Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a Memory-Like Manner

ABSTRACTThe ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH,Escherichia colimust maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of theE. coliDedA family with 62% amino acid identity and partially redundant functions. We have shown thatE. coliwith ΔyqjAand ΔyghBmutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ΔyqjAmutant (but not ΔyghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parentE. coli. Overexpression ofyqjArestores growth at alkaline pH, but only when more than ∼100 mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival ofE. coliat alkaline pH.IMPORTANCEThe ability to survive under alkaline conditions is important for many species of bacteria.Escherichia colican grow at pH 5.5 to 9.5 while maintaining a constant cytoplasmic pH of about 7.6. Under alkaline conditions, bacteria rely upon proton-dependent transporters to maintain a constant cytoplasmic pH. The DedA/Tvp38 protein family is a highly conserved but poorly characterized family of membrane proteins. Here, we show that the DedA/Tvp38 protein YqjA is critical forE. colito survive at pH 8.5 to 9.5. YqjA requires sodium and potassium for this function. At low cation concentrations, osmolytes, including sucrose, can facilitate rescue ofE. coligrowth by YqjA at high pH. These data are consistent with YqjA functioning as an osmosensing cation-dependent proton transporter.

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Introduction of a Carboxyl Group in the First Transmembrane Helix of Escherichia coliF1Fo ATPase Subunit c and Cytoplasmic pH Regulation

Journal of Bacteriology ◽

10.1128/jb.183.5.1524-1530.2001 ◽

2001 ◽

Vol 183 (5) ◽

pp. 1524-1530 ◽

Cited By ~ 13

Author(s):

Phil C. Jones

Keyword(s):

Ph Regulation ◽

Transmembrane Helix ◽

Binding Pocket ◽

Side Chain ◽

Ion Binding ◽

Cytoplasmic Ph ◽

Wild Type ◽

Atpase Subunit ◽

Subunit C ◽

E Coli

ABSTRACT The multicopy subunit c of the H+-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic α helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp61 in E. coli, centered in the second transmembrane helix is essential for H+ transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+pumping for cytoplasmic pH regulation. In this study, Ile28was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+ pumping and passive H+ transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.

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Indole Pulse Signalling Regulates the Cytoplasmic pH ofE. coliin a Memory-Like Manner

10.1101/425538 ◽

2018 ◽

Author(s):

Ashraf Zarkan ◽

Santiago Caño Muñiz ◽

Jinbo Zhu ◽

Kareem Al Nahas ◽

Jehangir Cama ◽

...

Keyword(s):

Stationary Phase ◽

Ph Regulation ◽

Critical Role ◽

Bacterial Cells ◽

Cytoplasmic Ph ◽

E Coli ◽

Degree Of Protection ◽

Bacterial Cell Membrane ◽

Ph Range ◽

Phase Entry

SUMMARYBacterial cells are critically dependent upon pH regulation. Most proteins function over a limited pH range and the pH gradient across the bacterial cell membrane is central to energy production and transduction1. Here we demonstrate that indole plays a critical role in the regulation of the cytoplasmic pH ofE. coli. Indole is an aromatic molecule with diverse signalling roles that in bacteria is produced from tryptophan by the enzyme tryptophanase (TnaA)2. Two modes of indole signalling have been described: persistent and pulse signalling. The latter is illustrated by the brief but intense elevation of intracellular indole during stationary phase entry3,4. We show thatE. colicells growing under conditions where no indole is produced maintain their cytoplasmic pH at 7.8 ± 0.2. In contrast, under conditions permitting indole production, pH is maintained at 7.2 ± 0.2. Experiments where indole was added experimentally to non-producing cultures showed that pH regulation results from pulse, rather than persistent, indole signalling. Furthermore, the application of an artificial pulse of either of two non-biological proton ionophores (DNP or CCCP) caused a similar effect, suggesting that the relevant property of indole in this context is its ability to conduct protons across the cytoplasmic membrane5. Additionally, we show that the effect of the indole pulse that occurs normally during stationary phase entry in rich medium remains as a “memory” to maintain the correct cytoplasmic pH until entry into the next stationary phase. The indole-mediated reduction in cytoplasmic pH may explain why indole providesE. coliwith a degree of protection against stresses, including some bactericidal antibiotics.

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Importance of Glutathione for Growth and Survival of Escherichia coli Cells: Detoxification of Methylglyoxal and Maintenance of Intracellular K+

Journal of Bacteriology ◽

10.1128/jb.180.16.4314-4318.1998 ◽

1998 ◽

Vol 180 (16) ◽

pp. 4314-4318 ◽

Cited By ~ 64

Author(s):

G. P. Ferguson ◽

I. R. Booth

Keyword(s):

Escherichia Coli ◽

Intracellular Ph ◽

Parent Strain ◽

Cytoplasmic Ph ◽

Growth And Survival ◽

E Coli ◽

Enhanced Sensitivity ◽

Escherichia Coli Cells ◽

Highly Sensitive

ABSTRACT The role of the tripeptide glutathione in the growth and survival of Escherichia coli cells has been investigated. Glutathione-deficient mutants leak potassium and have a reduced cytoplasmic pH. These mutants are more sensitive to methylglyoxal than the parent strain, indicating that in the absence of glutathione-dependent detoxification, acidification of the cytoplasm cannot fully protect cells. However, increasing the intracellular pH of the glutathione-deficient strain resulted in enhanced sensitivity to methylglyoxal. This suggests that acidification of the cytoplasm can provide some protection to E. coli cells in the absence of glutathione. In the presence of the Kdp system, glutathione-deficient mutants are highly sensitive to methylglyoxal. This is due to the higher intracellular pH in these cells. In the absence of methylglyoxal, the presence of the Kdp system in a glutathione-deficient strain also leads to an extended lag upon dilution into fresh medium. These data highlight the importance of glutathione for the regulation of the K+ pool and survival of exposure to methylglyoxal.

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Cytoplasmic pH Response to Acid Stress in Individual Cells of Escherichia coli and Bacillus subtilis Observed by Fluorescence Ratio Imaging Microscopy

Applied and Environmental Microbiology ◽

10.1128/aem.00354-12 ◽

2012 ◽

Vol 78 (10) ◽

pp. 3706-3714 ◽

Cited By ~ 58

Author(s):

Keith A. Martinez ◽

Ryan D. Kitko ◽

J. Patrick Mershon ◽

Haley E. Adcox ◽

Kotiba A. Malek ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Subtilis ◽

Acid Stress ◽

Adherent Cells ◽

Cytoplasmic Ph ◽

Fluorescence Ratio ◽

Isolated Cells ◽

Content Type ◽

E Coli ◽

Standard Curve

ABSTRACTThe ability ofEscherichia coliandBacillus subtilisto regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. TheE. colicytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In anE. colibiofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells.B. subtiliscells were acid shifted from pH 7.5 to pH 6.0. InB. subtilis, unlike the case withE. coli, cytoplasmic pH showed no “overshoot” but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individualB. subtiliscells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

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Endotoxin Alteration of the Mucopolysaccharide Blood Vessel Coating in Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050755 ◽

1974 ◽

Vol 32 ◽

pp. 148-149

Author(s):

D. E. Philpott ◽

A. Takahashi

Keyword(s):

Skeletal Muscle ◽

Endothelial Cells ◽

Salivary Gland ◽

Carotid Artery ◽

Basement Membrane ◽

Coronary Vessels ◽

Ruthenium Red ◽

E Coli ◽

Old Rats ◽

The Brain

Two month, eight month and two year old rats were treated with 10 or 20 mg/kg of E. Coli endotoxin I. P. The eight month old rats proved most resistant to the endotoxin. During fixation the aorta, carotid artery, basil arartery of the brain, coronary vessels of the heart, inner surfaces of the heart chambers, heart and skeletal muscle, lung, liver, kidney, spleen, brain, retina, trachae, intestine, salivary gland, adrenal gland and gingiva were treated with ruthenium red or alcian blue to preserve the mucopolysaccharide (MPS) coating. Five, 8 and 24 hrs of endotoxin treatment produced increasingly marked capillary damage, disappearance of the MPS coating, edema, destruction of endothelial cells and damage to the basement membrane in the liver, kidney and lung.

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Ribosome Structural Organization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051670 ◽

1974 ◽

Vol 32 ◽

pp. 332-333

Author(s):

James A. Lake

Keyword(s):

Ribosomal Proteins ◽

Structural Organization ◽

Three Dimensional ◽

Small Subunit ◽

Structural Studies ◽

X Ray Diffraction ◽

Specific Antibodies ◽

X Ray ◽

E Coli ◽

Complete Sequences

The understanding of ribosome structure has advanced considerably in the last several years. Biochemists have characterized the constituent proteins and rRNA's of ribosomes. Complete sequences have been determined for some ribosomal proteins and specific antibodies have been prepared against all E. coli small subunit proteins. In addition, a number of naturally occuring systems of three dimensional ribosome crystals which are suitable for structural studies have been observed in eukaryotes. Although the crystals are, in general, too small for X-ray diffraction, their size is ideal for electron microscopy.

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Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060490 ◽

1967 ◽

Vol 25 ◽

pp. 96-97

Author(s):

Manfred E. Bayer

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Electron Microscope ◽

Uranyl Acetate ◽

E Coli ◽

Receptor Sites ◽

Rigid Cell Wall ◽

Host Cell Surface ◽

Bacterial Viruses ◽

Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

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Emigration of neutrophils in the central nervous system

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077517 ◽

1983 ◽

Vol 41 ◽

pp. 788-789

Author(s):

John L.Beggs ◽

John D. Waggener ◽

Wanda Miller ◽

Jane Watkins

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Osmium Tetroxide ◽

White Blood Cells ◽

Arterial Perfusion ◽

E Coli ◽

Intracisternal Injection ◽

The Central Nervous System ◽

Brain And Spinal Cord ◽

Interendothelial Junctions

Studies using mesenteric and ear chamber preparations have shown that interendothelial junctions provide the route for neutrophil emigration during inflammation. The term emigration refers to the passage of white blood cells across the endothelium from the vascular lumen. Although the precise pathway of transendo- thelial emigration in the central nervous system (CNS) has not been resolved, the presence of different physiological and morphological (tight junctions) properties of CNS endothelium may dictate alternate emigration pathways.To study neutrophil emigration in the CNS, we induced meningitis in guinea pigs by intracisternal injection of E. coli bacteria.In this model, leptomeningeal inflammation is well developed by 3 hr. After 3 1/2 hr, animals were sacrificed by arterial perfusion with 3% phosphate buffered glutaraldehyde. Tissues from brain and spinal cord were post-fixed in 1% osmium tetroxide, dehydrated in alcohols and propylene oxide, and embedded in Epon. Thin serial sections were cut with diamond knives and examined in a Philips 300 electron microscope.

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Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060696 ◽

1967 ◽

Vol 25 ◽

pp. 136-137

Author(s):

J. P. Petrali ◽

E. J. Donati ◽

L. A. Sternberger

Keyword(s):

Endoplasmic Reticulum ◽

Hela Cells ◽

Mammalian Cells ◽

Specific Antiserum ◽

Electron Microscopic ◽

E Coli ◽

Cytoplasmic Membranes ◽

Viral Progeny ◽

Ultrathin Sections ◽

Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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