pH Stress and Response in Bacteria

Research Interests of Joan L. Slonczewski

Biology Department
Kenyon College
Gambier OH 43022
(740) 427-5397



Our current research focuses on extreme pH stress in Escherichia coli and other enteric bacteria. Much is made of the fact that "extremophiles," such as thermophilic archeans, can grow under extreme conditions of temperature or pH. But our work has shown that even enteric bacteria can survive for extended periods under conditions considered extreme, such as pH 2 or pH 10, resuming growth when favorable conditions return.

Recently our research has extended to look at the gram-positive model system, Bacillus subtilis. Surprisingly little is known about pH regulation in gram-positive bacteria. B. subtilis is a useful model for the pathogen B. anthracis, causative agent of anthrax.

We study the physiology and genetic response of E. coli and Bacillus subtilis to pH change. Current projects include:


pH Regulated Genes in Escherichia coli


The following review is adapted from an article in ASM News, March 1992.

In recent years great excitement has been generated about bacterial genes which respond to environmental changes such as heat shock, osmotic shock, and anaerobiosis. Since pathogens experience major environmental changes as they enter the human system, is not surprising that some of these genes play important roles in pathogenesis.

The pH of the growth medium, or external pH, has been shown to regulate gene expression in several enteric bacteria, including virulence genes mediated by toxR in Vibrio cholerae. Some of the acid-shock genes in Salmonella may turn out to assist this organism to grow within macrophages, possibly by preventing lysosomal acidification. Interestingly, acid also induces virulence in the plant pathogen Agrobacterium tumefaciens.

Study of pH-regulated genes is at last answering questions about pH homeostasis, an important capability of many enteric bacteria. Furthermore, we increasingly recognize that pH interacts in important ways with other environmental and metabolic pathways involving anaerobiosis, Na+ and K+ levels, DNA repair, and amino acid degradation. It may turn out that pH- regulated systems will offer clues to the evolution of virulence genes from multivalent environmental sensors.

Bacterial Response to pH Change

The ability of bacteria to respond to pH change metabolically has long been recognized. Fermentation pathways show pH dependence in many species. In the forties Helen Epps and Ernest Gale showed that both Escherichia coli and Micrococcus luteus generate various amino acid decarboxylases at low external pH and deaminases at high external pH -- in each case, acting to neutralize the growth medium. The genetic basis for some of these responses is now understood.

With respect to motility, several kinds of pH taxis have been characterized in E. coli and Salmonella typhimurium. These include repellent response to external acid and to membrane- permeable weak acids, and attractant response to base and membrane-permeable weak bases. In Robert Macnab's lab I showed that signal transduction for acid and weak-acid taxis involves methylation of the methyl-accepting chemotaxis protein MCP I. This finding suggests a molecular basis for pH detection, although it does not involve gene expresssion.

pH-Regulated Gene Expression

Until recently, few studies focused on pH regulation of gene expression. For genetic screening, my group at Kenyon developed plate and liquid media containing a range of sulfonic-acid buffers specific for the range of pH to be fixed; this is an important consideration, for even with 100 mM of an appropriate buffer growth of Escherichia coli can change the plate pH by half a unit. We used these media to screen pools of Mud lac operon fusions for pH-regulated activity. The acid-inducible locus exa(cadA) and the alkaline-inducible alx were both identified in this manner.

In addition, we showed that a number of lac fusions previously isolated by anaerobic induction were also dependent on pH, for example hyd (hydrogenase) in Salmonella. Other well- known loci in E. coli show induction at high pH: Etana Padan and Shimon Schuldiner found that heat-shock genes are induced at high external pH, and that components of the SOS response are induced by internal alkalinization.

Acidic induction of cadAB

The acid-inducible locus exa turned out to be an allele of cadA, encoding lysine decarboxylase. George Bennett's group constructed similar fusions to cadA and to adi, arginine decarboxylase. These fusions show several hundred-fold induction in acidic medium. Anaerobic growth increases induction several-fold across the pH range. This enormous output of decarboxylases protects the cell by removing acid from a protein- rich medium; the resulting alkalinization is the basis of the well-known M eller Broth test.

The anaerobic enhancement of decarboxylase makes sense given that fermentative growth can generate enough acid to retard growth. David Clark's lab has evidence that lactate dehydrogenase may also be induced anaerobically by acid, acting to decrease the output of acidic endproducts.

The acidic induction of the cad operon has been dissected by Eric Olson's group. Acid induction of cadAB requires CadC, which is independently transcribed. The CadC-regulated promoter is located upstream of cadB. Bennett has evidence that CadB may act to exchange lysine and its alkaline product cadaverine across the cell membrane.

CadC Resembles ToxR

The amino acid sequence of the regulator CadC shows striking homology to known transcriptional regulators, in particular ToxR of Vibrio cholerae. ToxR is a membrane-bound protein which binds DNA to induce transcription of cholera toxin and other virulence genes. Virginia Miller and John Mekalanos have shown that, like cadA, ToxR-dependent genes are induced strongly by acid. Moreover, ToxR is now known to induce at least one metabolic enzyme, aldehyde dehydrogenase. It is tempting to speculate that virulence genes may evolve from environmental regulators of metabolism.

Like ToxR, CadC has both a putative membrane-spanning domain and a putative DNA-binding domain; thus it is believed to act both as environmental sensor and as a transcription regulator. The working model proposed for CadC action suggests that an external sensory domain of the molecule is exposed to the periplasmic space, where external proton concentration can be detected directly or indirectly, while the amino-terminal DNA- binding domain acts to turn on transcription at the cadAB promoter. If this model is correct, CadC will provide a useful system for mutagenesis to study the molecular mechanism by which pH affects a sensory molecule.

Other Regulator Genes

While acidic induction of cadA requires the linked regulator locus cadC, the induction of cadA by lysine involves an unlinked regulator, cadR. A second unlinked regulator of the cad operon, exaR, was recently identified by Olson as a Tn10 insertion which partly deregulates cadA. My lab has preliminary evidence that exaR is required for aerobic growth of E. coli below pH 6. In a strain containing exaR::Tn10, the aerobic expression of cadA::lac is increased to the anaerobic expression levels across the pH range, while acid induction is unaffected.

Michael Leonardo in David Clark's group and Bradley Hersch in my group have evidence that exaR regulates a number of fermentation genes, including alcohol dehydrogenase, lactate dehydrogenase, and fumarate reductase. The exaR::Tn10 is closely linked to the nar operons, but Tn10 insertions in n

exaR may be the first known case of an anaerobic regulator required for aerobic growth in E. coli at low pH. It might act by repressing metabolic activities such as lactate dehydrogenase which protect the cell from acidic fermentation at low external pH, but retard cell growth under aerobic conditions.

Several other regulator genes are known to mediate pH- dependent expression. earA, identified by John Foster, represses acid-induced transcription of aniG in Salmonella. My group identified inaR, which represses protonophore-dependent induction of a weak acid-inducible locus in E. coli (see below.) Camille LaCroix in my lab identified a regulator of alkaline-sensitive expression, alxC, which represses alx.

Internal pH-regulated Genes

An interesting consideration is that, in theory, bacterial regulators could detect pH in two different compartments: the cytoplasm (internal pH), and the periplasm, whose pH presumably would equal that of the external medium. When I first began to look at pH-regulated genes, I predicted that expression of particular genes would be found to respond specifically to internal or external pH. Since then, we and others have found this to be the case: pH-dependent genes respond specifically to internal or external pH change. The internal alkaline induction of the SOS response is a particularly interesting case, since stability of DNA could be affected by internal alkalinization.

In our lab Tania Gonzalez found a number of proteins in two- dimensional SDS gels which were induced by benzoate, a membrane- permeable weak acid which depresses internal pH. She also identified the locus ina which shows pH-dependent expression only in the presence of membrane-permeable weak acids or uncouplers. Under certain conditions the expression of ina correlates with internal acidification, although other effects of the weak acids cannot be ruled out.

Inducible Acid Tolerance

Do pH-regulated genes play a role in internal pH homeostasis? For a long while pH homeostasis in E. coli was generally believed to be constitutive, in contrast to the pH- dependent ATPase in Streptococcus faecalis, for example.

Recently however two different kinds of inducible pH homeostasis have been demonstrated: acid tolerance, and the sodium-proton antiporter NhaA. Both cases are dauntingly complex, involving several different stimuli and gene loci.

Foster's group discovered in Salmonella typhimurium that growth in moderately acid medium (pH 5.5-6.0) induces genes whose products enable cells to retain viability under more extreme acid conditions (below pH 4) where growth is not possible. Close to 100% of acid-tolerant (or acid-adapted) cells can recover from extreme-acid exposure and grow at neutral pH. The inducible survival mechanism is called acid tolerance response. The retention of viability by acid-tolerant cells correlates with improved pH homeostasis at low external pH; thus, acid tolerance may represent inducible pH homeostasis. Unlike constitutive pH homeostasis, acid tolerance requires a functional proton- translocating ATPase as well as the regulator fur, although its iron uptake function is not involved.

Acid tolerance may be significant for pathogenesis because Salmonella faces extreme acidification during uptake by phagocytic cells, which the bacteria then colonize. Some of the acid tolerance functions may protect the cell from effects of acidification. Alternatively, Brett Finlay finds that Salmonella may actually prevent or reverse acidification of its environment within the phagocyte; perhaps some of the acid tolerance genes function in this regard.

Extreme acid tolerance is not confined to Salmonella. Pam Small finds that clinical isolates of E. coli and Shigella exhibit acid tolerance below pH 2, either constitutively or under control of the stationary-phase regulator KatF.


Na+/H+ Exchange at High External pH

At high external pH, Padan and Schuldiner have found in E. coli an inducible system for internal pH homeostasis, the sodium- proton antiporter NhaA. nhaA is induced at high external pH in the presence of high sodium. In the presence of 10 mM NaCl, the induction of nhaA increases with pH over precisely the range in which external pH exceeds E. coli internal pH (maintained at 7.4-7.9). The NhaA antiporter acts to acidify the cytoplasm through electrogenic proton/sodium exchange.

A strain deleted for nhaA fails to grow at high pH in the presence of 100 mM NaCl. In this deletion background, Padan and Schuldiner identified a second antiporter locus nhaB. This antiporter is constitutive and electroneutral. An nhaA nhaB deletion strain is extremely sensitive to high pH. This suggests that E. coli has both a constitutive mechanism for internal acidification (over moderate pH values) and an inducible electrogenic mechanism (at high pH, in the presence of Na+). Interestingly, this confirms the observation of Anna Castle and Robert Macnab that sodium transport is electroneutral at acid-to- neutral pH and becomes electrogenic above pH 7.5.

How does the cell detect external alkalinization? Schuldiner and Padan have recently identified a positive regulator NhaR which shows homology to OxyR and LysR. This protein may be part of the alkaline signal transduction system.

Sodium-proton antiporters function in pH homeostasis in a number of species, especially alkalophiles. In a particularly intriguing experiment, Padan transferred an antiporter gene from the alkalophile Bacillus alkalophilus into the nhaA nhaB deletion strain, where it restored growth at high pH. Terry Krulwich believes that B. alkalophilus may have as many as three sodium- proton antiporters, and that the number of antiporters may relate to the alkalophilicity of a species.
K+ transport at Low External pH

The work of Ian Booth and others has long suggested that potassium transport may play a role in pH homeostasis in E. coli under acid conditions, possibly a K+/H+ antiporter analogous to the Na+/H+ antiporter at high pH. The study is complicated however by the existence of multiple potassium transport systems, both inducible and constitutive. It may well be that both constitutive and inducible pH homeostasis mechanisms exist in the acid range, as they do in the alkaline range.

Donald Dosch and Wolfgang Epstein constructed a strain in which the three major K+ uptake systems are deleted. In my lab we find that this strain requires 100 mM K+ for good growth at pH 6, but only 10 mM K+ at pH 8.5. Furthermore, the internal pH of the K+ uptake-deficient strain is depressed in low K+ medium. At present we are using this strain background to test the effects of internal acidification on gene expression, while avoiding the side effects of membrane-permeable weak acids. We also hope to identify additional loci involved in the relationship between K+ and pH, possibly as mutations which compensate for the K+ defect.

Clearly, pH interacts with a bewildering number of other environmental effects on gene expression. The antiporter nhaA requires both sodium and high pH for induction. For the cad operon, a specific protein CadC mediates pH regulation, while different regulators mediate induction by anaerobiosis or by lysine. ToxR mediates response to osmolarity, temperature, and amino acids, as well as pH; it induces a growing list of virulence factors and metabolic enzymes. Fur regulates ferric siderophores, Shiga-like toxin, and acid tolerance -- all of which are known or potential virulence factors.

In addition, the mechanisms of pH homeostasis now appear considerably more complex than was once thought, involving both inducible and constitutive systems, which studies of pH-regulated gene expression are at last helping to identify. Some references (see also Publications):

Aliabadi, Z., Y. K. Park, J. L. Slonczewski, and J. W. Foster. 1988. Novel regulatory loci controlling oxygen- and pH- regulated gene expression in Salmonella typhimurium. J. Bacteriol. 170:842-851.

Auger, E. A., K. E. Redding, T. Plumb, L. C. Childs, S.-Y. Meng, and G. N. Bennett. 1989. Construction of lac fusions to the inducible arginine and lysine decarboxylase genes of Escherichia coli K12. Mol. Microbiol. 3:609-620.

Bingham, R. J., K. S. Hall, and J. L. Slonczewski. 1990. Alkaline induction of a novel gene locus, alx, in Escherichia coli. J. Bacteriol. 172:2184-2186.

Foster, J. W., and H. K. Hall. 1991. Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium. J. Bacteriol. 173:5130-5135.

Karpel, R. T. Alon, G. Glaser, S. Schuldiner, and E. Padan. In press. Expression of a sodium proton antiporter (NhaA) in Escherichia coli is induced by Na+ and Li+ ions. J. Biol. Chem.

Parsot, C., and J. J. Mekalanos. 1991. Expression of the Vibrio cholerae gene encoding aldehyde dehydrogenase is under control of ToxR, the cholera toxin transcriptional activator. J. Bacteriol. 173:2842-2851.

Slonczewski, J. L., T. N. Gonzalez, F. M. Bartholomew, and N. J. Holt. 1987. Mu d-directed lacZ fusions regulated by low pH in Escherichia coli. J. Bacteriol. 169:3001-3006.

Watson, N., D. S. Dunyak, E. L. Rosey, J. L. Slonczewski, and E. R. Olson. In press. The Escherichia coli cad operon: Identification of elements involved in its transcriptional regulation by external pH. J. Bacteriol.