Several members of the family Enterobacteriaceae were examined for differences in extreme acid survival strategies. A surprising degree of variety was found between three related genera. The minimum growth pH of Salmonella typhimurium was shown to be significantly lower (pH 4.0) than that of either Escherichia coli (pH 4.4) or Shigella flexneri (pH 4.8), yet E. coli and S. flexneri both survive exposure to lower pH levels (2 to 2.5) than S. typhimurium (pH 3.0) in complex medium. S. typhimurium and E. coli but not S. flexneri expressed low-pHinducible log-phase and stationary-phase acid tolerance response (ATR) systems that function in minimal or complex medium to protect cells to pH 3.0. All of the organisms also expressed a pH-independent general stress resistance system that contributed to acid survival during stationary phase. E. coli and S. flexneri possessed several acid survival systems (termed acid resistance [AR]) that were not demonstrable in S. typhimurium. These additional AR systems protected cells to pH 2.5 and below but required supplementation of minimal medium for either induction or function. One acid-inducible AR system required oxidative growth in complex medium for expression but successfully protected cells to pH 2.5 in unsupplemented minimal medium, while two other AR systems important for fermentatively grown cells required the addition of either glutamate or arginine during pH 2.5 acid challenge. The arginine AR system was only observed in E. coli and required stationary-phase induction in acidified complex medium. The product of the adi locus, arginine decarboxylase, was responsible for arginine-based acid survival.

Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an ␣ level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F 1 F o and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

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Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.In order to colonize the human gastrointestinal tract, the enteric bacterium Escherichia coli must be able to grow between pH 4.5 and pH 9 (7). Over this wide pH range, E. coli preserves enzyme activity, as well as protein and nucleic acid stability, by maintaining the cytoplasmic pH in the range from pH 7.2 to 7.8 (26,27,32). E. coli responds rapidly to intracellular pH change; after acidification of the external environment, the intracellular pH of E. coli begins to recover within 1 min, and full recovery occurs within 5 min (28). The efficiency with which E. coli maintains pH homeostasis has been attributed to a combination of constitutive and regulated mechanisms, but the essential requirements remain poorly understood (7,9,14,18,28). Some components of pH homeostasis act in the presence of chloramphenicol, whereas others require ongoing protein synthesis (10).Previously, cytoplasmic pH has been measured using 31 P nuclear magnetic resonance (NMR) of titratable phosphate and methylphosphonate (28) and through transmembrane equilibration of radiolabeled permeant acids (32). Both methods have limitations. Radiolabeled permeant acids have low sensitivity, and they measure only the transmembrane pH difference; they do not measure cytoplasmic pH independent of external pH.31 P NMR requires highly concentrated cell suspensions, typically suspensions with optical densities at 600 nm (OD 600 ) of 20 to 200.The advent of highly pH-sensitive fluorescent proteins...

Escherichia coli K-12 strains and Shigellaflexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor Ci38. The clone also conferred acid resistance on S.flexneri rpoS::TnlO but not on Salmonella typhimurium. E. coli and S.flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10-to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::TnJO showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed <0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::TnlO strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::TnlO and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.

Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetaterepressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in which E. coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.

The intracellular pH ofEscherichia coli cells, respiring onendogenous energy sources, was monitored-continuously by 31P NMR over an extracellular pH range-between 5.3 and 9. pH homeostasis was found to be good over the entire range, with the data conforming to the simple relationship intracellular pH = 7.6 + O.1(external pH -7.6) so that the extreme values observed for intracellular pH were 7.4 and 7.8 at external pH 5.5 and 9, respectively. As well as inorganic phosphate, we employed the pH-sensitive NMR probe methylphosphonate, which was taken up by glycerol-grown cells and was nontoxic; its pK. of 7.65 made it an ideal probe for measurement of cytoplasmic pH and alkaline external pH.Given the pH sensitivity of-most enzymatic reactions, the ability to regulate cytoplasmic pH is likely to be a valuable attribute for any cell, and especially for a free-living unicellular organism such as a bacterium that encounters substantial variation in the pH of its environment.A number of studies have been made of the dependence of the internal pH (pHin) ofbacteria upon the value ofthe external pH (pHext) (1)(2)(3)(4)(5)(6)(7)(8) reported may stem partly from the difficulty of keeping cells adequately energized at alkaline pH, but there are also questions regarding the assumption that membrane-permeant basic probes such as methylamine equilibrate passively (9). 31P NMR has been employed successfully for measurement of pHit in bacteria (5, 10), but the chemical shift of inorganic phosphate (Pd), the species usually monitored, is pH insensitive above pH 8, because the pK, is 6.8 at physiological ionic strengths. Any failure ofregulation at alkaline pH would therefore not be readily detected by phosphate chemical shift experiments. For our studies both of molecular mechanisms of pH regulation (11, 12) and of pH taxis of bacteria (13, 14) we felt it was important to establish the degree ofhomeostasis ofa population of cells as pHe was progressively adjusted over a wide range.We have used 3P NMR to monitor both pHint and pHext rapidly and continuously, employing for the alkaline region a synthetic probe, methylphosphonate [MeP; CH3PO(OH)2], which is taken up by the cells, has a pH-sensitive chemical shift that is well resolved from the shifts ofphosphorus metabolites, and has a pK, of 7.6 that enables measurement of both pHmnt and pHext;for acid pHeit we employed the resonance of P,. Our results indicate that homeostasis ofpH in respiring Escherichia coli cells is extremely good (7.6 ± 0.2) over a pH,, range of about 5.5 to 9.MATERIALS AND METHODS Measurement of 31P NMR Spectra. 31P NMR spectra were obtained at 145.8 MHz on a Bruker WH360 wide-bore spectrometer. Free induction decays were accumulated in 2-min blocks, using 35Q pulses at a repetition rate of 10 s-'. The low magnetic field drift (<0.01 ppm hr-') obviated the use ofa field frequency lock. In experiments with cells, the absolute chemical shift scale was chosen such that extracellular MeP and Pi gave the same value for pHeit at the beginning of the experimen...

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.