Chapter 96

pH-Regulated Genes and Survival at Extreme pH

JOAN L. SLONCZEWSKI and JOHN W. FOSTER

INTRODUCTION

Microbial responses to pH are of interest for many reasons (reviewed in references 19, 57, 96, and 98). Many species require homeostasis of internal pH for growth (100, 132, 153). The transmembrane pH difference (ΔpH) is a component of the proton potential, which drives processes of transport, motility, and coupling of respiration (see chapter 19). Anaerobic metabolism is regulated by pH (83).

Most enteric bacteria, including Escherichia coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium), are neutrophiles; they grow best at approximately neutral pH, but can also grow in moderate acid or base, with a positive or negative ΔpH. Thus, their mechanisms of pH homeostasis may prove more complex than those of acidophiles or alkaliphiles (62, 74, 96). Pathogenic strains of E. coli, S. typhimurium, Shigella flexneri, and Vibrio cholerae encounter extreme pH both within and outside human hosts. During pathogenesis, cells are exposed to low pH in the stomach (48) or within the phagosomes and phagolysosomes of intestinal epithelial cells or macrophages (34). Consequently, low pH induces virulence factors that contribute to pathogenesis, such as the virulence regulator ToxR in Vibrio spp. (reviewed by Miller et al. [90]) or acid shock proteins (1, 2). Acid-regulated promoters may find applications in biotechnology, as expression switches in cloning vectors (146).

FUNCTION OF pH IN CELLS

In interpreting genetic responses to pH (Table 1), a number of complicating factors must be considered, including internal pH homeostasis, the transmembrane proton potential, and the pH-dependent uptake of inducers such as weak acids. Several classes of pH response can be distinguished.

Table 1pH-responsive genes in enteric bacteriaa

pH Homeostasis

E. coli regulates its internal pH at 7.4 to 7.8 during growth over the external pH range of 5.0 to 9.0 (100, 132, 153). At external pH >7.6, an inverted ΔpH must be maintained, partly balanced by the relatively large electrical potential. During anaerobic growth, when the transmembrane electrical potential is greatly reduced, the ΔpH provides the cell’s main component of proton motive force (70).

The mechanisms of pH homeostasis in E. coli have proven remarkably elusive (for reviews, see references 19 and 98). Electron transport components pump hydrogen ions and contribute to the proton gradient, but their role in regulating internal pH is unknown. Unlike Streptococcus faecalis, in which the proton-translocating ATPase regulates internal pH (73), E. coli does not require ATPase to maintain internal pH during growth (70). Transport of K⁺ is required for pH homeostasis at external pH <7 (10, 30, 113, 150). The rapid recovery of internal pH upon a shift of external pH by one or two units indicates that some aspects of pH homeostasis must be constitutive (131, 153), although some inducible components may exist as well (38; discussed below). E. coli and S. typhimurium can survive several hours (without growth) with internal pH decreased below pH 6 (40, 133).

Classes of pH Response

The study of pH response is complicated by the possibility of several different classes of pH effect. External acid (89, 130) and external base (15, 67) are found to mediate distinct responses from different sets of genes, as discussed below. External pH can be detected directly by proteins containing a periplasmic domain, such as ToxR (92) or the lysine decarboxylase regulator CadC (149), whereas internal pH might be detected by a cytoplasmic protein. External pH could also be sensed indirectly, mediated by changes in internal pH (132).

A change in internal pH within the range 6.5 to 7.5 might directly alter DNA conformation at a regulatory site, for example by the formation of triple-helical H-DNA (61). The influence of external pH on internal pH can be increased by the presence of membrane-permeant weak acids which conduct hydrogen ions down a gradient and depress internal pH, or by weak bases which increase internal pH (71, 111, 150). Acidification of the cytosol can cause the gene to respond indirectly to external acidification, which drives uptake of the weak acid in the protonated form. This effect may be particularly significant under growth conditions in which weak acids accumulate as end products. The hydrophobic moiety of some weak acids may affect expression of various genes in a pH-independent manner (72).

Since pH is a component of the proton potential, metabolic defects that disrupt the proton motive force may result in pH sensitivity. An example is cya (adenyl cyclase) mutants, which grow poorly in alkaline media (3). The cya mutants appear to have deficient ΔΨ and show defective amino acid transport at high external pH, where ΔΨ would be the major component of the proton potential.

Finally, effects on gene regulation need to be distinguished from physiological responses such as chemotaxis, in which changes in pH or concentration of weak acids or bases are detected by a chemoreceptor without altering gene expression per se (71, 111, 131). This chapter focuses primarily on gene expression.

SCREENING METHODS

A number of methods have been devised to screen E. coli and S. typhimurium for pH-dependent mutants. It is important to design such screens carefully so as to rule out secondary effects related to pH.

It would be hard to overemphasize the importance of appropriate buffering systems for studying pH responses, and indeed genetic responses in general. A number of pH effects had been observed previously but ascribed to other causes. Examples include anaerobic induction of genes in media acidified during fermentative growth (4, 152), phosphate regulation due to the buffering effects of phosphate (86, 138), and effects due to differing concentrations of Na⁺ from N_aOH used to adjust the pH of buffers (E. Zinser and J. L. Slonczewski, unpublished data).

Both liquid and plate media should contain buffers with values of pK_a appropriate to maintain the desired pH, that is, generally no more than half a pH unit above or below (Table 2). The sulfonate "biological buffers" are particularly useful (31, 50, 112, 129), although breakdown of these compounds during autoclaving and uptake by cells cannot be ruled out. Substituted amines such as Tris and triethanolamine should be avoided, as they cross the membrane in the deprotonated form (i.e., act as weak bases) (111). Citrate is a common choice in the acid range, but it should be avoided in selection media, as it tends to select for citrate-specific gene expression (J. L. Slonczewski, unpublished data). Below pH 3, the actual concentration of hydronium ion becomes high enough to obviate the need for buffering of cell suspensions.

Table 2Buffers for study of pH regulationa

In Luria broth (LB) agar plates, at least 100 mM buffer may be needed to overcome respiratory alkalinization during growth to stationary phase, particularly on plate media (130). The pH needs to be adjusted before addition of agar, and the pH of solidified plates should be retested by using a flat-surface electrode. Below pH 5.5, extra agar must be included to compensate for acid inhibition of gelation.

Replica plating at pH values near the ends of the growth range (pH 5.5 or 8.5) has been used to identify lac fusions specifically inducible by external acid or base (130). It should be noted that pH-dependent expression may not necessarily increase indefinitely toward acid or base; a sharp peak of expression is observed in some cases. Thus, screening for pH induction should include several pH values, not just the extreme ends of the growth range. pH-dependent fusions to phoA for membrane-bound proteins have also been reported (135), but low external pH inactivates alkaline phosphatase.

The detection of loci specifically induced by internal pH change is more problematic. One approach has been to plate lacZ fusion pools on indicator media containing a membrane-permeant weak acid, such as benzoic acid or salicylic acid, which conduct protons and partly depress internal pH during growth (130). The weak acids, however, have potential side effects, including partial uncoupling of proton potential (122) and unknown effects of the hydrophobic moiety of the weak acid (72). One approach to confirm the internal pH sensitivity of weak acid-inducible lac fusions is transduction into a background defective for K⁺ transport(30) in which internal pH is generally depressed (150). In the K⁺-defective background, genes induced by internal pH perturbation should be induced during growth in low K⁺.

Acid resistance (survival in extreme acid) can be screened for by tube assays, in which independent isolates are diluted in broth at low pH (pH 2 to 3), incubated for 2 to 4 h, and then plated for growth at neutral pH (53). A microtiter well version of this assay has been devised for mass screening for loss of acid (or base) resistance (B. Hersh, E. Zinser, D. Blankenharn, and J. L. Slonczewski, submitted for publication; Zinser and Slonczewski, unpublished data). Colony overnight cultures are grown in microtiter wells without shaking, then diluted into LB (pH 2.5) for 2 h (or LB [pH 9.8] for 1 h), and transferred to LB plates by using a multiple-prong replicator. Mutants lacking acid tolerance (survival in acid, induced by moderate acid exposure) have been identified in S. typhimurium by plating on media containing the uncoupler dinitrophenol at pH <5 (40). Acid-sensitive cells are more sensitive to dinitrophenol, presumably because their pH homeostasis is impaired.

Screens for induction by anaerobiosis (4) or fermentative conditions such as proton suicide (151) have also yielded pH-regulated genes.

ACID-INDUCIBLE GENES IN E. COLI

Genes in E. coli whose expression is induced or enhanced by acid are listed in Table 1. Those studied in greater depth in S. typhimurium are discussed later in this chapter, as are virulence factors.

Amino Acid Decarboxylases

In an early study (44), Gale and Epps found relatively high activities for arginine, ornithine, lysine, and histidine decarboxylases in cultures grown in acid media, whereas amino acid deaminases were high in alkaline media. Thus, decarboxylases and deaminases neutralize the medium under acid and alkaline conditions, respectively. Decarboxylase activity enhances growth in acid media; a double mutant for arginine- and ornithine-degradative decarboxylases grows poorly below pH 6 (11). The neutralizing effect is presumably the basis of the well-known Moeller broth test for decarboxylase activity. The pH-dependent decarboxylases are those of the degradative class, as opposed to the biosynthetic decarboxylases, which synthesize polyamines and are pH independent (reviewed by Tabor and Tabor [139]).

The genetic circuit of lysine decarboxylase, cadBA at min 93.5, is understood in some detail (Fig. 1) (9, 88, 121, 130, 149). cadA (exaA) encodes the degradative lysine decarboxylase, which decarboxylates lysine to produce the alkaline end product cadaverine (121, 140). cadB encodes the putative cadaverine transporter (88), a membrane-soluble protein with homology to the Pseudomonas aeruginosa arginine-ornithine antiporter arcD (84). CadB excretes the alkaline product cadaverine, thus alkalizing the external medium (Fig. 1). CadB may also participate in uptake of lysine, although the majority of lysine uptake occurs via transporter/regulator LysP (CadR) (135, 140).

Fig. 1Regulation of lysine decarboxylase and cadaverine transport: a mechanism to reverse external acidification.

Source:

The cadBA operon is coinduced by external acid, anaerobiosis, and lysine. cadBA expression requires the presence of its substrate lysine, which derepresses the regulator LysP/CadR (135, 140). LysP also functions as a lysine transporter; lysP mutants were selected by resistance to thiosine, a toxic lysine analog. Expression of cadBA is several hundredfold higher at external pH 5 to 6 than at pH 8; anaerobic growth increases expression across the pH range severalfold (9, 130). Acid induction is mediated by the positive regulator CadC, whose locus is closely linked to cadBA but possesses a constitutive promoter (149).

CadC is a membrane-bound protein with an amino-terminal DNA-binding domain closely related to the acid-dependent ToxR in V. cholerae (27, 92). Its sequence resembles the output domains of bacterial response regulators, class RO_II (101). The putative binding site for CadC at the cadBA promoter has been characterized (88, 149). The periplasmic domain of CadC detects both external pH and lysine as positive regulators and possibly cadaverine as a negative regulator (27, 94). One of the lysyl-tRNA synthetase genes, lysU, also is induced by acid and anaerobiosis (21, 81). lysU maps downstream of cadBA, and its promoter shows some similarity to that of cadBA.

Thus, optimal cad expression requires acid, lysine, and anaerobiosis—precisely the conditions in which lysine decarboxylase would be the most useful to the cell. The anaerobic regulator of cadBA has not been defined, although the DNA-binding protein Hns (encoded by hns/osmZ/bglY) plays some role (126). Mutations in hns derepress both cad and adi (arginine decarboxylase) at neutral pH.

The degradative arginine decarboxylase gene adi (17) similarly is induced by acid and anaerobiosis (9, 136). The transporter for the Adi enzyme product agmatine has not yet been mapped. The degradative ornithine decarboxylase gene speF (6, 69) and the ornithine-putrescine antiporter gene potE (11, 68) have been sequenced. The three degradative decarboxylases Adi, Cad, and SpeF all share at least 30% amino acid sequence homology and appear to derive from a common decarboxylase transporter locus (136). By contrast, the biosynthetic decarboxylases show little sequence homology to their particular amino acid counterparts and are not pH regulated.

Fermentation Pathways

Like the degradative decarboxylases, fermentation genes show anaerobic acid regulation. The anaerobic pH dependence makes sense because pathways which generate lesser amounts of acidic end products, such as lactate dehydrogenase (LDH), encoded by ldhA (86, 144), are more advantageous at low external pH. LDH is induced severalfold by acid during anaerobic growth; thus, lactic acid, rather than acetic acid plus formic acid, builds up as an end product. During aerobic growth at low pH (pH 5), a smaller induction is observed. Acid induction of ldhA requires growth on a fermentable sugar; anaerobic growth on gluconate produces no induction. Some evidence of regulation by phosphate (86) was later explained by the effects of buffering pH during growth.

In the absence of alternate electron acceptors, the formate-hydrogenlyase (FHL) system converts the formate produced from pyruvate into CO₂ and H₂ (13, 16, 18, 103, 104, 118). This conversion, evolving molecular hydrogen and carbon dioxide gases, serves in part to decrease internal acid levels during fermentation, and like LDH, FHL is induced by acid. At least 15 genes are involved, including hyc for hydrogenase and redox carriers, fdhF for the formate dehydrogenase, and hyp for other components (see chapter 18). The rpoN/ntrA-dependent system is coordinately regulated by formate, oxygen, nitrate, and cyclic AMP (cAMP), as well as by pH.

Maximal expression of FHL is seen during anaerobic growth at pH 6.5, in the presence of added formate (118). During aerobic growth, FHL expression is completely repressed across the external pH range of 6 to 8. The presence of 30 to 50 mM formate, however, partly induces hycB at acid pH; addition of cAMP restores nearly maximal expression in acid, while at high pH, expression remains near zero. Nitrate represses hycB expression during anaerobic growth, but again, addition of formate restores expression, most dramatically in the acid range. Acid may act by potentiating the transport of formate, which is taken up by a proton symport-based mechanism (118). Similarly, acid enhances the induction of butane-2,3-diol formation by acetate in Klebsiella aerogenes (145).

Membrane-Permeant Weak Acids

Aside from their role in specific fermentation pathways, membrane-permeant weak acids can affect cellular internal pH by crossing the cell membrane, primarily as the hydrophobic protonated form. Their net uptake is driven by the transmembrane pH difference ΔpH (70, 122). At low external pH, a high concentration of deprotonated weak acid can exhaust the cell’s buffering and proton export capacity and depress internal pH, thereby diminishing the ΔpH. Weak acids sufficiently hydrophobic to cross the membrane in the deprotonated form will also partly collapse the electrical potential and therefore the total proton potential. The degree of this "uncoupler" effect (122) will depend upon the pK_a and the membrane solubility of the weak acid. The uncoupler 2,4-dinitrophenol induces a number of proteins on two-dimensional sodium dodecyl sulfate gels, including heat shock proteins (43a). Finally, some weak acids may interact with the cell by virtue of the chemical structure of the hydrophobic moiety; such effects may or may not be pH dependent (72).

A striking case of regulation by weak acids is the multiple drug resistance system marRAB, near the transcription terminator at 34 min (22, 23, 46, 56, 117). Salicylic acid and other benzoic acid derivatives induce mar-dependent resistance (24, 115) possibly mediated by repression of porins which permit transport of antibiotics (24, 116). Mutations in marR confer constitutive expression of marA, which mediates low-level resistance to tetracycline, nalidixic acid, and other antibiotics (29, 43, 46).

A locus unlinked to mar, designated inaA, was independently identified as a lac fusion inducible by benzoic acid (130). inaA shows pH-dependent induction by various benzoate derivatives, including the uncoupler dinitrophenol (150). Constitutive mutations in inaA::lac strains mapped to the regulator gene marR (117, 150). In a marRAB deletion strain, inaA loses induction by weak acids but retains induction by the superoxide generator paraquat, mediated by soxRS (55).

The growth phase-dependent σ ³⁸, encoded by rpoS (katF), regulates genes that protect E. coli from environmental stresses such as starvation, hyperosmolarity, oxidative damage, and UV irradiation (75, 128), as well as extreme acid or base (53, 133). lac fusions to rpoS and to katE (encoding HPII hydroperoxidase) are induced by acetate (123), benzoate derivatives (93), and possibly other weak-acid products of growing cells. External pH has no effect on rpoS expression (Slonczewski, unpublished data), but rpoS plays a major role in extreme acid and base resistance (see below).

ACID-INDUCIBLE GENES IN S. TYPHIMURIUM

The Salmonella homologs of ompF and ompC, as well as cadA, are acid regulated (41, 42). Other acid-regulated genes have been identified only in S. typhimurium (Table 1). S. typhimurium may have a greater number of acid-regulated genes overall than does E. coli (60). Some Salmonella genes which do have E. coli homologs, such as the gene encoding FHL, are subject to different regulatory proteins (see below).

The periplasmic protein AniG, unique to Salmonella sp., was the first primarily pH-regulated locus to be discovered in S. typhimurium (4, 5). Induction of aniG by acid is mediated by the membrane-bound negative regulator earA (37). Anaerobiosis raises overall expression severalfold, as it does for decarboxylases. Expression requires exogenous d-mannose as a coinducer, presumably acting on EarA (4, 37). A second regulator of aniG, called earB, maps upstream of earA. aniG also responds to a variety of other environmental stress conditions, including osmolarity, oxygen, and heat. DNA topology and supercoiling at the aniG promoter may participate in these responses (66). The function of aniG and its regulators remains unknown.

The FHL system encoded by hyd in S. typhimurium shows acid induction with several coinducers, as does FHL in E. coli (4). In addition, however, activation of hyd in S. typhimurium requires the iron-regulatory protein Fur (42, 58). Regulation of hyd by Fur may involve the iron-sulfur center of hydrogenases (107). But even in the presence of high Fe²⁺ and formate concentrations, maximum expression of hyd still requires external acid. Furthermore, Fur is essential for the acid tolerance response, independent of iron or hydrogenase activity (see below).

Several other acid-regulated genes of unknown function have been identified by lacZ fusions in S. typhimurium (Table 1) (42). A number of pH-dependent loci in S. typhimurium, such as the pag (PhoP-activated) loci expressed within macrophages, have been found to be virulence factors (see below).

VIRULENCE FACTORS

Virulence factors are coordinately regulated by a variety of environmental signals such as temperature, osmolarity, and anaerobiosis, as well as pH (reviewed by Miller et al. [90]). A growing number of acid-inducible virulence factors have been identified in E. coli, S. typhimurium, and other enteric pathogens (Table 1). Some appear to function in intracellular survival in lysosomes (31, 33). Survival in extreme acid may also function in pathogenesis by increasing viability in the stomach.

The ToxR virulence regulon in V. cholerae shows striking evolutionary and regulatory relationships with metabolic genes (92; reviewed by Betley et al. [14]). ToxR controls expression of the cholera toxin locus ctxAB and the major pilus gene tcpA, as well as at least 14 additional genes involved in pathogenesis (28). ToxR is a sensor/regulator which combines a carboxy-terminal input domain (sensor) with an amino-terminal DNA-binding output domain (response regulator) (for a review of signal transduction, see reference 137). The structure of this combined sensor/regulator, with its N- and C-terminal domains reversed, is remarkably similar to that of E. coli pH sensor CadA (149). Like CadA, ToxR senses external pH; in fact, acid induction is commonly used to confirm new members of the ToxR regulon (102). Coregulation occurs not by anaerobiosis but by temperature, osmolarity, and amino acids. Interestingly, a metabolic gene, aldA (encoding aldehyde dehydrogenase), has also been assigned to the ToxR regulon (102). Perhaps virulence regulons have evolved from metabolic circuits designed to cope with pH stress and other fundamental environmental challenges.

Salmonella spp. are facultative intracellular parasites capable of growth within macrophages (33, 34, 80). Survival within phagosomes requires five pag genes (12, 32, 91) regulated by PhoQ and PhoP, a two-component regulatory system (101). Low pH induces PhoP/Q regulated genes two- to threefold invitro. Manipulating the phagosomal pH of macrophages results in 70-fold induction of pag gene expression (7). Thus, the low pH of phagosomes may trigger virulence. Several genes on the resident virulence plasmid of Salmonella dublin are induced by low pH and iron, potentially contributing to the dissemination of the organism to various tissues and organs (147).

In Yersinia pestis, the virulence factor pH 6 antigen gene (psaA), regulated by psaE, is involved in pathogenesis of bubonic plague (77, 78). Expression of psaA requires pH values below 6.7 and temperatures above 36°C. Sequence analysis and electron microscopy indicate that psaA participates in expression of fimbriae during growth in host macrophages.

SURVIVAL IN EXTREME ACID

Enteric bacteria can survive exposure to extreme acid, at pH values considerably lower than the lower limit for growth, retaining colony-forming potential at neutral pH. The conditions and degrees of survival, or "acid resistance," vary among species and strains and may require various growth conditions such as low pH and stationary phase (39, 51, 53). Various terms are used; habituation (51), acid adaptation, and acid tolerance response (ATR) (39) generally refer to survival of cultures grown at moderately low pH (pH 5 to 6). Cultures grown to stationary phase that survive extreme pH even when grown originally at neutral pH are said to be acid resistant or show "stationary phase acid resistance" (53). It is most helpful to emphasize the distinction between conditions in which survival does or does not require prior exposure to moderate acid.

Acid-Inducible Acid Tolerance

S. typhimurium LT2 growing logarithmically in minimal medium will experience a logarithmic death phase when external pH is shifted below 4.0 (40). A steep increase in the rate of cell death is associated with a critical value of internal pH, below about 5.5 (40). The cell may be able to tolerate lower values of internal pH if (i) acid shock proteins have been synthesized or (ii) a strong rpoS allele is present (79, 133).

Exposure of log-phase cultures to mild acid (pH 5.5 to 6.0) induces adaptation, protecting the organism from more severe acid stress (pH 3.3) over a period of several hours. Optimum induction of ATR occurs between pH 5.5 and 6.0 (39). The critical feature of adaptation to mild acid is the synthesis of protective acid shock proteins at pH 3.3 to 4.5 (35). Functions of the mild acid shock stage may include inducible pH homeostasis (40) as well as changes in the composition of outer membrane proteins and cell surface hydrophobicity (76, 82).

A second phase of ATR, a more severe acid shock induced below pH 4.5, can be distinguished from the mild acid shock stage triggered at pH 5.5 to 6.0 (36, 40). The severe acid shock stage was demonstrated by chloramphenicol inhibition of protein synthesis immediately after pH 5.8 adaptation. Under these conditions, the cell does not become resistant to subsequent exposure to extreme acid (below pH 3.3). Thus, mild acid shock shock at pH 5.8 enables the synthesis of important survival proteins at pH 3.3 (40). But when cells are shifted instead to pH 4.3, barely above the growth limit, and incubated for 10 to 20 min prior to addition of chloramphenicol, cells produce the required proteins and survive subsequent exposure at pH 3.3 (36).

Fifty-two proteins show increased expression during mild or severe acid exposure, as observed on two-dimensional sodium dodecyl sulfate-polyacrylamide gels (36, 40). Some of these can be induced by uncouplers such as dinitrophenol or benzoic acid, which suggests that they detect internal acidification. For comparison, in E. coli, acid adaptation protects cells from internal acidification by weak acids (51). Fourteen of the severe acid shock proteins in S. typhimurium are induced transiently, their expression declining within an hour after a shift to pH 4.3. There is some cross-protection between acid shock and other global stimulons: acid shock induces cross-protection to heat, osmolarity, and H₂O₂, whereas stationary-phase starvation cross-protects from acid (79, 82, 134). In a study of global regulation of S. typhimurium gene expression within the macrophage, a number of acid shock proteins were induced (1, 2), although the total is only a fraction of those induced at low pH in supplemented glucose medium.

Acid Tolerance Genes in S. typhimurium

Table 3 lists known genetic loci associated with acid tolerance. The Mg²⁺-dependent H⁺-translocating ATPase (F₀F₁) is required for ATR induced in mild acid; addition of the F₀ inhibitor N,N '-dicyclohexylcarbodiimide makes Atp^– cells acid sensitive (40). The H⁺-translocating ATPase may participate in the inducible pH homeostasis system induced by mild acid, which enables cells adapted at pH 5.8 to maintain a higher internal pH during extreme acid exposure than unadapted cells do. But atp mutants do survive extreme acid exposure after incubation at pH 4.3, the pH value at which the severe acid shock proteins are expressed. Thus, some of the severe acid shock proteins may actually protect cells from the effects of lowered internal pH.

Table 3Genetic loci associated with acid tolerance in S. typhimurium

Several regulators affect acid tolerance. The iron regulator Fur is required for acid tolerance induced by either mild or severe acid (41), and adequate iron availability is needed (H. K. Hall and J. W. Foster, unpublished data; reviewed in reference 57). Thus, acid tolerance is one of a growing number of global systems requiring Fur. AtbR negatively regulates atrB and at least 10 other genes of unknown function (42). Transposon insertion in atbR results in constitutive acid tolerance. Another regulator, AtrE (OxrG), activates three mild-acid-induced aci genes (4, 42).

Selection based on resistance to dinitrophenol (see Screening Methods) reveals several acid tolerance defects (38). Mutations in atrD or atrF affect iron metabolism, causing the overproduction of the iron-scavenging compound enterochelin as well as the products of several other iron-repressed genes. The atrF mutation was mapped within the ent cluster, possibly involved in transport of the iron-enterochelin complex. Another atr mutation was identified as polA (DNA polymerase I), which in E. coli is induced by low pH (60). Thus, acid tolerance may include enhancement of DNA repair, which has also been implicated in acid resistance of E. coli (52, 110).

Stationary-Phase Acid Resistance

The starvation-induced stationary phase (128) invokes cross-protection to a variety of other stresses, including acid (85). Stationary phase often includes or precedes acid exposure, such as during ingestion by animal hosts. In humans, ingested bacteria typically remain in stomach acid at pH <3 for up to 2 h before colonizing the intestinal tract (48). S. typhimurium may also enter a nongrowing (or slowly growing) state within the macrophage, where phagolysosomes lower the pH (2).

Stationary-phase acid resistance has been studied most closely in E. coli and Shigella flexneri (53, 133). These species can survive at lower pH values than S. typhimurium, as low as pH 1.5, when grown to stationary phase in complex media. Components of complex media which contribute to acid resistance, such as glutamic acid and arginine, have been identified (82a). Recently stationary-phase acid resistance (as low as pH 3) has also been shown in S. typhimurium (79).

Survival of aerobic stationary-phase cultures of E. coli in extreme acid is independent of growth pH (Fig. 2) (133) and requires the growth phase-dependent regulator σ ³⁸, encoded by rpoS (75, 143). Most laboratory clinical isolates of E. coli show strong acid resistance (53) unless they contain rpoS mutations, which arise frequently in laboratory strains (51, 133). But rpoS strains grown anaerobically in moderate acid are fully resistant to extreme acid (Fig. 2). Thus, anaerobiosis provides an alternative signal for stationary-phase acid resistance.

Fig. 2Extreme acid resistance of stationary-phase E. coli as a function of growth pH. Cultures were grown aerobically (circles) or anaerobically (triangles) to stationary phase in LB with buffers of appropriate pKa (see Table 2) and then either resuspended or diluted 1,000-fold in LB (pH 2.5) for 2 h. Percent survival is based on colony counts on LB at neutral pH. (?, ?), E. coli MC4100; (,), E. coli JLS9300 (rpoS).

Source: (Modified with permission from reference 133.)

Overall, it is clear that several pathways independently enable survival in extreme acid. These include acid-inducible acid tolerance, the σ ³⁸ regulon, aspects of stationary phase independent of σ ³⁸, and anaerobic acid growth to stationary phase. The mechanisms of acid resistance are unknown, although pH homeostasis as well as protection from internal acidification are important possibilities. Ion transport via porins, possibly PhoE, may be involved (119). Two other loci required for extreme acid survival, xasA and xasB, have been identified (Hersh et al., submitted).

Several strains of virulent S. typhimurium are dramatically more acid resistant than the laboratory strain LT2, in part because LT2 contains a partly defective allele of rpoS (45, 79). These strains have inducible acid tolerance as well as higher levels of stationary-phase survival in extreme acid. Mutations in fur cause both avirulence and loss of ATR in some strains (45). Insertion of pairs of atr mutations that successfully reduce the acid tolerance response also reduce the oral virulence of the parent strains (M. Wilmes-Riesenberg, B. Bearson, J. Foster, and R. Curtiss III, unpublished data).

BASE-INDUCIBLE GENES

Some well-studied regulons, including those for the heat shock response (141), and porins such as OmpC (59), show induction by external alkaline shift (Table 1). Internal alkalinization appears to induce the SOS functions (124). It is possible that SOS induction functions to counteract the alkaline destabilization of DNA.

Sodium Proton Antiporters

The best characterized base-inducible system in E. coli is that of the sodium proton antiporter NhaA (47, 63, 67, 142). Sodium concentration in E. coli is regulated by sodium/hydronium exchange, increasingly electrogenic above external pH 7.2 (20, 105, 125, 142), via two antiporters, NhaA and NhaB (see chapter 74 and reference 99). NhaA is required for growth at high pH in the presence of high sodium. The gene nhaA (ant) was revealed by loss-of-function mutations in strains deficient for sodium-dependent transport of melibiose or serine (63, 95, 97, 127) and mutations with increased antiporter activity, antup, allowing growth at higher pH in the presence of Na⁺ (49, 87). At low external pH, Na⁺/H⁺ exchange is mediated by NhaB, an antiporter discovered in a Δ nhaA background (105, 106). The regulation of NhaB by pH is not yet fully characterized.

Expression of nhaA is low in the acid range but increases sevenfold over the external pH range of 7.0 to 9.0 (67). Regulation is mediated by NhaR (109), a positive regulator in the LysR family. NhaA detects external pH directly (47). Replacement of His-226 with arginine confers a lowered pH range of activity (pH 6.5 to 7.5) and loss of activity above pH 7.5; mutant strains become sensitive to sodium ion above external pH 7.5. The His-226 is proposed to be part of the pH sensor domain of the antiporter.

Electron Transport Chains

Whereas anaerobic systems are inducible by acid, aerobic systems may be induced by base. An intriguing case is that of the two operons cyoABCDE (encoding cytochrome o, a terminal quinol oxidase with low O₂ affinity) and cydAB (encoding cytochrome d terminal quinol oxidase) (8, 54, 114). The cyo and cyd operons respond to environmental stimuli, participating in coordinate regulation by fnr and arcA (26, 64, 65; see chapter 17). The cyo complex is predominant in oxygen-rich conditions, whereas cyd, with a higher affinity for oxygen, is favored as oxygen becomes limiting. With cytochrome o expressed, the H⁺/e^– ratio is near 2, whereas with cytochrome d expressed, the ratio is near 1 (108, 148). Thus, cytochrome o oxidase causes greater acidification than does cytochrome d.

During aerobic growth, cyo is induced severalfold as external pH increases from 6.0 to 7.5, whereas cyd expression is unaffected (25). During anaerobic growth, however, cyo induction is abolished. Moreover, in an fnr deletion background, cyo is more strongly induced by base, whereas cyd is induced by acid. Thus, expression of cyo predominates aerobically at high pH, where external acidification enhances growth, whereas cyd expression predominates anaerobically at low pH, where external acidification needs to be minimized.

SURVIVAL IN EXTREME BASE

Stationary-phase cultures of E. coli can survive several hours of exposure to extreme base with little loss of viability (133). Base resistance drops off above pH 10, just one unit higher than the alkaline limit for growth, whereas acid resistance can occur as much as three units below the acid limit for growth. Nevertheless, base resistance may have clinical significance, as enteric bacteria are exposed to alkaline secretions from the pancreas shortly after passing through the pyloric valve into the intestine.

Base resistance, like acid resistance, requires σ ³⁸ (133). It is interesting that one regulator governs both acid and base resistance, since enteric bacteria are likely to be exposed to extreme acid and extreme base in succession. For acid-grown cultures, anaerobiosis also increases base resistance by an order of magnitude. Thus, in a sense, base resistance offers another example of anaerobiosis-acid coregulation. Base resistance is also highly sensitive to Na⁺, consistent with the sodium sensitivity of alkaline growth (97, 105). Growth at moderate alkaline pH (pH 8) increases survival of base-treated cells by an order of magnitude (120, 133).

CONCLUSIONS

The literature on pH-dependent gene expression is a challenge to assess, because so much of the data have come from studies directed primarily at some other topic, such as metabolism or virulence mechanisms. Many of the genes induced by acid or base encode products which help the cell grow in moderate acid or base or to survive extreme acid or base. Often they are coinduced by some other environmental condition, such as anaerobiosis or high sodium. Often pH acts as a signal for conditions of broader significance to the cell, such as pathogenesis.

The known components of internal pH homeostasis during acid growth, such as the potassium transporters, appear to be constitutive. Several other genetic systems enhance growth in moderate acid, usually coinduced by anaerobiosis. These include the decarboxylase porter systems, which generate and excrete basic amines; LDH, which produces lactic acid in preference to formic plus acetic acid; FHL, leading to consumption of formic acid; and cytochrome oxidase d, in preference to cytochrome oxidase o, limiting transport of hydrogen ions. Most of these systems appear to limit or even reverse external acidification rather than to enhance internal pH homeostasis. Thus, E. coli appears to try to practice "environmental management."

Complementary to the acid-anaerobiosis connection, is there an alkaline-aerobiosis connection for gene regulation? In complex media, aerobic metabolism tends to consume acids, resulting in elevation of pH; for example, growth of E. coli on unbuffered LB plates raises the plate pH from 5.8 to pH 8.0 (130). This connection may be a factor in the regulation of cyo. Another important connection appears between high pH and sodium ion. Sodium proton antiporters play a role in excretion of Na⁺ and pH homeostasis at high pH (67). The survival of nongrowing cells in extreme base is also retarded by Na⁺.

Survival of cells at pH outside the growth range involves a large number of gene systems, both pH dependent and pH independent. Stationary-phase growth clearly enhances survival in both acid and base. A single regulator, rpoS, is crucial for stationary-phase induction of survival in both extreme acid and extreme base (133). It is unusual to find one component which appears to protect cells against both pH extremes. It may be advantageous for enteric bacteria to activate both protections simultaneously, because bacteria ingested and exposed to the acid stomach contents are subsequently exposed to the alkaline secretions from the pancreas.

Most if not all of the loci identified as externally induced by low pH also require coinduction (Table 1). One possible mechanism for coinduction is that a signal receptor possesses a pH-sensitive binding site for the coinducer molecule (e.g., mannose for aniG). A very different case is that of weak acids, whose uptake by the cell is in equilibrium with the transmembrane ΔpH. Acid-enhanced uptake of formic acid has also been proposed to explain the coinduction of FHL by acid and formate (118).

The coinducer for many pH-regulated genes may actually be a product of the cell’s own metabolism, such as weak acid excreted during fermentation. Such products may act, in a sense, as autoinducers. If an end product has a negative consequence for an organism at high pH but not low pH (or vice versa), a gene designed to deal with that consequence needs to be expressed only at the critical pH. For example, as pH of the medium decreases, production of formic acid has a progressively negative effect on the cell due to increased intracellular accumulation. Induction of the FHL system by low pH and formate will serve to decrease the formate concentration and help retard acidification.

Nowhere is the role of pH as an environmental sensor more evident than in pathogenesis (90). The ToxR regulon provides a particularly dramatic example of acid-inducible virulence (92) and of joint regulation of metabolic and virulence genes via a common acid-dependent regulatory protein (102). The homology between ToxR and CadC exemplifies the evolutionary relationship between virulence regulators and metabolic pH sensors (149). Eventually a better understanding of pH sensors in virulence may lead to therapeutic strategies which target these functions.