Production of QS signals by the wild type and the complemented strains but not the <i>lon</i> mutant.

(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 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.</p

Segregation of morphologically distinct colony types from the <i>lon</i> mutant.

(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).</p

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)</p

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 (PDF)</p

Structural alignment of three homologs of ATP-dependent Lon protease in <i>B</i>. <i>glumae</i>.

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)</p

Acyl-homoserine lactone (acyl-HSL) levels were lower in the <i>lon</i> 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)</p

Supplementation of 10 μM C8-HSL in <i>lon</i> mutant cultures did not increase the level of oxalate biosynthetic protein to the wild-type level.

(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 lon mutant to the wild type level.</p

Cell viability and extracellular pH of the <i>lon</i> mutant of <i>B</i>. <i>glumae</i>.

(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.</p

Population density of BGR1, <i>lon</i>::Gm, and <i>lon</i>::Gm/<i>lon</i> 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)</p