Improved acid stress survival of Lactococcus lactis expressing the histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524

Abstract

Degradative amino acid decarboxylation pathways in bacteria generate secondary metabolic energy and provide resistance against acid stress. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 was functionally expressed in the heterologous host Lactococcus lactis NZ9000, and the benefits of the newly acquired pathway for the host were analyzed. During growth in M17 medium in the pH range of 5-6.5, a small positive effect was observed on the biomass yield in batch culture, whereas no growth rate enhancement was evident. In contrast, a strong benefit for the engineered L. lactis strain was observed in acid stress survival. In the presence of histidine, the pathway enabled cells to survive at pH values as low as 3 for at least 2 h, conditions under which the host cells were rapidly dying. The flux through the histidine decarboxylation pathway in cells grown at physiological pH was under strict control of the electrochemical proton gradient (pmf) across the membrane. Ionophores that dissipated the membrane potential (ΔΨ) and/or the pH gradient (ΔpH) strongly increased the flux, whereas the presence of glucose almost completely inhibited the flux. Control of the pmf over the flux was exerted by both ΔΨ and ΔpH and was distributed over the transporter HdcP and the decarboxylase HdcA. The control allowed for a synergistic effect between the histidine decarboxylation and glycolytic pathways in acid stress survival. In a narrow pH range around 2.5 the synergism resulted in a 10-fold higher survival rate.

FIGURE 1.

The histidine decarboxylation pathway is controlled by the pmf. Histamine production by resting cells expressing the HDC pathway. Cells were incubated at an A₆₀₀ of 1 in 100 mm KPi (A) and 200 mm KPi (B) (pH 5.0) containing 5 mm histidine without further additions (A and B, ●) in the presence of 25 μm CCCP (A, ○; B, ■), 10 mm glucose (A, ▾), and in the presence of 1 μm valinomycin (B, ○), 1 μm nigericin (B, ▾), 1 μm valinomycin, and 1 μm nigericin (B, △).

FIGURE 2.

The histidine decarboxylation pathway generates pmf. A, internal pH. 10 μl of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded cells of L. lactis NZ9000 expressing the HDC pathway were resuspended in 3 ml of 200 mm KPi (pH 5.0) buffer and incubated for 5 min at 30 °C. Histidine, glucose, and nigericin were added at final concentrations of 5 mm, 5 mm, and 1 μm, respectively, at the indicated times. B, membrane potential. 10 μl of cells expressing hdcAPB resuspended at ∼20 mg protein/ml were added to 3 ml of 200 mm KPi (pH 5.0). Histidine and valinomycin were added at final concentrations of 5 mm and 1 μm, respectively.

FIGURE 3.

Histidine/histamine exchange is controlled by the pmf. Cells of L. lactis NZ9000 ΔlysQ expressing HdcP were loaded with 5 mm histamine and resuspended to an A₆₀₀ of 2 in 200 mm KPi (pH 6.0) buffer. Fifteen seconds before addition of 1.5 μm ¹⁴C-histidine, either no further additions were made (●), or 5 μm valinomycin (△), 5 μm nigericin (▾), 5 μm valinomycin, and 5 μm nigericin (○), 25 μm CCCP (■), and 10 mm glucose (□) was added to the cells.

FIGURE 4.

pH profile of purified HdcA (A) and of cells of L. lactis NZ9000 expressing the HDC pathway (B). A, 0.5 μg of purified HdcA, was added to 500 μl of 200 mm ammonium acetate, adjusted to the indicated pH values, and containing 50 mm L-histidine at 37 °C. Histidine decarboxylation was stopped at 5, 10, 15, 30, 45, and 60 min by incubating 50-μl samples for 5 min at 95 °C. Histamine concentrations were measured as described under “Experimental Procedures.” HdcA of S. thermophilus was purified from E. coli as described before (28). B, cells of L. lactis expressing hdcAPB were resuspended to an A₆₀₀ of 1 in 100 mm KPi buffer or KPi/citrate buffer, adjusted to the indicated pHs containing 5 mm histidine with no further additions (left panel) in presence of 25 μm CCCP (center panel) and in the presence of 0.2% Triton X-100 (right panel). Error bars represent mean ± S.D. of three independent experiments.

FIGURE 5.

Growth of L. lactis NZ9000 pNZ-PhdcAPB in GM17 medium adjusted to pH 6.5 (♢ and ♦), 6.0 (□ and ■), 5.5 (△ and ▴), or 5.0 (○ and ●) in the presence (♢, □, △, and ○) or absence (♦, ■, ▴, and ●) of 10 mm histidine.

FIGURE 6.

Acid stress survival by histidine decarboxylation in L. lactis. A, L. lactis NZ9000 harboring pNZ-PhdcAPB (HDC+) or the empty vector pNZ8048 (HDC-) was grown in GM17 to stationary phase and washed and resuspended in GM17 adjusted to pH 3 with or without 25 mm histidine (His+ or His-, respectively). After 2-h incubation, 10 μl of droplets of serial dilutions were put on GM17 agar plates to determine colony-forming units (CFU)/ml. Dilution factors are indicated at the top. B, survival of L. lactis NZ9000 expressing the active (pNZ-hdcAPB, ■) or inactive pathway (pNZ-hdcA-S82S-PB, □) resuspended in GM17 containing 10 mm histidine adjusted to pH 3. At the indicated times, CFU/ml were determined as described above.

FIGURE 7.

Acid stress survival in the presence of histidine and glucose. A, cells of L. lactis NZ9000 harboring pNZ-hdcAPB were incubated in M17 containing 10 mm histidine and adjusted to pH 2.7 (□ and ■), pH 2.5 (△ and ▴), or pH 2.4 (○ or ●) in presence (■, ▴, and ●) and absence (□, △, and ○) of 10 mm glucose. B, survival of L. lactis NZ9000 pNZ-hdcA-S82A-PB in M17 containing 10 mm histidine and adjusted to pH 3.5 (□ and ■), pH 3.25 (△ and ▴), and pH 3.0 (○ or ●) in the presence (■, ▴, and ●) or absence (□, △, and ○) of 10 mm glucose. Survival of the cells was determined at the indicated times as described in the legend for Fig. 4.

FIGURE 8.

Interaction between histidine and glucose metabolism. A, histamine production rates by resting cells of L. lactis NZ9000 pNZ-hdcAPB in 100 mm KPi/citrate buffer at the indicated pH and containing 5 mm histidine in the presence (black bars) or absence (gray bars) of 10 mm glucose. B, lactate production rates by resting cells of L. lactis NZ9000 harboring pNZ-hdcAPB (black bars) and pNZ-hdcA-S82A-PB (gray bars) in 100 mm KPi/citrate buffer at the indicated pHs and containing 10 mm glucose and 5 mm histidine. Histamine and lactate production rates were determined from the initial linear part of the time curves. Inset, time curves of lactate production at pH 3.5 (○ or ●) and pH 4.0 (△ and ▴) by cells harboring pNZ-hdcAPB (● and ▴) or pNZ-hdcA-S82A-PB (● and ▴). Error bars represent mean ± S.D. of three different experiments. C, histamine production (○ and ●) and glucose consumption (▴) by L. lactis NZ9000 pNZ-hdcAPB in 100 mm KPi (pH 5.0), 10 mm histidine, in the presence (○) or absence (●) of 2 mm glucose.