The AR properties of pathogenic E. coli
strains such as O157:H7 contribute to the low infectious dosages of these organisms by allowing small numbers to pass the stomach acidity barrier. Because of this, AR is considered to be an important virulence factor. Our laboratory has shown that commensal and O157 strains of E. coli
possess three stationary-phase-dependent AR systems that protect cells under extremely acidic conditions (13
). The present results confirm that there are only three discernible systems. The oxidative or glucose-repressed AR system is controlled by the alternative sigma factor ςS
, cAMP, and CRP. This RpoS-dependent system is activated by glutamate or glutamine during adaptation, but these amino acids have no effect if they are added during challenge. The arginine-dependent system is under positive control by the CysB protein, while the most effective AR system (GAD) is regulated partly by RpoS, but its main control by acidic pH occurs through an as yet unknown regulator.
Both decarboxylase systems are clearly induced by acidic conditions, although the gadBC
genes are partially induced simply by entry into stationary phase, as is the RpoS-dependent system. Induction of GAD by acidic pH was previously shown (29
); however, the present study illustrates that both gadA
, encoding the two isoforms of GAD, are regulated in similar fashions, with ςS
controlling stationary-phase induction but not controlling induction. Expression of gadA
is affected predominantly by acidic pH whereas expression of gadB
is affected primarily by entry into stationary phase. The data also indicate that both GAD enzymes are required for optimal AR at pH 2, since the loss of either one makes cells much more sensitive to pH 2 but has little obvious effect at pH 2.5. A surprising finding was that induction of the GAD enzymes alone is insufficient for glutamate-dependent AR, since log-phase cells grown at pH 5.5 produced large amounts of GadA and GadB but failed to survive pH 2.5. This suggests that additional genes required to survive pH stress must be induced during stationary phase. The participation of other genes was predicted because when one closely examines how the decarboxylase systems should work, the systems have the appearance of futile proton cycles. For example, glutamic acid at pH 2.5 outside the cell is protonated. After transport, it will deprotonate because of the higher intracellular pH. The subsequent consumption of protons by decarboxylation would seem to just compensate for the protons released when glutamate enters the cell, so that there would be no net removal of intracellular protons. Thus, while it is clear that inducible GAD and arginine decarboxylase systems play important roles in E. coli
AR, the way in which they actually accomplish AR is unknown.
A GAD-dependent AR system has also been reported in Lactococcus lactis
. The system in this organism is controlled by a gene called gadR
) and is induced by low pH, glutamate, and chloride ions. However, the addition of NaCl to E. coli
cultures did not affect the induction of GAD, suggesting a distinct difference in regulation.
New insights were also obtained into the control of the oxidative AR system by pH and complex media. This system ordinarily requires both RpoS- and CRP-dependent gene products and is glucose repressed. It was not clear why induction of this system required growth in complex medium at pH 5.5, since RpoS levels were high in cells grown to stationary phase in LB or minimal medium at pH 5.5 or 8 (data not shown). In addition, neither CRP nor cAMP levels appear to change dramatically under these conditions. The results presented indicate that pH control involves the synthesis of an inhibitor made at pH 8 but not pH 5.5. This inhibitor appears to interfere with the activity rather than with the synthesis of the system. Its influence is removed by washing pH 8 cells prior to acid challenge. The identity of the inhibitor is under investigation.
The complex-medium requirement for inducing the RpoS-dependent oxidative AR system is due to the presence of glutamate and glutamine in yeast extract. Glutamate and glutamine appear to activate a preformed RpoS-dependent system that is produced simply due to entry into stationary phase. Growth at pH 5.5 or 8 will partially consume glutamate and glutamine, making the oxidative system more reliant on CRP, although growth at pH 8 also appears to produce an inhibitor of this system. The fact that glutamate and glutamine can restore AR to a crp mutant or to glucose-repressed cells supports the theory that the RpoS-dependent system can stand alone in protecting cells against pH stress. CRP is not essential for AR. Because CRP dependence can be circumvented by adding excess activator (glutamate) of the RpoS-dependent system but not by removing the inhibitor, it is conceivable that a CRP-dependent pathway may contribute to intracellular glutamate synthesis. In the absence of glucose, this putative pathway might produce enough glutamate to allow the RpoS-dependent AR system to function. Once the oxidative system is induced and active, the way in which it protects cells at pH 2 in minimal media remains a mystery. One could envision intracellular glutamate serving as a counterion for K+ entering the cell due to an RpoS-dependent K+/H+ antiport system, but there is no evidence for this.
Another question raised about RpoS-dependent AR as a result of these studies is why glutamine masks the system in a gadC
mutant. It must be more than coincidence that activation of the oxidative system in cells grown at pH 8 requires the addition of glutamate or glutamine and that this activation involves GadC. It has been shown that acidic environments will cause a decrease in intracellular glutamate levels, so that one might predict that the RpoS-dependent AR system may depend upon this (16
). However, too much intracellular glutamate might be deleterious. If this is true, excess glutamate might be siphoned from the cell via the GAD system. In this model, exogenous glutamine would be transported into the cell and converted to glutamate via glutaminase. If the GadC antiporter is required to release excess glutamate but is missing, the resulting glutamate accumulation might be too high for AR purposes. Ordinarily, the GadC antiporter is not required for the RpoS-dependent AR system. It is only necessary when glutamine is added at the challenge pH (pH 2.5) or when the CRP subsystem is nonfunctional and extra glutamate is needed from the medium during adaptation. GadC may only provide an overflow for excess internal glutamate or a conduit to build glutamate concentrations if they are not high enough in the cell. It should be noted that although evidence indicating roles for medium components affecting log-phase acid habituation were published previously, those log-phase systems are clearly different from the more efficient stationary-phase systems presented here (21
One question concerning the role of AR in the virulence of pathogenic E. coli
involves how these systems are induced in nature. Recent evidence reported by Diez-Gonzales et al. (6
) supports the hypothesis that AR can be induced in E. coli
growing in the intestinal tracts of cattle. However, it was suggested that VFAs present in the intestinal contents induced a system that may be unique in protecting cells at pH 2. The results presented here indicate that lowered pH alone (no VFAs) can induce a system suitable to protect cells to pH 2 and that this system is the glutamate-dependent system. It is still possible that VFAs contribute to induction in the intestinal environment by decreasing pH but that the VFAs are not essential.
An earlier report from our laboratory has shown that, once induced, all three AR systems in commensal or O157:H7 strains will persist for at least 1 month under refrigerated conditions (14
). Thus, O157 strains with AR systems induced by growth in the gastrointestinal tracts of cattle do not need to grow in contaminated foods prior to ingestion in order to infect at low infectious doses. Based on these findings, we are currently investigating strategies designed to subvert AR in these organisms.