Glutathione reductase from Lactobacillus sanfranciscensis DSM20451T: contribution to oxygen tolerance and thiol exchange reactions in wheat sourdoughs

Abstract

The effect of the glutathione reductase (GshR) activity of Lactobacillus sanfranciscensis DSM20451(T) on the thiol levels in fermented sourdoughs was determined, and the oxygen tolerance of the strain was also determined. The gshR gene coding for a putative GshR was sequenced and inactivated by single-crossover integration to yield strain L. sanfranciscensis DSM20451(T)DeltagshR. The gene disruption was verified by sequencing the truncated gshR and surrounding regions on the chromosome. The gshR activity of L. sanfranciscensis DSM20451(T)DeltagshR was strongly reduced compared to that of the wild-type strain, demonstrating that gshR indeed encodes an active GshR enzyme. The thiol levels in wheat doughs fermented with L. sanfranciscensis DSM20451 increased from 9 microM to 10.5 microM sulfhydryl/g of dough during a 24-h sourdough fermentation, but in sourdoughs fermented with L. sanfranciscensis DSM20451(T)DeltagshR and in chemically acidified doughs, the thiol levels decreased to 6.5 to 6.8 microM sulfhydryl/g of dough. Remarkably, the GshR-negative strains Lactobacillus pontis LTH2587 and Lactobacillus reuteri BR11 exerted effects on thiol levels in dough comparable to those of L. sanfranciscensis. In addition to the effect on thiol levels in sourdough, the loss of GshR activity in L. sanfranciscensis DSM20451(T)DeltagshR resulted in a loss of oxygen tolerance. The gshR mutant strain exhibited a strongly decreased aerobic growth rate on modified MRS medium compared to either the growth rate under anaerobic conditions or that of the wild-type strain, and aerobic growth was restored by the addition of cysteine. Moreover, the gshR mutant strain was more sensitive to the superoxide-generating agent paraquat.

FIG. 1.

Thiol levels of SDS extracts from chemically acidified wheat doughs and doughs fermented with L. sanfranciscensis DSM20451^T and L. sanfranciscensis DSM20451^TΔgshR. Shown are the means ± standard deviations of the results from five independent experiments. Data differing significantly (P < 0.05) from those for chemically acidified doughs are marked with asterisks.

FIG. 2.

Detection of GshR acivity in crude cellular extracts of L. sanfranciscensis DSM20451^T, L. pontis LTH2587, and L. reuteri BR11 after separation of crude cellular extracts by SDS-PAGE. The location of GshR, with a predicted relative molecular mass of 48,614 kDa, is indicated.

FIG. 3.

Thiol levels in sourdoughs fermented with L. reuteri BR11, L. reuteri BR11ΔcyuC, and L. pontis LTH2587 and chemically acidified doughs. Shown are the means ± standard deviations of the results from three independent experiments. Data differing significantly (P < 0.05) from those for chemically acidified doughs are marked with asterisks, and significant differences (P < 0.05) between doughs fermented with L. reuteri BR11 and BR11ΔcyuC are marked with an “x.” SH, sulfhydril.

FIG. 4.

Growth rates of L. sanfranciscensis DSM20451^T (triangles) and DSM20451^TΔgshR (circles) under aerobic conditions (black symbols) and anaerobic conditions (open symbols). Experiments were carried out in mMRS without cysteine (A) and in mMRS containing 0.5 g liter⁻¹ cysteine (B). The results shown are representative of three independent experiments.

FIG. 5.

Growth rates of L. sanfranciscensis DSM20451^TΔgshR (A) and L. sanfranciscensis DSM20451^T (B) in mMRS without the addition of cysteine. Zero (•), 30 (○), or 49 (▾) mM paraquat was added to the media. The results shown are representative of two independent experiments.