A Single-Nucleotide Insertion in a Drug Transporter Gene Induces a Thermotolerance Phenotype in Gluconobacter frateurii by Increasing the NADPH/NADP+ Ratio via Metabolic Change

Abstract

Thermotolerant microorganisms are beneficial to the fermentation industry because they reduce the need for cooling and offer other operational advantages. Previously, we obtained a thermally adapted Gluconobacter frateurii strain by experimental evolution. In the present study, we found only a single G insertion in the adapted strain, which causes a frameshift in a gene encoding a putative drug transporter. A mutant derivative strain with the single G insertion in the transporter gene (Wild-G) was constructed from the wild-type strain and showed increased thermotolerance. We found that the thermotolerant strains accumulated substantial intracellular trehalose and manifested a defect in sorbose assimilation, suggesting that the transporter is partly involved in trehalose efflux and sorbose uptake and that the defect in the transporter can improve thermotolerance. The ΔotsAB strain, constructed by elimination of the trehalose synthesis gene in the wild type, showed no trehalose production but, unexpectedly, much better growth than the adapted strain at high temperatures. The ΔotsAB mutant produced more acetate as the final metabolite than the wild-type strain did. We hypothesized that trehalose does not contribute to thermotolerance directly; rather, a metabolic change including increased carbon flux to the pentose phosphate pathway may be the key factor. The NADPH/NADP⁺ ratio was higher in strain Wild-G, and much higher in the ΔotsAB strain, than in the wild-type strain. Levels of reactive oxygen species (ROS) were lower in the thermotolerant strains. We propose that the defect of the transporter causes the metabolic flux to generate more NADPH, which may enhance thermotolerance in G. frateuriiIMPORTANCE The biorefinery industry has to ensure that microorganisms are robust and retain their viability and function at high temperatures. Here we show that Gluconobacterfrateurii, an industrially important member of the acetic acid bacteria, exhibited enhanced thermotolerance through the reduction of trehalose excretion after thermal adaptation. Although intracellular trehalose may play a key role in thermotolerance, the molecular mechanisms of action of trehalose in thermotolerance are a matter of debate. Our mutated strain that was defective in trehalose synthase genes, producing no trehalose but a larger amount of acetic acid as the end metabolite instead, unexpectedly showed higher thermotolerance than the wild type. Our adapted and mutated thermotolerant strains showed increased NADPH/NADP⁺ ratios and reductions in ROS levels. We concluded that in G. frateurii, trehalose does not contribute to thermotolerance directly; rather, the metabolic change increases the NADPH/NADP⁺ ratio to enhance thermotolerance.

Keywords: Gluconobacter; NADPH/NADP+ ratio; acetic acid bacteria; drug transporter; reactive oxygen species; thermal adaptation; thermotolerance; trehalose.

FIG 1

(Top) Genomic organization of the transporter gene (locus tag GLF_2756) flanking regions. (Bottom) Sequence alignment showing the mutations in thermally adapted strains CHM43 AD-T and CHM43 AD-G.

FIG 2

Comparison of growth characteristics of G. frateurii CHM43 parental and mutated strains at various temperatures. (A) Growth curves of the mutant strains obtained by UV mutagenesis (UVA10 [△, solid black line] and UVB1 [●, dashed black line]), the parental strain, CHM43 (□, solid gray line); and the thermally adapted strain, CHM43 AD-G (♢, solid gray line). Cells were cultivated in 50 ml of a 10% (wt/vol) sorbitol medium at 30, 37, or 38.5°C. (B) Growth curves for the Wild-G (○, black line), parental (□, gray line), and CHM43 AD-G (♢, gray line) strains cultured under the same growth conditions as those described for panel A.

FIG 3

Effects of disruption and complementation of the transporter gene on bacterial growth at various temperatures. The parental strain, CHM43, harboring pBBR1MCS-4 (□, solid gray line), Wild-G harboring pBBR1MCS-4 (○, solid black line), Wild ΔsteP harboring pBBR1MCS-4 (△, solid black line), Wild-G harboring pCNM2 (promoterless steP) (●, dashed black line), and Wild ΔsteP harboring pCNM2 (promoterless steP) (▲, dashed black line) were cultured in 50 ml of a 10% (wt/vol) sorbitol medium containing 500 μg/ml ampicillin at 30, 37, or 38.5°C. (A) Growth curves of strains CHM43/pBBR1MCS-4, Wild-G/pBBR1MCS-4, and Wild-G/pCNM2 (promoterless steP). (B) Growth curves of strains CHM43/pBBR1MCS-4, Wild ΔsteP/pBBR1MCS-4, and Wild ΔsteP/pCNM2 (promoterless steP).

FIG 4

Measurement of extracellular (A) and intracellular (B) trehalose concentrations by an enzymatic assay. Wild-type strain CHM43 harboring pBBR1MCS-4 (white bars), strain Wild-G harboring pBBR1MCS-4 (gray bars), the Wild ΔsteP strain harboring pBBR1MCS-4 (hatched bars), and the Wild ΔsteP strain harboring pCNM2 (promoterless steP) (stippled bars) were cultivated in a 10% (wt/vol) sorbitol medium containing 500 μg/ml ampicillin for 30 h (to the stationary phase) at 30, 37, or 38.5°C. Trehalose concentrations in the samples were measured with the Glucose (GO) assay kit after treatment with trehalase. Data are means ± standard deviations from three independent cultivation experiments.

FIG 5

Comparison of growth characteristics of the G. frateurii parental strain, CHM43, strain Wild-G, and the Wild ΔotsAB strain in a sorbose medium. CHM43 (A), Wild-G (B), and the Wild ΔotsAB strain (C) were cultivated in 50 ml of a 0.1-to-10% (wt/vol) sorbose medium at 30°C. The sorbose concentrations were 0.1% (△, solid black line), 0.5% (●, solid gray line), 1.0% (□, dashed black line), 2.0% (▲, solid gray line), 5.0% (○, solid black line), and 10% (■, dashed gray line).

FIG 6

Comparison of the growth characteristics of the G. frateurii CHM43 parental strain, the Wild-G strain, and mutated strains at various temperatures. Strains CHM43 (□, solid gray line), Wild-G (○, solid gray line), Wild ΔsteP (△, solid black line), Wild ΔotsAB (□, dashed black line), and Wild-G ΔotsAB (○, solid black line) were cultivated in 50 ml of a 10% (wt/vol) sorbitol medium at various temperatures.

FIG 7

Sugar metabolism related to sorbose utilization and trehalose production in G. frateurii CHM43. Fructose 6P, fructose 6-phosphate; fructose 1,6-bisP, fructose 1,6-bisphosphate; G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; 6PG, 6-phosphogluconate; ribulose 5P, ribulose 5-phosphate; trehalose 6P, trehalose 6-phosphate; GLDH, glycerol dehydrogenase (GLF_2776–2777); SR, sorbose reductase (GLF_2768); SDH, sorbitol dehydrogenase (GLF_2449); PGMase, phosphoglucomutase (GLF_2713); G1P UTase, G1P uridylyltransferase (GLF_1122); OtsA, α,α-trehalose phosphate synthase (GLF_0035); OtsB, trehalose 6-phosphatase (GLF_0036); SteP, sugar-transporting/exporting permease (GLF_2756); G6PDH, G6P dehydrogenase (GLF_1023); 6PGDH, 6PG dehydrogenase (GLF_1943); PDC, pyruvate decarboxylase (GLF_1518); ALDH, aldehyde dehydrogenase (GLF_0658). G. frateurii CHM43 possesses neither phosphofructokinase nor trehalase.

FIG 8

Intracellular NADPH/NADP⁺ ratios (A) and intracellular ROS levels (B) in the CHM43, Wild-G, and Wild ΔotsAB strains. The cells were cultivated in a 10% (wt/vol) sorbitol medium at 37°C until the late-log phase (12 to 13 h). NADP⁺ and NADPH contents and ROS levels were measured as described in Materials and Methods. Data are means ± standard deviations from three independent cultivation experiments.