YD1, a derivative of El Tor strain N16961 (the V. cholerae
genome strain), was constructed by standard means (3
) to study whether VCA0526 played a role in V. cholerae
acid resistance. In YD1, an in-frame deletion removes all but 10 codons of vca0526
. This mutation, whose presence was confirmed by Southern analysis, does not impair growth in vitro in Luria broth (LB).
Several experimental methods for measuring acid resistance in bacteria have been reported (6
). The extreme acid resistance test protocol is thought to mimic conditions present in the highly acidic environment of the human stomach. With this method, stationary-phase organisms are challenged in low-pH minimal media containing arginine or glutamate. While the viability of wild-type E. coli
is not significantly reduced after a pH 2.5 challenge (9
), we found that V. cholerae
does not survive this acid challenge when the media contained either glutamate or arginine. Wild-type El Tor N16961 cells were not detectable after 1 h of incubation in pH 2.5 media. In fact, even in pH 5 minimal media supplemented with arginine or glutamate, approximately 1% of wild-type cells survived for 30 min (Fig. ). These observations are consistent with clinical studies that suggest that the infectious dose of V. cholerae
is much larger than that of pathogenic E. coli
). We found that V. cholerae
can survive a pH 5 acid challenge when minimal media are supplemented with 1% Casamino Acids. However, N16961 and YD1 did not differ in their sensitivity to challenge in pH 5 minimal media supplemented with 1% Casamino Acids (Fig. ). This finding suggests that the putative V. cholerae
ClC channel encoded by vca0526
may not function in stationary-phase cells in the same manner as the E. coli
FIG. 1. Survival of stationary-phase N16961 and YD1 in pH 5.0 minimal medium (40 mM KCl, 80 mM KH2PO4, 1.7 mM sodium citrate, 20 mM glucose, using H3PO4 to adjust to pH 5.0) alone (MM), or minimal medium supplemented with 1 mM arginine, 1 mM glutamate, or 1% (more ...)
We developed an assay using exponential-phase cells to further investigate YD1 acid sensitivity. In this assay system, cells were grown in LB to an optical density at 600 nm of ~0.5 and then were placed in lower-pH LB challenge media. Cell viability, determined by plating, was evaluated after 60 min of incubation in the low-pH challenge media. There was minimal reduction (approximately twofold) in N16961 viability after incubation at pH 4.4 when the challenge medium pH was adjusted with HCl (Fig. ). In contrast, there was an ~330-fold reduction in the viability of YD1 at this time. Acid sensitivity of YD1 was attributable to the deletion of vca0526, since the expression of this gene from a plasmid (pCLC) in YD1 restored the acid resistance of this strain (Fig. ).
FIG. 2. Survival of mid-log-phase N16961, YD1, and YD1(pClC) in LB adjusted to pH 4.4 with either HCl, H3PO4, or HNO3, or to pH 4.9 with an organic acid cocktail (OA) after 60 min of incubation. Cells were grown in LB to an optical density at 600 nm of around (more ...)
The acid sensitivity of YD1 was influenced by the acid used to adjust the pH of the challenge media. For example, in contrast to the marked sensitivity of YD1 to HCl-adjusted media, pH 4.4, the viability of this strain was hardly reduced after incubation in media adjusted to this pH with either H3
(Fig. ). YD1 was somewhat more susceptible to acid than N16961 when the pH of the challenge media was adjusted to 4.2 with either H3
(data not shown); however the significance of this observation is difficult to interpret given the fact that the viability of N16961 is greatly reduced (<0.1% survival) in media adjusted to pH 4.2 with either of these acids. N16961 was more sensitive to acid conditions in the presence of organic acids (a cocktail of butyric acid, acetic acid, and propionic acid at concentrations that approximate those in the human intestine; see the Fig. legend) (2
). For example, less than 0.1% of these cells survived challenge in pH 4.7 LB supplemented with organic acids. Approximately 50% of these cells survived after 60 min in media adjusted to pH 4.9 with organic acids. There was no difference in the susceptibilities of YD1 and N16961 to this acid challenge (Fig. ).
Our findings reveal that the V. cholerae ClC channel encoded by vca0526, like the E. coli channels EriC and MriT, plays some role in acid resistance. However, while the mechanistic basis for the acid sensitivity of the E. coli mriT eriC deletion mutant is known, for YD1 it is not. Surprisingly, in our assay, the anion component of the acid used to adjust the pH of the challenge media influenced the acid sensitivity of YD1. Similar observations have not been reported for the E. coli ClC deletion mutant. Since we used exponentially growing cells and rich media to study acid sensitivity of the V. cholerae ClC channel deletion mutant, we cannot directly compare our findings with those obtained from studies of the acid sensitivity of the E. coli eriC deletion mutant. However, if the V. cholerae ClC channel acts as an electrical shunt, as was proposed for the E. coli ClC channel, it is difficult to explain the anion specificity that we observed. The amount of Cl− added to LB challenge media to adjust the pH to 4.4 does not significantly alter the Cl− concentration of the media.