Physiological and Transcriptional Response of Lactobacillus casei ATCC 334 to Acid Stress

Lactobacillus casei is an aciduric, rod-shaped, facultatively heterofermentative lactic acid bacterium (LAB) that can be isolated from a variety of environments including raw and fermented milk and meat or plant products, as well as the oral, intestinal, and reproductive tracts of humans and animals (24). Like other LAB species, L. casei produces lactic acid as a major end product of carbohydrate fermentation during growth, and strains of L. casei are used as acid-producing starter cultures in the preparation of fermented foods, as health-enhancing probiotic cultures, and for the production of l(+)-lactic acid (18, 47, 52). In each of these applications, the industrial performance of L. casei strains is dependent, in one way or another, on their acidurance. During fermentation, L. casei transports lactic acid outside the cell as lactate ion via an electrogenic proton-lactate symporter. As the pH of the medium (pHo) decreases or the concentration of lactate increases, the concentration of protonated (undissociated) lactic acid in the medium also increases. The undissociated form of lactic acid is membrane soluble and thus can enter the cytoplasm by simple diffusion (27). Metabolically active bacteria maintain a pH gradient (ΔpH) where the intracellular pH (pHi) is more alkaline than the pHo (4, 25), so diffusion of acid into the cytoplasm results in rapid dissociation and release of protons and anions inside the cell. If the rate of proton accumulation exceeds the cytoplasmic buffering capacity and capabilities of efflux systems, the pHi begins to fall and eventually reaches a critical point where the ΔpH can no longer be maintained and cellular functions are impaired (4, 22). Furthermore, intracellular accumulation of acid anions may be of greater importance than proton release in the inhibition of cell growth (8, 40, 41). Thus, low pH and organic acid resistance are vitally important physiological attributes of L. casei and other LAB species, in relation to both lifestyle and commercial utility, and there is considerable interest in the mechanisms used by these cells to combat acid-related stress conditions (4, 11, 22, 51). Interestingly, differences in acidurance among LAB species have been linked to the activity and pH optima of the proton-translocating (H+)-ATPase, and this enzyme complex plays a major role in pHi regulation by these cells (11, 22, 48). However, many LAB species do not utilize the H+-ATPase to maintain a near-neutral pHi in response to acid stress. Instead, these species (including L. casei) maintain a relatively constant ΔpH as the pHo falls by allowing the pHi to decrease (25, 22, 40, 43, 45). This capability is thought to reduce the energy demand for proton translocation through the H+-ATPase (45) and has been shown to help prevent intracellular accumulation of organic acid anions (40). In addition to H+-ATPase activity, acidurance in a LAB is also known to involve a variety of stress protection mechanisms (11, 30, 51). In particular, many cells display an inducible acid tolerance response (ATR) that protects them from acid killing. First reported by Goodson and Rowbury (19), the ATR is usually triggered by brief exposure to a sublethal pHo (“acid adaptation”), which, like other stress responses, results in transient induction of specific proteins and physiological changes that enhance cell survival during subsequent exposure to a lethal pHo. The ATR has been detected in numerous bacteria, including L. casei and other species of LAB (20, 21, 30, 31, 50, 51). Characterization of the ATR has facilitated the identification of enzymes, proteins, and macromolecular changes (e.g., alteration of cytoplasmic membrane lipid content) that allow bacteria to combat the negative consequences of cytoplasmic acidification (11, 30, 51). Work to dissect the L. casei ATR has detected 9 to 11 acid-regulated proteins (21) and shown that cytoplasmic membrane fatty acid (CMFA) content is altered to increase the proportion of C18:1 and cyclopropane fatty acid (FA) C19:0 in response to acidification (15). However, information on the mechanisms used by L. casei to modulate ATR and CMFA content, as well as the identities and functions of acid-regulated proteins, is still lacking. In this study, we optimized conditions for ATR induction in L. casei ATCC 334 and then analyzed its effects on membrane lipid composition and global gene expression. As expected, the membrane lipid composition of acid-adapted cells showed a dramatic increase in the ratio of saturated to unsaturated membrane FAs and cyclopropane FA content. Comparisons between the transcriptome of cells grown at pH 6.0 (control) to that of acid-adapted (5 or 20 min at pH 4.5) or acid-adapted and then acid-challenged (20 min at pH 4.5 and then 10 min at pH 2.0) cells showed differential expression of numerous genes in acid-treated versus control cells. Overall, functional predictions for these genes indicated that acid adaptation induced a stringent-type response that was accompanied by other functions, particularly malolactic fermentation and intracellular accumulation of histidine, which were important for enhanced acidurance. Validation of microarray data was provided by follow-up experiments that showed that L. casei survival at pH 2.5 was improved at least 100-fold by chemical induction of the stringent response with α-methylglucoside or by the addition of 30 mM malate or 30 mM His to the acid challenge medium.