This page intentionally left blankBacterial Physiology and MetabolismRecent determination of the genome sequences for a wide range of bacteriahas made an in-depth knowledge of prokaryotic metabolic function evenmore essential in order to give biochemical, physiological and ecologicalmeaning to the genomic information. Clearly describing the importantmetabolic processes that occur in prokaryotes under different conditionsand in different environments, this advanced text provides an overview ofthe key cellular processes that determine bacterial roles in the environment,biotechnology and human health. Prokaryotic structure and compositionare described as well as the means by which nutrients are transported intocells across membranes. Discussion of biosynthesis and growth is followedby detailed accounts of glucose metabolism through glycolysis, the TCAcycle, electron transport and oxidative phosphorylation, as well as othertrophic variations found in prokaryotes including the use of organic com-pounds other than glucose, anaerobic fermentation, anaerobic respiration,chemolithotrophy and photosynthesis. The regulation of metabolismthrough control of gene expression and enzyme activity is also covered, aswell as the survival mechanisms used under starvation conditions.Professor Byung Hong Kim is an expert on anaerobic metabolism, organicdegradation and bioelectrochemistry. He graduated from KyungpookNational University, Korea, and obtained a Ph.D. from University CollegeCardiff. He has carried out research at several universities around theworld, with an established career in the Korea Institute of Science andTechnology. He has been honoured by the Korean Government, whichdesignated his research group a National Research Laboratory, theBioelectricity Laboratory, and has served as President of the KoreanSociety for Microbiology and Biotechnology. Professor Kim wrote theclassic Korean microbiology text on Microbial Physiology and has publishedover 100 refereed papers and reviews, and holds over 20 patents relating toapplications of his research in environmental biotechnology.Professor Geoffrey Michael Gadd is an authority on microbial interactionswith metals, minerals and radionuclides and their applications in environ-mental biotechnology. He holds a personal Chair in Microbiology at theUniversity of Dundee and is the Head of the Division of Molecular andEnvironmental Microbiology in the College of Life Sciences. He has pub-lished over 200 refereed scientific papers, books and reviews and hasreceived invitations to speak at international conferences in over 20 coun-tries. Professor Gadd has served as President of the BritishMycological Societyand is an elected Fellow of the Institute of Biology, the American Academy ofMicrobiology, the Linnean Society, and the Royal Society of Edinburgh. Hehas received the Berkeley Award fromthe British Mycological Society and theCharles Thom award from the Society for Industrial Microbiology for hisoutstanding research contributions to the microbiological sciences.Bacterial Physiology and MetabolismByung Hong KimKorea Institute of Science and TechnologyGeoffrey Michael GaddUniversity of DundeeCAMBRIDGE UNIVERSITY PRESSCambridge, New York, Melbourne, Madrid, Cape Town, Singapore, So PauloCambridge University PressThe Edinburgh Building, Cambridge CB2 8RU, UKFirst published in print formatISBN-13 978-0-521-84636-3ISBN-13 978-0-521-71230-9ISBN-13 978-0-511-39322-8 B. H. Kim and G. M. Gadd 20082008Information on this title: www.cambridge.org/9780521846363This publication is in copyright. Subject to statutory exception and to the provision of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press.Cambridge University Press has no responsibility for the persistence or accuracy of urls for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate.Published in the United States of America by Cambridge University Press, New Yorkwww.cambridge.orgpaperbackeBook (EBL)hardbackTo our familiesHyungock Hong, Kyoungha Kim and Youngha KimandJulia, Katie and Richard GaddContents in brief1 Introduction to bacterial physiology and metabolism page 12 Composition and structure of prokaryotic cells 73 Membrane transport nutrient uptake and proteinexcretion 354 Glycolysis 605 Tricarboxylic acid (TCA) cycle, electron transport andoxidative phosphorylation 856 Biosynthesis and microbial growth 1267 Heterotrophic metabolism on substrates other thanglucose 2028 Anaerobic fermentation 2529 Anaerobic respiration 29810 Chemolithotrophy 35411 Photosynthesis 38612 Metabolic regulation 40813 Energy, environment and microbial survival 482ContentsPreface page xxi1 Introduction to bacterial physiology andmetabolism 1Further reading 42 Composition and structure of prokaryotic cells 72.1 Elemental composition 72.2 Importance of chemical form 82.2.1 Five major elements 82.2.2 Oxygen 92.2.3 Growth factors 102.3 Structure of microbial cells 102.3.1 Flagella and pili 102.3.2 Capsules and slime layers 122.3.3 S-layer, outer membrane and cell wall 122.3.3.1 S-layer 132.3.3.2 Outer membrane 132.3.3.3 Cell wall and periplasm 172.3.4 Cytoplasmic membrane 212.3.4.1 Properties and functions 212.3.4.2 Membrane structure 222.3.4.3 Phospholipids 232.3.4.4 Proteins 262.3.5 Cytoplasm 272.3.6 Resting cells 29Further reading 303 Membrane transport nutrient uptake andprotein excretion 353.1 Ionophores: models of carrier proteins 353.2 Diffusion 373.3 Active transport and role of electrochemical gradients 373.4 ATP-dependent transport: ATP-binding cassette(ABC) pathway 383.5 Group translocation 393.6 Precursor/product antiport 403.7 Ferric ion (Fe(III)) uptake 413.8 Export of cell surface structural components 433.8.1 Protein transport 433.8.1.1 General secretory pathway (GSP) 433.8.1.2 Twin-arginine translocation (TAT) pathway 453.8.1.3 ATP-binding cassette (ABC) pathway 463.8.2 Protein translocation across the outer membrane inGram-negative bacteria 463.8.2.1 Chaperone/usher pathway 473.8.2.2 Type I pathway: ATP-binding cassette (ABC) pathway 473.8.2.3 Type II pathway 473.8.2.4 Type III pathway 493.8.2.5 Type IV pathway 503.8.2.6 Type V pathway: autotransporter and proteins requiringsingle accessory factors 51Further reading 524 Glycolysis 604.1 EMP pathway 614.1.1 Phosphofructokinase (PFK): key enzyme of the EMPpathway 614.1.2 ATP synthesis and production of pyruvate 634.1.3 Modified EMP pathways 644.1.3.1 Methylglyoxal bypass 644.1.3.2 Modified EMP pathways in archaea 654.1.4 Regulation of the EMP pathway 664.1.4.1 Regulation of phosphofructokinase 664.1.4.2 Regulation of pyruvate kinase 674.1.4.3 Global regulation 674.2 Glucose-6-phosphate synthesis: gluconeogenesis 674.2.1 PEP synthesis 674.2.2 Fructose diphosphatase 684.2.3 Gluconeogenesis in archaea 684.2.4 Regulation of gluconeogenesis 694.3 Hexose monophosphate (HMP) pathway 694.3.1 HMP pathway in three steps 694.3.2 Additional functions of the HMP pathway 704.3.2.1 Utilization of pentoses 714.3.2.2 Oxidative HMP cycle 714.3.3 Regulation of the HMP pathway 714.3.4 F420-dependent glucose-6-phosphate dehydrogenase 714.4 EntnerDoudoroff (ED) pathway 724.4.1 Glycolytic pathways in some Gram-negative bacteria 724.4.2 Key enzymes of the ED pathway 724.4.3 Modified ED pathways 72x CONTENTS4.4.3.1 Extracellular oxidation of glucose by Gram-negativebacteria 724.4.3.2 Modified ED pathways in archaea 744.5 Phosphoketolase pathways 744.5.1 Glucose fermentation by Leuconostoc mesenteroides 754.5.2 Bifidum pathway 774.6 Use of radiorespirometry to determine glycolytic pathways 78Further reading 805 Tricarboxylic acid (TCA) cycle, electron transportand oxidative phosphorylation 855.1 Oxidative decarboxylation of pyruvate 855.2 Tricarboxylic acid (TCA) cycle 865.2.1 Citrate synthesis and the TCA cycle 875.2.2 Regulation of the TCA cycle 885.3 Replenishment of TCA cycle intermediates 885.3.1 Anaplerotic sequence 885.3.2 Glyoxylate cycle 895.3.2.1 Regulation of the glyoxylate cycle 905.4 Incomplete TCA fork and reductive TCA cycle 915.4.1 Incomplete TCA fork 915.4.2 Reductive TCA cycle 925.5 Energy transduction in prokaryotes 935.5.1 Free energy 935.5.1.1 G00from the free energy of formation 945.5.1.2 G00from the equilibrium constant 945.5.1.3 G fromG00955.5.1.4 G00fromG0955.5.2 Free energy of an oxidation/reduction reaction 955.5.2.1 Oxidation/reduction potential 955.5.2.2 Free energy fromE00965.5.3 Free energy of osmotic pressure 975.5.4 Sum of free energy change in a series of reactions 975.6 Role of ATP in the biological energy transduction process 985.6.1 High energy phosphate bonds 995.6.2 Adenylate energy charge 1005.6.3 Phosphorylation potential (Gp) 1015.6.4 Interconversion of ATP and proton motive force (p) 1015.6.5 Substrate-level phosphorylation (SLP) 1025.7 Proton motive force (p) 1025.7.1 Proton gradient and membrane potential 1025.7.2 Acidophilicity and alkalophilicity 1035.7.3 Proton motive force in acidophiles 1035.7.4 Proton motive force and sodium motive forcein alkalophiles 104CONTENTS xi5.8 Electron transport (oxidative) phosphorylation 1055.8.1 Chemiosmotic theory 1055.8.2 Electron carriers and the electron transport chain 1055.8.2.1 Mitochondrial electron transport chain 1055.8.2.2 Electron carriers 1075.8.2.3 Diversity of electron transport chains in prokaryotes 1085.8.2.4 Inhibitors of electron transport phosphorylation (ETP) 1105.8.2.5 Transhydrogenase 1105.8.3 Arrangement of electron carriers in theH-translocating membrane 1115.8.3.1 Q-cycle and Q-loop 1115.8.3.2 Proton pump 1125.8.4 ATP synthesis 1125.8.4.1 ATP synthase 1125.8.4.2 H/O ratio 1135.8.4.3 H/ATP stoichiometry 1145.8.5 Uncouplers 1145.8.6 Primary H (Na) pumps in fermentative metabolism 1155.8.6.1 Fumarate reductase 1155.8.6.2 Na-dependent decarboxylase 1155.8.6.3 p formation through fermentation product/Hsymport 1165.9 Other biological energy transduction processes 1165.9.1 Bacterial bioluminescence 1165.9.2 Electricity as an energy source in microbes 117Further reading 1186 Biosynthesis and microbial growth 1266.1 Molecular composition of bacterial cells 1266.2 Assimilation of inorganic nitrogen 1276.2.1 Nitrogen fixation 1286.2.1.1 N2-fixing organisms 1286.2.1.2 Biochemistry of N2 fixation 1296.2.1.3 Bioenergetics of N2 fixation 1326.2.1.4 Molecular oxygen and N2 fixation 1326.2.1.5 Regulation of N2 fixation 1346.2.2 Nitrate reduction 1356.2.3 Ammonia assimilation 1376.3 Sulfate assimilation 1396.4 Amino acid biosynthesis 1406.4.1 The pyruvate and oxaloacetate families 1406.4.2 The phosphoglycerate family 1416.4.3 The 2-ketoglutarate family 1416.4.4 Aromatic amino acids 1416.4.5 Histidine biosynthesis 1456.4.6 Regulation of amino acid biosynthesis 145xii CONTENTS6.5 Nucleotide biosynthesis 1456.5.1 Salvage pathway 1456.5.2 Pyrimidine nucleotide biosynthesis through a de novopathway 1486.5.3 De novo synthesis of purine nucleotides 1496.5.4 Synthesis of deoxynucleotides 1496.6 Lipid biosynthesis 1526.6.1 Fatty acid biosynthesis 1526.6.1.1 Saturated acyl-ACP 1536.6.1.2 Branched acyl-ACP 1546.6.1.3 Unsaturated acyl-ACP 1546.6.1.4 Cyclopropane fatty acids 1566.6.1.5 Regulation of fatty acid biosynthesis 1566.6.2 Phospholipid biosynthesis 1566.6.3 Isoprenoid biosynthesis 1596.7 Heme biosynthesis 1596.8 Synthesis of saccharides and their derivatives 1616.8.1 Hexose phosphate and UDP-sugar 1616.8.2 Monomers of murein 1636.8.3 Monomers of teichoic acid 1646.8.4 Precursor of lipopolysaccharide, O-antigen 1646.9 Polysaccharide biosynthesis and the assembly of cellsurface structures 1656.9.1 Glycogen synthesis 1656.9.2 Murein synthesis and cell wall assembly 1676.9.2.1 Transport of cell wall precursor componentsthrough the membrane 1676.9.2.2 Murein synthesis 1676.9.2.3 Teichoic acid synthesis 1676.9.2.4 Cell wall proteins in Gram-positive bacteria 1696.9.3 Outer membrane assembly 1696.9.3.1 Protein translocation 1696.9.3.2 Lipopolysaccharide (LPS) translocation 1696.9.3.3 Phospholipid translocation 1706.9.4 Cytoplasmic membrane (CM) assembly 1706.10 Deoxyribonucleic acid (DNA) replication 1706.10.1 DNA replication 1706.10.1.1 RNA primer 1716.10.1.2 Okazaki fragment 1726.10.1.3 DNA polymerase 1726.10.2 Spontaneous mutation 1736.10.3 Post-replicational modification 1736.10.4 Chromosome segregation 1736.11 Transcription 1746.11.1 RNA synthesis 1746.11.2 Post-transcriptional processing 174CONTENTS xiii6.12 Translation 1756.12.1 Amino acid activation 1766.12.2 Synthesis of peptide: initiation, elongation andtermination 1766.12.2.1 Ribosomes 1776.12.2.2 Initiation and elongation 1776.12.2.3 Termination 1786.12.3 Post-translational modification and protein folding 1786.13 Assembly of cellular structure 1816.13.1 Flagella 1816.13.2 Capsules and slime 1826.13.3 Nucleoid assembly 1826.13.4 Ribosome assembly 1826.14 Growth 1826.14.1 Cell division 1836.14.1.1 Binary fission 1836.14.1.2 Multiple intracellular offspring 1846.14.1.3 Multiple offspring by multiple fission 1856.14.1.4 Budding 1876.14.2 Growth yield 1876.14.3 Theoretical maximum YATP 1896.14.4 Growth yield using different electron acceptors andmaintenance energy 1896.14.5 Maintenance energy 192Further reading 1937 Heterotrophic metabolism on substratesother than glucose 2027.1 Hydrolysis of polymers 2027.1.1 Starch hydrolysis 2027.1.2 Cellulose hydrolysis 2037.1.3 Other polysaccharide hydrolases 2047.1.4 Disaccharide phosphorylases 2057.1.5 Hydrolysis of proteins, nucleic acids and lipids 2067.2 Utilization of sugars 2067.2.1 Hexose utilization 2067.2.2 Pentose utilization 2077.3 Organic acid utilization 2087.3.1 Fatty acid utilization 2087.3.2 Organic acids more oxidized than acetate 2107.4 Utilization of alcohols and ketones 2137.5 Amino acid utilization 2147.5.1 Oxidative deamination 2157.5.2 Transamination 2157.5.3 Amino acid dehydratase 2157.5.4 Deamination of cysteine and methionine 216xiv CONTENTS7.5.5 Deamination products of amino acids 2177.5.6 Other amino acids 2207.6 Degradation of nucleic acid bases 2207.7 Oxidation of aliphatic hydrocarbons 2237.8 Oxidation of aromatic compounds 2257.8.1 Oxidation of aromatic amino acids 2257.8.2 Ortho and meta cleavage, and the gentisate pathway 2277.8.3 Oxygenase and aromatic compound oxidation 2297.9 Utilization of methane and methanol 2297.9.1 Methanotrophy and methylotrophy 2297.9.2 Methanotrophy 2307.9.2.1 Characteristics of methanotrophs 2307.9.2.2 Dissimilation of methane by methanotrophs 2337.9.3 Carbon assimilation by methylotrophs 2357.9.3.1 Ribulose monophosphate (RMP) pathway 2357.9.3.2 Serineisocitrate lyase (SIL) pathway 2367.9.3.3 Xylulose monophosphate (XMP) pathway 2407.9.4 Energy efficiency in C1 metabolism 2417.10 Incomplete oxidation 2417.10.1 Acetic acid bacteria 2417.10.2 Acetoin and butanediol 2427.10.3 Other products of aerobic metabolism 243Further reading 2448 Anaerobic fermentation 2528.1 Electron acceptors used in anaerobic metabolism 2528.1.1 Fermentation and anaerobic respiration 2528.1.2 Hydrogen in fermentation 2528.2 Molecular oxygen and anaerobes 2538.3 Ethanol fermentation 2558.4 Lactate fermentation 2578.4.1 Homolactate fermentation 2578.4.2 Heterolactate fermentation 2578.4.3 Biosynthesis in lactic acid bacteria (LAB) 2598.4.4 Oxygen metabolism in LAB 2608.4.5 Lactate/H symport 2608.4.6 LAB in fermented food 2608.5 Butyrate and acetonebutanolethanol fermentations 2638.5.1 Butyrate fermentation 2638.5.1.1 Phosphoroclastic reaction 2638.5.1.2 Butyrate formation 2648.5.1.3 Lactate fermentation by Clostridium butyricum 2658.5.1.4 Clostridium butyricum as a probiotic 2688.5.1.5 Non-butyrate clostridial fermentation 2688.5.2 Acetonebutanolethanol fermentation 2698.5.3 Fermentation balance 271CONTENTS xv8.6 Mixed acid and butanediol fermentation 2728.6.1 Mixed acid fermentation 2728.6.2 Butanediol fermentation 2738.6.3 Citrate fermentation by facultative anaerobes 2758.6.4 Anaerobic enzymes 2778.7 Propionate fermentation 2788.7.1 Succinatepropionate pathway 2788.7.2 Acrylate pathway 2808.8 Fermentation of amino acids and nucleic acid bases 2818.8.1 Fermentation of individual amino acids 2818.8.2 Stickland reaction 2868.8.3 Fermentation of purine and pyrimidine bases 2878.9 Fermentation of dicarboxylic acids 2878.10 Hyperthermophilic archaeal fermentation 2878.11 Degradation of xenobiotics under fermentative conditions 289Further reading 2909 Anaerobic respiration 2989.1 Denitrification 2999.1.1 Biochemistry of denitrification 2999.1.1.1 Nitrate reductase 3009.1.1.2 Nitrite reductase 3029.1.1.3 Nitric oxide reductase and nitrous oxide reductase 3029.1.2 ATP synthesis in denitrification 3029.1.3 Regulation of denitrification 3039.1.4 Denitrifiers other than facultatively anaerobicchemoorganotrophs 3049.1.5 Oxidation of xenobiotics under denitrifying conditions 3069.2 Metal reduction 3069.2.1 Fe(III) and Mn(IV) reduction 3069.2.2 Microbial reduction of other metals 3099.2.3 Metal reduction and the environment 3099.3 Sulfidogenesis 3109.3.1 Biochemistry of sulfidogenesis 3129.3.1.1 Reduction of sulfate and sulfur 3129.3.1.2 Carbon metabolism 3139.3.2 Electron transport and ATP yield in sulfidogens 3179.3.2.1 Incomplete oxidizers 3179.3.2.2 Complete oxidizers 3189.3.3 Carbon skeleton supply in sulfidogens 3189.3.4 Oxidation of xenobiotics under sulfidogenic conditions 3209.4 Methanogenesis 3209.4.1 Methanogens 3209.4.1.1 Hydrogenotrophic methanogens 3209.4.1.2 Methylotrophic methanogens 3229.4.1.3 Aceticlastic methanogens 322xvi CONTENTS9.4.2 Coenzymes in methanogens 3229.4.3 Methanogenic pathways 3249.4.3.1 Hydrogenotrophic methanogenesis 3249.4.3.2 Methylotrophic methanogenesis 3259.4.3.3 Aceticlastic methanogenesis 3269.4.4 Energy conservation in methanogenesis 3279.4.5 Biosynthesis in methanogens 3289.5 Homoacetogenesis 3309.5.1 Homoacetogens 3309.5.2 Carbon metabolism in homoacetogens 3309.5.2.1 Sugar metabolism 3309.5.2.2 Synthesis of carbon skeletons for biosynthesisin homoacetogens 3339.5.3 Energy conservation in homoacetogens 3349.6 Dehalorespiration 3349.6.1 Dehalorespiratory organisms 3359.6.2 Energy conservation in dehalorespiration 3369.7 Miscellaneous electron acceptors 3369.8 Syntrophic associations 3379.8.1 Syntrophic bacteria 3379.8.2 Carbon metabolism in syntrophic bacteria 3399.8.3 Facultative syntrophic associations 3409.9 Element cycling under anaerobic conditions 3409.9.1 Oxidation of aromatic hydrocarbons under anaerobicconditions 3419.9.2 Transformation of xenobiotics under anaerobic conditions 343Further reading 34510 Chemolithotrophy 35410.1 Reverse electron transport 35410.2 Nitrification 35510.2.1 Ammonia oxidation 35610.2.2 Nitrite oxidation 35710.2.3 Anaerobic nitrification 35810.3 Sulfur bacteria and the oxidation of sulfur compounds 35810.3.1 Sulfur bacteria 35810.3.2 Biochemistry of sulfur compound oxidation 36010.3.3 Carbon metabolism in colourless sulfur bacteria 36210.4 Iron bacteria: ferrous iron oxidation 36210.5 Hydrogen oxidation 36410.5.1 Hydrogen-oxidizing bacteria 36410.5.2 Hydrogenase 36410.5.3 Anaerobic H2-oxidizers 36510.5.4 CO2 fixation in H2-oxidizers 36610.6 Carbon monoxide oxidation: carboxydobacteria 36610.7 Chemolithotrophs using other electron donors 367CONTENTS xvii10.8 CO2 fixation pathways in chemolithotrophs 36810.8.1 Calvin cycle 36810.8.1.1 Key enzymes of the Calvin cycle 37010.8.1.2 Photorespiration 37210.8.2 Reductive TCA cycle 37310.8.3 Anaerobic CO2 fixation through the acetyl-CoA pathway 37410.8.4 CO2 fixation through the 3-hydroxypropionate cycle 37510.8.5 Energy expenditure in CO2 fixation 37710.9 Chemolithotrophs: what makes them unable to useorganics? 377Further reading 37911 Photosynthesis 38611.1 Photosynthetic microorganisms 38611.1.1 Cyanobacteria 38711.1.2 Anaerobic photosynthetic bacteria 38711.1.3 Aerobic anoxygenic phototrophic bacteria 38811.2 Photosynthetic pigments 38911.2.1 Chlorophylls 39011.2.2 Carotenoids 39011.2.3 Phycobiliproteins 39211.2.4 Pheophytin 39211.2.5 Absorption spectra of photosynthetic cells 39311.3 Photosynthetic apparatus 39411.3.1 Thylakoids of cyanobacteria 39411.3.2 Green bacteria 39511.3.3 Purple bacteria 39511.3.4 Heliobacteria and aerobic anoxygenic phototrophicbacteria 39511.4 Light reactions 39611.4.1 Properties of light 39711.4.2 Excitation of antenna molecules and resonance transfer 39711.4.3 Electron transport 39811.4.3.1 Photosystem I and II in cyanobacteria 39811.4.3.2 Green sulfur bacteria 39811.4.3.3 Purple bacteria 40011.4.3.4 Aerobic anoxygenic photosynthetic bacteria 40111.5 Carbon metabolism in phototrophs 40111.5.1 CO2 fixation 40111.5.2 Carbon metabolism in photoorganotrophs 40211.5.2.1 Purple bacteria, heliobacteria and aerobicanoxygenic photosynthetic bacteria 40211.5.2.2 Green sulfur bacteria 40311.5.2.3 Cyanobacteria 40311.6 Photophosphorylation in halophilic archaea 403Further reading 405xviii CONTENTS12 Metabolic regulation 40812.1 Mechanisms regulating enzyme synthesis 40812.1.1 Regulation of transcription by promoter structureand sigma () factor activity 40912.1.2 Induction of enzymes 41112.1.2.1 Inducible and constitutive enzymes 41112.1.2.2 Enzyme induction 41212.1.2.3 Positive and negative control 41312.1.3 Catabolite repression 41312.1.3.1 Carbon catabolite repression by the cAMPCRPcomplex 41412.1.3.2 Catabolite repressor/activator 41512.1.3.3 Carbon catabolite repression in Gram-positivebacteria with a low GC content 41712.1.4 Repression and attenuation by final metabolic products 41912.1.4.1 Repression 41912.1.4.2 Attenuation 42012.1.5 Regulation of gene expression by multiple end products 42312.1.6 Termination and antitermination 42412.1.6.1 Termination and antitermination aided by protein 42412.1.6.2 Termination and antitermination aided by tRNA 42612.1.6.3 Termination and antitermination aided bymetabolites 42812.1.7 Two-component systems with sensor-regulator proteins 42812.1.8 Autogenous regulation 42812.1.9 Post-transcriptional regulation of gene expression 43012.1.9.1 RNA stability 43012.1.9.2 mRNA structure and translational efficiency 43112.1.9.3 Modulation of translation and stability of mRNAby protein 43112.1.9.4 Modulation of translation and stability of mRNAby small RNA and small RNA-proteincomplex: riboregulation 43312.2 Global regulation: responses to environmental stress 43512.2.1 Stringent response 43712.2.2 Response to ammonia limitation 43912.2.3 Response to phosphate limitation: the pho system 44112.2.4 Regulation by molecular oxygen in facultativeanaerobes 44212.2.4.1 arc system 44312.2.4.2 fnr system 44412.2.4.3 RegB/RegA system in purple non-sulfurphotosynthetic bacteria 44512.2.5 Oxidative stress 44512.2.6 Heat shock response 44612.2.7 Cold shock response 448CONTENTS xix12.2.8 Quorum sensing 45212.2.9 Response to changes in osmotic pressure 45312.2.10 Other two-component systems 45412.2.11 Chemotaxis 45512.2.12 Adaptive mutation 45712.3 Regulation through modulation of enzymeactivity: fine regulation 45912.3.1 Feedback inhibition and feedforward activation 45912.3.2 Enzyme activity modulation through structuralchanges 46012.3.2.1 Phosphorylation 46112.3.2.2 Adenylylation 46112.3.2.3 Acetylation 46212.3.2.4 Other chemical modifications 46312.3.2.5 Regulation through physical modification anddissociation/association 46312.4 Metabolic regulation and growth 46412.4.1 Regulation in central metabolism 46412.4.2 Regulatory network 46412.4.3 Growth rate and regulation 46612.5 Secondary metabolites 46612.6 Metabolic regulation and the fermentation industry 46712.6.1 Fermentative production of antibiotics 46712.6.2 Fermentative amino acid production 467Further reading 46813 Energy, environment and microbial survival 48213.1 Survival and energy 48213.2 Reserve materials in bacteria 48313.2.1 Carbohydrate reserve materials: glycogen and trehalose 48313.2.2 Lipid reserve materials 48413.2.2.1 Poly--hydroxyalkanoate (PHA) 48513.2.2.2 Triacylglyceride (TAG) 48613.2.2.3 Wax ester and hydrocarbons 48613.2.3 Polypeptides as reserve materials 48713.2.4 Polyphosphate 48813.3 Resting cells 48913.3.1 Sporulation in Bacillus subtilis 49013.3.2 Cysts 49013.3.3 Viable but non-culturable (VBNC) cells 49013.3.4 Nanobacteria 49213.3.5 Programmed cell death (PCD) in bacteria 492Further reading 493Index 496xx CONTENTSPrefaceKnowledge of the physiology and metabolism of prokaryotes under-pins our understanding of the roles and activities of these organismsin the environment, including pathogenic and symbiotic relation-ships, as well as their exploitation in biotechnology. Prokaryoticorganisms include bacteria and archaea and, although remainingrelatively small and simple in structure throughout their evolutionaryhistory, exhibit incredible diversity regarding their metabolism andphysiology. Such metabolic diversity is reflective of the wide range ofhabitats where prokaryotes canthrive andinmany cases dominate thebiota, and is a distinguishing contrast with eukaryotes that exhibit amore restricted metabolic versatility. Thus, prokaryotes can be foundalmost everywhere under a wide range of physical and chemicalconditions, including aerobic to anaerobic, light and dark, low tohigh pressure, low to high salt concentrations, extremes of acidityand alkalinity, and extremes of nutrient availability. Some physiolo-gies, e.g. lithotrophy and nitrogen fixation, are only found in certaingroups of prokaryotes, while the use of inorganic compounds, suchas nitrate and sulfate, as electron acceptors in respiration is anotherprokaryotic ability. The explosion of knowledge resulting from thedevelopment and application of molecular biology to microbial sys-tems has perhaps led to a reduced emphasis on their physiology andbiochemistry, yet paradoxically has enabled further detailed analysisand understanding of metabolic processes. Almost in a reflection ofthe bacterial growth pattern, the number of scientific papers hasgrown at an exponential rate, while the number of prokaryoticgenome sequences determined is also increasing rapidly. This pro-ductionof genome sequences for a wide range of organisms has madean in-depth knowledge of prokaryotic metabolic function even moreessential in order to give biochemical, physiological and ecologicalmeaning to the genomic information. Our objective in writing thisnew textbook was to provide a thorough survey of the prokaryoticmetabolic diversity that occurs under different conditions and indifferent environments, emphasizing the key biochemical mechan-isms involved. We believe that this approach provides a useful over-view of the key cellular processes that determine bacterial andarchaeal roles in the environment, biotechnology and human health.We concentrate on bacteria and archaea but, where appropriate, alsoprovide comparisons with eukaryotic organisms. It should be notedthat many important metabolic pathways found in prokaryotes alsooccur in eukaryotes further emphasizing prokaryotic importance asresearch models in providing knowledge of relevance to eukaryoticprocesses.This book can be considered in three main parts. In the first part,prokaryotic structure and composition is described as well as themeans by which nutrients are transported into cells acrossmembranes. Discussion of biosynthesis and growth is followed bydetailed accounts of glucose metabolism through glycolysis, the TCAcycle, electron transport and oxidative phosphorylation, largelybased on the model bacterium Escherichia coli. In the second part,the trophic variations found in prokaryotes are described, includingthe use of organic compounds other than glucose, anaerobic fermen-tation, anaerobic respiration, chemolithotrophy and photosynthesis.In the third part, the regulation of metabolism through control ofgene expression and enzyme activity is covered, as well as the survi-val mechanisms used by prokaryotes under starvation conditions.This text is relevant to advanced undergraduate and postgraduatecourses, as well as being of use to teachers and researchers in micro-biology, molecular biology, biotechnology, biochemistry and relateddisciplines.We would like to express our thanks to all those who helped andmade this book possible. We appreciate the staff of AcademyPublisher (Seoul, Korea) who re-drew the figures for the book, andthose at Cambridge University Press involved at various stages of thepublication process, including Katrina Halliday, Clare Georgy, DawnPreston, Alison Evans and Janice Robertson. Special thanks also go toDiane Purves in Dundee, who greatly assisted correction, collation,editing and formatting of chapters, and production of the index, andDr Nicola Stanley-Wall, also in Dundee, for the cover illustrationimages. Thanks also to all those teachers and researchers in micro-biology around the world who have helped and stimulated usthroughout our careers. Our families deserve special thanks fortheir support and patience.Byung Hong KimGeoffrey Michael Gaddxxii PREFACE1Introduction to bacterialphysiology and metabolismThe biosphere has been shaped both by physical events and by inter-actions with the organisms that occupy it. Among living organisms,prokaryotes are much more metabolically diverse than eukaryotesand can also thrive under a variety of extreme conditions whereeukaryotes cannot. This is possible because of the wealth of genes,metabolic pathways and molecular processes that are unique toprokaryotic cells. For this reason, prokaryotes are very important inthe cycling of elements, including carbon, nitrogen, sulfur and phos-phorus, as well as metals and metalloids such as copper, mercury,selenium, arsenic and chromium. A full understanding of the com-plex biological phenomena that occur in the biosphere thereforerequires a deep knowledge of the unique biological processes thatoccur in this vast prokaryotic world.After publication in 1995 of the first full DNA sequence of a free-living bacterium, Haemophilus influenzae, whole genome sequencesof hundreds of prokaryotes have now been determined and manyothers are currently being sequenced (www.genomesonline.org/).Our knowledge of the whole genome profoundly influences allaspects of microbiology. Determination of entire genome sequences,however, is only a first step in fully understanding the properties ofan organism and the environment in which the organism lives. Thefunctions encoded by these sequences need to be elucidated to givebiochemical, physiological and ecological meaning to the informa-tion. Furthermore, sequence analysis indicates that the biologicalfunctions of substantial portions of complete genomes are so farunknown. Defining the role of each gene in the complex cellularmetabolic network is a formidable task. Inaddition, genomes containhundreds to thousands of genes, many of which encode multipleproteins that interact and function together as multicomponentsystems for accomplishing specific cellular processes. The productsof many genes are often co-regulated in complex signal transductionnetworks, and understanding how the genome functions as a wholepresents an even greater challenge. It is also known that for a sig-nificant proportion of metabolic activities, no representative geneshave been identified across all organisms, such activities beingtermed orphan to indicate they are not currently assigned to anygene. This also represents a major future challenge and will requireboth computational and experimental approaches.It is widely accepted that less than 1% of prokaryotes have beencultivated in pure culture under laboratory conditions. Developmentof new sequencing techniques has allowed us to obtain genomicinformation fromthe multitudes of unculturable prokaryotic speciesand complex microbial populations that exist in nature. Such infor-mation might provide a basis for the development of newcultivationtechniques. Elucidation of the function of unknown genes througha better understanding of biochemistry and physiology could ulti-mately result in a fuller understanding of the complex biologicalphenomena occurring in the biosphere.Unlike multicellular eukaryotes, individual cells of unicellularprokaryotes are more exposed to the continuously changing envir-onment, and have evolved unique structures to survive under suchconditions. Chapter 2 describes the main aspects of the compositionand structure of prokaryotic cells.Life can be defined as a reproduction process using materialsavailable fromthe environment according to the genetic informationpossessed by the organism. Utilization of the materials available inthe environment necessitates transport into cells that are separatedfrom the environment by a membrane. Chapter 3 outlines transportmechanisms, not only for intracellular entry of nutrients, but also forexcretion of materials including extracellular enzymes and materialsthat form cell surface structures.Many prokaryotes, including Escherichia coli, can grow in a simplemineral salts medium containing glucose as the sole organic com-pound. Glucose is metabolized through glycolytic pathways and thetricarboxylic acid (TCA) cycle, supplying all carbon skeletons, energyin the form of ATP and reducing equivalents in the form of NADPHfor growth and reproduction. Glycolysis is described in Chapter 4with emphasis on the reverse reactions of the EMP pathway andon prokaryote-specific metabolic pathways. When substratesother than glucose are used, parts of the metabolic pathwaysare employed in either forward or reverse directions. Chapter 5describes the TCA cycle and related metabolic pathways, and energytransduction mechanisms. Chapter 6 describes the biosyntheticmetabolic processes that utilize carbon skeletons, ATP and NADPH,the production of which is discussed in the previous chapters. Thesechapters summarize the biochemistry of central metabolism that isemployed by prokaryotes to enable growth on a glucosemineralsalts medium.The next five chapters describe metabolism in some of thevarious trophic variations found in prokaryotes. These are the useof organic compounds other than glucose as carbon and energysources (Chapter 7), anaerobic fermentation (Chapter 8), anaerobicrespiratory processes (Chapter 9), chemolithotrophy (Chapter 10) andphotosynthesis (Chapter 11). Some of these metabolic processes are2 I NTRODUCTI ON TO BACTERI AL PHYSI OLOGY AND METABOLI SMprokaryote specific, while others are found in both prokaryotes andeukaryotes.Prokaryotes only express a proportion of their genes at any giventime, just like eukaryotes. This enables them to grow in the mostefficient way under any given conditions. Metabolism is regulatednot only through control of gene expression but also by controllingthe activity of enzymes. These regulatory mechanisms are discussedin Chapter 12. Finally, the survival of prokaryotic organisms understarvation conditions is discussed in terms of storage materials andresting cell structures in Chapter 13.This book has been written as a text for senior students at under-graduate level and postgraduates in microbiology and related sub-jects. A major proportion of the book has been based on reviewpapers published in various scientific journals including those listedbelow:Annual Review of MicrobiologyAnnual Review of BiochemistryCurrent Opinion in MicrobiologyFEMS Microbiology ReviewsJournal of BacteriologyMicrobiology and Molecular Biology Reviews (formerly MicrobiologyReviews)Nature Reviews MicrobiologyTrends in Microbiology.The authors would also like to acknowledge the authors of the bookslisted below that have been consulted during the preparation ofthis book.Caldwell, D. R. (2000). Microbial Physiology and Metabolism, 2nd edn. Belm, CA:Star Publishing Co.Dawes, D. A. (1986). Microbial Energetics. Glasgow: Blackie.Dawes, I. W. & Sutherland, I. W. (1992). Microbial Physiology, 2nd edn. BasicMicrobiology Series, 4. Oxford: Blackwell.Gottschalk, G. (1986). Bacterial Metabolism, 2nd edn. New York: Springer-Verlag.Ingraham, J. L., Maaloe, O. & Neidhardt, F. C. (1983). Growth of the Bacterial Cell.Sunderland, MA: Sinauer Associates Inc.Mandelstam, J., McQuillin, K. & Dawes, I. (1982). Biochemistry of BacterialGrowth, 3rd edn. Oxford: Blackwell.Moat, A. G., Foster, J. W. & Spector, M. P. (2002). Microbial Physiology, 4th edn.New York: Wiley.Neidhardt, F. C. & Curtiss, R. (eds.) (1996). Escherichia coli and Salmonella: Cellularand Molecular Biology, 2nd edn. Washington, DC: ASM Press.Neidhardt, F. C., Ingraham, J. L. & Schaechter, M. (1990). Physiology of theBacterial Cell: A Molecular Approach. Sunderland, MA: Sinauer Associates Inc.Stanier, R. J., Ingraham, J. L., Wheelis, M. K. &Painter, P. R. (1986). The MicrobialWorld, 5th edn. Upper Saddle River, NJ: Prentice-Hall.White, D. (2000). The Physiology and Biochemistry of Prokaryotes, 2nd edn. Oxford:Oxford University Press.I NTRODUCTI ON TO BACTERI AL PHYSI OLOGY AND METABOLI SM 3FURTHER READI NGGeneralDowns, D. M. (2006). Understanding microbial metabolism. Annual Review ofMicrobiology 60, 533559.Galperin, M. Y. (2004). All bugs, big and small. Environmental Microbiology 6,435437.Klamt, S. & Stelling, J. (2003). Two approaches for metabolic pathway analy-sis? Trends in Biotechnology 21, 6469.Papin, J. A., Price, N. D., Wiback, S. J., Fell, D. A. & Palsson, B. O. (2003).Metabolic pathways in the post-genome era. Trends in Biochemical Sciences28, 250258.Park, S., Lee, S., Cho, J., Kim, T., Lee, J., Park, J. & Han, M. J. (2005). Globalphysiological understanding and metabolic engineering of microorgan-isms based on omics studies. Applied Microbiology and Biotechnology 68,567579.Postgate, J. R. (1992). Microbes and Man, 3rd edn. Cambridge: CambridgeUniversity Press.DiversityCrawford, R. L. (2005). Microbial diversity and its relationship to planetaryprotection. Applied and Environmental Microbiology 71, 41634168.DeLong, E. F. (2001). Microbial seascapes revisited. Current Opinion inMicrobiology 4, 290295.Fernandez, L. A. (2005). Exploring prokaryotic diversity: there are other mole-cular worlds. Molecular Microbiology 55, 515.Fredrickson, J. & Balkwill, D. (2006). Geomicrobial processes and biodiversityin the deep terrestrial subsurface. Geomicrobiology Journal 23, 345356.Pedros-Alio, C. (2006). Marine microbial diversity: can it be determined?Trends in Microbiology 14, 257263.Rappe, M. S. & Giovannoni, S. J. (2003). The uncultured microbial majority.Annual Review of Microbiology 57, 369394.EcologyGadd, G. M., Semple, K. T. &Lappin-Scott, H. M. (2005). Micro-organisms and EarthSystems: Advances in Geomicrobiology. Cambridge: Cambridge University Press.Galperin, M. Y. (2004). Metagenomics: from acid mine to shining sea.Environmental Microbiology 6, 543545.Geesey, G. G. (2001). Bacterial behavior at surfaces. Current Opinion inMicrobiology 4, 296300.Ivanov, M. V. & Karavaiko, G. I. (2004). Geological microbiology. Microbiology-Moscow 73, 493508.Johnston, A. W. B., Li, Y. & Ogilvie, L. (2005). Metagenomic marine nitrogenfixation feast or famine? Trends in Microbiology 13, 416420.Karl, D. (2002). Nutrient dynamics in the deep blue sea. Trends in Microbiology10, 410418.Riesenfeld, C. S., Schloss, P. D. & Handelsman, J. (2004). Metagenomics: geno-mic analysis of microbial communities. Annual Review of Genetics 38, 525552.4 I NTRODUCTI ON TO BACTERI AL PHYSI OLOGY AND METABOLI SMShively, J. M., English, R. S., Baker, S. H. & Cannon, G. C. (2001). Carbon cycling:the prokaryotic contribution. Current Opinion in Microbiology 4, 301306.Tyson, G. W. & Banfield, J. F. (2005). Cultivating the uncultivated: a commu-nity genomics perspective. Trends in Microbiology 13, 411415.EvolutionAltermann, W. & Kazmierczak, J. (2003). Archean microfossils: a reappraisalof early life on Earth. Research in Microbiology 154, 611617.Arber, W. (2000). Genetic variation: molecular mechanisms and impact onmicrobial evolution. FEMS Microbiology Reviews 24, 17.Boucher, Y., Douady, C. J., Papke, R. T., Walsh, D. A., Boudreau, M. E., Nesbo, C. L.,Case, R. J. & Doolittle, W. F. (2003). Lateral gene transfer and the origins ofprokaryotic groups. Annual Review of Genetics 37, 283328.Groisman, E. A. & Casadesus, J. (2005). The origin and evolution of humanpathogens. Molecular Microbiology 56, 17.Koch, A. L. (2003). Were Gram-positive rods the first bacteria? Trends inMicrobiology 11, 166170.Koch, A. L. & Silver, S. (2005). The first cell. Advances in Microbial Physiology 50,227259.Moran, N. (2003). Tracing the evolution of gene loss in obligate bacterialsymbionts. Current Opinion in Microbiology 6, 512518.Orgel, L. E. (1998). The origin of life a reviewof facts and speculations. Trendsin Biochemical Sciences 23, 491495.Ouzounis, C. A., Kunin, V., Darzentas, N. & Goldovsky, L. (2006). A minimalestimate for the gene content of the last universal common ancestor exobiology from a terrestrial perspective. Research in Microbiology 157,5768.Rainey, P. B. & Cooper, T. F. (2004). Evolution of bacterial diversity and theorigins of modularity. Research in Microbiology 155, 370375.Sallstrom, B. & Andersson, S. G. E. (2005). Genome reduction in thea-proteobacteria. Current Opinion in Microbiology 8, 579585.Trevors, J. T. (1997). Bacterial evolution and metabolism. Antonie vanLeeuwenhoek 71, 257263.Trevors, J. T. (2003). Origin of the first cells on Earth: a possible scenario.Geomicrobiology Journal 20, 175183.vander Meer, J. R. &Sentchilo, V. (2003). Genomic islands and the evolution ofcatabolic pathways in bacteria. Current Opinion in Biotechnology 14, 248254.Weinbauer, M. G. & Rassoulzadegan, F. (2004). Are viruses driving microbialdiversification and diversity? Environmental Microbiology 6, 111.GenomicsBoucher, Y., Nesbo, C. L. & Doolittle, W. F. (2001). Microbial genomes: dealingwith diversity. Current Opinion in Microbiology 4, 285289.Clayton, R. A., White, O. & Fraser, C. M. (1998). Findings emerging fromcomplete microbial genome sequences. Current Opinion in Microbiology 1,562566.Conway, T. &Schoolnik, G. K. (2003). Microarray expression profiling: captur-ing a genome-wide portrait of the transcriptome. Molecular Microbiology 47,879889.FURTHER READI NG 5Doolittle, R. F. (2005). Evolutionary aspects of whole-genome biology. CurrentOpinion in Structural Biology 15, 248253.Francke, C., Siezen, R. J. & Teusink, B. (2005). Reconstructing the metabolicnetwork of a bacteriumfromits genome. Trends in Microbiology 13, 550558.Glaser, P. & Boone, C. (2004). Beyond the genome: from genomics to systemsbiology. Current Opinion in Microbiology 7, 489491.Groisman, E. A. & Ehrlich, S. D. (2003). Genomics: a global view of gene gain,loss, regulation and function. Current Opinion in Microbiology 6, 479481.Koonin, E. V. (2004). Comparative genomics, minimal gene-sets and the lastuniversal common ancestor. Nature Reviews Microbiology 1, 127136.Nelson, K. E. (2003). The future of microbial genomics. EnvironmentalMicrobiology 5, 12231225.Puhler, A. &Selbitschka, W. (2003). Genome research on bacteria relevant foragriculture, environment and biotechnology. Journal of Biotechnology 106,119120.Ward, N. & Fraser, C. M. (2005). How genomics has affected the concept ofmicrobiology. Current Opinion in Microbiology 8, 564571.Extreme environmentsCowan, D. A. (2004). The upper temperature for life where do we draw theline? Trends in Microbiology 12, 5860.Deming, J. (2002). Psychrophiles and polar regions. Current Opinion inMicrobiology 5, 301309.Javaux, E. J. (2006). Extreme life on Earth: past, present and possibly beyond.Research in Microbiology 157, 3748.Mock, T. & Thomas, D. N. (2005). Recent advances in sea-ice microbiology.Environmental Microbiology 7, 605619.Simonato, F., Campanaro, S., Lauro, F. M., Vezzi, A., DAngelo, M., Vitulo, N.,Valle, G. & Bartlett, D. H. (2006). Piezophilic adaptation: a genomic point ofview. Journal of Biotechnology 126, 1125.Steven, B., Leveille, R., Pollard, W. H. & Whyte, L. G. (2006). Microbial ecologyand biodiversity in permafrost. Extremophiles 10, 259267.New areasAnderson, N. L., Matheson, A. D. & Steiner, S. (2000). Proteomics: applicationsin basic and applied biology. Current Opinion in Biotechnology 11, 408412.Chen, L. & Vitkup, D. (2007). Distribution of orphan metabolic activities.Trends in Biotechnology 25, 343348.Dufrene, Y. F. (2002). Atomic force microscopy, a powerful tool in microbio-logy. Journal of Bacteriology 184, 52055213.Whitfield, E. J., Pruess, M. & Apweiler, R. (2006). Bioinformatics databaseinfrastructure for biotechnology research. Journal of Biotechnology 124,629639.6 I NTRODUCTI ON TO BACTERI AL PHYSI OLOGY AND METABOLI SM2Composition and structureof prokaryotic cellsLike all organisms, microorganisms grow, metabolize and replicateutilizing materials available from the environment. Such materialsinclude those chemical elements required for structural aspects ofcellular composition and metabolic activities such as enzyme regu-lation and redox processes. To understand bacterial metabolism, it istherefore helpful to know the chemical composition of the cell andcomponent structures. This chapter describes the elemental compo-sition and structure of prokaryotic cells, and the kinds of nutrientsneeded for biosynthesis and energy-yielding metabolism.2.1 Elemental compositionFrom over 100 natural elements, microbial cells generally onlycontain 12 in significant quantities. These are known as major ele-ments, and are listed in Table 2.1 together with some of their majorfunctions and predominant chemical forms used by microorganisms.They include elements such as carbon (C), oxygen (O) and hydro-gen (H) constituting organic compounds like carbohydrates.Nitrogen (N) is found in microbial cells in proteins, nucleic acidsand coenzymes. Sulfur (S) is needed for S-containing amino acids suchas methionine and cysteine and for various coenzymes. Phosphorus(P) is present in nucleic acids, phospholipids, teichoic acid andnucleotides including NAD(P) and ATP. Potassium is the major inor-ganic cation (K), while chloride (Cl) is the major inorganic anion.Kis required as a cofactor for certain enzymes, e.g. pyruvate kinase.Chloride is involved in the energy conservation process operated byhalophilic archaea (Section 11.6). Sodium (Na) participates in sev-eral transport and energy transduction processes, and plays a crucialrole in microbial growth under alkaline conditions (Section 5.7.4).Magnesium(Mg2) forms complexes with phosphate groups includingthose found in nucleic acids, ATP, phospholipids and lipopolysacchar-ides. Several microbial intracellular enzymes, e.g. monomeric alkalinephosphatase, are calcium dependent. Ferrous and ferric ions play acrucial role in oxidationreduction reactions as components of elec-tron carriers such as Fe-S proteins and cytochromes.In addition to these 12 major elements, others are also foundin microbial cells as minor elements (Table 2.2). All the metals listedin Table 2.2 are required for specific enzymes. It is interesting tonote that the atomic number of tungsten is far higher than thatof the other elements and that this element is only required inrare cases.2.2 Importance of chemical form2.2.1 Five major elementsThe elements listed in Tables 2.1 and 2.2 need to be supplied or bepresent in the chemical forms that the organisms can use. Carbonis the most abundant element in all living organisms. Prokaryotesare broadly classified according to the carbon sources they use:organotrophs (heterotrophs) use organic compounds as their carbonsource while CO2 is used by lithotrophs (autotrophs). These groupsTable 2.1. Major elements found in microbial cells with their functions and predominant chemical formsused by microorganismsElementAtomicnumberChemical forms usedby microbes FunctionC 6 organic compounds, CO, CO2 major constituents of cell materialin proteins, nucleic acids, lipids,carbohydrates and othersO 8 organic compounds, CO2, H2O, O2H 1 organic compounds, H2O, H2N 6 organic compounds, NH4, NO3, N2S 16 organic sulfur compounds, SO42,HS, S0, S2O32proteins, coenzymesP 15 HPO42 nucleic acids, phospholipids, teichoicacid, coenzymesK 19 K major inorganic cation, compatiblesolute, enzyme cofactorMg 12 Mg2 enzyme cofactor, bound to cell wall,membrane and phosphate estersincluding nucleic acids and ATPCa 20 Ca2 enzyme cofactor, bound to cell wallFe 26 Fe2, Fe3 cytochromes, ferredoxin,Fe-S proteins, enzyme cofactorNa 11 Na involved in transport and energytransductionCl 17 Cl major inorganic anion8 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSare divided further according to the form of energy they use: chemo-trophs (chemoorganotrophs and chemolithotrophs) depend on che-mical forms for energy while phototrophs (photoorganotrophs andphotolithotrophs) utilize light energy (organo refers to an organicsubstance while litho refers to an inorganic substance).Nitrogen sources commonly used by microbes include organicnitrogenous compounds such as amino acids, and inorganic formssuch as ammonium and nitrate. Gaseous N2 can serve as a nitrogensource for a limited number of nitrogen-fixing prokaryotes. Nitrogenfixation is not known in eukaryotes. Some chemolithotrophs can useammonium as their energy source (electron donor, Section 10.2)while nitrate can be used as an electron acceptor by denitrifiers(Section 9.1).Sulfate is the most commonly used sulfur source, while othersulfur sources used include organic sulfur compounds, sulfide, ele-mental sulfur and thiosulfate. Sulfide and sulfur can serve as electrondonors in certain chemolithotrophs (Section 10.3), and sulfate andelemental sulfur are used as electronacceptors and reduced to sulfideby sulfidogens (Section 9.3).2.2.2 OxygenOxygen in cells originates mainly from organic compounds, water orCO2. Molecular oxygen (O2) is seldom used in biosynthetic processes.Some prokaryotes use O2 as the electron acceptor, but some cannotgrow in its presence. Thus, organisms can be grouped according totheir reaction with O2 into aerobes that require O2, facultative anae-robes that use O2when it is available but can also growin its absence,and obligate anaerobes that do not use O2. Some obligate anaerobescannot grow and/or lose their viability in the presence of O2 whileothers can tolerate it. The former are termed strict anaerobes and thelatter aerotolerant anaerobes.Table 2.2. Minor elements found in microbial cells with their functions and predominant chemical formused by microorganismsElementAtomicnumberChemical formused by microbes FunctionMn 23 Mn2 superoxide dismutase, photosystem IICo 27 Co2 coenzyme B12Ni 28 Ni hydrogenase, ureaseCu 29 Cu2 cytochrome oxidase, oxygenaseZn 30 Zn2 alcohol dehydrogenase, aldolase, alkaline phosphatase,RNA and DNA polymerase, arsenate reductaseSe 34 SeO32 formate dehydrogenase, glycine reductaseMo 42 MoO42 nitrogenase, nitrate reductase, formate dehydrogenase,arsenate reductaseW 74 WO42 formate dehydrogenase, aldehyde oxidoreductase2. 2 I MPORTANCE OF CHEMI CAL FORM 92.2.3 Growth factorsSome organotrophs such as Escherichia coli can grow in simple mediacontaining glucose and mineral salts, while others, like lactic acidbacteria, require complex media containing various vitamins, aminoacids andnucleic acidbases. This is because the latter organisms cannotsynthesize certain essential cellular materials from only glucose andmineral salts. These required compounds should therefore be suppliedin the growth media: such compounds are known as growth factors.Growth factor requirements differ between organisms with vitaminsbeing the most commonly required growth factors (Table 2.3).2.3 Structure of microbial cellsMicroorganisms are grouped into either prokaryotes or eukaryotesaccording to their cellular structure. With only a few exceptions,prokaryotic cells do not have subcellular organelles separated fromthe cytoplasm by phospholipid membranes such as the nuclear andmitochondrial membranes. Organelles like the nucleus, mitochon-dria and endoplasmic reticulum are only found in eukaryotic cells.The detailed structure of prokaryotic cells is described below.2.3.1 Flagella and piliMotile prokaryotic cells have an appendage called a flagellum(plural,flagella) involved in motility, and a similar but smaller structure, thefimbria (plural, fimbriae). Fimbriae are not involved in motility andare composed of proteins.The bacterial flagellum consists of three parts. These are a basalbody, a hook and a filament (Figure 2.1). The basal body is embeddedin the cytoplasmic membrane and cell surface structure andTable 2.3. Common growth factors required by prokaryotes and their major functionGrowth factor Functionp-aminobenzoate part of tetrahydrofolate, a one-carbon unit carrierBiotin prosthetic group of carboxylase and mutaseCoenzyme M methyl carrier in methanogenic archaeaFolate part of tetrahydrofolateHemin precursor of cytochromes and hemoproteinsLipoate prosthetic group of 2-keto acid decarboxylaseNicotinate precursor of pyridine nucleotides (NAD, NADP)Pantothenate precursor of coenzyme A and acyl carrier proteinPyridoxine precursor of pyridoxal phosphateRiboflavin precursor of flavins (FAD, FMN)Thiamine precursor of thiamine pyrophosphateVitamin B12 precursor of coenzyme B12Vitamin K precursor of menaquinone10 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSconnected to the filament through the hook. In Gram-negative bac-teria the basal body consists of a cytoplasmic membrane ring, aperiplasmic ring and an outer-membrane ring through which thecentral rod passes. The diameter of the rings can be 2050 nmdepending on the species. The cytoplasmic ring of the basal body isassociated with additional proteins known as the Mot complex. TheMot complex rotates the basal body with the entire flagellum con-suming a proton motive force (or sodiummotive force). The cytoplas-mic membrane ring is therefore believed to function as a motor withthe Mot complex. A more detailed description of motility is given inSection 12.2.11. In addition to the Mot complex, the basal body isassociated with an export apparatus through which the buildingblocks of the filament are transported.The hook connects the central rod of the basal body to the fila-ment and is composed of a single protein called the hook protein. Thefilament, with a diameter of 1020 nm, can be dissolved at pH 34with surfactants to a single protein solution of flagellin. The mole-cular weight of flagellin varies from 20 to 65 kD depending on thebacterial species. The hook and the filament are tube-shaped andthe flagellin moves through the tube to the growing tip of the fila-ment. The tip of the filament is covered with filament cap protein.Flagellin can be exported to the medium in mutants defective inexpression of this protein.Figure 2:1 The structure ofthe flagellum in Gram-negative bacteria.(J. Bacteriol. 180:10091022, 1998)Three rings of the basal body areembedded in the cytoplasmicmembrane, murein layer and outermembrane. The outer filament isconnected to the basal bodythrough the hook. The cytoplasmicmembrane ring of the basal body isassociated with the Mot complex.This complex functions like a motorrotating the flagellum, thusrendering the cell motile. Energy forthis rotation is provided in the formof the proton motive (or sodiummotive) force.2. 3 STRUCTURE OF MI CROBI AL CELLS 11The number and location of flagella vary depending on the bac-terial species. In some prokaryotes they are located at one or bothpoles, while the entire cell surface may be covered with flagella inothers.The fimbria, also known as the pilus (plural, pili), is observed inmany Gram-negative bacteria but rarely in Gram-positive bacteria.Fimbriae have been proposed as the fibrils that mediate attachmentto surfaces. For this reason, the term pilus should be used only todescribe the F-pilus, the structure that mediates conjugation.Fimbriae are generally smaller in length (0.220 mm) and width(314 nm) than flagella. Fimbriae help the organism to stick to sur-faces of other bacteria, to host cells of animals and plants, and to solidsurfaces. Different kinds of fimbriae are known which depend onthe species as well as the growth conditions for a given organism.Fimbriae consist of a major protein with minor proteins called adhe-sins that facilitate bacterial attachment to surfaces by recognizingthe appropriate receptor molecules. They are classified as type Ithrough to type IV according to such receptor recognition properties.Adhesive properties are inhibited by sugars such as mannose, galac-tose and their oligomers, suggesting that the receptors are carbo-hydrate in nature.Afibril bigger in size than fimbriae occurs in many Gram-negativebacteria that harbour the conjugative F-plasmid. This is called theF-pilus or sex pilus and mediates attachment between mating cellsfor the purpose of transmitting DNA from the donor cell by means ofthe F-pilus to a recipient cell. The F-pilus recognizes a receptor mole-cule on the surface of the recipient cell and after attachment, theF-pilus is depolymerized so that there is direct contact betweenthe cells for DNA transmission.2.3.2 Capsules and slime layersMany prokaryotic cells are covered with polysaccharides. In somecases the polymers are tightly integrated with the cell while in othersthey are loosely associated. The former is called a capsule, and thelatter a slime layer (Figure 2.2). Slime layer materials can diffuse intothe medium with their structure and composition being dependenton growth conditions. An important role for these structures isadhesion to host cells for invasion or to a solid surface to initiateand stabilize biofilmformation. These structures are also responsiblefor resistance to phagocytosis, thereby increasing virulence. In somebacteria the capsule functions as a receptor for phage. Since thepolysaccharides are hydrophilic, they can also protect cells fromdesiccation.The termglycocalyx can be used to describe extracellular structuresincluding the capsule and S-layer, the latter being described below.2.3.3 S-layer, outer membrane and cell wallUnicellular prokaryotes have elaborate surface structures. Theseinclude the S-layer, outer membrane and cell wall. The cell wallMC CWCMSLCFigure 2:2 Diagram of thesurface structure of aprokaryotic cell showing thecapsule and slime layer.C, capsule; CM, cytoplasmicmembrane; CW, cell wall; MC,micro-capsule; SL, slime layer.12 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSdetermines the physical shape of prokaryotic cells in most cases.Prokaryotes can be classified into four groups according to theircell wall structure. These are mycoplasmas, Gram-negative bacteria,Gram-positive bacteria and archaea. Cell walls are not found inmycoplasmas which are obligate intracellular pathogens. Murein isthe sole building block of the cell wall in Gram-negative bacteriawhich also have an outer membrane. Murein and teichoic acid con-stitute the cell wall in Gram-positive bacteria which do not possess anouter membrane. Some archaea have cell walls that do not containmurein, while others are devoid of cell walls.2.3.3.1 S-layerA protein or glycoprotein layer is found on the surface of all prokar-yotic cells except mycoplasmas. This is called an S-layer (Figure 2.3).All prokaryotic cells studied are surrounded by this layer, but thisproperty can be lost in some laboratory strains. This suggests thatthis layer is indispensable in natural environments. The proposedfunctions of the S-layer are (1) protection from toxic compounds,(2) adhesion to solid surfaces, (3) a phage receptor, (4) a physicalstructure to maintain cell morphology, and (5) a binding site forcertain extracellular enzymes.Strains devoid of an S-layer are less resistant to murein- andprotein-hydrolyzing enzymes, and more prone to release from bio-films with environmental changes such as pH. The S-layer serves as acell wall in some archaea. Amylase is bound to the S-layer in Bacillusstearothermophilus.The protein forming the S-layer is one of the most abundantproteins in bacterial cells, comprising 510% of total cellular protein.In a fast-growing organism, this protein needs to be synthesized andexported very efficiently. The promoter and signal sequence of thisprotein has therefore been studied for use in foreign protein produc-tion using bacteria.2.3.3.2 Outer membraneGram-negative bacteria are more resistant to lysozyme, hydrolyticenzymes, surfactants, bile salts and hydrophobic antibiotics thanGram-positive bacteria. These properties are due to the presence ofthe outer membrane in Gram-negative bacteria (Figure 2.3). The outermembrane (OM) is different in structure from the cytoplasmicmembrane (CM). The CM consists of phospholipids while lipopoly-saccharide (LPS) forms the outer leaflet of the OM with the innerleaflet composed of phospholipids. LPS provides a permeability bar-rier against the hydrophobic compounds listed above. In addition tothese lipids, the OM contains protein and lipoprotein (Table 2.4).Lipopolysaccharide (LPS) consists of three components: lipid A,core polysaccharide and repeating polysaccharide. The repeatingpolysaccharide is referred to as O-antigen. Lipid A is embedded in themembrane to form the lipid layer, and the sugar moieties extend intothe surface. The sugar moieties of LPS consist of hexoses, hexosamines,2. 3 STRUCTURE OF MI CROBI AL CELLS 13Figure 2:3 Cell surfacestructures of prokaryotic cells.(a) Archaea, (b) Gram-positivebacteria, (c) Gram-negativebacteria.S, S-layer; CM, cytoplasmicmembrane; CW, cell wall; OM,outer membrane; PG,peptidoglycan (murein).14 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSdeoxyhexoses and keto-sugars with different structures depending onthe species and on the culture conditions. Figure 2.4 shows the LPSstructures found in Salmonella typhimurium and Escherichia coli.O-antigen mutants of pathogens are less virulent and show adifferent phage susceptibility, suggesting that the O-antigen isinvolved in pathogenesis and phage recognition processes. The struc-ture of O-antigen varies with changes in growth conditions such astemperature, and this polysaccharide may therefore protect the cellduring such changes. Lipid A consists of 67 molecules of 3-hydroxyfatty acids bound to phosphorylated glucosamine (Figure 2.5).Figure 2:4 Lipopolysaccharidestructure in Salmonellatyphimurium and Escherichiacoli.Lipopolysaccharide (LPS) consistsof lipid A, core polysaccharide andrepeating polysaccharide. Lipid Aforms a bilayer membrane withthe lipid part of phospholipid andthe sugar part extending intothe surface. The repeatingpolysaccharide is involved inpathogenesis and is called O-antigen.LPS contains unique sugarsL-glycero-D-mannoheptose (Hep)and 2-keto-3-deoxyoctonate(KDO), and rare sugars such asabequose (Abe) and colitose (Col).Galactose (Gal), glucose (Glc),mannose (Man) and rhamnose(Rha) can also be present.Table 2.4. Outer membrane (OM) components and their functions in Escherichia coliComponent FunctionPhospholipid inner leafletLipopolysaccharide outer leaflet, hydrophilic in nature providing a barrier against hydrophobiccompounds. Stabilization of the surface structure by bonding with metalions such as Mg2Lipoprotein lipid part is embedded in the OM hydrophobic region, and the sugar part iscovalently bound to murein which stabilizes the OMOM protein A maintenance of OM stability, receptor for amino acids and peptides, F-pilusin recipient cell for conjugationPorin three different porins, OmpC, OmpF and PhoE, each consisting of threepeptides, act as specific and non-specific channels for hydrophilic solutesReceptor proteins for sugars, amino acids, vitamins, etc.Other proteins enzymes such as phospholipase, protease, etc., extracellular protein exportmachinery2. 3 STRUCTURE OF MI CROBI AL CELLS 15Due tophosphate andcarboxylic moieties inLPS, the Gram-negativebacterial cell surface is negatively charged. Divalent cations such asMg2 cross-link LPS, thus stabilizing OM structure. The OM becomesmore permeable to hydrophobic compounds when the cations areremoved using chelating agents such as EDTA. In addition to LPS andphospholipids, the OM contains various proteins which have variousfunctions including lipoprotein (Figure 2.6).Active transport of nutrients takes place across the cytoplasmicmembrane. Nutrients therefore need to cross the OM, and for this theOMhas porins and receptors. Porins are hydrophilic channels formedby three peptides spanning the OM, allowing the diffusion of solutesof molecular weight less than 600 Da. Escherichia coli has three porins,OmpC, OmpF and PhoE OmpC and OmpF being non-specific. OmpFis slightly bigger than OmpC, the synthesis of which is stimulatedFigure 2:6 The structure ofthe outer membrane of Gram-negative bacteria.(Angew Chem. 91:394407, 1979)Omp A, outer membrane protein A.Figure 2:5 Lipid A structurein Salmonella typhimurium.3-hydroxy fatty acids are bound toglucosamine and the corepolysaccharide is linked to carbon 6as indicated by the dotted line.16 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSunder high osmotic pressure when the synthesis of the former isrepressed(Section12.2.9). PhoEis synthesizedunder phosphate-limitedconditions and is specific for anions. The pore size of porins variesdepending on the species, and is regulated by the molecular arrange-ment around it. Nutrients with a higher molecular weight cross the OMthroughreceptor proteins. Thespeedof diffusionacross theOMthroughnon-specific porins and receptors is dependent on the concentrationgradient, but thespeedincreases onlyuptothesaturationconcentrationthrough the porins and receptors specific for that nutrient.OmpA functions as a receptor for various amino acids and pep-tides and for F-pili. Various other receptors are identified for nucleo-sides, vitamins and other nutrients. Lipoprotein stabilizes OMstructure. The lipid end of the molecule is embedded in the lipidarea of OM, while the protein end is covalently bound to murein.A lipoprotein in a plant pathogenic bacterium, Erwinia chrysanthemi,has pectin methylesterase activity.Like other microbes, many Gram-negative bacteria can use poly-mers as their carbon and energy sources (Section 7.1) and excretehydrolyzing enzymes into their external environment. The OM hasproteins responsible for the translocation of extracellular proteins(Section 3.8.2). Cytochromes and ferric reductase are located in theOM in Fe(III)-reducing bacteria such as Shewanella putrefaciens andGeobacter sulfurreducens (Section 9.2.1).2.3.3.3 Cell wall and periplasmWith a fewexceptions, prokaryotic cells have a cell wall that providesthe physical strength to maintain their shapes. Murein is the maincomponent of the cell wall of bacteria. The cell wall in Gram-negativebacteria is much thinner than in Gram-positive bacteria, which havea complex cell wall with other polymers and do not possess an outermembrane (Figure 2.3).Murein(peptidoglycan) is a polymer witha backbone of -1,4-linkedN-acetylglucosamine and N-acetylmuramate (Figure 2.7), the lactylgroup of which is cross-linked through tetrapeptides (Figure 2.8).Some Gram-positive bacteria, including Lactobacillus acidophilus, areresistant to lysozyme since they have murein O-acetylated at the 6-OHof N-acetylmuramate.D-glutamate and D-alanine occupy the 2nd and 4th positions ofthe tetrapeptide in most cases, and L-serine or glycine the 1st positiondepending on the species. Various amino acids are found in the 3rdposition including L-ornithine, L-lysine, L,L-diaminopimelate, meso-diaminopimelate and rarely L-homoserine. The 3rd amino acid in themurein tetrapeptide is a key criterion used for bacterial classifica-tion. The degree of cross-linking differs depending on the species,and is low at the growing tip of a single bacterial cell. Murein withreduced cross-linking is more susceptible to hydrolyzing enzymes.Gram-positive bacteria do not have an outer membrane but have amuch thicker cell wall containing teichoic acid, lipoteichoic acid andlipoglycan in addition to murein.2. 3 STRUCTURE OF MI CROBI AL CELLS 17The structure of teichoic acid differs depending on the bacterialspecies. Teichoic acids are polymers of ribitol phosphate, glycerolphosphate and their derivatives. In some cases, the hydroxyl groupsof the poly alcohols are bonded with amino acids or sugars. Teichoicacid is linked to murein through a linkage unit (Figure 2.9). Whilemurein is a structure rendering physical strength, teichoic acid hassome physiological functions. Due to the phosphate in teichoic acids,the surface of Gram-positive bacteria is negatively charged, attractingcations including Mg2 which stabilize cell wall structure. An auto-lytic enzyme is necessary to hydrolyze murein at the growing tip forgrowth of a cell. This enzyme is attached to teichoic acid, ensuring itis not released into the medium and enabling control of activity.Teichoic acid is also a receptor for certain phages. Gram-positivebacteria synthesize teichuronic acid in place of teichoic acid underphosphate-limited conditions.Lipoteichoic acid has a similar structure to teichoic acid. A diacyl-glycerol-containing sugar is attached to the terminal polyalcoholphosphate. This lipid part of lipoteichoic acid is embedded in thecytoplasmic membrane, while the polyalcohol phosphate partly con-stitutes the cell wall.The Gram-positive bacterial cell wall contains various proteins,including the autolytic enzymes mentioned above. They are attachedto the cell wall through the action of an enzyme called sortase(Section 6.9.2.4) after they are excreted through the cytoplasmicmembrane. Their functions include (1) metabolism of cell surfacestructures, (2) invasion into the host, (3) hydrolysis of polymers suchas proteins and polysaccharides and (4) adhesion to solid surfaces.Figure 2:7 Basic unit ofmurein in Escherichia coli.The backbone of murein is -1,4-linked N-acetylglucosamine andN-acetylmuramate. A tetrapeptideis covalently bonded to the lactylgroup of N-acetylmuramate.18 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSMycobacterium species possess a distinctive staining propertycalled acid-alcohol fastness. This property is due to the presence ofa high concentration of mycolic acid in their cell wall. This cell wallhas some similar properties to the outer membrane of Gram-negativebacteria and has a porin-like structure.The term periplasm is used to describe a separate compartmentbetween the outer membrane and the cytoplasmic membrane inGram-negative bacteria. Murein (cell wall) is contained within thiscompartment. The periplasm is in a gel state and contains proteinsand oligosaccharides. Under high osmotic pressure conditions,Gram-negative bacteria accumulate an oligosaccharide known asosmoregulated periplasmic glucan, which buffers the osmolarity.A variety of proteins are found in the periplasm including sensorproteins (Section 12.2.10), enzymes for the synthesis of cell surfacecomponents (Section 6.9), transporters (Section 3.8), solute-bindingproteins, regulatory proteins (Section 12.2.9), part of the electrontransport system(Sections 5.8.2, 5.8.3, 9.1.1), and hydrolytic enzymessuch as -lactamase, amylase and alkaline phosphatase. Pseudomonasaeruginosa releases outer membrane vesicles containing autolyticenzymes into the medium, causing the lysis of other bacteria.Members of the family Mycoplasmataceae and the class Chlamydiaeare obligate intracellular pathogens of humans and other animalsand do not have cell wall structures. However, the genome ofFigure 2:8 The structure ofmurein: the main componentof the eubacterial cell wall.The backbone of mureinconsists of a repeat of -1,4-linkedN-acetylglucosamine andN-acetylmuramate that is bondedwith a tetrapeptide. Thetetrapeptides of neighbouringbackbones are cross-linked tomake a net-like structure.(a) Escherichia coli; (b) Staphylococcusaureus; (c) Micrococcus luteus(Micrococcus lysodeikticus).2. 3 STRUCTURE OF MI CROBI AL CELLS 19Chlamydiae includes a complete set of genes for the synthesis ofmurein. They are also susceptible to antibiotics such as -lactamsand D-cycloserine which inhibit murein synthesis. It is not knownhow these biosynthetic genes are repressed or how the antibioticsinhibit growth. These organisms have cysteine-rich proteins in theirouter membrane. Disulfide bonds in the proteins render the cellsresistant to osmotic pressure.The phylum Planctomycetes is one of the major divisions of thedomain bacteria and is phylogenetically deep-rooted. These bacterialack murein in their cell wall, whichmainly comprises protein, and isprobably an S-layer.The archaeal cell wall is different from the bacterial cell wall. Themain components of the archaeal cell wall include pseudomurein(pseudopeptidoglycan), sulfonated polysaccharide and glycoprotein.Figure 2:9 Structure ofteichoic acid.(Microbiol. Mol. Biol. Rev.63:174229, 1999)Teichoic acid is a polymer of ribitolphosphate or glycerol phosphate.In some Gram-positive bacteria,the poly-alcohols are bondedwith alanine, galactose andN-acetylglucosamine. Linkage unitslink teichoic acid to murein.20 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSPseudomurein is found in the cell wall of Methanobacterium species.The structure of pseudomurein is different from that of murein.N-acetyltalosaminuronate is linked to N-acetylglucosamine througha -1,3-linkage in the place of -1,4-linked N-acetylmuramate.Consequently lysozyme does not hydrolyze pseudomurein. D-aminoacids are not found in pseudomurein. Members of the genusMethanosarcina have a cell wall consisting of polysaccharide, and thehalophilic archaeon Halococcus has a cell wall of sulfonated polysac-charide. Members of the genus Halobacterium and hyperthermophilicarchaea have glycoprotein as their cell wall, probably as an S-layer.2.3.4 Cytoplasmic membrane2.3.4.1 Properties and functionsThe cell contents (cytoplasm) need to be isolated from the externalenvironment, but at the same time nutrients must be transportedinto the cell. The cytoplasmic membrane mediates not only thesefunctions but also other important physiological activities. Theseinclude solute transport (Chapter 3), oxidative phosphorylationthrough electron transport (Section 5.8), photosynthetic electrontransport in photosynthetic prokaryotes (Section 11.4), maintenanceof electrochemical gradients and ATP synthesis (Section 5.8.4), moti-lity (Section 12.2.11), signal transduction (Section 12.2), synthesis ofcell surface structures and protein secretion (Section 3.8). The cyto-plasmic membrane consists of phospholipid (3550%) and protein(5065%). The phospholipid is responsible for the isolation propertyof the membrane with the various proteins being involved in the restof the membrane functions.Phospholipids have unique properties which determine the isola-tion of the cytoplasm. They are amphipathic compounds havinghydrophobic hydrocarbon chains (tails) and a hydrophilic headgroup in a single molecule. When an amphipathic compound ismixed with water, the hydrophilic heads and water interact witheach other and the hydrophobic tails do so in such a way that themolecules form micelles or liposomes (Figure 2.10). Because of thisproperty, a damaged cytoplasmic membrane can restore its structurespontaneously, and when the cells are broken the membrane formssmaller vesicles (Figure 2.11).When cells are broken with physical methods such as sonicationor the French press, inside-out vesicles are formed, while right-side-out vesicles are formed when the protoplast is osmotically lyzed afterthe cell wall is removed using enzymes. Vesicles are a useful tool tostudy membranes without the interference of cytosolic activities.Phospholipid forms bothinner and outer leaflets of the cytoplasmicmembrane, but the membrane is asymmetrical due to proteins presentinthe membrane. The phospholipid bilayer membrane is permeable tohydrophobic solutes and water but not to charged solutes and poly-mers. Membrane proteins transport these inandout of the cell. Thoughwater can diffuse through the membrane, the diffusion rate is too low2. 3 STRUCTURE OF MI CROBI AL CELLS 21to explain the rapid water flux which counters osmotic shock. Thisrapid flux is mediated by a protein, aquaporin.2.3.4.2 Membrane structureThe hydrocarbon part of the cytoplasmic membrane phospholipids isin a semi-solid state at physiological temperature. The proteins in themembrane are clustered in functional aggregates, with the aggre-gates floating around within the membrane. This membrane struc-ture is called a fluid mosaic model (Figure 2.12). Thin sections of thecytoplasmic membrane stained with metal compounds show twolines of strong electron absorption separated by a less electrondense zone (Figure 2.3). The electron dense area is the metal-bindinghead group separated by the hydrocarbon part which binds lessmetal. This structure is consistent with the behaviour of an amphi-pathic phospholipid.The fluidity of the membrane is determined by the meltingpoint of the hydrocarbon part of the phospholipid. PhospholipidFigure 2:10 Diagram of amicelle and liposomeformed when amphipathicphospholipid is mixed withwater.Figure 2:11 Membranevesicle formation from brokencells.(Dawes, E. A. 1986, MicrobialEnergetics, Figure 1.1. Blackie &Son, Glasgow)Right-side-out vesicles are obtainedwhen enzymically preparedprotoplasts are burst by osmoticshock, while physical methodswhich break cells result in inside-out vesicles.CM, cytoplasmic membrane; CW,cell wall; OM, outer membrane.22 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLScontaining more unsaturated fatty acid has a lowmelting point and isfound in the cytoplasmic membrane of psychrophiles. Thermophileshave a high degree of saturation in their membrane that manifestsitself as impaired transport function at low temperature. Freeze-fractured membranes show integral as well as peripheral proteins(Figure 2.3).2.3.4.3 PhospholipidsLipids found in the bacterial cytoplasmic membrane are shown inFigure 2.13. Phospholipids are the major lipid component in thecytoplasmic membrane. Sterols are universal lipids in eukaryoticcells, but are found only in obligate intracellular pathogenic myco-plasmas among prokaryotic cells. Hopanoids, members of the iso-prenoid family, are widespread in many bacteria (Figure 2.13g).Glycerophospholipids are dominant phospholipids in bacteria,and sphingolipids are found in certain bacterial cytoplasmicmembranes. Sphingolipids (Figure 2.13f) comprise more than 50%of cytoplasmic phospholipids in the obligate anaerobe Prevotellamelaninogenicus (formerly Bacteroides melaninogenicus). Other bacteriaknown to have sphingolipids include Prevotella ruminicola, Bdello-vibrio bacterivorus, Flectobacillus major, Bacteroides fragilis,Sphingobacterium spiritivorum and Porphyromonas gingivalis (formerlyBacteroides gingivalis).As shown in Figure 2.13, membrane phospholipids contain fattyacids with a carbon number of 1020, but mainly 1620. The obli-gately anaerobic fermentative bacterium Sarcina ventriculi containsfatty acids with carbon numbers of 1418 in its cytoplasmic mem-brane, but ,!-dicarboxylic acids with carbon numbers of 2836become the major fatty acids in its membrane in hostile environ-ments such as those of lowpH, high ethanol concentration, etc. Sincethe dicarboxylic acids form diglycerol tetraesters, this moleculespans the thickness of the membrane and reduces its fluidity.Diglycerol tetraesters are found in other anaerobes including mem-bers of Desulfotomaculum, Butyrivibrio, and thermophilic Clostridiumspecies. Diglycerol tetraethers are also found in archaea, as discussedlater.Figure 2:12 Schematicdiagram of the cytoplasmicmembrane.Protein comprises about 5065%ofthe membrane. The proteins in themembrane are clustered infunctional aggregates, and theaggregates float around within themembrane. This membranestructure is called a fluid mosaicmodel. Sterols are found only inobligate intracellular pathogenicmycoplasmas among prokaryotesas in eukaryotic cells (b), but not inmost prokaryotes (a). Hopanoids, amember of the isoprenoid family,are widespread in many bacteria.2. 3 STRUCTURE OF MI CROBI AL CELLS 23Plasmalogens are found in strictly anaerobic bacteria(Figure 2.13c). The C-1 of glycerol in this phospholipid has an etherlinkage to a long chain alcohol. Plasmalogens are not found in aero-bic, facultative anaerobic and microaerophilic bacteria. They areknown in species of the genera Bacteroides, Clostridium, Desulfovibrio,Peptostreptococcus, Propionibacterium, Ruminococcus, Selenomonas andVeillonella among others.To maintain membrane fluidity, more saturated fatty acids arefound in cells growing at higher temperature. In addition to tempera-ture, fatty acid composition is influenced by other environmentalfactors such as pH, osmotic pressure and organic solvents. Fatty acidscontaining a cyclopropane ring are found in many bacteria. These areknown as lactobacillic acids and were first identified in Lactobacillusarabinosus. They are formed under certain growth conditions or acertain growth phase. They are not present in vegetative cells ofAzotobacter vinelandii, but cysts of this organism possess them asmajor fatty acids.Archaea possess different phospholipids from bacteria. Archaeacomprise three physiologically distinct groups of organisms. Theseare halophiles and methanogens (both belong to Euryarchaeota), andhyperthermophiles (Crenarchaeota). Though they are distinct in theirphysiological characteristics and habitats, they share important phy-logenetic characters different from bacteria, including unique ether-linked phospholipids, ribosomal structure, and the lack of murein intheir cell walls.Figure 2:13 Phospholipids inbacteria.Hydrophobic tails of phospholipidsin the bacterial cytoplasmicmembrane include saturated,unsaturated, branched andcyclopropane fatty acids.Phosphatidyls are the mostcommon lipid in the bacterialcytoplasmic membrane. In mostphospholipids, fatty acids form anester linkage with glycerol, and inClostridium butyricum ether formsare found as plasmalogens.Sphingolipids are the mainphospholipids in certainanaerobes and hopanoids arefound in many bacteria.(a) Phosphatidylethanolamine;(b) phosphatidylglycerol;(c) phosphatidylethanolamineplasmalogen;(d) phosphatidylcholine;(e) phosphatidylinositol;(f) sphingomyelin; (g) hopanoids.24 COMPOSI TI ON AND STRUCTURE OF PROKARYOTI C CELLSThe hydrophobic tails of the phospholipids in archaea are isoprenoidalcohols ether-linked to glycerophosphate to form monoglycerol-dietheror diglycerol-tetraether (Figure 2.14). The alcohols are either 20 or 25 car-bons long in monoglycerol-diethers. The di-diphytanyl-diglycerol-tetraetherFigure 2:14 Phospholipids in archaea.The hydrophobic tails of the phospholipids in archaea are isoprenoid alcohols, either 20 (phytanyl) or 25 (sesterterpanyl) carbonslong and fully saturated. They are ether-linked to polyalcohols, mainly glycerol (a, b, c), and tetritol (f) and nonitol (g) are also found inarchaeal phospholipids.Phospholipids in halophilic archaea: complex lipids consisting of (a) 2,3-di-O-phytanyl-sn-glycerol (b), 2-O-sesterterpanyl-3-O-phytanyl-sn-glycerol, and (c) 2,3-di-O-sesterterpanyl-sn-glycerol, the C1 of which is linked to phosphate compounds, oligosaccharidesor sulfonated oligosaccharides.The common phospholipids in methanogenic archaea are (a) 2,3-di-O-phytanyl-sn-glycerol and (d) di-diphytanyl-diglycerol-tetraether.The C40 molecule (diphytanol) comprises two phytanols linked head-to-head as in (d). Oligosaccharides and phosphate compounds arelinked to the C1 of glycerol. In some methanogenic archaeal phospholipids, tetritol replaces glycerol.Di-diphytanyl-diglycerol-tetraether is the major phospholipid in hyperthermophilic archaea. Cyclopentanes are found in theirhydrophobic tails (e). Glycerol is the common polyalcohol in the tetraether (RHin (e)), and rarely nonitol (g) occupies one end of thetetraether compounds (R C6H13O6 in (e)).2. 3 STRUCTURE OF MI CROBI AL CELLS 25contains a C40dialcohol (diphyt