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...

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels

Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived <15%. Cells grown at pH 9.0 survived 40 to 100% at pH 10, whereas cells grown at pH 7.0 survived <5%. Thus, growth in a moderate acid or base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K ؉ /H ؉ antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids.Bacillus subtilis can grow over several log units of environmental pH while maintaining cytoplasmic pH within a relatively narrow range that preserves protein and nucleic acid stability (19, 50, 55a). Environmental pH is important for the pathogenesis of related Bacillus species, such as the food-borne pathogen Bacillus cereus, which encounters acidic environments in the gastrointestinal tract and in food products where organic acids are used as preservatives (9, 64). B. cereus shows an acid tolerance response in which vegetative growth in a moderate acid induces proteins that enable survival under extreme acid conditions (64). In Bacillus anthracis, the lethal factor toxin undergoes a low-pH-driven structural change as it passes through acidic vesicles, which allows it to translocate into the cytosol (45). The Bacillus thuringiensis toxin is activated by alkaline pH upon entering the midgut of insect larvae (13), and bacterial growth is inhibited by acids (46).Cytoplasmic pH homeostasis has been studied extensively in B. subtilis, which maintains cytoplasmic pH within approximately pH 7.3 to pH 7.6 during vegetative growth over a range of environmental pH, from pH 6.0 to pH 9.0 (14, 50, 55a). At high external pH, cytoplasmic pH homeostasis involves Na ϩ /H ϩ antiporters as well as other Na ϩ transport components (14,26,50,67,68). Low pH triggers spore germi...

SUMMARY Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical Major Histocompatibility Complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homologue, HLA-DOB revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with Human Hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.

Background: In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated.