot. W and M denote the wild type and *lon* mutant, respectively.

Low levels of oxalate biosynthetic enzymes produced by the lon mutant

Because the lon mutant produced no detectable QS signal, we measured the capacity for oxalate biosynthesis, which is known to be QS-dependent, in the lon mutant. We found that the oxalate level was significantly lower in the lon mutant than in the wild type (Fig 4A). Reduced production of oxalate by the lon mutant was in accordance with the observed increase in pH of the lon mutant (Figs 2C and 4A). To determine whether the cause of the low oxalate level in the lon mutant was due to the low expression of obcA and obcB, which are responsible for oxalate biosynthesis, we measured the expression levels of both genes in the wild type and the lon mutant. The expression of the obcA and obcB genes was significantly lower in the lon mutant than in the wild type and the complemented strains (Fig 4B). We also analyzed the levels of ObcA and ObcB by western blotting using ObcA and ObcB antibodies at the time points corresponding to the mid-log, early stationary, and mid-stationary growth phases after subculture (6, 12, and 24 hours, respectively). ObcA and ObcB were not detected in the lon mutant at 6 hours but were detected at 12 hours after subculture (Fig 4C). Oxalate production was significantly lower in the lon mutant than in the wild type, even though the ObcA and ObcB were accumulated in the lon mutant during growth (Fig 4A and 4C). To investigate whether Lon affects the activities of ObcA and ObcB, we measured specific oxalate biosynthetic activity in the wild type and lon mutant. Specific oxalate biosynthetic activity was reduced by 68.86% in the lon mutant compared with the wild type (Fig 4D). When specific oxalate biosynthetic activity was converted into the units per CFU, the oxalate biosynthetic activity was significantly lower in the lon mutant than in the wild type (S4 Fig).

Fig 4 — (A) Oxalate in the culture supernatant of each strain was measured at a designated time point. The wild type biosynthesized approximately 20 mM oxalate, whereas the *lon* mutant produced very little oxalate throughout its growth. (B) Expression of *obcA* and *obcB* in each strain during mid-log phase was determined by qRT-PCR. W, M, and C denote the wild type, *lon* mutant, and complemented strains, respectively. The letters (a, b, and c) above each mean indicate significant differences based on a one-way ANOVA, followed by Tukey’s post-hoc analysis. A value of p < 0.05 indicates significant differences among the strains. (C) Western blot analysis using ObcA and ObcB antibodies at 6, 12, and 24 h after subculture indicated lower levels of ObcA and ObcB in the *lon* mutant than in the wild type. Ma, W, M, and C denote the molecular markers, the wild type, *lon* mutant, and complemented strains, respectively. (D) The specific oxalate biosynthetic activity of each strain during early stationary phase was determined. W, A, M, and C denote the wild type, *obcA* mutant, *lon* mutant, and complemented strains, respectively. The letters (a, b, and c) above each mean represent significant differences based on a one-way ANOVA, followed by Tukey’s post-hoc analysis. A value of p < 0.05 represented significant differences among strains.

Reduced oxalate biosynthetic enzyme activity in the lon mutant was not due to low production of C8-HSL

As obcA and obcB are expressed in a QS-dependent manner in B. glumae, and the lon mutant produced no detectable amounts of QS signal, we evaluated whether oxalate biosynthetic capacity can be recovered after exogenous addition of C8-HSL to the lon mutant culture medium. When levels of ObcA and ObcB and oxalate biosynthetic activity were measured in the lon mutant cells grown in LB with the addition of 10 μM C8-HSL, levels of ObcA and ObcB and oxalate biosynthetic activity in the lon mutant did not recover to wild-type levels (Fig 5A and 5B). Oxalate production was monitored during growth in LB buffered with 100 mM HEPES (pH 7.0) and supplemented with up to 10 μM C8-HSL for 2 days after subculture, but the lon mutant did not produce oxalate as much as the wild type did (Fig 5C).

Fig 5 — (A) The amount of ObcAB protein in the wild type, *lon* mutant, and complemented strains during early stationary phase was determined via western blot using anti-ObcA and anti-ObcB antibodies. Ma, W, M, and C denote molecular markers, the wild type, *lon* mutant, and complemented strain, respectively. (B) The specific oxalate biosynthetic activity of each strain during early stationary phase was determined. W, M, M*, A, and C denote the wild type, *lon* mutant, *lon* mutant supplemented with 10 μM C8-HSL, *obcA* mutant, and complemented strain, respectively. The letters (a, b, c, and d) above each mean represent significant differences based on a one-way ANOVA followed by Tukey’s post-hoc analysis. A value of p < 0.05 indicates significant differences among strains. (C) Growth for 2 days after subculture in the LB supplemented with 100 mM HEPES (pH 7.0) and 1 ~ 10 μM C8-HSL did not recover oxalate biosynthesis in the *lon* mutant to the wild type level.

Influence of Lon protease on the TofR level

As addition of excessive QS signal to the lon mutant did not recover the QS-dependent oxalate production to wild-type levels, we hypothesized that Lon protease might influence the activity of the QS signal receptor, TofR. To determine whether Lon protease affects TofR levels, we measured total TofR levels in the lon mutant. TofR levels were 1.81- and 1.6-fold higher in the lon mutant and the tofI/lon double mutant, respectively, than in the wild type (Fig 6A). Although no QS signals were detected in the tofI mutant or lon mutant, TofR levels in the tofI mutant were 12.21% of its wild type and 6.72% of its lon mutant (Fig 6A). When lon and tofR were individually expressed with or without C8-HSL in E. coli, C8-HSL did not influence total levels of TofR (Fig 6B). However, TofR levels were reduced by 4.9-fold when the two genes were co-expressed without C8-HSL compared to the individual expression of the tofR gene (Fig 6B). Addition of C8-HSL to E. coli cultures co-expressing the two genes resulted in a 2.2-fold increase in TofR compared with the same cultures grown without C8-HSL (Fig 6B).

Fig 6 — (A) TofR protein was detected in the B. *glumae* strains grown in the early stationary phase via western blot analysis using an anti-TofR antibody. Ma, W, I, I^c, M, IM, R, and TofR-His denote the molecular marker, the wild type, *tofI* mutant, chemically complemented strain of *tofI* mutant with addition of 1 μM C8-HSL, *lon* mutant, *tofI*/*lon* double mutant, *tofR* mutant, and TofR-His overexpressed in BL21(DE3), respectively. The densities (pixels/inch) of the TofR bands are presented alongside the blot. (B) Western blot analysis using an anti-His antibody showed that Lon influences the levels of TofR, and this influence depends on the presence of C8-HSL. The TofR-His, His-Lon-His, and TofR-His plus His-Lon-His were overexpressed in the BL21(DE3) via addition of 1 mM IPTG with or without 4 μM C8-HSL. The symbols * and & denote addition of 4 μM C8-HSL and co-expression of two proteins, respectively. The densities (pixels/ inch) of TofR bands are shown alongside the blot.

Interaction of Lon protease with ObcA and ObcB but not with TofI

As the lon mutant exhibited low oxalate biosynthetic activity and produced significantly less QS signals compared with the wild type, we hypothesized that Lon protease interacts with ObcA, ObcB, and TofI, affecting their activities. Immunoprecipitation was performed using anti-ObcB and anti-Lon antibodies, ObcA protein was detected, which indicated that ObcA interacts with ObcB and Lon in the wild type strain (Fig 7A). In addition to the 59.11 kDa band of ObcA, another positive band was detected with a size of approximately 52 kDa (Fig 7A). ObcB protein was pulled down using an anti-Lon antibody in an affinity pulldown experiment, which showed that ObcB and Lon interacted in the wild type strain (Fig 7B). To determine whether Lon interacts with TofI, we performed immunoprecipitation using an anti-Lon antibody and detected TofI-HA using an anti-HA antibody by western blot analysis (S5 Fig). TofI-HA was not detected in the immunoprecipitated sample using the anti-Lon antibody (S5 Fig), indicating that Lon and TofI did not interact.

Fig 7 — (A) The ObcA protein was pulled down from the total cell lysate of the wild type strain grown in the early stationary phase using anti-ObcB and anti-Lon antibodies in affinity pulldown experiments. Western blotting was performed using an anti-ObcA antibody to detect ObcA in the immunoprecipitated samples. * denote additional unidentified positive signals. (B) The ObcB protein was detected from the total cell lysate of the wild -ype strain grown in the early stationary phase using an anti-Lon antibody. Ma, Input, IP, and WB denote molecular markers, total lysate of the wild type, immunoprecipitation, and western blotting, respectively.

Discussion

Continuous colony differentiation and abnormal growth due to the loss of Lon protease allowed us to determine that Lon protease is vital to the integrity of cellular functions. It has been reported that the null mutant of lon formed large and small colonies in A. tumefaciens, but its large colonies were homogeneous after subculture [14]. Although the lon mutant has a longer doubling time than that of the wild type A. tumefaciens, they reached similar maximum population densities [14]. This study is the first to report that the lon mutant colonies are continuously segregated morphologically, and that this mutant cannot grow to its maximum capacity. We explored whether several physiological issues caused by a deficiency of Lon protease threaten bacterial survival in vitro.

In general, heterogeneity of the bacterial population is induced by nutrient exhaustion or stress, and it occurs to allow adaptation of the bacteria to environmental changes [23]. Heterogeneous populations may exhibit phenotypic differences within a genetically identical population, or genetic heterogeneity may be present [23]. Persistent colony differentiation was observed in the lon mutant of B. glumae under all tested culture conditions; therefore, heterogeneity of the lon mutant did not appear to be caused by the external environments. The BLONB, generated from BLONN, did not return to the BLONN and was not complemented with pLa4 containing the lon gene; other genetic changes appear to have occurred in the BLONB, although we did not identify any mutation in the BLONB genome. The BLONN maintained a homogeneous population only via genetic complementation, indicating that Lon protease plays an essential role in normal growth and cellular integrity.

The primary biological function of Lon protease is control of protein quality by aiding degradation of unfolded proteins and unstable proteins such as global regulators, thereby preventing the accumulation of abnormal proteins [1]. Protein quality control is also important for stress responses; in particular, Lon plays a role in environmental stress responses in E. coli [1]. Expression of the lon gene in E. coli is dependent on the σ³² transcription factor (RpoH) and is activated by heat shock [24]. These stress-induced proteins are essential for removing damaged polypeptides from stressed cells. High demand for Lon, which suggests that the lon gene is upregulated under certain stress conditions, explains the reduced growth of the lon mutant under thermal stress [24]. As the promoter region of the lon gene in B. glumae had no recognition sequence for sigma factors, Lon in B. glumae is unlikely to belong to the heat regulon, as observed in E. coli.

In addition to proteolytic activity, chaperone-like functions of Lon have also been reported in eukaryotic cells. The human Lon protease LONP1 is a mitochondrial matrix protein that, in conjunction with mitochondrial HSP70, promotes protein folding [25]. Lon plays a vital role in enhancing cell survival by maintaining the stability of the Hsp60–mtHsp70 complex [26]. The interactions of Lon with ObcA and ObcB in B. glumae might be due to Lon acting as a chaperone for ObcA and ObcB. The ObcA and ObcB proteins were detected in samples obtained by affinity pulldown using an anti-Lon antibody. Lon clearly interacts with ObcA and ObcB; however, the biochemical mechanism by which Lon functions as a chaperone is not understood and should be addressed in future works. The appearance of additional unidentified positive signals of approximately 52 kDa, as shown in Fig 7A, suggests that ObcA might undergo posttranslational processing. However, the purpose of posttranslational processing of ObcA remains unknown.

The B. glumae Lon protease has significant homology with the Lon proteases of other bacteria, and the organization of the lon locus is conserved. The locus encoded by clpX and hupB in the flanking region of lon is consistent with those of P. aeruginosa, P. putida, E. coli, and A. tumefaciens. The Lon protease of these four different bacteria had 81% (accession number_AAG05192.1), 67% (AAN67915.1), 70% (AAC73542.1), and 65% (WP_080866091.1) identity with B. glumae Lon (bglu_1g13520), respectively (S6 Fig). However, despite these similarities, the function of B. glumae Lon is quite different from the functions of Lon in these bacteria. The P. aeruginosa Lon negatively controls the LasR/LasI system via degradation of LasI, which directs the synthesis of 3-oxo-dodecanoyl-HSL [15]. Lon negatively regulates the expression of rhlR/rhlI via the degradation of LasI [15]. The lon mutant of P. putida produces significantly higher QS signals than those produced by the wild type [16]. However, in B. glumae, no QS signals were detected from the lon mutant, even though the TofI level in the lon mutant was approximately 71.1% of the wild type level. Lon is unlikely to function as a chaperon for TofI activity, as TofI does not interact with Lon in B. glumae. However, Lon might affect other proteins that are involved in AI biosynthesis, such as acyl carrier protein (ACP) or enzymes associated with S-adenosylmethionine (SAM). Whether Lon affects the biosynthesis of two substrates of TofI at any level is an interesting topic for future research.

No recovery of the QS-dependent phenotype in the lon mutant with excessive addition of C8-HSL QS signal suggests that TofR might not function properly as a receptor protein of C8-HSL in the lon mutant. It has been reported that ClpB and Lon protease of E. coli are responsible for degradation of TraR, a receptor protein for the QS signal, in A. tumefaciens [27]. However, there is no evidence that ClpB or Lon protease of A. tumefaciens directly affects the TraR level dependent on the presence of the QS signal. In contrast to TraR of A. tumefaciens, we showed that TofR appears to be degraded by the Lon protease of B. glumae in E. coli when C8-HSL is unavailable. As overexpressed TofR in the absence of C8-HSL was completely insoluble, degradation of TofR by Lon protease in vitro was not demonstrable.

Our results revealed previously unidentified roles of Lon protease in bacteria. Continuous segregation and growth defects of the lon mutant of B. glumae are unique among the phenotypes reported in bacteria. The negative role of Lon protease in bacterial QS is contradictory to the previously known activities of this enzyme in other bacteria. Our findings raise fundamental questions about the functions of bacterial Lon protease, including the mechanism underlying continuous segregation of colony morphology. Our findings suggest that Lon protease plays essential roles in QS and morpho-physiological traits, and that it is a potential target for the development of control agents for the rice pathogen B. glumae.

Supporting information

S1 Fig. Structural alignment of three homologs of ATP-dependent Lon protease in B. glumae.

Domain names were marked in the black text boxes. The e-values and accession numbers below the representing domains were obtained from NCBI Conserved Domains.

(PDF)

Click here for additional data file.^{(123.5KB, pdf)}

S2 Fig. Population density of BGR1, lon::Gm, and lon::Gm/lon grown in LB supplemented with 100 mM HEPES (pH 7.0).

(A) The lon mutant had a significantly lower initial growth rate than the wild type, even in buffered LB medium. (B) The population crash of the lon mutant was rescued after growing in LB supplemented with 100 mM HEPES (pH 7.0).

(PDF)

Click here for additional data file.^{(124.5KB, pdf)}

S3 Fig. Acyl-homoserine lactone (acyl-HSL) levels were lower in the lon mutant than the wild type, even when cells were pooled at 10⁹ CFU/mL and cultured for an additional 24 h in LB supplemented with 100 mM HEPES (pH 7.0).

Thin-layer chromatography (TLC) analysis was performed for acyl-HSLs extracted from the lon mutant (3.57 × 10⁸ CFU/mL) and the pooled lon mutant (3.57 × 10⁹ CFU/mL) grown for 24 h in LB supplemented with 100 mM HEPES (pH 7.0). The acyl-HSL sensor strain CV026 was used to visualize C6-HSL and C8-HSL. W, M, and M2 denote the wild type, lon mutant, and pooled lon mutant, respectively.

(PDF)

Click here for additional data file.^{(212.5KB, pdf)}

S4 Fig. Oxalate biosynthetic component activity (units per CFU).

The letters (a, b, and c) above each mean represent significant differences based on a one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc analysis. A value of p < 0.05 indicates significant differences among strains.

(PDF)

Click here for additional data file.^{(75KB, pdf)}

S5 Fig. No detection of TofI-HA in the immunoprecipitated sample using an anti-Lon antibody.

Strain S2HA carrying the TofI-HA clone, pTOFI6, was used to determine interaction between Lon and TofI.

(PDF)

Click here for additional data file.^{(74.5KB, pdf)}

S6 Fig. Similarities among ATP-dependent Lon proteases of B. glumae, Pseudomonas aeruginosa, Pseudomonas putida, Escherichia coli, and Agrobacterium tumefaciens.

Comparison between ATP-dependent Lon proteases of B. glumae BGR1 (bglu_1g13520), P. aeruginosa PAO1 (AAG05192.1), P. putida KT2440 (AAN67065.1), E. coli K-12 substr. MG1655 (AAC73542.1), and A. tumefaciens C58 (WP_080866091.1).

(PDF)

Click here for additional data file.^{(165.7KB, pdf)}

S1 Table. The strains and plasmids used in this study.

(DOCX)

Click here for additional data file.^{(42.6KB, docx)}

S2 Table. The primers used in this study.

(DOCX)

Click here for additional data file.^{(37.2KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of the Education (2021R1I1A1A01040314) (E.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0257257.r001

Decision Letter 0

Ya-Wen He

9 Aug 2021

PONE-D-21-24187

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

PLOS ONE

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Reviewer #1: Comments to the Author

Summary:

This paper by Eunhye Goo* & Ingyu H describes experiments to decipher the roles of Lon protease for morpho-physiological traits, including colony morphology, growth, QS system, and oxalate biosynthesis in the rice pathogen Burkholderia glumae.

Overall I think that the study provides some interesting insights, but there are some areas in the manuscript where the authors should clarify to help the reader understand. Further follow up and some clarifications would strengthen the paper.

Major comments are detailed below.

1. In the methods section, “Measurements of oxalate levels and oxalate biosynthetic activity assay” and “Autoinducer assay”, the authors should briefly describe how you performed these experiments in this study.

2. In the results section, the authors should indicate how you came to this conclusion shown in Line 174-180. In addition, please consider if you need a figure to show the structural alignment of these homologous proteins.

3. Line 189-191, The authors state: “Such segregation of the BLONB and BLONN types did not change in M9 minimal medium supplemented with 0.2% glucose (Fig. 1E), nutrient broth medium (Fig. 1F), King’s B medium (Fig. 1G), and LB supplemented with 100mM HEPES (pH 7.0) (Fig. 1H).” Please make sure whether these figures are complete. And looking at these figures, the results do not appear to be consistent. How do the authors explain the obvious difference between Fig. 1A and 1E-1F, or between Fig. 1B and 1E-1F? Please indicate how you distinguished between BLONN and BLONB in Fig. 1A, 1B, and 1E-1H, since it’s a a little hard for readers to judge.

4. After diving thorough the Methods section, I did not find out what are the relevant approaches used in the measurement of cell viability and extracellular pH assay. Please help the reader to understand the assay. There is an obvious difference of the initial bacteria density shown in Fig. 2A and Fig. 2B. So, when exactly were the samples taken for analysis? In addition, the font and axis value should be consistent.

5. Line 204-205, The authors state: “Environmental pH increased during growth of the lon mutant, which resulted in a population crash (Fig. 2).” Please clarify this statement, since I don’t see the results of “a population crash” from Fig. 2.

6. Line 251-257, the description of the results is too general to highlight the work shown in Fig. 5A-C. Please describe the results objectively to help the reader help to understand how you get the conclusion.

7. In the manuscript, hypotheses are proposed in Line 222 and 260 lacking a proper information to understand the point of these experiment. It would be helpful for readers to indicate the function of tofI and TofR and the correlation between tofI or TofR and Lon protease in the introduction or the corresponding result section.

8. Line 314-316, the authors state: “As the promoter region of the lon gene in B. glumae had no recognition sequence for sigma factors, Lon in B. glumae is unlikely to belong to the heat regulon, as observed in E. coli.” Is there any evidence of this? As pointed out in Line 312-313, “the lon gene is upregulated under certain stress conditions”. So did the authors test the expression of lon gene under heat shock?

9. Line 314-316, this statement of the similarities of the Lon protease among four different bacteria lacks proper presentation of alignment results.

Mirror comments:

1. Figure legends should briefly describe the technique and/or experimental design used. The result section can be unnecessary.

2. There are few recent studies in the references. Please consider update references of discuss section.

3. Please unify the primers’ name in Line 105 ( “Tp or TP”).

4. Line 191, “100mM” should be “100 mM”.

Reviewer #2: Reviewing comments for PONE-D-21-24187

Lon protease plays a negative regulatory role in the quorum sensing (QS) system of P. aeruginosa and P. putida but the mechanisms remain unknown. This study wants to investigate the role of Lon protease in growth capacity, failed QS signal biosynthesis and colony size variation etc.

I have the following comments.

• Major concerns

1) In Results part (row 172-180), can you use a supplementary figure to explain the difference and show this diagram among 13520, 31260 and 33380?

2) For Figure 1A, what medium did you use? From Figure 1B, why there is no BLONN but only BLONB and BLONS were found from BLONB? Have you tried to grow BLONB in different medium and same results were obtained?

3) For Figure 2B and 2C, how about the cell number and pH in complemented strain?

4) For Figure 3A, strong signal can be detected in wild type when the number can be more than 109 (left). However, the signal is quite weak for the wild type on the right part when the strain number is 3.48 x 109. Why did they show such significant difference? Similarly, the signal is also very weak for complemented strain when the number is more than 3.03 x 109 (right side on Figure 3A). Is this caused by different exposing time or others?

5) For the ‘failure of QS signal biosynthesis in the lon mutant (row 215-227), can the author make a conclusion for the analysis?

6) In Figure 4D, the author has checked the oxalate biosynthetic activity in obcA mutant. How about the activity in obcB mutant? Similar as obcA?

7) In figure 5, we can conclude that adding with C8-HSL can not rescue the reduced oxalate biosynthetic enzyme activity. Have you ever tried C6-HSL?

8) For Figure 7A, is the additional signal band produced by phosphorylation?

9) In Figure 2S, how about the HSL level if we extend the time to 48h or even 72 hrs?

10) What’s the relationship between tofI and tofR? Can the author add some literatures to explain in the main text to benefit the understanding? What’s the relationship between tofI and QS signal? Please also cite a paper to briefly explain it in the result part (row 221).

• Minor concerbs

Row mistakes or errors suggestions

86 Gm in the main text and figures, ‘gm’ is used. Please use the same word in the manuscript.

222 tofI and tofR What are their full names?

279-282 Revise the sentence

388, 396 etc ‘available from….’ Pls use the same format for all cited papers.

465-475 BLONB etc denote the B-Big, N-normal and S-small size in the figure legend

486 produced than consider to revise the sentence here

535 the * and & both of them denote the addition of 4uM C8-HSL?

38 AHL if possible, pls use HSL in the main text

**********

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PLoS One. 2021 Sep 15;16(9):e0257257. doi: 10.1371/journal.pone.0257257.r002

Author response to Decision Letter 0

22 Aug 2021

Reviewer #1: Comments to the Author

Summary:

This paper by Eunhye Goo* & Ingyu Hwang describes experiments to decipher the roles of Lon protease for morpho-physiological traits, including colony morphology, growth, QS system, and oxalate biosynthesis in the rice pathogen Burkholderia glumae.

Major comments are detailed below.

- Oxalate was measured using an oxalate assay kit (Trinity Biotech) according to the manufacturer’s instructions. Briefly, oxalate was converted into carbon dioxide and hydrogen peroxide using oxalate oxidase, and the production of hydrogen peroxide was measured via reaction with 3-(dimethylamino) benzoic acid, during which it formed a blue compound catalyzed by peroxidase. Absorbance at 590 nm was measured using a microplate reader (PerkinElmer); 0.5 mM oxalate was used as a standard. The absorbance of the sample was divided by the absorbance of standard and multiplied by the appropriate dilution factor. The reaction buffer used in the assay measuring oxalate biosynthetic activity consisted of 200 mM Tris-Cl (pH 8.0), 10 mM EDTA, 20 mM CoCl2, 2 mM acetyl-CoA, and 200 mM oxaloacetate. The total cell lysate was added to the reaction mixture, followed by incubation at 37°C for 10 min. The level of biosynthesized oxalate from the reaction was measured using an oxalate assay kit. Units were calculated as the concentration of biosynthesized oxalate divided by the amount of total protein and reaction time. Autoinducer was extracted from the 500 µL culture-free supernatant using the same amount of ethyl acetate and dried. The dried pellet was dissolved in 10 µL dimethyl sulfoxide. Autoinducer was developed via the thin layer chromatography (TLC), using 70 % methanol in the TLC tank. LB agar with biosensor strain Chromobacterium violaceum CV026 overlaid was used to develop the TLC plate to visualize C8-HSL and C6-HSL. We have added a description to the Methods section.

2. In the results section, the authors should indicate how you came to this conclusion shown in Line 205-211. In addition, please consider if you need a figure to show the structural alignment of these homologous proteins.

- We have added a Supplementary Figure (S1 Fig) to show the structural alignment of three homologs of ATP-dependent Lon protease in B. glumae.

3. Line 222-224, The authors state: “Such segregation of the BLONB and BLONN types did not change in M9 minimal medium supplemented with 0.2% glucose (Fig. 1E), nutrient broth medium (Fig. 1F), King’s B medium (Fig. 1G), and LB supplemented with 100mM HEPES (pH 7.0) (Fig. 1H).” Please make sure whether these figures are complete. And looking at these figures, the results do not appear to be consistent. How do the authors explain the obvious difference between Fig. 1A and 1E-1F, or between Fig. 1B and 1E-1F? Please indicate how you distinguished between BLONN and BLONB in Fig. 1A, 1B, and 1E-1H, since it’s a little hard for readers to judge.

- Colony differentiation was consistently showed in M9 minimal medium supplemented with 0.2 % glucose (Fig. 1E), nutrient broth medium (Fig. 1F), King’s B medium (Fig. 1G), and LB supplemented with 100 mM HEPES (pH 7.0) (Fig. 1H). BLONB was bigger than BLONN in all tested media, and was darker, than BLONN under a dissecting microscope. Fig. 1A and 1E–1H were obtained from BLONN subcultured in different media. Fig. 1B shows that the BLONB segregated into BLONB and BLONS only, not BLONN, which did not change in all tested media (data now shown). We have added an explanations on lines 216–218.

- Cells were inoculated in 2 mL LB broth with appropriate antibiotics and grown at 37°C at 250 rpm for 18 h. Overnight cultures were washed twice with fresh LB broth, and turbidity was adjusted to an optical density (OD) of 0.05 at 600 nm using a BioSpectrometer (Eppendorf) followed by 2 mL subculture in each of glass test tubes (PYREX) for all assays. To compare initial growth rate of wild type and mutant, an OD600 of 0.05 was diluted 20 times followed by subculture (Figs. 2A and S1A Fig). Aliquots of 100 µL from each sample were serially diluted and 10 µL each of three repeats was spotted on LB agar medium to monitor colony-forming units (CFUs) at the designated time point. LB agar plates were incubated at 37°C for 24 h to allow colonies to grow. CFUs were counted under a dissecting microscope and multiplied by the appropriated dilution factor to calculate CFU/mL. In an extracellular pH assay, the culture supernatant was sampled from each vial at the designated time point and the pH was measured using a pH meter (Lab 860, SCOTT Instruments). We have added a description of the measurement of cell viability and the extracellular pH assay in the Methods section. Although we adjusted the optical densities of overnight cultures to approximately similar cell densities for growth studies, there remains some differences in the initial cell numbers. However, we believe that these discrepancies do not affect our growth studies because all cells were at late stationary phase when they were diluted and subcultured. To clearly visualize the biological significance, we used different axis values in Fig. 2A and Fig. 2B, but the same font size is consistently used.

5. Line 237-238, The authors state: “Environmental pH increased during growth of the lon mutant, which resulted in a population crash (Fig. 2).” Please clarify this statement, since I don’t see the results of “a population crash” from Fig. 2.

- The population of the lon mutant gradually decreased after 12 hours of subculture, and viable cells were not detected after 3 days of subculture. As per your advice, we have added the results of the population crash at 4 days after subculture in Fig. 2B.

6. Line 285-290, the description of the results is too general to highlight the work shown in Fig. 5A-C. Please describe the results objectively to help the reader help to understand how you get the conclusion.

There were no differences in the levels of ObcA and ObcB proteins in the lon mutant between no C8-HSL and supplementation with 10 µM C8-HSL, although the gene expression of obcA and obcB is dependent on the C8-HSL-mediated QS system in B. glumae. The oxalate biosynthetic activity of the lon mutant was not increased with the addition of 10 µM C8-HSL. Even with supplementation of C8-HSL up to 10 µM, oxalate production was not recovered to the wild-type levels in lon mutant cells grown in LB buffered with 100 mM HEPES (pH 7.0). We have expanded our description of these results in the revised manuscript.

7. In the manuscript, hypotheses are proposed in Line 254-255 and 296-297 lacking a proper information to understand the point of these experiment. It would be helpful for readers to indicate the function of tofI and TofR and the correlation between tofI or TofR and Lon protease in the introduction or the corresponding result section.

- B. glumae possesses a LuxI/R type QS system, which uses C8-HSL synthesized by TofI, QS signal synthase, and its cognate receptor TofR to regulate the expression of QS-dependent genes. We determined whether the lack of QS signals in the lon mutant was due to the low expression of the tofI gene (line 254-255). We have added additional information in the Results section to help readers understand the rationale of these experiments.

8. Line 350-352, the authors state: “As the promoter region of the lon gene in B. glumae had no recognition sequence for sigma factors, Lon in B. glumae is unlikely to belong to the heat regulon, as observed in E. coli.” Is there any evidence of this? As pointed out in Line 348-349, “the lon gene is upregulated under certain stress conditions”. So did the authors test the expression of lon gene under heat shock?

- The recognition sequence of sigma-70, sigma-32, and sigma-54 in E. coli has been previously reported (reference 24 in manuscript). There was no recognition sequence of sigma-70, sigma-32, and sigma-54 in the promoter region of the lon gene in B. glumae. Expression of the lon gene in E. coli is activated by heat shock (reference 24 in manuscript). We tested the expression of Lon in the wild-type of B. glumae under heat stress conditions via Western blotting using an anti-Lon antibody, but it was not upregulated by heat shock (data now shown).

9. Line 368-370, this statement of the similarities of the Lon protease among four different bacteria lacks proper presentation of alignment results.

- We have added a Supplementary Figure (S6 Fig) to present the structural alignment of Lon protease homologs of four different bacteria.

Mirror comments:

1. Figure legends should briefly describe the technique and/or experimental design used. The result section can be unnecessary.

- Amended

2. There are few recent studies in the references. Please consider update references of discuss section.

- Amended

3. Please unify the primers’ name in Line 118 ( “Tp or TP”).

- We have changed TP to Tp.

4. Line 224, “100mM” should be “100 mM”.

- Amended

Reviewer #2: Reviewing comments for PONE-D-21-24187

I have the following comments.

• Major concerns

1) In Results part (row 203-211), can you use a supplementary figure to explain the difference and show this diagram among 13520, 31260 and 33380?

- We have added a Supplementary Figure (S1 Fig) to show the structural alignment of three homologs of ATP-dependent Lon protease in B. glumae.

- LB medium was used for Figure 1A. This information has been added to the figure legend. The BLONB was segregated into BLONB and BLONS only, not BLONN, which did not change in all tested media (data now shown).

3) For Figure 2B and 2C, how about the cell number and pH in complemented strain?

- The complemented strain showed the same patterns of cell population and extracellular pH as the wild type.

- The visualizing signals in an autoinducer assay can depend on the loading amount of extracted samples and cell number of biosensor Chromobacterium violaceum CV026. Because the left and right sides of Fig. 3A were determined with different TLC plates, it is difficult to compare them directly.

5) For the ‘failure of QS signal biosynthesis in the lon mutant (row 247-260), can the author make a conclusion for the analysis?

- QS signal biosynthesis is closely related to cell density. Because the cell density of the lon mutant does not rise to the maximum population, pooled samples were then further cultured to see if the QS signal was biosynthesized, but the QS signal was not detected. Lon protease can affect other proteins involved in QS signal biosynthesis because QS signal synthase TofI was detected in the lon mutant. These comments have been included in the Discussion.

6) In Figure 4D, the author has checked the oxalate biosynthetic activity in obcA mutant. How about the activity in obcB mutant? Similar as obcA?

- The obcB mutant did not produce oxalate (reference 20 in manuscript). Oxalate biosynthetic activity in the obcB mutant might be similar to that of the obcA mutant.

7) In figure 5, we can conclude that adding with C8-HSL can not rescue the reduced oxalate biosynthetic enzyme activity. Have you ever tried C6-HSL?

- The obcA and obcB gene expression is dependent on the C8-HSL-mediated QS system in B. glumae. The addition of C6-HSL to the lon mutant culture did not change any phenotypes, including the oxalate biosynthetic enzyme activity and growth.

8) For Figure 7A, is the additional signal band produced by phosphorylation?

- We did not obtain direct evidence of an additional positive signal produced by phosphorylation, but ObcA may be phosphorylated based on our phosphorylation prediction results.

9) In Figure 2S, how about the HSL level if we extend the time to 48h or even 72 hrs?

- Autoinducer was not detected in the lon mutant grown in LB supplemented with HEPES (pH 7.0) for 72 hours. We expected that the same results might be reproduced from pooled cells of the lon mutant grown for 72 hours.

- B. glumae uses C8-HSL synthesized by TofI, QS signal synthase, and its cognate receptor TofR to regulate the expression of QS-dependent genes. We determined whether the lack of QS signals in the lon mutant was due to the low expression of the tofI gene (line 254-255). We have added additional information in the Results section to help readers understand the rationale of these experiments.

• Minor concerns

Row mistakes or errors suggestions

99 Gm in the main text and figures, ‘gm’ is used. Please use the same word in the manuscript.

- Amended

254-255 tofI and tofR What are their full names?

- The tofI is a QS signal synthase, and tofR is the QS signal receptor in B. glumae. We have added this information to the Results section.

315-317 Revise the sentence

- We have revised this sentence accordingly.

429, 439 etc ‘available from….’ Pls use the same format for all cited papers.

- Amended

524-534 BLONB etc denote the B-Big, N-normal and S-small size in the figure legend

- Amended

545 produced than consider to revise the sentence here

- Amended

597 the * and & both of them denote the addition of 4uM C8-HSL?

- The symbol * denotes the addition of 4 µM C8-HSL, and the symbol & denotes co-expression of Lon and TofR proteins. For example, TofR* & Lon indicates co-expression of TofR and Lon supplemented with 4 µM C8-HSL.

547 AHL if possible, pls use HSL in the main text

- We have changed AHL to acyl-HSL.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(33.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0257257.r003

Decision Letter 1

Ya-Wen He

27 Aug 2021

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

PONE-D-21-24187R1

Dear Dr. Goo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Ya-Wen He, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Thanks the authors to revise it and explain it in details. This version is much readable now. I strongly recommend for publishing in this journal.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Gu Keyu

PLoS One. doi: 10.1371/journal.pone.0257257.r004

Acceptance letter

Ya-Wen He

2 Sep 2021

PONE-D-21-24187R1

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

Dear Dr. Goo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Structural alignment of three homologs of ATP-dependent Lon protease in B. glumae.

Domain names were marked in the black text boxes. The e-values and accession numbers below the representing domains were obtained from NCBI Conserved Domains.

(PDF)

Click here for additional data file.^{(123.5KB, pdf)}

S2 Fig. Population density of BGR1, lon::Gm, and lon::Gm/lon grown in LB supplemented with 100 mM HEPES (pH 7.0).

(PDF)

Click here for additional data file.^{(124.5KB, pdf)}

(PDF)

Click here for additional data file.^{(212.5KB, pdf)}

S4 Fig. Oxalate biosynthetic component activity (units per CFU).

(PDF)

Click here for additional data file.^{(75KB, pdf)}

S5 Fig. No detection of TofI-HA in the immunoprecipitated sample using an anti-Lon antibody.

Strain S2HA carrying the TofI-HA clone, pTOFI6, was used to determine interaction between Lon and TofI.

(PDF)

Click here for additional data file.^{(74.5KB, pdf)}

S6 Fig. Similarities among ATP-dependent Lon proteases of B. glumae, Pseudomonas aeruginosa, Pseudomonas putida, Escherichia coli, and Agrobacterium tumefaciens.

(PDF)

Click here for additional data file.^{(165.7KB, pdf)}

S1 Table. The strains and plasmids used in this study.

(DOCX)

Click here for additional data file.^{(42.6KB, docx)}

S2 Table. The primers used in this study.

(DOCX)

Click here for additional data file.^{(37.2KB, docx)}

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(33.7KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

Eunhye Goo

Ingyu Hwang

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Abstract

Introduction

Materials and methods

Bacterial strains and growth conditions

Cell viability and extracellular pH assay

Fig 2. Cell viability and extracellular pH of the lon mutant of B. glumae.

Construction of the B. glumae lon::Gm, lon::Gm/lon, tofI::Sp/lon::Gm double mutant, and obcA::Tp

Measurements of oxalate levels and oxalate biosynthetic activity assay

RNA extraction and quantitative reverse-transcription PCR (qRT-PCR)

Overexpression of Lon and TofR and co-expression of Lon and TofR

Immunoblotting using anti-ObcA, anti-ObcB, anti-TofR, anti-HA, and anti-His antibodies

Coimmunoprecipitation (Co-IP) and western blot analysis

Autoinducer assay

Statistical analysis

Results

Continuous segregation of the lon mutant into morphologically distinct colonies

Fig 1. Segregation of morphologically distinct colony types from the lon mutant.

Impaired growth of the lon mutant

Failure of QS signal biosynthesis in the lon mutant

Fig 3. Production of QS signals by the wild type and the complemented strains but not the lon mutant.

Low levels of oxalate biosynthetic enzymes produced by the lon mutant

Fig 4. Reduced production of oxalate by the lon mutant of B. glumae.

Reduced oxalate biosynthetic enzyme activity in the lon mutant was not due to low production of C8-HSL

Fig 5. Supplementation of 10 μM C8-HSL in lon mutant cultures did not increase the level of oxalate biosynthetic protein to the wild-type level.

Influence of Lon protease on the TofR level

Fig 6. Lon protease affects the level of TofR.

Interaction of Lon protease with ObcA and ObcB but not with TofI

Fig 7. Lon protease interacted with ObcA and ObcB.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Ya-Wen He

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Author response to Decision Letter 0

Decision Letter 1

Ya-Wen He

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Acceptance letter

Ya-Wen He

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Associated Data

Supplementary Materials

Data Availability Statement

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