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Previously we showed that glutathione (GSH) can protect Lactococcus lactis against oxidative stress (Y. Li et al., Appl. Environ. Microbiol. 69:5739-5745, 2003). In the present study, we show that the GSH imported by L. lactis subsp. cremoris SK11 or produced by engineered L. lactis subsp. cremoris NZ9000 can protect both strains against a long-term mild acid challenge (pH 4.0) and a short-term severe acid challenge (pH 2.5). This shows for the first time that GSH can protect a gram-positive bacterium against acid stress. During acid challenge, strain SK11 containing imported GSH and strain NZ9000 containing self-produced GSH exhibited significantly higher intracellular pHs than the control. Furthermore, strain SK11 containing imported GSH had a significantly higher activity of glyceraldehyde-3-phosphate dehydrogenase than the control. These results suggest that the acid stress resistance of starter culture can be improved by selecting L. lactis strains capable of producing or importing GSH.Lactococcus lactis is a neutrophilic bacterium whose optimal growth occurs within an extracellular pH range of 6.3 to 6.9 (9). Growth of L. lactis is typified by the generation of acidic end products (mainly lactic acid), which results in medium acidification and subsequent acid stress. Acid stress has detrimental effects on the cellular physiology of L. lactis, including damage to the cell membrane and inhibition of enzymes and transport systems (14). Low pH is therefore considered a growth-limiting factor for L. lactis grown in milk or weakly buffered media. Consequently, the capability of L. lactis to survive, grow, and metabolize actively at a low pH will greatly influence its industrial performance as a starter.A number of acid stress resistance mechanisms in L. lactis have been identified and characterized. The primary mechanism of L. lactis for surviving low pH is to control the intracellular pH (pH i ) by membrane-bound F o F 1 ATPase, which translocates protons to the environment at the expense of ATP (9, 21). Other mechanisms include generation of alkaline substances by amino acid catabolism (e.g., deamination) (6, 24). L. lactis also develops a complex adaptive response to acid stress which is dependent on the synthesis of proteins such as heat shock proteins and proteinases (9). Although the native acid stress resistance mechanisms in L. lactis were extensively studied, improving the acid stress resistance of L. lactis by introducing a xenobiotic compound whose metabolism is not directly related to acid stress resistance has not been investigated.Glutathione (␥-Glu-Cys-Gly) (GSH) is the major nonprotein thiol compound in living cells. The major physiological role of GSH in living organisms is to maintain a redox balance (4). However, recent studies showed that GSH is also involved in bacterial acid stress resistance (23), osmotic-stress resistance (28), chlorine compound defense (5), and toxic electrophile detoxification (10). Most of these new physiological roles of GSH were found in gram-negative bacteria, such ...

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