Mechanisms and Regulation of Carbohydrate Transport in Bacteria

Mechanisms and Regulation of Carbohydrate Transport in Bacteria

Milton H. Saier, Jr.

Department of Biology, The John Muir College, University of California, San Diego, La Jolla, California

Table of Contents

Cover image

Title page

Copyright

Dedication

Inside Front Cover

Preface

Acknowledgments

Chapter 1: Introduction

Publisher Summary

Chapter 2: Mechanisms of Carbohydrate Transport

Publisher Summary

A GLYCEROL PERMEASE: FACILITATED DIFFUSION

B LACTOSE PERMEASE: SUGAR:H+ SYMPORT

C MELIBIOSE PERMEASE: SUGAR : Na+ SYMPORT

D MALTOSE PERMEASE: PRIMARY ACTIVE TRANSPORT

Chapter 3: Group Translocation Catalyzed by the Phosphoenolpyruvate: Sugar Phosphotransferase System

Publisher Summary

A GENERAL FEATURES OF THE PHOSPHOTRANSFERASE SYSTEM

B BIOCHEMICAL AND GENETIC NOMENCLATURE OF THE PTS

C PROPERTIES AND MECHANISM OF ACTION OF ENZYME I

D PROPERTIES OF HPr

E PROPERTIES AND MECHANISM OF ACTION OF ENZYME IImtl

F EVOLUTION OF THE PHOSPHOTRANSFERASE SYSTEM

Chapter 4: Mechanisms of Inducer Exclusion

Publisher Summary

A REGULATION OF TRANSPORT BY THE MEMBRANE POTENTIAL

B REGULATION OF TRANSPORT BY INTRACELLULAR SUGAR PHOSPHATES

C PTS-MEDIATED REGULATION OF CARBOHYDRATE UPTAKE

D REGULATION OF PTS-MEDIATED CARBOHYDRATE UPTAKE BY COMPETITION FOR PHOSPHO-HPr

Chapter 5: Mechanisms of Adenylate Cyclase Regulation in Gram-Negative Bacteria

Publisher Summary

A REGULATION OF ADENYLATE CYCLASE BY THE MEMBRANE POTENTIAL

B REGULATION BY INTRACELLULAR SUGAR PHOSPHATES

C PTS-MEDIATED REGULATION OF ADENYLATE CYCLASE

Chapter 6: Involvement of Protein Kinases in the Regulation of Carbohydrate Transport and Metabolism

Publisher Summary

A FUTILE CYCLES OF SUGAR UPTAKE AND EFFLUX IN E. COLI AND S. LACTIS

B PHYSIOLOGICAL CHARACTERIZATION OF INDUCER EXPULSION

C HPr KINASE AND HPr(ser)P PHOSPHATASE IN S. PYOGENES

D PROPERTIES OF BACTERIAL PROTEIN KINASES IN S. TYPHIMURIUM

E ISOCITRATE DEHYDROGENASE PHOSPHORYLATION AND THE REGULATION OF CARBON METABOLISM

F EVOLUTION OF CYCLIC AMP AND PROTEIN KINASES AS REGULATORY AGENTS FOR THE CONTROL OF CARBON AND ENERGY METABOLISM

Chapter 7: Exogenous Induction of Certain Carbohydrate Permeases in Bacteria

Publisher Summary

A EXOGENOUS INDUCTION OF THE HEXOSE PHOSPHATE AND PHOSPHOGLYCERATE PERMEASES

B AUTOGENOUS INDUCTION OF THE PROTEINS OF THE PHOSPHOTRANSFERASE SYSTEM

C POSSIBLE INVOLVEMENT OF TRANSPORT PROTEINS IN TRANSCRIPTIONAL REGULATION

Chapter 8: Permease Classification and Mechanisms: Conclusions and Future Perspectives

Publisher Summary

A DIVERSITY OF PERMEASE MECHANISMS IN LIVING CELLS

B CLASSIFICATION OF CARBOHYDRATE TRANSPORT SYSTEMS IN E. COLI AND S. TYPHIMURIUM

C CONCLUSIONS

Bibliography

Index

Copyright

COPYRIGHT © 1985, 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.

Orlando, Florida 32887

United Kingdom Edition published by

ACADEMIC PRESS INC. (LONDON) LTD.

24–28 Oval Road, London NW1 7DX

Library of Congress Cataloging in Publication Data

Saier, Milton H.

Mechanisms and regulation of carbohydrate transport in bacteria.

Bibliography: p.

Includes index.

1. Bacteria--Physiology. 2. Microbial metabolism.

3. Carbohydrates--Metabolism. 4. Biological transport--Regulation. I. Title.

QR92.C3S25 1985 589.9′019248 84-16949

ISBN 0-12-614780-9 (alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA

85 86 87 88 9 8 7 6 5 4 3 2 1

Dedication

To my parents, Milton and Lucelia Saier, on the occasion of their fiftieth wedding anniversary

Inside Front Cover

Man’s mind stretched to a new idea never goes back to its original dimensions.

Oliver Wendell Holmes

Preface

In 1980, we published a fairly detailed review in Microbiological Reviews entitled Carbohydrate Transport in Bacteria (Dills et al., 1980). This review article, completed at the end of 1979, summarized and evaluated the literature then available concerning the mechanisms and regulation of carbohydrate transport in prokaryotic organisms. The present monograph was initiated with the intent of updating that review. However, within the elapsing five-year period so much progress has been made in understanding transport and its regulation at the molecular level that a review article of normal length could not possibly provide an in-depth analysis of all of the new information.

In this volume we shall see (1) that important structural and topographical features of several sugar permeases in Escherichia coli have been elucidated; (2) that the details of the energy-coupling processes have been clearly delineated for most (but not all) types of bacterial carbohydrate permease systems; and (3) that mechanistic details of the translocation processes have now been proposed. The genes encoding representative transport proteins within each major class of permease, those specific for lactose, melibiose, mannitol, maltose, and probably glycerol, have all been cloned, and at the time this volume was prepared, the nucleotide sequences of the lacY and mtlA genes encoding the lactose and mannitol permeases had been sequenced and analyzed. These two permeases have been purified to homogeneity and their vectorial reactions have been characterized in proteoliposomes consisting solely of phospholipids and a single transport protein. It can therefore be considered established that both of these permease proteins catalyze sugar transport in the absence of any other protein constituent of the cell. Mechanistic features of the two permeases have come to light as a result of combined kinetic, biochemical, genetic, and biophysical approaches. Comparable information is now available concerning outer membrane porins. In the first three chapters this information, together with the structural advances, will be evaluated.

Great strides have been made in understanding the mechanisms by which carbohydrate uptake and efflux are regulated. Five years ago, a specific model by which the phosphotransferase system regulates other uptake systems was available, but no direct biochemical evidence substantiated this model. The recent advances of molecular genetics and the more refined biochemical technology now available have allowed the establishment of this model as detailed in Chapter 4 and have revealed some unexpected features of the regulatory process. This system appears to be the best-characterized example of macromolecular mediation of information transfer from the external cell surface to multiple targets inside the cell.

The discussion in Chapters 4 and 5 also reveals that while the existence of several additional regulatory processes is well established, their mechanistic details are less well defined. In some cases, however, specific postulates have been put forth to explain these processes, and substantial evidence has been presented in their support. Since bioelectric, chemical, and macromolecular regulatory mechanisms are evidently operative, a number of parallels can be drawn with analogous processes in higher eukaryotes.

Perhaps the most startling advance over the last five years was the discovery of the unanticipated involvement of metabolite-activated protein kinases in the regulation of transport and the accumulation of cytoplasmic inducers in gram-positive bacteria. These advances, as well as the current literature on the involvement of similar catalytic agents in carbon metabolic control in gram-negative bacteria, are reviewed in Chapter 6. It is proposed that cyclic AMP and protein kinases first evolved to coordinate carbohydrate metabolism in prokaryotes and that subsequent diversification of these regulatory processes to more complicated processes in eukaryotes followed.

Not all inducers of carbohydrate permeases in gram-negative bacteria act from cytoplasmic locations. Some act from the periplasmic space in processes which are apparently mediated by integral transmembrane signaling devices. Both catalytic and structural models have been proposed, and the application of genetic engineering approaches already well under way should yield definitive information concerning these mechanisms in the near future (Chapter 7). Knowledge of these mechanisms may provide evidence concerning the evolutionary origins of transmembrane signaling processes such as those mediated by hormone receptors, cell adhesion macromolecules, and immunoglobulin receptors in animal cells.

Finally, in Chapter 8, an attempt is made to explain the diversity of transport systems throughout the living world and to point out the probable structural, functional, and evolutionary relationships of these systems to one another. The known carbohydrate transport systems in enteric bacteria are also classified according to mechanism. A survey of the list of permeases compared to the list of exogenous carbon sources which can be utilized by Escherichia coli and Salmonella typhimurium suggests that most of the carbohydrate transport systems in these organisms have already been characterized. It is probable that although considerable variation within each of the five principal classes of transport processes discussed in Chapters 2 and 3 will emerge, no fundamentally new mechanisms will come to light. A major task of the microbial biochemist is, therefore, to integrate our newly obtained structural information with functional studies aimed at defining the solute translocation mechanisms which serve as molecular gates separating the compartments of the living and the nonliving.

Milton Saier

Acknowledgments

I wish to thank the following colleagues for valuable discussions, for permission to cite unpublished results, and for suggestions regarding this monograph:

A. Apperson

K. Basu

P. Bavoil

J. Beckwith

K. Beyreuther

J. Brass

A. Danchin

J. Desai

J. Deutscher

S. Dills

R. Doolittle

B. Erni

S. Froshauer

P. Gay

S. Ghosh

E. Gilson

F. C. Grenier

J. L. Guan

W. Hengstenberg

M. Hofnung

G. R. Jacobson

H. R. Kaback

W. W. Kay

J. Kyte

C. A. Lee

J. E. Leonard

J. London

M. Neuhaus

M. J. Newman

R. A. Nicholas

H. Nikaido

M. J. Novotny

T. Osumi

M. Pfahl

D. Printz

J. Reizer

H. V. Rickenberg

G. T. Robillard

H. Rosenberg

J. Rosenbusch

H. Schindler

M. Schwartz

I. Stuiver

L. E. Tanney

G. Tenn

J. Thompson

R. Tuttle

J. Y. J. Wang

E. B. Waygood

T. H. Wilson

Particular thanks are extended to Leanne Sorenson for invaluable assistance in the preparation of this manuscript and Lynn Green, who drew the illustrations. Work in the author’s laboratory was supported by NIH Grants 2-RO1-AM21994-0401 and 5-RO1-AI14176-07MBC.

1

Introduction

Publisher Summary

This chapter describes the carbohydrate transport process. Carbohydrates are transported into bacterial cells by a variety of mechanisms. There has been tremendous progress in defining these mechanisms and characterizing the proteins that catalyze the vectorial reactions. Conceptually, these proteins are thought to function by either of two general processes: (1) they may translocate their hydrophilic solutes through aqueous pores or (2) they may transport the solute by a carrier-type mechanism. It is presumed that in both cases, all or part of the hydration shell of the solute is lost as it passes through the membrane the hydration shell is restored upon reentry into the aqueous solution on the other side of the membrane. The product of the carbohydrate transport process, or a primary metabolite derived from it, usually serves as an inducer of the sugar catabolic enzyme system. The inhibition of sugar uptake by any one of the mechanisms is referred to as inducer exclusion.

Carbohydrates are transported into bacterial cells by a variety of mechanisms. Within the last 4 years tremendous progress has been made in defining these mechanisms and characterizing the proteins which catalyze the vectorial reactions. Conceptually, these proteins are thought to function by either of two general processes as shown in Fig. 1.1. They may translocate their hydrophilic solutes through aqueous pores (Fig. 1.1, I), or they may transport the solute by a carrier-type mechanism (Fig. 1.1, II). It is presumed that in both cases all or part of the hydration shell of the solute is lost as it passes through the membrane but that the hydration shell is restored upon re-entry into the aqueous solution on the other side of the membrane. While a pore-type mechanism may involve zero, one, two, or more solute binding sites, a carrier-type mechanism usually invokes a single site, which can shuttle between states localized to the two sides of the membrane.

Fig. 1.1 Schematic depiction of two fundamentally different transport processes. Solute (S) with its hydration shell (W) becomes associated with a membrane protein which replaces all or part of the hydration shell. The molecule then passes through the membrane (I) via a pore-type mechanism in which the solute passes from one complexation site of the protein to another before re-entry into the aqueous environment on the opposite side of the membrane, or (II) by a carrier-type mechanism in which the solute passes through the hydrophobic barrier of the membrane complexed with a moiety of the carrier protein. The fundamental difference lies in the fact that only in the latter case does part of the protein shuttle across the membrane with the solute. Transport mechanisms can be envisaged which incorporate elements of both mechanisms. [Adapted from Mitchell (1979).]

In order to enter the cytoplasm of the gram-negative bacterial cell, an extracellular hydrophilic molecule must pass through both the outer and the inner membranes of the cell envelope (Fig. 1.2). Passage through the outer membrane is normally accomplished via hydrophilic proteinaceous pores. The properties of the best characterized carbohydrate-translocating porins in Escherichia coli are summarized in Table 1.1 (Lugtenberg and Van Alphen, 1983). Among these proteins are the ompF and ompC porins, which are fairly nonspecific with respect to their substrate specificities. Oligosaccharides of less than 500 daltons can generally pass through these pores, and amino acids, phosphorylated carbohydrates, and salts are also effectively transported. Synthesis of these proteins is dependent on the osmolarity of the growth medium. By contrast, the phoE, tsx and lamB porins exhibit substrate specificity, preferentially transporting inorganic phosphate and phosphorylated carbohydrates, nucleosides, and maltodextrins, respectively. The specificities of these three porins presumably result from the presence of specific solute-binding sites within the pores, i.e., highly stereospecific arrangements of the aminoacyl residues, which comprise the pores. Induction characteristics of these proteins are in accord with the specificity studies, both of which suggest a specialized function for each protein (Table 1.1). The lamB porin (maltoporin) will be discussed at length in Chapter 2, Section D as it comprises part of the maltodextrin transport system. Other outer membrane proteins, which function in the transport of vitamin B12 and complexed ferric ions, have been identified (Lugtenberg and Van Alphen, 1983).

TABLE 1.1

Characteristics of Outer Membrane Transport Proteins in E. coli

aThe ompF, tsx, and lamB porins have been reported to be subject to catabolite repression, emphasizing the roles of these channel proteins in carbohydrate transport across the outer membrane.

b—, Not known

Fig. 1.2 Schematic depiction of the cell envelope of a gram-negative bacterial cell. The figure depicts the two membranes, the inner or plasma membrane which houses the energy-dependent carbohydrate-specific permeases shown in Fig. 2.1 as well as the outer or lipopolysaccharide-containing membrane which houses the porin proteins which are relatively nonspecific with respect to their substrates (Table 1.1). The peptidoglycan layer is presumed to be porous, so that small hydrophilic molecules can easily diffuse from the outer membrane across the periplasmic space to the cytoplasmic membrane. [From Saier (1979).]

Recently the amino acid sequences of several of the major outer membrane proteins of E. coli have been examined for sequence homology (Nikaido and Wu, 1984). These proteins included the ompF, ompC, and phoE porins as well as the lamB (malM) porin, which encodes maltoporin (see Table 1.1). Significant local homology was observed for these proteins as well as for two other outer membrane proteins, those encoded by the ompA and tolC genes. These findings either suggest a common evolutionary origin for these different proteins or reflect a common mechanism by which these proteins are exported to the outer membrane.

By contrast with the transport proteins of the outer membrane, those in the inner, cytoplasmic membrane (Fig. 1.2) generally function by stereospecific mechanisms exhibiting a high degree of specificity as expected for a catalytic protein. These distinct transport processes are depicted in Fig. 2.1, in Chapter 2. The one exception to this rule is the glycerol permease. Glycerol has been shown to enter the E. coli cell by facilitated diffusion through a nonstereospecific hydrophilic pore of inner diameter equal to about 0.4 nm. The kinetic characteristics of glycerol transport have been well defined (Chapter 2, Section A). The lactose (lac) permease protein (Chapter 2, Section B), which catalyzes lactose: H+ symport, has been solubilized from the membrane, purified to homogeneity, and reconstituted in artificial phospholipid membranes. The gene encoding this protein has been cloned and sequenced so that the primary amino acid sequence of the lac permease is known. Kinetic and chemical studies have led to postulates regarding the translocation process, and evidence is emerging that this protein functions by a carrier-type mechanism. Such a postulated mechanism involves a shuttling of the substrate-binding site of the permease between two states, each accessible to only one side of the membrane (Fig. 1.1). A distinct system, similar in several respects to the lactose permease, is the melibiose (mel) permease. This transport system catalyzes sugar: Na+ symport, but it is also capable of catalyzing sugar: H+ and sugar: Li+ symport (Chapter 2, Section C). The activity of this protein has also been reconstituted in an artificial membrane. It appears to function by a mechanism analogous to that of the lac carrier, but with certain interesting kinetic differences. The tertiary structure of this protein probably resembles that of the lactose permease.

In contrast to the other systems discussed in this volume, maltose and maltodextrins cross the cytoplasmic membrane by a high-affinity active transport system whose activity depends on the integrity of four distinct proteins (Chapter 2, Section D). A channel-mediated translocation process seems likely for this multicomponent system. In order to ensure efficient scavenging activity at low maltodextrin concentrations, the bacteria synthesize a proteinaceous hydrophilic pore in the outer membrane (maltoporin), which, together with the periplasmic maltose-binding protein, functions to translocate maltooligosaccharides across this structure. Much is now known about the structure and function of maltoporin.

Finally, like the lac permease, the mannitol permease (the mannitol Enzyme II of the phosphotransferase system) has been solubilized from the membrane, it has been purified to homogeneity, and its transport function has been reconstituted in artificial proteoliposomes (Chapter 3, Section E). The soluble energy coupling proteins of the system have also been purified and subjected to critical analyses (Chapter 3, Sections C and D). Detailed biochemical and genetic experiments have resulted in the identification of most of the PTS proteins (Chapter 3, Sections A and B). The gene encoding the mannitol Enzyme II protein has been cloned and sequenced so that the primary amino acid sequence of the protein is known. The mannitol Enzyme II and the lactose permease are therefore the two bacterial carbohydrate transport systems for which the structures and transport functions are best understood.

As a consequence of extensive biochemical and genetic analyses performed on the proteins of the phosphotransferase system, the structures of these proteins have been correlated with function (Fig. 1.3). Group translocation of a sugar (mannitol or glucitol in Fig. 1.3) probably involves five sequential phosphoryl transfer reactions and four distinct protein phosphorylation sites. All such sites involve histidyl residues in the phosphocarrier proteins of the PTS. The first two phosphorylation sites are associated with the general energy coupling proteins of the PTS, Enzyme I, and HPr. The second two sites are associated either with a single Enzyme II (as in the case of the mannitol system) or with an Enzyme II–III pair (as in the case of the glucitol system).

Fig. 1.3 Sequential protein phosphorylation reactions of the phosphoryl transfer chains for mannitol (mtl) and glucitol (gut) in E. coli. The figure shows five phosphoryl transfer reactions, which result in the phosphorylation of four distinct protein sites. Phosphoenolpyruvate (PEP) phosphorylates Enzyme I on the N-3 position of a histidyl residue, HPr is phosphorylated on the N-1 position of a histidyl residue; the first phosphorylation site on the Enzyme IImtl and the phosphorylation site on IIIgut are N-3 histidyl residues, while the second phosphorylation site on the Enzyme IImtl and the phosphorylation site on Enzyme IIgut are thought to be N-1 histidyl residues. The last step involves transfer of the phosphoryl moiety from the N-1 position of a histidyl residue in the Enzyme II to the incoming sugar. The Enzyme IIgut–IIIgut pair is shown to be structurally and functionally equivalent to the Enzyme IImtl. It is proposed that all Enzymes II and Enzyme II–III pairs of the PTS exhibit similar structural features, involve the same sequence of phosphoryl transfer reactions, and catalyze sugar transport by essentially the same mechanism as illustrated in the figure. These speculative proposals result from their common evolutionary ancestry.

In the last section of