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

Dr. Elena RomancencoDepartment 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:

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.

     Saprophytes—live on dead organic matter      Parasites—nutrients 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 (O2) serves as the final e- acceptor of the ETC

O2 is reduced to H2O 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 Kreb’s 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

CHO’s 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 respirationUtilizes same 3 coupled pathways as Aerobic RespirationUsed as an alternative to aerobic respiration

Final electron acceptor something other than oxygen:

NO3- : Pseudomonas, Bacillus.

SO4-: Desulfovibrio

CO3-: methanogens

In Facultative organismsIn 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

Pathwaysinvolved

Final e- acceptor ATP yield

Aerobic respiration

Glycolysis, TCA, ET

O2 38

Anaerobic respiration

Glycolysis, TCA, ET

NO3-, So4

-2, CO3

-3

variable

Fermentation

Glycolysis Organic 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

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: 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 Kreb’s cycle. These reactions include:

1. Deamination—the amino group is removed, converted to an ammonium ion, and excreted.

2. Decarboxylation—the   ---COOH group is removed3. Dehydrogenation—a 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 fissionmode: 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- 27-30 mins

Mycobacterium tuberculosis - 792-932 mins

Treponema pallidum -1980 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 - 107-109 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 & H2O 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)

– H2O: 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: assoc’d w/ PRO (i.e., Fe:PRO); common

component of enzymes.

Nutrients

2 types1. 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 diffusiono simple diffusiono facilitated diffusion

o Active transport

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

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Some pathogens can multiply in the refrigerator: Listeria monocytogenes

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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

Most 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.

82Similar 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 = O2, N2, & CO2

O2 - 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 – O2 present, require O2 for growth e.g. Pseudomonas aeruginosa

Obligate aerobe – 20% O2: only grows with O2 Microaerophile – 4% O2: best growth with small amount O2

e.g. Campylobacter spp, Helicobacter spp

Strict (Obligate) Anaerobes – O2 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 O2 Aerotolerant anaerobe: anaerobes that “tolerate” +/or survive in O2, but do NOT

utilize O2 during E* metabolism e.g. Clostridium perfringens

Facultative Anaerobe – grows both in presence & absence of O2; but grows BEST under Aerobic conditions; considered to be aerobic organism; O2 present – aerobic respiration for E*; O2 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

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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 6.5-6.8

Alkalophiles: grow BEST at high pH (alkaline: pH 10.0)

V. cholerae - pH 8.4-9.2

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

Biological catalysts Highly specific Extremely efficient Increase reaction rates 108-1010 times High turnover numbers Proteins or RNA (ribozymes)

Enzymes

Uptake of nutrients by bacteria

Passive diffusionsimple diffusionFacilitated diffusion

Active transport

Enzymes - catalysts that speed up and direct chemical reactions A. Enzymes are substrate specific

Lipases Lipids Sucrases Sucrose Ureases Urea Proteases Proteins DNases DNA

Sucrose Sucrase Lipids Lipase DNA DNase Proteins Protease 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. Oxidoreductases oxidation & reduction

2. Hydrolases hydrolysis 3. Ligases synthesis

Oxidoreductase

Oxidation reduction in which hydrogen or oxygen are gained or lost

Cytochrome oxidase, lactate dehydrogenase

Transferase Transfer of functional groups, e.g. amino, acetyl or phosphate groups

Acetate kinase, alanine deaminase

Hydrolase Hydrolysis – addition of water

Lipase, sucrase

Lyase Removal of atoms without addition of water

Oxalate decarboxylase, isocitrate lyase

Isomerase Rearrangement of atoms within a molecule

Glucose phosphate isomerase, alanine racemase

Ligase Joining of two molecules Acetyl-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