Microbial physiology. Microbial metabolism. Enzymes. Nutrition. Bioenergetics. Bacterial growth and multiplication. Dr. Elena Romancenco Department of Microbiology

Microbial physiology. Microbial metabolism. Enzymes. Nutrition. Bioenergetics. Bacterial growth and multiplication.

Microbial metabolism the Greek metabole, meaning change. Metabolism - the sum of the biochemical reactions required for energy generation AND the use of energy to synthesize cell material from small molecules in the environment.

Why do we must know the metabolism of bacteria? Because we want to know how to inhibit or stop bacteria growth and want to control their metabolism.

Metabolism Two components: Anabolism - biosynthesis building complex molecules from simple ones requires ENERGY (ATP) Catabolism - degradation breaking down complex molecules into simple ones generates ENERGY (ATP) 3 Biochemical Mechanisms Utilized Aerobic Respiration Anaerobic Respiration Fermentation

Catabolic reactions or sequences produce energy as ATP adenosine triphosphate, which can be utilized in anabolic reactions to build cell material from nutrients in the environment.

METABOLIC DIVERSITY Bacterial metabolism is classified into nutritional groups on the basis of three major criteria: nutritional groups 1. Source of energy, used for growth 2. Source of carbon, and 3. Sours of electron donors used for growth.

1. ENERGY SOURCE a. Phototrophs can use light energy b. Chemotrophs must obtain energy from oxidation-reduction of external chemical compounds

2. CARBON SOURCE a. Autotrophs can draw carbon from carbon dioxide b. Heterotrophs carbon from organic compounds c. Mixotrophic – carbon is obtained from both organic compounds and by fixing carbon dioxide

These requirements can be combined: 1. Photoautotrophs - light energy, carbon from 2. Photoheterotrophs light energy, carbon from organic compounds 3. Chemoautotrophs energy from chemical compounds, carbon from CO2 4. Chemoheterotrophs energy from chemical compounds, carbon from organic compounds

CHEMOHETEROTROPHS Energy and carbon both come from organic compounds, and the same compound can provide both. Specifically, their energy source is electrons from hydrogen atoms in organic compounds. Saprophyteslive on dead organic matter Parasitesnutrients from a living host This group ( more precisely chemoorganoheterotrophic) includes most bacteria as well as all protozoa, fungi, and animals. All microbes of medical importance are included in this group.

Microbial physiology. Microbial metabolism. Bioenergetics. Enzymes. Nutrition. Bacterial growth and multiplication.

Energy – capacity to do work or cause change Endergonic reactions – consume energy Exergonic reactions – release energy

Energy Production 3 Biochemical Mechanisms Utilized Aerobic Respiration Anaerobic Respiration Fermentation

Aerobic and anaerobic respiration Aerobic respiration – terminal electron acceptor is oxygen Anaerobic respiration – terminal electron acceptor is an inorganic molecule other than oxygen (e.g. nitrogen)

Aerobic Respiration Molecular Oxygen (O 2 ) serves as the final e - acceptor of the ETC O 2 is reduced to H 2 O Energy-generating mode used by aerobic chemoheterotrophs General term applied to most human pathogens Energy source = Oxidation of organic compounds Carbon Source = Organic Carbon 3 Coupled Pathways Utilized Glycolysis Krebs Cycle or Tricarboxylic Acid Cycle or Citric Acid Cycle Respiratory Chain or Electron Transport Chain (ETC)

1. Glycolysis (splitting of sugar) Carbohydrate (CHO) Catabolism Oxidation of Glucose into 2 molecules of Pyruvic acid CHOs are highly reduced structures (thus, H-donors); excellent fuels Degradation of CHO thru series of oxidative reactions End Products of Glycolysis: 2 Pyruvic acid 2 NADH2 2 ATP

Glycolysis

2. Krebs Cycle (Citric Acid Cycle,TCA) Series of chemical reactions that begin and end with citric acid 1. Initial substrate – modified end product of Glycolysis 2 Pyruvic Acid is modified to 2Acetyl-CoA, which enters the TCA cycle 2. Circuit of organic acids – series of oxidations and reductions Eukaryotes – Mitochondrial Matrix Prokaryotes – Cytoplasm of bacteria & Cell Membrane Products: 2 ATP 6 NADH2 2 FADH2 4 CO2

TCA cycle

3. Electron Transport System Occurs within the cell membrane of Bacteria Chemiosomotic Model of Mitchell 34 ATP

Electron transport system

Overview of aerobic respiration

Anaerobic respiration Utilizes same 3 coupled pathways as Aerobic Respiration Used as an alternative to aerobic respiration Final electron acceptor something other than oxygen: NO 3 - : Pseudomonas, Bacillus. SO 4 - : Desulfovibrio CO 3 - : methanogens In Facultative organisms In Obligate anaerobes Lower production of ATP because only part of the TCA cycle and the electron transport chain operate.

Fermentation Incomplete oxidation of glucose or other carbohydrates in the absence of oxygen Uses organic compounds as terminal electron acceptors Effect - a small amount of ATP Production of ethyl alcohol by yeasts acting on glucose Formation of acid, gas & other products by the action of various bacteria on pyruvic acid

Fermentation

Fermentation may result in numerous end products 1. Type of organism 2. Original substrate 3. Enzymes that are present and active

Fermentation End Products

Metabolic strategies Pathways involved Final e- acceptor ATP yield Aerobic respiration Glycolysis, TCA, ET O2O2 38 Anaerobic respiration Glycolysis, TCA, ET NO 3 -, So 4 -2, CO 3 -3 variable Fermentatio n GlycolysisOrganic molecules 2

Many pathways of metabolism are bi-directional or amphibolic Metabolites can serve as building blocks or sources of energy Pyruvic acid can be converted into amino acids through amination Amino acids can be converted into energy sources through deamination Glyceraldehyde-3-phosphate can be converted into precursors for amino acids, carbohydrates and fats

Redox reactions Always occur in pairs. There is an electron donor and electron acceptor which constitute a redox pair. Released energy can be captured to phosphorylate ADP or another compound.

Basic reaction Biological reaction 35 : electron removal : electron uptake

ATP 3 part molecule consisting of adenine – a nitrogenous base ribose – a 5-carbon sugar 3 phosphate groups Removal of the terminal phosphate releases energy Adenosine Tri Phosphate ADP + energy + phosphate ATP contains energy that can be easily released (high-energy or unstable energy bond) Required for anabolic reactions

ATP

Formation of ATP 1. substrate-level phosphorylation 2. oxidative phosphorylation, ( reduced chemicals) 3. Photophosphorylation (reduced chlorophyll molecules) Uses of ATP: Energy for active transport Energy for movement Energy for synthesis of cellular components ALL SYNTHESIS REACTIONS INVOLVE USE OF ENERGY

Substrate-level phosphorylation

Phosphorylation of glucose by ATP

Lipid Metabolism Lipids are essential to the structure and function of membranes Lipids also function as energy reserves, which can be mobilized as sources of carbon 90% of this lipid is triacyglycerol triacyglycerol lipase glycerol + 3 fatty acids The major fatty acid metabolism is β-oxidation

Lipid catabolism Lipids are broken down into their constituents of glycerol and fatty acids Glycerol is oxidised by glycolysis and the TCA cycle Lipids are broken down to 2 carbon acyl units where they enter the TCA cycle

Protein Catabolism

PROTEIN CATABOLISM Intact proteins cannot cross bacterial plasma membrane, so bacteria must produce extracellular enzymes called proteases and peptidases that break down the proteins into amino acids, which can enter the cell. Many of the amino acids are used in building bacterial proteins, but some may also be broken down for energy. If this is the way amino acids are used, they are broken down to some form that can enter the Krebs cycle. These reactions include: 1. Deaminationthe amino group is removed, converted to an ammonium ion, and excreted. 2. Decarboxylationthe ---COOH group is removed 3. Dehydrogenationa hydrogen is removed Tests for the presence of enzymes that allow various amino acids to be broken down are used in identifying bacteria in the lab.

Catobolism of organic food molecules Proteins and carbohydrates are degraded by secreted enzymes – proteases and amylases Amino acids must be deaminated for further oxidation

Microbial physiology. Microbial metabolism. Enzymes. Bioenergetics. Nutrition. Bacterial growth and multiplication.

Growth and multiplication mode: Binary fission mode: Binary fission

Bacterial Cell Division 1. Replication of chromosome 2. Cell wall extension 3. Septum formation 4. Membrane attachment of DNA pulls into a new cell.

It is an increase in all the cell components, which ends in multiplication of cell leading to an increase in population. It involves - an increase in the size of the cell & an increase in the number of individual cells. Bacteria divide by binary fission. Growth

Generation time Interval of time between two cell divisions OR The time required for a bacterium to give rise to 2 daughter cells under optimum conditions Also called population doubling time.

Generation time Coliform bacilli like E.coli & other medically important bacteria – 20 mins Staphylococcus aureus mins Mycobacterium tuberculosis mins Treponema pallidum mins

Colony – formed by bacteria growing on solid media. (20-30 cell divisions) Each bacterial colony represents a clone of cells derived from a single parent cell. Turbidity – liquid media cells/ml Biofilm formation – thin spread over an inert surface. Growth form in Laboratory

Solid medium Colony

Liquid medium

Bacterial biofilm

Cell Counts... many ways 2 methods – Total cell count - Viable cell count Bacterial counts

Total Count Total number of cells in the sample = living + dead. Can be obtained by : Direct counting under microscope using counting chambers. Counting in an electronic device – Coulter counter.

Counting chambers

Over method Direct counting using stained smears - by spreading a known volume of culture over a measured area of slide. Opacity measurements using an absorptiometer/ nephalometer. Chemical assays of cell components.

Turbidity- a spectrophotometer measures how much light gets through

Compared to known controls, MacFarland controls

Viable Cell Count Measures the number of living cells. Methods – Surface colony count Dilution method Plating method Number of colonies that develop after incubation gives an estimate of the viable count.

Plate counts

When a bacterium is added to a suitable liquid medium and incubated, its growth follows a definite course. If bacteria counts are made at intervals after inoculation & plotted in relation to time, a growth curve is obtained. Shows 4 phases : Lag, Log or Exponential, Stationary Decline. Bacterial Growth Curve

Phases of Growth Curve 1. Lag phase – No increase in number but there may be an increase in the size of the cell. 2. Log OR Exponential phase – cells start dividing and their number increases exponentially.

3. Stationary phase – cell division stops due to depletion of nutrients & accumulation of toxic products. - equilibrium exists between dying cells and the newly formed cells, so viable count remains stationary 4. Phase of Decline – population decreases due to the death of cells – autolytic enzymes. Phases of Growth Curve

Morphological & Physiological alterations during growth Lag phase – maximum cell size towards the end of lag phase. Log phase – smaller cells, stain uniformly Stationary phase – irregular staining, sporulation and production of exotoxins & antibiotics Phase of Decline –involution forms(with ageing)

Availability of Nutrients & H 2 O Temperature Atmosphere – O2 & CO2 H-ion concentration Moisture & drying Osmotic effects Radiation Mechanical & sonic stress. Factors Affecting Bacterial Growth

Bacterial Nutrition Water constitutes 80% of the total weight of bacterial cells. Proteins, polysaccharides, lipids, nucleic acids, mucopeptides & low molecular weight compounds make up the remaining 20%.

Moisture & Drying Water – essential ingredient of bacterial protoplasm. Hence drying is lethal to cells. Effect of drying varies : T. pallidum – highly sensitive Staphylococci sp– stand for months Spores – resistant to desiccation, may survive for several decades.

Nutrients Functions – Generation of energy – Synthesis of cellular materials – Essential nutrients (basic bioelements needed for bacterial cell growth) – H 2 O: universal solvent; hydrolyzing agent – Carbon: food & E* source; in form of prot., sugar, lipid – Nitrogen: for prot. syn; nucleic acid syn (purines & pyrimidines) – Sulfur (sulfate): AA syn (i.e., Cystine) – Phosphate: key component of DNA & RNA, ATP, and inner & outer membrane phospholipids – Minerals: assocd w/ PRO (i.e., Fe:PRO); common component of enzymes.

Nutrients 2 types 1. Macronutrients – needed in large quantities for cellular metabolism & basic cell structure C, H, O, N 2. Micronutrients – needed in small quantities; more specialized (enzyme & pigment structure & function) Mn, Zn – Fastidious Bacteria: microbes that require other complex - nutrients/growth factors ( i.e., Vitamins or AAs)

Vary in the temperature requirements. Temperature range – growth does not occur above the maximum or below the minimum. Optimum Temperature – growth occurs best, 37ºC for most pathogenic bacteria. Temperature

Uptake of nutrients by bacteria o Passive diffusion o simple diffusion o facilitated diffusion o Active transport

Psychrophiles: -10 to 20 C Psychrotrophs: 0 to 30 C Mesophiles: 10 to 48 C e.g. most bacterial pathogens Thermophiles: 40 to 72 C Hyperthermophile: 65 to 110 C 77

Some pathogens can multiply in the refrigerator: Listeria monocytogenes 78

Neutral or slightly alkaline pH (7.2 – 7.6) – majority of pathogenic bacteria grow best. acidic pH – Lactobacilli alkaline pH -Vibrio cholerae H-ion Concentration

Osmotic Pressure or Osmolarity M ost bacteria require an isotonic environment or a hypotonic environment for optimum growth. Osmotolerant - organisms that can grow at relatively high salt concentration (up tp 10%). Halophiles - bacteria that require relatively high salt concentrations for growth, like some of the Archea that require sodium chloride concentrations of 20 % or higher.

82 Similar effect with sugars

Radiation X rays & gamma rays exposure – lethal Mechanical & Sonic Stress May be ruptured by mechanical stress. Radiation, stress

Some bacteria require certain organic compounds in minute quantities – Growth Factors OR Bacterial Vitamins. It can be : Essential – when growth does not occur in their absence. Accessory – when they enhance growth, without being absolutely necessary for it. Growth Factors

Identical with eukaryotic nutrition Vitamin B complex – thiamine riboflavine nicotinic acid pyridoxine folic acid & Vit.B 12 Growth Factors

Primary gases = O 2, N 2, & CO 2 O 2 - greatest impact on microbial growth (even if the microorganism does not require it) Aerobic respiration – terminal electron acceptor is oxygen. Anaerobic respiration – terminal electron acceptor is an inorganic molecule other than oxygen (e.g. nitrogen). Presence or Absence of Gases

Strict (Obligate) Aerobes – O 2 present, require O2 for growth e.g. Pseudomonas aeruginosa Obligate aerobe – 20% O 2 : only grows with O 2 Microaerophile – 4% O 2 : best growth with small amount O 2 e.g. Campylobacter spp, Helicobacter spp Strict (Obligate) Anaerobes – O 2 depleted, grow in the absence of O2 & may even die on exposure to O2 e.g. Bacteroides fragilis Obligate anaerobe: only grows in absence of O 2 Aerotolerant anaerobe: anaerobes that tolerate +/or survive in O 2, but do NOT utilize O 2 during E* metabolism e.g. Clostridium perfringens Facultative Anaerobe – grows both in presence & absence of O 2 ; but grows BEST under Aerobic conditions; considered to be aerobic organism; O 2 present – aerobic respiration for E*; O 2 absent – anaerobic pathways (fermentation) e.g. Staphylococcus spps Capnophilic organism – requires high CO2 levels eg Neisseria spps Depending on the O2 requirement

Oxygen-related growth zones in a standing test tube

Oxygen is readily converted into radicals (singlet oxygen, superoxide, hydrogen peroxide, hydroxyl radical) Most important detoxifying enzymes are superoxide dismutase and catalase Cells differ in their content of detoxifying enzymes and hence, ability to grow in the presence of oxygen 90

Classification of gram-positive cocci Staphylococci are catalase + Streptococci are catalase - Staphylococci Streptococci 91

pH Majority of bacteria grow BEST at neutral or slightly alkaline pH pH 7.0 – 7.4 => this is near most normal body fluids Acidophiles: grow BEST at low pH (acid: pH 0 – 1.0) T.B. - pH Alkalophiles: grow BEST at high pH (alkaline: pH 10.0) V. cholerae - pH

Microbial physiology. Microbial metabolism. Enzymes. Nutrition. Bioenergetics. Bacterial growth and multiplication.

Biological catalysts Highly specific Extremely efficient Increase reaction rates times High turnover numbers Proteins or RNA (ribozymes) Enzymes

Uptake of nutrients by bacteria Passive diffusion simple diffusion Facilitated diffusion Active transport

Enzymes - catalysts that speed up and direct chemical reactions A. Enzymes are substrate specific Lipases Lipids SucrasesSucrose UreasesUrea ProteasesProteins DNasesDNA

SucroseSucrase LipidsLipase DNADNase ProteinsProtease removes a Hydrogen Dehydrogenase removes a phosphate phosphotase Naming of Enzymes - most are named by adding ase to the substrate

Naming of Enzymes Grouped based on type of reaction they catalyze 1. Oxidoreductasesoxidation & reduction 2. Hydrolaseshydrolysis 3. Ligasessynthesis

OxidoreductaseOxidation reduction in which hydrogen or oxygen are gained or lost Cytochrome oxidase, lactate dehydrogenase TransferaseTransfer of functional groups, e.g. amino, acetyl or phosphate groups Acetate kinase, alanine deaminase HydrolaseHydrolysis – addition of water Lipase, sucrase LyaseRemoval of atoms without addition of water Oxalate decarboxylase, isocitrate lyase IsomeraseRearrangement of atoms within a molecule Glucose phosphate isomerase, alanine racemase LigaseJoining of two moleculesAcetyl-CoA synthetase, DNA ligase Types of enzymes

Enzyme Components Holoenzyme - whole enzyme 2 Parts 1. Apoenzyme - protein portion 2. Coenzyme (cofactor) - non-protein

Coenzymes Many are derived from vitamins 1. Niacin NAD (Nicotinamide adenine dinucleotide) 2. Riboflavin FAD (Flavin adenine dinucleotide) 3. Pantothenic Acid CoEnzyme A

Enzyme components Cofactors may be metal ions Cofactors may accept or donate atoms removed from the substrate or donated to the substrate Cofactors may act as electron carriers Often derived from vitamins e.g. NAD and NADP – electron carries derived from nicotinic acid