: Prokaryotic Cell Growth And Microbial Metabolism 4-2 Microbial Growth Bacterial growth Pure culture Environmental factors Nutritional factors Culture media/atmosphere Measuring bacterial growth 4-3 Bacterial Growth Binary fission Generation time Phases of growth 4-4 Principles of bacterial growth (4.1) Prokaryotic cells divide by binary fission One cell divides into two Two into four etc. 4-5 Principles of bacterial growth (4.1) Prokaryotic cells divide by binary fission One cell divides into two Two into four etc. Cell growth is exponential Doubling of population with each cell division Exponential growth has important health consequences Generation time Time it takes for population to double a.k.a doubling time Varies among species 4-6 Generation Time Ex. Escherichia coli doubles every 20 minutes Ex. Mycobacterium tuberculosis doubles every 12 to 24 hours 4-7 Biofilms - Prokaryotic growth in nature (4.3) 4-8 Insert image Growth curve (4.6) 4-9 Lag phase Number of cells does not increase Cells prepare for growth tooling up 4-10 Lag phase Number of cells does not increase Cells prepare for growth tooling up Log phase Period of exponential growth Doubling of population with each generation Produce primary metabolites Compounds required for growth Cells enter late log phase Synthesize secondary metabolites used to enhance survival antibiotics 4-11 Stationary phase Overall population remains relatively stable Cells exhausted nutrients Cell growth = cell death dying cell supply metabolites for replicating cells 4-12 Stationary phase Overall population remains relatively stable Cells exhausted nutrients Cell growth = cell death dying cell supply metabolites for replicating cells Death phase Total number of viable cells decreases Decrease at constant rate Death is exponential Much slower rate than growth 4-13 Phase of prolonged decline Once nearly 99% of all cells dead, remaining cells enter prolonged decline Marked by very gradual decrease in viable population Phase may last months or years Most fit cells survive Each new cell more fit than previous 4-14 Pure Culture Colony Streak-plate method 4-15 Obtaining pure culture (4.4) Culture media can be liquid or solid Liquid is broth media Used for growing large numbers of bacteria Solid media is broth media with addition of agar Agar marine algae extract Liquefies at temperatures above 95C Solidifies at 45C Remains solid at room temperature and body temperature Bacteria grow in colonies on solid media surface All cells in colony descend from single cell Approximately 1 million cells produce 1 visible colony 4-16 Streak-plate method (4.5) Simplest and most commonly used in bacterial isolation Object is to reduce number of cells being spread Solid surface dilution Each successive spread decreases number of cells per streak 4-17 Environmental Factors on Growth As group, prokaryotes inhabit nearly all environments Some live in comfortable habitats Some live in harsh environments Most of these are termed extremophiles and belong to domain Archaea Major conditions that influence growth Temperature Oxygen pH Water availability 4-18 Temperature (4.8) Each species has well defined temperature range Within range lies optimum growth temperature Prokaryotes divided into 5 categories Psychrophile Optimum temperature -5C to 15C, found in polar regions Psychrotroph 20C to 30C, important in food spoilage Mesophile 25C to 45C, more common, disease causing Thermophiles 45C to 70C, common in hot springs Hyperthermophiles 70C to 110C, usually members of Archaea, found in hydrothermal vents (Table 4.3) 4-20 Oxygen Obligate aerobes, absolute requirement for oxygen use for energy production Obligate anaerobes, no multiplication in presence of oxygen may cause death Facultative anaerobes, grow better with oxygen use fermentation in absence of oxygen Microaerophiles, require oxygen in lower concentrations higher concentration inhibitory Aerotolerant anaerobes, indifferent to oxygen Do not use oxygen to produce energy 4-21 pH Bacteria survive within pH range Neutrophiles Multiply between pH of 5 to 8 Maintain optimum near neutral Acidophiles Thrive at pH below 5.5 Maintains neutral internal pH, pumping out protons (H+) Alkalophiles Grow at pH above 8.5 Maintain neutral internal pH through sodium ion exchange Exchange sodium ion for external protons 4-22 Water Availability All microorganisms require water for growth Water is not available in all environments In high salt environments Bacteria increase internal solute concentration Synthesize small organic molecules Osmotolerant bacteria tolerate high salt environments Bacteria that require high salt for cell growth are termed halophiles 4-23 Effects of solute concentration on cells (4.9) 4-24 Nutritional Factors on Growth Growth of prokaryotes depends on nutritional factors as well as physical environment Main factors to be considered are: Required elements Growth factors Energy sources Nutritional diversity 4-25 Required Elements Major elements Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, calcium and iron Essential components for macromolecules Organisms classified based on carbon usage Heterotrophs Use organism carbon as nutrient source Autotrophs Use inorganic carbon (CO 2 ) as carbon source Trace elements Cobalt, zinc, copper, molybdenum and manganese Required in minute amounts Assist in enzyme function 4-26 4-27 Growth Factors Some bacteria cannot synthesize some cell constituents These must be added to growth environment Referred to as growth factors Organisms can display wide variety of factor requirements Some need very few while others require many These termed fastidious 4-28 Energy Sources Organisms derive energy from sunlight or chemical compounds Phototrophs Derive energy from sunlight Chemotrophs Derive energy from chemical compounds Organisms often grouped according to energy source 4-29 Nutritional Diversity Organisms thrive due to their ability to use diverse sources of carbon and energy Photoautotrophs Use sunlight and atmospheric carbon (CO 2 ) as carbon source Called primary producers (Plants) Chemolithoautotrophs a.k.a chemoautotrophs or chemolitotrophs Use inorganic chemicals for energy and use CO 2 as carbon source Photoheterotrophs Energy from sunlight, carbon from organic compounds Chemoorganoheterotrophs a.k.a chemoheterotrophs or chemoorganotrophs Use organic compounds for energy and carbon source Most common among humans and other animals 4-30 4-31 Laboratory Cultivation Knowing environmental and nutritional factors makes it possible to cultivate organisms in the laboratory Organisms are grown on culture media Media is classified as complex media or chemically defined media 4-32 Complex Media Contains a variety of ingredients There is no exact chemical formula for ingredients Can be highly variable Examples include Nutrient broth Blood agar Chocolate agar 4-33 Chemically Defined Media Composed of precise amounts of pure chemical Generally not practical for routine laboratory use Invaluable in research Each batch is chemically identical Does not introduce experimental variable Special Types of Culture Media Used to detect or isolate particular organisms Are divided into selective and differential media 4-34 Selective Media Inhibit the growth of unwanted organisms Allow only sought after organisms to grow Example Thayer-Martin agar For isolation of Neisseria gonorrhoeae cornstarch added to bind toxic fatty acids - Antimicrobials added to inhibit other microbes MacConkey agar For isolation of Gram negative bacteria differential for lactose fermentation - Crystal violet inhibits Gram-positive bacteria, bile salts inhibit non-intestinal bacteria 4-35 Differential media (4.11) Contains substance that bacteria change in recognizable way Example Blood agar Certain bacteria (eg Streptococcus) produce hemolysin to break down RBC Hemolysis MacConkey agar Contains pH indicator to identify bacteria that produce acid beta hemolysis (eg Streptococcus pyogenes) alpha hemolysis 4-36 MacConkey agar selective for Gram-negative rods residing in the intestinal tract. Red shows lactose fermentation; bile salts and dyes inhibit non intestinal species (4.11) 4-37 Providing Appropriate Atmospheric Conditions Bacteria can be grouped by oxygen requirement - Capnophiles - Microaerophiles - Anaerobes 4-38 Capnophiles Require increased CO 2 Achieve higher CO 2 concentrations via Candle jar CO 2 incubator 4-39 Capnophiles Require increased CO 2 Achieve higher CO 2 concentrations via Candle jar CO 2 incubator Microaerophiles Require higher CO 2 than capnophile Incubated in gastight jar Chemical packet generates hydrogen and CO 2 Anaerobes 4-40 Anaerobes (4.13) Die in the presence of oxygen Even if exposed for short period of time Incubated in anaerobe jar Chemical reaction converts atmospheric oxygen to water Organisms must be able to tolerate oxygen for brief period Reducing agents in media achieve anaerobic environment Agents react with oxygen to eliminate it Sodium thyoglycolate Anaerobic chamber also used for cultivation 4-41 4-42 Detecting Bacterial Growth Variety of techniques to determine growth Numbers of cells Total mass Detection of cellular products 4-43 Direct Cell Count Useful in determining total number of cells Does not distinguish between living and dead cells Methods include Direct microscopic count Use of cell counting instruments 4-44 Direct microscopic count (4.15) One of the most rapid methods Number is measured in a known volume Liquid dispensed in specialized slide Counting chamber Viewed under microscope Cells counted Limitation Must have at least 10 million cells per mL to gain accurate estimate 4-45 Cell counting instruments (4.16) Count cells in suspension Cells pass counter in single file Measure changes in environment Coulter counter Detects changes in electrical resistance Flow cytometer Measures laser light 4-46 Viable Cell Count Used to quantify living cells Cells able to multiply Valuable in monitoring bacterial growth Often used when cell counts are too low for other methods Methods include Plate counts Membrane filtration Most probable numbers 4-47 Plate counts (4.17) Measures viable cells growing on solid culture media Count based on assumption that one cell gives rise to one colony Number of colonies = number of cells in sample Ideal number to count between 30 and 300 colonies Sample diluted in 10- fold increments Plate count methods pour-plates spread-plates methods (4.18) 4-49 Membrane filtration (4.19) Used with relatively low numbers Known volume of liquid passed through membrane filter Filter pore size retains organism Filter is placed on appropriate growth medium and incubated Cells are counted 4-50 Most probable numbers (MPN) (4.20) Statistical assay Series of dilution sets created Each set inoculated with 10x less sample than previous set Sets incubated and results noted Results compared to MPN table Table gives statistical estimation of cell concentration 4-51 Biomass Measurement Cell mass can be determined via Turbidity Total weight Amounts of cellular chemical constituents 4-52 Turbidity (4.21) Measures with spectrophotometer Measures light transmitted through sample Measurement is inversely proportional to cell concentration Must be used in conjunction with other test once to determine cell numbers Limitation Must have high number of cells 4-53 Total Weight Tedious and time consuming Not routinely used Useful in measuring filamentous organisms Wet weight Cells centrifuged down and liquid growth medium removed Packed cells weighed Dry weight Packed cells allowed to dry at 100C 8 to 12 hours Cells weighed 4-54 Detecting Cell Products Acid production pH indicator detects acids that result from sugar breakdown Gas production Gas production monitored using Durham tube Tube traps gas produced by bacteria ATP Presence of ATP detected by use of luciferase Enzyme catalyzes ATP dependent reaction If reaction occurs ATP present bacteria present 4-55 Microbial Metabolism Cells must accomplish two fundamental tasks to grow Synthesize new components biosynthesis Harvest energy The sum total of chemical reactions of biosynthesis and energy-harvesting is termed metabolism 4-56 Principles of Metabolism Metabolism is broken down into two components Anabolism Catabolism Catabolism Degradative reactions Reactions produce energy from the break down of larger molecules Anabolism Reactions involved in the synthesis of cell components Anabolic reactions require energy Anabolic reactions utilize the energy produced from catabolic reactions 4-57 Scheme of Metabolism Three key pathways Central metabolic pathways Glycolysis Tricarboxcylic acid cycle Pentose phosphate pathway Central pathways are catabolic and provide Energy Reducing power Precursor metabolites 4-58 Glycolysis a.k.a Embden-Myerhoff pathway Oxidizes glucose to two molecules of pyruvate Pentose phosphate pathway (PPP) Breaks down glucose Produces molecules for biosynthesis Works in conjunction with glucose degrading pathways Tricarboxylic acid cycle (TCA) Before entering cycle, pyruvate enters transition step Pyruvate formed in glycolysis and PPP Cycle turns twice to complete oxidation of one glucose molecule 4-59 CO 2 GLUCOSE (f) Fermentation Reduces pyruvate or a derivative (e) Respiration Uses the electron transport chain to convert reducing power to proton motive force (d) TCA cycle Incorporates an acetyl group and releases CO 2 Acetyl-CoA Pyruvate Reducing power Reducing power Reducing power Reducing power Reducing power Acids, alcohols, and gases Yields Precursor metabolites Precursor metabolites ATP substrate-level phosphorylation (c) Transition step CO 2 ATP oxidative phosphorylation Precursor metabolites + Biosynthesis Yields ATP substrate-level phosphorylation (b) Pentose phosphate pathway (less commonly used than glycolysis) Initiates the oxidation (a) Glycolysis Oxidizes glucose to pyruvate CO 2 ~~ ~~ ~~ Scheme of Metabolism (6.10) 4-60 4-61 4-62 TB A New Target, a New Drug (Science 14January2005) Multidrug Resistant Tuberculosis (MDR-TB) outbreaks in NYC in early 1990s stimulated world governments to support drug development TB kills 2 million each year, no new drugs in 40 years Research by Adries et al in Science 14January2005 reveals a new TB drug TB drug should: Be highly active to reduce treatment time Kill persistent bacteria than might reactivate after treatment Show activity against MDR-TB strains Be specific for M. turberculosis and compatible with TB drugs Method: Medium throughput screening on live bacteria rather than high throughput screening of specific targets (enzymes) All hits were in the diarylquinoline family R found most effective active against drug-resistant strains excellent pharmacokinetics and pharmacodynamics Target identified by whole genome sequencing of resistant strains Mutations mapped to genes for the C subunit of ATP synthase 4-63 4-64 From Science 4-65 ATP synthase is highly conserved, present in virtually all cells Mycobacterial specificity could be caused by: 1) differences in C subunit structure 2) mycobacterial processing of R prodrug R may be effective against other mycobacterial diseases M. ulcerans Buruli ulcer, an emerging disease other mycobacterial diseases include: leprosy M. leprae MAC disease M. avium, M. intracellulare (MAC = Mycobacterium avium complex) 4-66 Respiration vs. Fermentation Respiration uses reducing power to generate ATP NADH and FADH 2 transfer electrons to produce proton motive force Allows for recycling of electron carriers Electrons join with terminal electron acceptor Oxygen in aerobic respiration Anaerobic respiration uses another inorganic molecule Fermentation is partial oxidation of glucose Produces very little ATP Uses pyruvate or derivative as terminal electron acceptor Other organisms may use other organic molecules as terminal electron acceptor 4-67 Metabolic Pathways Pathways modify organic molecules to form High energy intermediates to synthesize ATP Intermediates to generate reducing power Intermediates and end products as precursor metabolites Pathways Glycolysis Pentose Phosphate Pathway Tricarboxylic Acid Cycle 4-68 Glycolysis Primary pathway to convert one glucose to two pyruvate 10 step process Pathway generates Two 3 C pyruvate molecules Net gain of two ATP 2 ATP expended to break glucose 4 ATP harvested Two molecules reducing power NADH 6 different precursor metabolites 5 intermediates and pyruvate 4-69 ATP ADP Glucose Step 1: ATP is expended to add a phosphate group. Step 2: A chemical rearrangement occurs. Step 3: ATP is expended to add a phosphate group. Step 4: The 6-carbon molecule is split into two 3-carbon molecules. Step 5: A chemical rearrangement of one of the molecules occurs. Glucose 6-phosphate Fructose 1,6-bisphosphate Glyceraldehyde 3-phosphate Dihydroxyacetone phosphate Fructose 6-phosphate ~~ ~ ~~ ~ 4-70 ~ ~~ ~ ~~ ~ NADH + H + NAD + Step 5: A chemical rearrangement of one of the molecules occurs. Step 6: The addition of a phosphate group is coupled to an oxidation, generating NADH and a high-energy phosphate bond. Step 7: ATP is produced by substrate-level phosphorylation. Step 8: A chemical rearrangement occurs. Step 10: ATP is produced by substrate-level phosphorylation. Step 9: Water is removed, causing the phosphate bond to become high-energy. Glyceraldehyde 3-phosphate 1,3-bisphospho- glycerate 3-phospho- glycerate 2-phospho- glycerate Phospho- enolpyruvate Pyruvate H2OH2O ~ H2OH2O NADH + H + NAD + ~~ ~ ~~ ~ 4-71 Pentose Phosphate Pathway Generates 5 and 7 carbon sugars Also produces glyceraldehyde 3-phosphate Can go into glycolysis for further breakdown Major contributor to biosynthesis Produces reducing power in NADPH Two vital precursor metabolites 4-72 Transition Step Links glycolysis to Tricarboxylic Acid Cycle Modifies 3-C pyruvate from glycolysis to 2-C acetyl CoA CO 2 is removed through decarboxylation Remaining 2-C acetyl group joined to coenzyme A Forms Acetyl CoA NAD+ is reduced to NADH Each pyruvate enters transition step Reaction occurs twice for one glucose Yield from transition step Reducing power NADH Precursor metabolites Acetyl CoA 4-73 Tricarboxylic Acid Cycle Completes the oxidation of glucose Incorporates acetyl CoA from transition step Releases CO 2 in net reaction Cycle turns once for each acetyl CoA Two turns for each glucose molecule Cycle produces 2 ATP 6 NADH 2 FADH 2 2 precursor metabolites 4-74 ATP FADH 2 FAD NADH NAD + NADH NAD + Transition step: An oxidation generates NADH, CO 2 is removed, and coenzyme A is added. Step 1: The acetyl group is transferred to initiate a round of the cycle. Step 2: A chemical rearrangement occurs. Step 5: The energy released when CoA is removed is harvested to produce ATP. Step 8: Oxidation generates NADH. Pyruvate Acetyl-CoA CoA + P i H2OH2O Citrate Isocitrate CO 2 Succinyl-CoA Succinate Fumarate Malate Oxaloacetate NADH + H + NADH ~~ ~ CO 2 + H + CoA CO 2 -ketoglutarate Step 3: CO 2 is removed and an oxidation generates NADH. Step 4: An oxidation generates NADH, CO 2 is removed, and coenzyme A is added. Step 6: An oxidation generates FADH 2 Step 7: A molecule of water is added. 4-75 Respiration Uses NADH and FADH 2 to synthesize ATP Oxidative phosphorylation Occurs in electron transport chain Generates proton motive force Combined with ATP synthase Uses energy in proton motive force to synthesize ATP 4-76 Electron Transport Chain Group of membrane-embedded electron carriers Arrangement of carriers aids in production of proton motive force Four types of electron carriers Flavoproteins Iron-sulfur proteins Quinones Cytochromes 4-77 Mechanism of Proton Motive Force Certain carriers accept protons and electrons, some accept only electrons Pump protons across membrane Creates a proton gradient (proton motive force) Arrangement of carriers causes protons to be shuttled across membrane 4-78 Electron Transport Chain of Mitochondria Chain consists of following components Complex I a.k.a NADH dehydrogenase Complex II a.k.a succinate dehydrogenase Coenzyme Q Complex III Cytochrome C Complex IV Each carrier accepts electrons from previous carrier In process, protons are pumped across membrane 4-79 Electron transport chain of mitochondria (6.19) 4-80 Electron Transport Chain of Prokaryotes Respiration is either aerobic or anaerobic In aerobic respiration some prokaryotes have enzymes equivalent to complex I and II of mitochondria Do not have enzyme equivalents of complex III or cytochrome c Use quinones instead (ubiquinone) Shuttles electrons directly to terminal electron acceptor Oxygen acts as acceptor when available 4-81 H+H H + substance10 H + NAD Succinate CoQ reductase H2OH2O 1/2 O 2 3 ADP + 3 P i NADH + H + 3 ATP ATP synthase Active transport (one mechanism) Rotation of flagella Outside of cytoplasmic membrane Inside of cytoplasmic membrane Proton motive force is used to drive: Ubiquinone Prokaryotic cell Cytoplasmic membrane H + (0 or 4)H + (2 or 4) NADH dehydrogenaseUbiquinol oxidase 4-82 Electron Transport Chain in Prokaryotes Anaerobic respiration is less efficient Alternative electron carriers used Oxygen does not act as terminal electron acceptor Some bacteria use nitrate Nitrate converted to nitrite Nitrite converted to ammonia Sulfur-reducer bacteria use sulfate as terminal electron acceptor Quinone carrier (menaquinone) produces vitamin K 4-83 ATP Synthase Harvest energy from proton motive force to synthesize ATP Permits protons to flow back into cell Produces enough energy to phosphorylate ADP ATP One ATP is formed from entry of 3 protons 10 protons pumped out per NADH One NADH produces 3 molecules ATP 6 protons pumped out per FADH One FADH produces 2 molecules of ATP 4-84 ATP from Oxidative Phosphorylation ATP produced through re-oxidation of NADH and FADH 2 Maximum theoretical yield From glycolysis 2 NADH 6 ATP From transition step 2 NADH 6 ATP From TCA 6 NADH 18 ATP 2 FADH 2 4 ATP 4-85 Energy yield from aerobic respiration (6.21) 4-86 Total ATP Yield - Prokaryotic Aerobic Respiration Substrate phosphorylation 4 ATP Net 2 from glycolysis 2 ATP from TCA Oxidative phosphorylation 34 ATP 6 ATP from glycolysis (Re-oxidation of 2 NADH) 6 from transition step (Re-oxidation of NADH) 22 from TCA cycle (Re-oxidation of NADH and FADH 2 ) Total yield = 38 (theoretical maximum) Eukaryotic cells have theoretical maximum of 36 2 ATP spent crossing mitochondrial membrane 4-87 Fermentation Used by organisms that cannot respire Due to lack of suitable inorganic electron acceptor or lack of electron transport chain ATP produced only in glycolysis Other steps for consuming excess reducing power Recycles NADH Fermentation pathways use pyruvate or derivative as terminal electron acceptor 4-88 Fermentation pathways (6.22) 4-89 End products of fermentation include: Lactic acid Ethanol Butyric acid Propionic acid 2,3-Butanediol Mixed acids All are produced in a series of reactions to produce appropriate terminal electron acceptors 4-90 Fermentation pathway end products (6.23) 4-91 Anabolic Pathways - Synthesis of Subunits from Precursor Metabolites Pathways consume ATP, reducing power and precursor metabolites Macromolecules produce once subunits are synthesized 4-92 Lipid Synthesis Synthesis begins with transfer of acetyl group from acetyl CoA to acyl carrier protein Carrier holds fatty acid during elongation Fatty acid released when reaches required length 14, 16 or 18 carbons long Glycerol is synthesized from dihydroxyacetone phosphate 4-93 Amino Acid Synthesis Some precursors are formed in glycolysis, others in TCA cycle Glutamate synthesis essential for formation of other amino acids Synthesis incorporates ammonia with - ketoglutarate produce in TCA cycle Amino group from glutamate can be transferred to produce other amino acids Precursors for aromatic amino acids produced on pentose phosphate pathway and glycolysis 4-94 Nucleotide Synthesis Nucleotides synthesized as ribonucleotides and modified to deoxribonucleotides Replace OH group on 2 carbon of ribose and replace with hydrogen atom Remove oxygen