Ion Transport in Prokaryotes

Ion Transport in Prokaryotes provides an advance treatise on ion transport and prokaryotic organisms. This book is divided into three main topics—cation transport systems, anion transport systems, and plasmid-encoded transport systems. This compilation specifically discusses the proton transport and proton-motive force in prokaryotic cells, potassium transport in bacteria, and bioenergetic functions of sodium ions. The calcium transport in prokaryotes, phosphate transport in prokaryotes, and transport of organic acids in prokaryotes are also elaborated. This text likewise covers the chloride, nitrate, and sulfate transport in bacteria and bacterial magnesium, manganese, and zinc transport. This publication is recommended for biologists, specialists, and students interested in the bacterial ion transport system.

• Language: English
• Publisher: Academic Press
• Release date: Jun 28, 2014
• ISBN: 9781483272122

Book preview

Ion Transport in Prokaryotes

Edited by

Barry P. Rosen

Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan

Simon Silver

Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois

Table of Contents

Cover image

Title page

Copyright

Preface

Part I: Cation Transport Systems

Chapter 1: Proton Transport and Proton-Motive Force in Prokaryotic Cells

Publisher Summary

I INTRODUCTION

II GENERATION OF Δp IN PROKARYOTIC CELLS

III PROTON-TRANSLOCATING ATPASE (H+-ATPASE)

IV PROTON-TRANSLOCATING ELECTRON TRANSFER CHAINS, INORGANIC PYROPHOSPHATASE, AND BACTERIORHODOPSIN

V PROTON–SOLUTE COTRANSPORT SYSTEMS

VI UTILIZATION OF Δp IN PROKARYOTES OTHER THAN FOR ATP SYNTHESIS AND ACTIVE TRANSPORT

VII SUMMARY

VIII RECENT DEVELOPMENTS

ACKNOWLEDGMENTS

Chapter 2: Potassium Transport in Bacteria

Publisher Summary

I INTRODUCTION

II STREPTOCOCCUS FAECALIS

III ESCHERICHIA COLI

IV CYANOBACTERIA

V LESS FREQUENTLY STUDIED SPECIES

VI SUMMARY AND CONCLUSIONS

Chapter 3: Bacterial Sodium Transport: Bioenergetic Functions of Sodium Ions

Publisher Summary

I INTRODUCTION

II Na+ AS A SECONDARY COUPLING ION

III Na+ AS THE PRIMARY COUPLING ION

IV CONCLUSIONS

Chapter 4: Bacterial Magnesium, Manganese, and Zinc Transport

Publisher Summary

I INTRODUCTION

II MAGNESIUM TRANSPORT

III MANGANESE TRANSPORT

IV ZINC TRANSPORT

V NICKEL TRANSPORT

VI CADMIUM TRANSPORT

VII EPILOGUE

ACKNOWLEDGMENTS

Chapter 5: Calcium Transport in Prokaryotes

Publisher Summary

I INTRODUCTION

II SECONDARY TRANSPORT SYSTEMS

III PRIMARY CALCIUM TRANSPORT SYSTEMS

IV CONCLUSIONS

Part II: Anion Transport Systems

Chapter 6: Phosphate Transport in Prokaryotes

Publisher Summary

I INTRODUCTION

II PHOSPHATE TRANSPORT SYSTEMS OF ESCHERICHIA COLI

III PHOSPHATE TRANSPORT IN OTHER BACTERIA

IV GENETIC STUDIES AND THE IDENTIFICATION OF COMPONENTS OF THE PHOSPHATE TRANSPORT SYSTEMS

V TRANSPORT OF SOME PHOSPHATE ESTERS

VI THE Pst SYSTEM AS AN INTEGRAL PART OF THE pho REGULON OF ESCHERICHIA COLI

ACKNOWLEDGMENTS

Chapter 7: Chloride, Nitrate, and Sulfate Transport in Bacteria

Publisher Summary

I INTRODUCTION

II SULFATE TRANSPORT IN SALMONELLA TYPHIMURIUM

III SULFATE TRANSPORT IN DESULFOVIBRIO VULGARIS

IV SULFATE TRANSPORT IN PARACOCCUS DENITRIFICANS

V NITRATE TRANSPORT IN DENITRIFYING BACTERIA

VI CHLORIDE TRANSPORT IN HALOBACTERIA

Chapter 8: Transport of Organic Acids in Prokaryotes

Publisher Summary

I INTRODUCTION

II MONOCARBOXYLATE TRANSPORT

III DICARBOXYLATE TRANSPORT

IV TRICARBOXYLATE TRANSPORT

Part III: Plasmid-Encoded Transport Systems

Chapter 9: Plasmid-Encoded Ion Transport Systems

Publisher Summary

I ARSENATE TRANSPORT

II CADMIUM EXTRUSION

III CITRATE TRANSPORT

IV IRON TRANSPORT

V MERCURY(II) UPTAKE SYSTEM

VI SUMMARY

Index

Copyright

Cover: E. coli ML 308–225 membranes prepared from lysozyme-EDTA treated spheroplasts. Micrograph courtesy of Dr. V. Marchesi, National Cancer Institute, National Institutes of Health.

Copyright © 1987 by Academic Press, Inc.

all rights reserved.

no part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

ACADEMIC PRESS, INC.

1250 Sixth Avenue, San Diego, California 92101

United Kingdom Edition published by

ACADEMIC PRESS INC. (LONDON) LTD.

24–28 Oval Road, London NW1 7DX

Library of Congress Cataloging in Publication Data

Ion transport in prokaryotes.

Includes bibliographies and index.

1. Microbial metabolism. 2. Ion channels.

3. Biological transport, Active. I. Rosen, Barry P.

II. Silver, S. (Simon)

QR88.I66 1987 576′.1133 87-12652

ISBN 0-12-596935-X (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA

87 88 89 90 9 8 7 6 5 4 3 2 1

Preface

Although the topic of bacterial ion transport may seem focused and narrow, the need to limit ourselves to ion transport and prokaryotic organisms is the best measure of the rapid progress in this area in the past decade. The modern era of the field of transport began with the classic studies of lactose transport by Monod and co-workers (Rickenberg et al., 1956; Cohen and Monod, 1957). These studies introduced the word and concept of permease and initiated the use of genetics as a tool in transport research. Since then progress in bacterial transport has been revolutionary. Thirty years ago the very existence of highly specific transport systems was questioned; the evidence that these were composed of membrane proteins consisted of indirect arguments based on the analysis of mutants. Direct measurement of transport activity awaited the development of isotopic tracers. The use of radioactive substrates to characterize the kinetics of individual systems rapidly became standard.

Recognition of energy coupling mechanisms came later. By 1978, the book Bacterial Transport (edited by B. P. Rosen) reported progress in the understanding of a maturing science. The methodologies were diverse and generally appropriate. Several qualitatively different mechanisms of energy coupling were identified. Many transport systems, in particular those for carbohydrates and amino acids, were resolved at a rather sophisticated level. In that book, compiled almost a decade ago, the topic of ion transport was restricted to a single chapter, primarily because of a limited accumulation of knowledge. At that time, the chemiosmotic coupling hypothesis developed by Mitchell (1961, 1966, 1974) had been generally accepted in principle by the scientific community, as evidenced by his being awarded the 1978 Nobel Prize in Chemistry (Mitchell, 1979).

Progress in the past decade in this area has been phenomenal. It is no longer possible to consider all fundamental studies of transport, or even all areas of bacterial transport, in a single volume of reasonable size. We have elected to focus on ion transport systems in prokaryotes because these systems have had less exposure than those for organic compounds, especially sugars and amino acids. In addition, because cells cannot synthesize inorganic ions as they can organic compounds, ion transport is in a sense more basic than transport of organics. It seems reasonable to speculate that ion transport systems must have been early adaptations of the original living cellular organisms. All present-day organisms use a few basic types of transport systems with common themes of energy coupling; more unique and bizarre mechanisms have been later adaptations to specialized environments. In general, uphill transport of organic nutrients is coupled to downhill movement of ions with the ion gradients being established by primary ion pumps.

Our understanding of these chemiosmotically coupled systems—both primary proton pumps and secondary proton- and sodium-coupled cotransport systems—has been based on sound experimental evidence. New types of systems and coupling mechanisms have been identified. For example, two new types of sodium pumps have been reported, one directly coupled to the respiratory chain and the other to decarboxylase enzymes (see Skulachev, this volume). Several novel anion transport systems have been discovered (Part II). A new retinal protein, halorhodopsin, has been shown to catalyze light-driven chloride pumping in Halobacterium halobium (see Lanyi, this volume). An ATP-driven arsenical pump is responsible for plasmid-mediated arsenical resistance (Part III). A newly reported phosphate-sugar phosphate antiport system may prove to be a major sugar phosphate uptake mechanism (see Rosenberg, Part II).

As was true for studies of microbial physiology and biochemistry in general, the use of genetics, with the isolation and characterization of mutant organisms defective in a particular system, was absolutely required for the initial understanding of bacterial transport pathways. These genetic strategies have now been supplemented by the tools of recombinant DNA technology. The genetic determinants for many systems, including a few of those for ion transport, have been isolated. The nucleotide sequence of the genes has allowed deduction of the primary and secondary structure of transport proteins. This information has provided important new insights on mechanism and evolution. The degree of precision in hypotheses and experimental approaches has been radically advanced. This progress is most noticeable in the work described in the chapters on proton transport (Futai and Tsuchiya, Part I) and potassium transport (Walderhaug, et al; Part I). Using genetic and molecular biological tools we have gained more knowledge on the structure and function of the H+-translocating F0F1 ATPase in ten years of study of the Escherichia coli enzyme than in the previous forty years of biochemical analyses of the mitochondrial and chloroplast enzymes. The sequence homology between the Kdp K+ transport protein of Escherichia coli (Hesse et al., 1984) and the eukaryotic ion-motive Ca²+-ATPase of sarcoplasmic reticulum and Na+, K+-ATPase of the eukaryotic plasma membrane demonstrates an evolutionary relationship still discernible after two billion years. Evolutionary relationships are also apparent in relation to the unique plasmid-mediated transport systems for toxic ions (see Mobley and Summers, this treatise). Cells must pump out these toxic materials to maintain resistance; recombinant DNA technology has facilitated much understanding in this area. Genetic cloning has also allowed the overproduction of individual proteins, in this case the components of transport systems. By overexpressing the genes encoding a number of transport proteins, scientists have isolated and purified transport components reconstituting the cloned gene products in proteoliposomes. The ability to isolate single transport proteins in a functional form and to insert them into liposomes in the absence of other proteins has been one of the more gratifying accomplishments of the past decade. Some of the initial successes in this area are reported in this treatise. Considerably more progress is anticipated in the next decade.

What is left to be done? Quite a bit, in fact. We have no comprehension of the molecular mechanism of catalysis for even a single transport system. The progress in the past decade on the isolation and purification of transport proteins, on the one hand, and on primary amino acid sequences through cloning, on the other, has enabled us to tentatively put forth models of secondary structure. We must now determine the three-dimensional structure of these proteins and the manner in which their conformations change during the transport reaction. New approaches only now coming into use imply that a successor monograph ten years from now will of necessity be narrower in scope and more selective in topic in order to, by occasional example, describe in greater depth the general processes of transport.

The ability to map the topographical arrangement of amino acid residues of membrane proteins using antibodies directed against synthetic peptides will quickly tell us which parts of transport proteins are hidden or embedded and which are accessible to large external molecules on either side of the membrane. The use of small, highly specific site-labeling reagents will map functional domains within the proteins. The most powerful method for structure-function determinations is site-directed mutagenesis, which now allows the substitution of any single aminoacyl residue within a transport protein with any other of choice, as well as deletion or insertion of small segments of a protein. More massive reshuffling of transmembrane helical structures and functional domains is technically simple. Chimeric proteins are now being used to elucidate the manner in which proteins get into and through membranes. Potentially one could mix substrate binding domains from two different proteins to create novel transport systems. The impediment is our own lack of awareness of which shuffling steps are likely to illuminate the basic processes. As we gain the ability to isolate massive amounts of membrane proteins, we hope to see the concomitant application of X-ray diffraction to determine their tertiary structures. The eventual definition of mechanism demands familiarity with the three-dimensional structure of the proteins involved. This will be difficult with membrane proteins since methods for crystallizing them are scant.

The first progress in this direction has come from studies of bacteriorhodopsin (Henderson and Unwin, 1975; Engelman et al., 1980), a bacterial transport protein with many advantageous features for such studies. It is not only the simplest photosynthetic system, consisting of a single polypeptide, but it also forms natural two-dimensional crystals in the purple membrane patches of the halobacterial cytoplasmic membrane. Our comprehension of this model protein, however, is still limited today by the absence of adequate three-dimensional crystals. The first striking progress in obtaining three-dimensional crystals of a membrane protein has again been with a prokaryotic ion pump, the photosynthetic reaction center complex of Rhodopseudomonas viridis (Deisenhofer et al., 1984; Knapp et al., 1985). The trick for getting crystals may lie in having a small assemblage of polypeptides, including some that are intrinsic to the membrane, presumably containing the ion channels, and others which are peripheral to the membrane. The solubility properties of the extrinsic polypeptides may provide for the stacking interactions necessary for forming crystals. The next few years promise revolutionary advances.

Thus the time seems right for an advanced treatise on Ion Transport in Prokaryotes. The general area of research has progressed rapidly and has reached a new level of sophistication. Ion transport systems themselves are the subjects of numerous research projects so that a carefully focused approach is appropriate. We are also perched on the edge of a mighty explosion of knowledge, one which we anticipate will take five to ten years to reach a new plateau. A careful exposition of our present state of knowledge will be useful for future progress.

We are grateful to the authors of the individual chapters for their efforts and for the novel approaches and insight of their discourses.

BARRY P. ROSEN

SIMON SILVER

REFERENCES

Cohen, G. N., Monod, J. Bacterial permeases. Bacteriol. Rev. 1957; 21:169–194.

Deisenhofer, J., Epp, O., Miki, K., Huber, R., Michel, H. X-ray structure analysis of a membrane protein complex. Electron density map of 3 Å resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. J. Mol. Biol.. 1984; 180:385–398.

Engelman, D. M., Henderson, R., McLachlin, A. D., Wallace, B. A. Path of the polypeptide in bacteriorhodopsin. Proc. Natl. Acad. Sci. USA. 1980; 77:2023–2027.

Henderson, R., Unwin, P. N.T., Three-dimensional model of purple membrane obtained by electron microscopy. Nature 1975; 257:28–32

Hesse, J. E., Wieczorek, L., Altendorf, K., Reicin, A. S., Dorus, E., Epstein, W. Sequence homology between two membrane transport ATPases, the Kdp-ATPase of Escherichia coli and the Ca²+-ATPase of sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA. 1984; 81:4746–4750.

Knapp, E. W., Fischer, S. F., Zinth, W., Sander, M., Kaiser, W., Deisenhofer, J., Michel, H. Analysis of optical spectra from single crystals of Rhodopseudomonas viridis reaction centers. Proc. Natl. Acad. Sci. USA. 1985; 82:8463–8467.

Mitchell, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature. 1961; 191:141–148.

Mitchell, P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol. Rev. 1966; 41:445–502.

Mitchell, P. A chemiosmotic molecular mechanism for proton-translocating adenosine triphosphatases. FEBS Lett.. 1974; 43:189–194.

Mitchell, P. Keilin’s respiratory chain concept and its chemiosmotic consequences. Science. 1979; 206:1148–1158.

Rosen, B.P.Bacterial Transport.. New York: Marcell Dekker, Inc., 1978. [684].

Rickenberg, H. V., Cohen, G. N., Buttin, J., Monod, J. La galactoside-permease d’Escherichia coli. Ann. Inst. Pasteur. 1956; 91:829–857.

Part I

Cation Transport Systems

Outline

Chapter 1: Proton Transport and Proton-Motive Force in Prokaryotic Cells

Chapter 2: Potassium Transport in Bacteria

Chapter 3: Bacterial Sodium Transport: Bioenergetic Functions of Sodium Ions

Chapter 4: Bacterial Magnesium, Manganese, and Zinc Transport

Chapter 5: Calcium Transport in Prokaryotes

Proton Transport and Proton-Motive Force in Prokaryotic Cells

MASAMITSU FUTAI,     Department of Organic Chemistry and Biochemistry, The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan

TOMOFUSA TSUCHIYA,     Department of Microbiology, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700, Japan

Publisher Summary

This chapter discusses proton transport and proton-motive force (Δp) in prokaryotic cells. The vectorial movement of protons plays a central role in bioenergetics. Generation of a substantial Δp has been observed in both growing and nongrowing bacteria, suggesting that a chemi-osmotic mechanism functions in a wide variety of prokaryotes. Bacteria have at least four primary H+ -transport systems capable of forming a Δp. An obvious advantage of using bacteria is that genetic techniques can be applied easily. Mutants of each subunit have been isolated and the gene cluster for the enzyme has been cloned from E. coli. The primary structures of all the subunits of E. coli H+ -ATPase were determined from the DNA sequence and the essential residues in catalysis and proton transport were identified. The chapter discusses bacteriorhodopsin, which is the most extensively studied proton pump. The amino acid sequence and the higher ordered structure of this light-dependent proton pump are known presently.

I Introduction

II Generation of Δp in Prokaryotic Cells

A Measurement of Δp

B Values of Δψ, ΔpH, and Δp in Escherichia coli

C Values of Δp in Growing Bacteria

D Values of Δp in Prokaryotes Growing at Extreme pH Values

III Proton-Translocating ATPase (H+-ATPase)

A Synthesis and Hydrolysis of ATP in Bacteria

B Genetics of F0F1

C Structure of F0F1

D Mechanisms of Synthesis and Hydrolysis of ATP by F0F1

IV Proton-Translocating Electron Transfer Chains, Inorganic Pyrophosphatase, and Bacteriorhodopsin

A Electron Transfer Chains

B Proton-Translocating Inorganic Pyrophosphatase

C Bacteriorhodopsin

V Proton–Solute Cotransport Systems

A Evidence for H+–Solute Cotransport

B Stoichiometry

C Cotransport Carriers

VI Utilization of Δp in Prokaryotes Other Than for ATP Synthesis and Active Transport

A Utilization of Δp in Cellular Processes

B Participation of Δp in Genetic Processes

VII Summary

VIII Recent Developments

References

I INTRODUCTION

It is now well accepted that the same thermodynamic principle is involved in energy transduction in prokaryotic bacterial cells and eukaryotic cells. The electrochemical proton gradient plays a central role in both. A well-known example of this is in the synthesis of ATP by oxidative phosphorylation by mitochondrial inner membranes and bacterial plasma membranes. The overall mechanisms taking place in these energy-transducing membranes are now well explained by the chemiosmotic theory of Peter Mitchell (1961, 1979). The respiratory electron transfer chains localized in the membranes transport protons from the inside to the outside of the organelles or cells, resulting in the formation of an electrochemical gradient of protons. The proton-translocating ATPase complex (H+-ATPase) in these membranes phosphorylates ADP to form ATP coupled with transport of protons from out to in. The H+-ATPase complex can also function in the reverse direction, forming an electrochemical gradient by transporting protons to the outside of mitochondria or cells coupled with ATP hydrolysis. Essentially the same energy-coupling mechanism is operating during photophosphorylation in chloroplasts of plants and chromatophores of photosynthetic bacteria.

can be expressed as

where F is the Faraday constant, R is the gas constant, and T is the absolute temperature. Mitchell rearranged the above equation as

where Z is about 59 mV per pH unit at 37°, and Δp (also abbreviated as pmf) is the proton-motive force. Recently incorrect equations for Δp have been printed in some textbooks and literature, as already pointed out (Lowe and Jones, 1984). The Δp is formed by primary H+ transport systems such as the respiratory chain, photoelectron transfer chain, and H+-ATPase (Fig. 1). Thus, primary H+-transport systems convert light or chemical energy to a form directly usable in energy requiring reactions. The primary H+-transport system can be regarded as a biological equivalent of fuel cells, as discussed previously (Mitchell, 1966; Rosen and Kashket, 1978). The Δp can be an energy source for a wide variety of reactions other than ATP synthesis in bacteria (Fig. 1). Secondary active transport systems take up amino acids, sugars, or ions by coupling with transport of H+. H+ and solute can be transported in the same direction (symport) or in opposite directions (antiport). Charged solutes also move across the cell membrane in response to the membrane potential. The electrochemical gradient of protons can be converted to an electrochemical gradient of other ions such as Na+ or K+ through antiport carriers. An electrochemical gradient of Na+ can be an energy source for Na+–solute symport systems. The Δp also participates directly or indirectly in higher ordered bacterial reactions, such as mechanical movement, chemotaxis, and transfer of genetic materials between cells. Therefore proton transport or cellular circulation of protons is important for bacterial growth and survival.

FIG. 1 Proton-motive force in prokaryotes. The generation and utilization of the proton-motive force in different bacteria are summarized. It must be noted that not all the systems are found in any one species of bacteria. For example, bacteriorhodopsin and photoelectron transfer were found in H. halobium and photosynthetic bacteria, respectively. Anaerobes such as S. lactis and S. faecalis do not have electron transfer chains.

The field of bioenergetics has been transformed by introduction of the chemiosmotic theory and by studies on bacteria of diverse origins. is a common currency of energy transduction in both eukaryotic and prokaryotic cells. Bacteria found in extreme environments offer special advantages for analyzing H+ transport. The thermophilic bacterium PS3 has a stable H+-ATPase and respiratory chain which have been analyzed extensively under drastic conditions that would be impossible with the corresponding components of mesophilic organisms (Kagawa, 1978). A series of important experiments were carried out with this bacteria including isolation of H+-ATPase and synthesis of ATP by liposomes into which this enzyme had been incorporated. The extremely halophilic prokaryote Halobacterium halobium and its coupling with ATP synthesis.

Useful bacterial systems are mutants defective in energy coupling and simple membrane systems that form closed vesicles after disruption of cells (Kaback, 1974). Unfortunately molecular biologists have had only a limited interest in energy-transducing membranes until recently, except with respect to the lactose transport system (Kaback, 1983). However, great progress has been made in studies on the bioenergetics of Escherichia coli and other bacteria during the last 10 years. Genes for bioenergetically important proteins have been cloned and their DNA sequences have been determined. Mutationally altered residues in proton transport proteins such as H+-ATPase have been identified in a number of strains. Therefore, it became possible to analyze the mechanism of H+ translocation at a molecular level. These studies on proton transport in prokaryotes are important not only for understanding bacterial physiology but as model systems for understanding the corresponding systems in eukaryotes. Recent studies indicated that eukaryotic cells have other proton transport systems besides those in mitochondria or chloroplasts. It is noteworthy that H+-ATPase has been found in eukaryotic plasma membranes (Perlin et al., 1984; Scarborough, 1982), endoplasmic reticulum (Ress-Jones and Al-Awgati, 1984), coated vesicles (Forgac and Cantley, 1984; Stone et al., 1983), lysomes (Moriyama et al., 1984; Schneider, 1981), and chromaffin granules (Apps et al., 1982; Beers et al., 1982; Roisin et alor an electrochemical gradient of other ions have been reported in mammalian systems. Therefore, studies on the H+ transport mechanism in prokaryotes are very important for understanding a variety of cellular functions in eukaryotic cells.

This article tries to cover what is known about the generation and utilization of an electrochemical gradient of protons in bacteria. As discussed above, bacteria have a wide variety of proton transport systems. Thus it is impossible and beyond our ability to cover all the recent publications on this subject comprehensively in a limited space. We discuss the formation and roles of Δp in various bacteria and summarize the structure and mechanism of proton transport systems. We try to stress the advantage of combined biochemical and genetic approaches and make suggestions about what should be studied further. Emphasis is given to H+-ATPase and H+–solute cotransport systems, because they have been studied in detail. In many places we have cited review articles on specific topics rather than the original articles, as we thought that this would make our article more informative.

II GENERATION OF Δp IN PROKARYOTIC CELLS

A Measurement of Δp

H+ transport through a primary H+-transport system generates Δp. Precise determinations of the membrane potential (Δψ) and pH gradient (ΔpH) are essential for calculation of Δp. An accurate value of Δp is important for experimental evaluation of the chemiosmotic theory and for understanding the mechanism of proton transport. Proton translocation was first measured directly by Newmann and Jagendorf (1964) in chloroplasts and by Mitchell and Moyle (1965) in mitochondria. Since then proton translocation has been demonstrated in many systems with pH electrodes. However, the direct method is not convenient, as discussed previously (Fillingame, 1980; Rottenberg, 1979). Indirect methods of determining ΔpH and Δψ were reviewed by Rottenberg (1979) and also discussed critically in recent reviews (Ferguson and Sorgato, 1982; Fillingame, 1980). A common method for determining of Δψ is to measure the distribution of lipophilic ions such as triphenylmethylphosphonium or SCN− between external and internal aqueous compartments separated by a membrane. The ΔpH is estimated from the distribution of a weak base, such as methylamine or quinacrine, or a weak acid such as acetate, assuming that only the uncharged form can penetrate through the membrane. This determination also depends on the assumptions that only a trace amount of solute is bound to membranes and that the solute accumulates in the inner aqueous compartment. These assumptions must be carefully evaluated in individual systems, because different membrane systems differ in permeability and in affinity for the probe used. As discussed by Ferguson and Sorgato (1982), lipophilic phosphonium binds significantly to membranes, although a correction can be made for this binding using lysed mitochondria or heat-treated bacteria. Rottenberg (1979) also pointed out the importance of determination of the internal aqueous volume. Usually organelles or membrane vesicles have been separated from the external medium by centrifugation or filtration for assay of the amount of probe transported, but this separation should be carefully controlled. Flow dialysis has been employed to overcome this difficulty. Ramos et al. (1976) showed that flow dialysis gave the most reproducible ΔpH values over a wide range of concentrations of E. coli membranes: essentially the same values were obtained by flow dialysis with four weak acids, including acetate, whereas filtration or centrifugation gave much lower values and these differed depending on the probe used.

Fluorescent amines, such as quinacrine and 9-aminoacridine, are frequently used for measuring ΔpH. This method is quicker and more convenient than other assays and requires only a small amount of membranes. In this assay it is assumed that the membrane is permeable to only the unprotonated form and that only a trace amount of the probe binds to the membrane nonspecifically. Furthermore quenching of fluorescence is assumed to be due solely to internalization of the probe. Values for ΔpH in chloroplasts calculated from the quenching of 9-aminoacridine were similar to those obtained from the distribution of radioactive methylamine or ammonia (Schuldiner et al., 1972). However, anomalous values were obtained for bacterial systems by Michael and Konings (1978). Quinacrine is also a convenient probe for ΔpH measurement, although it cannot be used for quantitative determinations (Schuldiner et al., 1972).

Shift of the band of carotenoid is frequently used for determining the Δψ in chromatophore membranes of photosynthetic bacteria. As discussed previously, this method gives a higher value: carotenoid shift and butylphosphonium uptake in Rhodopseudomonas capsulata gave values for Δψ of 290 and 160 mV, respectively (Clark and Jackson, 1981). Ferguson and Sorgato (1982) concluded that Δψ is probably overestimated by measurement of the carotenoid shift, which may represent a combination of Δψ and charge separation in the membrane, although this has not been proved directly.

From a survey of the literature, Ferguson and Sorgato (1982) concluded that reasonable values of Δψ and ZΔpH for respiring nonphosphorylating mitochondria are −170 and −30 mV, respectively, given a net Δp of −200 mV, although they admitted that further studies are required to establish the exact values. Values for bacteria are discussed below.

B Values of Δψ, ΔpH, and Δ p in Escherichia coli

As discussed above, careful evaluation of controls is required for accurate determination of each component of Δp. Independent procedures were applied to E. coli cells and values of Δψ and ΔpH measured using uptake of permeant agents, ³¹P NMR, and microelectrodes were essentially similar (Table I). Felle et al. (1980) made giant cells of E. coli cultured in the presence of 6-amidinopenicillanic acid. They then inserted an electrode into these giant cells and obtained essentially the same values for Δψ at three different pH values as those determined by measuring uptake of tetraphenylphosphonium. Actual differences in the values obtained by the two methods were within 6 mV. Measurement of uptake of ⁸⁶Rb in the presence of valinomycin gave similar values to those obtained with tetraphenylphosphonium. Measurement of ³¹P NMR gave similar but somehow lower values of ΔpH for aerobic E. coli: ³¹P NMR measurement gave a ZΔpH of −90 mV at pH 6.0 (Navon et al., 1977), whereas other methods gave a value of about −100 mV (Zilberstein et al., 1979; Booth et al., 1979). The lower values obtained by measurement of ³¹P NMR may be due to lack of aeration during the measurements, as already pointed out (Fillingame, 1980). As clearly indicated in Table I, values of Δψ and ΔpH change with the pH of the medium. The value of ΔpH decreases substantially with increase of pH: the value at pH 6.0 is about −100 mV, whereas that at pH 8.0 is +10 to −30 mV. The value of Δψ increases with increase in the pH of the medium, although it is not able to compensate for decrease in ΔpH. Therefore values of Δp decrease substantially with increase in the pH of the medium. EDTA treatment of E. coli is necessary to obtain accurate values of Δψ using lipophilic cations, because the outer membranes of gram-negative bacteria are not freely permeable to these cations and EDTA treatment is necessary to remove this permeability barrier. In this connection it is interesting that Hirota et al. (1981a) recently isolated a lipopolysaccharide-defective mutant that is freely permeable to lipophilic tetraphenylphosphonium and found that the mutant cells without EDTA treatment gave essentially the same value of Δψ as wild-type cells treated with EDTA.

TABLE I

VALUES OF Δψ AND ΔpH IN Escherichia colia

aValues for mitochondria (Rottenberg, 1979) and E. coli obtained by other groups (Fillingame, 1980) can be found in previous reviews.

bKey to comments: A, Values are for resting cells treated with EDTA; B, Values during aerobic growth of uncA defective in H+-ATPase; C, value of wild-type cells after EDTA treatment; D, value of a lipopolysaccharide-deficient mutant without EDTA treatment; E, values during aerobic growth (exponential phase); F, values during anaerobic growth (exponential). Values during growths (E,F) were from cells which were treated with EDTA in the growth medium.

C Values of Δ p in Growing Bacteria

Physiologically, the value of Δp is the net result of extrusion of protons through primary H+ transport systems and their reentry into the cells through a number of systems including secondary active transport systems. Kashket and co-workers compared the generation of Δp in different bacteria in growing and resting conditions. In anaerobes such as Streptococcus lactis, H+ efflux is catalyzed by H+-ATPase. The growing bacteria had a fairly constant value of Δp of −143 to −133 mV in media of between pH 5 and 7 (Kashket, 1981b) and resting cells had a similar value (Kashket et al., 1980) (Table II). In both growing and resting cells the pH and K+ concentration of the medium are the main factors that determine the relative contributions of ΔpH and Δψ to the value of Δp. Kashket (1981a) also showed that growing cells in batch cultures with different carbon sources had constant values of Δp and Δψ, regardless of up to a 10-fold differences in their doubling times, suggesting that the systems for generation and utilization of Δp increased at the same rate in these bacteria. Ott et al. (1983) also studied the relation between Δp and the growth rate of a similar lactic Streptococcus species, S. cremoris. They examined a lactose-limited chemostat culture and in contrast to the results of Kashket, they found that the Δp was increased in cells with a low growth rate cultured at pH 7.0, although the rate was almost constant at pH 5. to 7. The difference between the results of two groups may be due to the growth conditions employed.

TABLE II

VALUES OF Δψ, ΔpH, AND Δp FOR VARIOUS BACTERIA

aKey to comments: Values are for resting cells; B, values from anaerobically growing cells; C, values from cells growing aerobically; D, approximate values of cells growing anaerobically; E, approximate values (estimated from the published figure) of cells growing aerobically; F, approximate values of cells growing anaerobically.

In facultative anaerobes such as Staphylococcus aureus, Δp is generated by H+-ATPase during anaerobic growth and by the respiratory chain during aerobic growth. In aerobic growing cultures of S. aureus the Δψ value was constant between pH 6 and 7 (medium pH), whereas ΔpH decreased with increase of pH and the overall Δp decreased with increase of pH (Kashket, 1981a) (Table II). The Δp of anaerobic cells was less dependent on pH; it was −192 mV at pH 6 and −164 mV at pH 7.2. The aerobic growth rate of this bacteria was not influenced by Δp, although it started to decrease in the late logarithmic phase.

In E. coli cells growing aerobically at about pH 7, Δp remained constant at about 210 mV. Similar values were obtained regardless of the carbon source or mutations affecting the H+-ATPase. The Δ pH decreased with increase in pH of the medium, whereas the Δψ remained constant at −161 mV between pH 6.3 and 7.2 (Table I). Therefore Δp was higher in cells growing at pH 6.3 than in those growing at pH 7.2 (Kashket, 1981b). In contrast to E. coli cells growing aerobically, those growing anaerobically did not take up tetraphenylphosphonium+ or SCN−, suggesting that they have no Δψ (Table I). Their Δp was much lower than that of aerobic cells and was composed of only chemical components. Similarly, Klebsiella pneumoniae did not have any Δψ when grown anaerobically. Furthermore, Δp is not influenced by the nitrogen sources. From these results it is clear that the degree of aerobiosis greatly affects the Δp of growing bacteria, whereas other growth conditions have very slight affects.

D Values of Δp in Prokaryotes Growing at Extreme pH Values

Most bacteria have maximal growth rates at neutral pH values and survive only within the range of pH 5 to 8.5. However, some organisms can grow in media of extreme pH values: these are acidophiles and alkalophiles which grow optimally at below pH 4 and above pH 10, respectively. A Δp is also established in these unusual organisms (Table II).

Acidophilic prokaryotes can be divided into two groups, acidophilic chemoautotrophs and heterotrophs. The mechanism of energy conservation in these bacteria is discussed in a recent review by Cobley and Cox (1983). In acidophiles, the cytoplasmic pH is close to neutrality and a high pH is maintained at an extremely acidic pH. The obligatory acidophilic chemoautotroph Thiobacillus ferrooxidans can grow at pH 1.5−3.5. At these extreme pH values, this bacteria uses oxidation of Fe²+ by O2 as the sole source of energy and produces a ΔpH of 4.5 units and total Δp of −256 mV. Respiratory inhibitors and uncouplers significantly decrease its Δp, suggesting that a chemiosmotic mechanism is involved in its acidophilic growth (Cox et al., 1979). The total Δp was measured in the acidophilic heterotrophs Thiobacillus acidophilus (Matin et al., 1982), and B. acidocaldarius (Krulwich et al., 1978). Consistent with the chemiosmotic mechanism, dinitrophenol completely abolished the ΔpH in Bacillus acidocaldarius. Thiobacillus acidophilus formed a lower Δp than T. ferrooxidans (Matin et al., 1982) (Table II).

Values of ΔpH and Δψ in alkalophilic bacteria were also measured. Bacillus alcalophilus grows on l-malate in medium of pH 9.0 to 11.5 with an optimum at pH 10.5, and at this optimal pH its doubling time is less than 1 hr. This organism maintains a cytoplasmic pH of about 9.5, which is lower than the pH of the alkaline medium. Thus it forms an interior acidic ΔpH of 30 mV (at pH 10.0) to 151 mV (at pH 11.5), and an interior negative Δψ of −84 mV (at pH 9.0) to −152 mV (at pH 11.5) (Guffanti et al., 1978). In spite of its low Δp (Table II) this organism shows normal oxidative phosphorylation involving H+-ATPase (Guffanti et al., 1978, 1981a).

III PROTON-TRANSLOCATING ATPASE (H+-ATPASE)

A Synthesis and Hydrolysis of ATP in Bacteria

1 BACTERIAL H+-ATPASE

Studies on the synthesis of ATP in bacteria are important and promising for understanding the role of Δp and the structure and mechanism of H+-ATPase. H+-ATPase, also called H+-translocating ATPase or ATP synthase, consists of two portions, F1 (peripheral membrane portion) and F0 (integral membrane portion). The catalytic portion, F1, consists of five different subunits, α, β, γ, δ, and ε. F1 with essentially the same subunit structure can be obtained from mitochondria, chloroplasts, and bacteria. F0 is a transmembrane protein complex, mediating H+ translocation between the inside and outside of the organelle or cell. The F0 portion has been less extensively characterized than the F1 portion, but the subunits of F0 (a, b, c) from E. coli have been unambiguously identified by biochemical and genetic studies. In this section we review the structure and mechanism of F0F1 and, especially, recent findings. For further information on aspects of this field that we discuss only briefly, the reader should refer to recent review articles on bacterial F0F1 (Bragg, 1984; Cox et al., 1984; Downie et al., 1979; Dunn and Heppel, 1981; Fillingame, 1981; Futai and Kanazawa, 1980, 1982, 1983; Gibson, 1983; Hoppe and Sebald, 1984; Kagawa, 1984a; Walker et al., 1984b; Senior and Wise, 1983; Vignais and Satre, 1984).

2 Δp AND ATP SYNTHESIS

The experimental approach initiated by Jagendorf and Uribe (1966) established that an artificially imposed Δp could drive ATP synthesis by chromatophores of R. rubrum (Leiser and Gromet-Elhanan, 1974) and plasma membrane vesicles of E. coli (Tsuchiya and Rosen, 1976a, b), suggesting that ATP synthesis by bacterial F0F1 is also coupled to Δp. Later Sone et al. (1977) and Rögner et al. (1979) showed that F0F1 from a thermophilic bacterium PS3 could synthesize ATP when it was incorporated into phospholipid vesicles and a Δp was applied artificially using a diffusion potential or electric field. Liposomes with F0F1 composed of eight subunits from E. coli (Negrin et al., 1980) or PS3 (Okamoto et al., 1977) were also capable of forming a Δp on hydrolysis of ATP. Thus eight subunits of F0F1, when properly assembled in liposomes, are sufficient for synthesis and hydrolysis of ATP coupled to Δp.

In aerobic bacteria F0F1 functions as either ATP synthase or a primary H+-pump ATPase forming Δp. In anaerobic bacteria such as S. faecalis, which have no electron transfer chain, F0F1 does not function as an ATP synthase physiologically, but behaves as a true H+ pump, extruding H+ to maintain a Δp (Maloney, 1982). The H+-ATPase in this organism is suggested to be responsible for regulation of the cytoplasmic pH (Kobayashi et al., 1982). However, even F0F1 of S. faecalis could synthesize ATP when a Δp and the phosphorylation potential, and a value of 3 H+/ATP was obtained for E. coli (Kashket, 1982) as for chloroplasts or mitochondria. Similar values were also obtained for Paracoccus denitrificans (McCarthy et al., 1981) and S. lactis (Maloney and Schattschneider, 1980), while values of 2−5 H+/ATP were reported for bacterial chromatophores (Baccarini-Melandri et al., 1981). These results suggest that Δp is the driving force of ATP synthesis, as shown in chloroplasts or mitochondria.

Mitchell (1966), assuming an H+/ATP ratio of 2, calculated that Δp of −210 mV would be required to maintain equal concentrations of ATP and ADP in mitochondria when the phosphate concentration was 10 mM. This Δp may consist of a pH alone of 3.5 units (inside alkaline) or a Δψ alone (inside negative) or of a combination of ΔpH and Δψ. A similar calculation assuming an H+/ATP ratio of 3 gives a Δp of −140 mV. This calculation gives an estimate of the Δp required for ATP synthesis, although the ATP/ADP ratio and phosphate concentration may differ in prokaryotes. As discussed above, most bacteria can form a Δp in the above range. Titration of ATP synthesis with Δp suggested that there is a threshold value of Δp. Sone et al. (1977) observed appreciable synthesis of ATP at a Δp above −200 mV in reconstituted vesicles from purified F0F1 of the thermophilic bacterium PS3 and phospholipids. Maloney (1977) observed a gated response of H+ entry and ATP synthesis at around −175 to −200 mV in S. lactis. An appreciable amount of ATP was synthesized in submitochondrial particles with an artificial Δp of −160 mV (Thayer and Hinkle, 1975). A similar value was also reported for chloroplasts energized with an artificial electric field (Witt, 1979).

3 ATP SYNTHESIS AT A Low Δp

Alkalophilic bacteria pose an interesting problem as to the role of Δp in oxidative phosphorylation. As discussed above, the internal pH of B. alcalophilus is 9.0 in medium of pH 11 and the total Δp was calculated to be −15 mV (Guffanti et al., 1978). Guffanti et al. (1981a) made right-side-out vesicles containing ADP and phosphate and kept their internal pH at 9.0. ATP synthesis was observed over a wide range of external pH values of up to pH 11.0 under conditions in which the total Δp was as low as −30 mV. These workers also titrated the steady state level of ATP synthesis with Δp and observed significant synthesis even with a Δp of less than −40 mV. Thus ATP synthesis in this organism appears to be carried out by an H+-ATPase that functions at low Δp with a high H+/ATP ratio. However, another possibility may be considered from the results with Bacillus firmus RAB (Guffanti et al., 1984). ATP synthesis by starved whole cells of this strain was observed at pH 9.0 when the cells were energized by addition of malate, but no synthesis was detected when a K+ diffusion potential mediated by valinomycin was created. In contrast, both treatments established a Δψ of −170 mV and stimulated α-aminoisobutyric acid uptake