FUNDAMENTAL CONCEPTS IN MICROBIOLOGYAEROBIC METABOLISM Metabolism : All the biochemical reactions that take place in cell

Metabolic task Function

Bringing nutrient into the cell

To transport nutrient across the cytoplasmic membrane and concentrate them in the cytoplasm

Catabolism To process the major nutrient and produce the 12 precursor metabolites, ATP and reducing power

Biosynthesis To synthesis all necessary small molecules, including building blocks for macromolecules from precursor metabolites

Polymerisation To link together building block, forming macromolecules, Eg. RNA, DNA, protein, polysaccharide and peptidoglycan

Assembly To assemble macromolecu;les into organelles

Bringing innutrients

Cell membrane Biosynthesis Polymerisation New cell

Catabolism Assembly

Bringing nutrients into the cells

• All nutrients pass through tiny water filled pores in the outer membrane formed by proteins called porin

• Nutrient of concentration higher than inside the cells will be passed through (taking across the cell envelope)

• Transporter protein (permease, facilitator or carrier)- bind to the nutrient in the periplasm

• Mechanism of transportation i. Transporter mediated facilitated diffusion II. Active transport – action of transporter via pump requiring ATP (proton gradient). III. Energy requiring process that concentrates nutrient in the cell - group translocation

Porin Cytoplasm

Transporter

Low concentrationof nutrient

High concentrationof nutrient

Catabolism Chemical changes/set of reaction that carbon or energy source undergo• Catabolite reactions produce 12 precursor metabolites for synthesis

Precursor metabolites Catabolic pathway that leads to its synthesis

Glucose-6-phosphate

Fructose-6-phosphate

Triose phosphate

3-phosphoglycerate

Phosphoenolpyruvate

Pyruvate

Acetyl Co-A

Α-ketoglutarate

Succinyl Co A

Oxaloacetate

Ribose 5-phosphate

Erythrose 4-phosphate

Glycolysis

Glycolysis

Glycolysis

Glycolysis

Glycolysis

Glycolysis

TCA cycle

TCA cycle

TCA cycle

TCA cycle

Pentose phosphate

Pentose phosphate

Reducing power : redox reactionATP : stored energy; compound that stores chemical energy (Adenosine triphosphate)

Energy used during ATP ADP Energy conserved other steps of metabolism during catabolism

Figure 1: Glycolysis. Glycolysis is a pathway of central metabolism that converts a molecule of glucose into two molecules of pyruvate with a net yield of 2 molecules of ATP and 2 molecules of NADH, along with 6 precursor metabolites (shown in colored boxes)

Figure 2: The TCA cycle. The tricarboxylic acid TCA cycle converts pyruvate into CO2, reducing power, ATP (by substrate-level phosphorylation), and 4 precursor metabolites, shown in colored boxes. (FADH2 is a carrier of reducing power capable of converting NAD+ into NADH.

Pentose phosphate

Figure 3: The pentose phosphate pathway. The pentose phosphate pathway is a part of central metabolism that forms 2 precursor metabolites, shown in boxes. The pathway begins with 1 intermediate of glycolysis and ends with another.

Biosynthesis – metabolic factory uses 3 products of catabolism• Precursor metabolites• ATP • Reducing power

Building blocks for macromolecules

Eg. Biosinthesis pathway :

asparagine methionine

TCA oxaloacetate aspartate threonineglycolysis pyruvate pyruvatepentose-phosphate pathway

lysine isoleucineRibose-5 phosphate histidine

Driving force that fuels biosynthesis – reducing power stored mostly in the form of NADPH

a

b

a) A branched biosynthesis pathway converts 2 precursor metabolites (pyruvate and oxaloacetate from glycolysis and the TCA cycle, respectively) into 6 amino acids (red) by 22 enzyme-catalyzed reactions (arrows). The pathway uses 3 molecules of ATP (yellow arrows) and 4 molecules of NADPH (green arrows).

b) An unbranched biosynthesis pathway converts a single precursor metabolite (ribose-5-phosphate from the pentose phosphate pathway) to a single amino acid (histidine) by 11 reactions. This pathway uses 1 ATP and 1 NADPH.

PolymerisationMolecular building blocks made by biosynthesis are joined together to

form macromolecules

Eg. Synthesis of DNA, RNA, proteins, polysaccharide and peptidoglycan

Macromolecules Building blocks

Protein 20 amino acids

Nucleic acid Nucleotides

RNA Adenine, guanine, cytosine, uracil, phosphate, ribose

DNA Adenine, guanine, cytosine, thymine, phosphate, deoxyribose

Polysaccharide Sugars

Peptidoglycan N-acetyl muramic acid, N-acetyl glucosamine, 5 amino acids

Lipid Fatty acid and other building blocks,

Polymerisation – catalysed by enzymes (protein) and protein structure is determined directly by DNA

Therefore, Polymerisation reaction indirectly determined by DNA.

• Polymerisation occurs by expanding chemical energy in the form of ATP

Assembly

Macromolecules assembled into cellular structuresAssembly may occur spontaneously (self-assembled), or

may be the result of reactions catalysed by enzymes

Eg.:

Self – assembled : Formation of flagella (from flagellin)

Reaction catalysed by enzymes : Formation of bacterial cell wall. short unit of peptidoglycans are released in periplasm assembled into intact cell wall

ANAEROBIC METABOLISM The critical difference between aerobic and anaerobic metabolism

lies in how ATP is generated. In aerobic metabolism, E.coli makes most of its ATP by aerobic

respiration, producing a proton gradient by an electron transport chain with oxygen as its terminal electron acceptor.

In the absence of oxygen the electron transport chain cannot function in this way.

Thus, aerobic respiration is impossible. There are 2 ways that cells can make ATP from organic nutrients in

the absence of oxygen. One, called anaerobic respiration – uses an electron transport

chain with a compound other than oxygen as the terminal electron acceptor.

The second, called fermentation, depends entirely on substrate-level phosphorylation.

Anaerobic Respiration In aerobic respiration, oxygen accepts electrons and is

reduced to water. In anaerobic respiration, another compound is reduced

by accepting these electrons. Compound that can act as a terminal electron acceptor

in anaerobic respiration include sulfate, nitrate, fumarate and trimethylamine oxide.

E. Coli, for example ,can use nitrate, fumarate or trimethylamine oxide as an electron acceptor if oxygen is not available.

Table 1: Some Terminal Electron Acceptor of Bacterial Electron Transport Chains

Type of Respiration Terminal Electron Acceptor

Reduced Product

Aerobic Respiration Oxygen (O2) Water (H2O)

Anaerobic Respiration

- Sulfate reduction Sulfate (SO42-) Hydrogen sulfide (H2S)

- Nitrate reduction Nitrate (NO3-) Nitrite (NO2

-)

- Fumarate reduction Fumarate (HOOC-CH=CH-COOH)

Succinate (HOOO-CH2-CH2-COOH)

-Denitrification Nitrate (NO3-) Nitrogen gas (N2)

Trimethylamine oxide reduction

Trimethylamine oxide Trimethylamine

FermentationFermentation is a form of anaerobic metabolism in which

all ATP is generated by substrate-level phosphorylation.Fermentation generates fewer molecules of ATP per

molecule of substrate than do aerobic and anaerobic respiration.

For example, E.coli derives about 28 ATP molecules from glucose by aerobic respiration but only about 3 by fermentation.

- 1 molecule of glucose is metabolized to produce 2 molecules of pyruvate, 2 molecules of ATP & 2 of NADH.- In order to reoxidize the 2 molecules of NADH (and thus allow fermentation to continue), pyruvate is reduced to lactic acid.

Nutritional classes of microorganism

Source of C atoms Chemical Rxn Light Energy

Organic compounds

Chemoheterotrophs Photoheterotrophs

CO2 Chemoautotrophs Photoautotrophs

Source of energy (ATP)

Heterotroph organic compounds as a source of carbon

Autotroph uses CO2 as a source of carbon

Genetic of microorganism• DNA structure• Replication of DNA• Regulation of gene expression

Genotype / Phenotype• Genotype cell genetic plan• Phenotype cell appearance and function

Mutation – Any chemical change in cell’s DNA• Base substitution mutation – changes a single pair of bases to different pair• Deletion mutation – removes a segment of DNA• Inversion mutation – reverses the order of a segment of DNA• Transposition mutation – moves a segment of DNA to a different position on the genome• Duplication mutation – adds an identical new segment of DNA next to the original one

Incidence of Mutation• Spontaneous mutation Natural course of microbial growth resulted mutation• Induced mutation Intentional chemical, physical or biological treatments

Induced mutation - treated with mutagens

Chemical mutagens : Eg. Nitrosoguanidine

Physical mutagen UV light, X rays, gamma radiation UV stimulates adjacent pyrimidine bases, usually T, to react with one another thymine dimer

Biological mutagen many carry fragments of DNA within their genome that are mutagenic, which moves from one part of the genome to another (transposable elements)

Selecting Mutants

Direct selection – create conditions that favour growth of the desired mutant strain

Indirect selection – counter selection, create conditions to prevent the growth of desired mutant. The growing cells are killed. The mutant will survive the lethal treatment which are isolated.

Site–directed mutagenesis – product of recombinant DNA technology (mutate one particular gene)

Genetic Exchange Among BacteriaGenetic exchange – transfer of genes from one cell to anotherIn bacteria – a portion of the DNA of one cell (the donor cell) is transferred to the other (recipient cell) merozygote

3 forms of genetic exchange in bacteria:

• Transformation : During transformation, DNA leaves one cell and exists for a time in the extracellular environment. Then it is taken into another cell, become incorporated into the genome. DNA fragment can become part of the resident chromosome.

• Conjugation : Carried out conjugative plasmids (plasmids able to transfer themselves to another cell) Eg.: F-plasmid (13 genes). One of the genes encodes a special pilus called sex pilus or the F-pilus F pilus allows F+ cells attach to F- cells

• Transduction : Transfer of chromosomal genes via virus that infect bacteria called bacteriophages, reproduce themselves. 2 kinds of transduction : Virulent phages (kills the host) Temperate phages (carried passively in the host without harming) Virulent – infect bacteria by attaching themselves to the surface of victim cells and rejecting their DNA Temperate – lysogenic cycle, phage DNA (prophage) exists as plasmid, incorporated in the host cell chromosome

• Transduction mediated by virulent phage – generalised transduction because it transfers any portion of the bacteria chromosome from one cell to another

• Prophage mediated specialised transduction – prophages are inserted only at a specific site on the bacterial chromosome

Conjugation Transduction

Recombinant DNA Technology• Techniques involve taking DNA from a cell, manipulate in vitro and putting it into another cell.• Recombination : processes of forming a new combination of genes by any means

Gene cloning – fundamental tool of recombinant DNA technology

• A process of obtaining a large number of copies of a gene from a single copy of the gene. Gene cloning involves 5 steps :

1. Obtaining a piece of DNA that carries the gene to be cloned2. Splicing that DNA into a cloning vector (a DNA molecule that a host cell will replicate)3. Putting the recombinant DNA (in this case the cloning vector with the desired gene spliced into it in an appropriate host cell)4. Testing to ensure that the gene has actually been put into the host cell5. Propagating the host cell to produce a clone of cells that carries the clone of genes

1. Obtaining a piece of DNA that carries the gene to be cloned

cell

DNA

DNA containing the gene to be clonedIs purified from intact cells

2. Splicing that DNA into a cloning vector (a DNA molecule that a host will replicate)

DNA molecules

DNA fragments

cloning vector moleculesEg. plasmid

Recombinant DNA

• Purified DNA is cut into pieces and spliced into cuts made in cloning vector DNA molecules• Cutting DNA using restriction endonuclease

3. Putting the recombinant DNA (in this case the cloning vector with the desired gene spliced into it in an appropriate host cell)

Recombinant DNA

Recombinant DNA molecules are put into host cells by transformation

4. Testing to ensure that the gene has actually been put into the host cell - host cells containing the gene to be cloned are identified by testing them for the presence of the gene product

5. Propagating the host cell to produce a clone of cells that carries the clone of genes - the colonies carrying the desired gene is propagated producing a clone of cells with the clone genes

Some application of recombinant DNA

Field Application Importance

Basic Biology DNA sequencing

Directed mutagenesis

Gene structure, function and relatedness between gene and microorganism

Medicine Therapeutic proteins

Gene therapy

Improved vaccines

Diagnosis

Veterinary medicine

Protein for treatment of diseases, genetic disorder

Effective vaccines

Rapid and accurate diagnosis

Industry Altering microorganisms Improve production

Agriculture Altering plants/farm animals Rapid breeding and disease resistance

criminal DNA fingerprinting Identify individual DNA

Genomics – study of an organism as revealed by the sequence of bases in its DNA

DNA sequencing- sequencing methods for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA.

DNA Assembling- to aligning and merging fragments of a much longerDNA sequence in order to reconstruct the original sequence

Annotation- The process of assigning function to DNA sequences

Microarray Technology- a means of determining which of an organism’s genes are expressed under various conditions

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