1. FUNDAMENTAL CONCEPTS IN MICROBIOLOGY
AEROBIC 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
Catabolism
Assembly
Bringing in
nutrients
Cell membrane
Biosynthesis
Polymerisation
New cell

2. 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

3. Porin
Cytoplasm
Transporter
Low concentration
of nutrient
High concentration
of nutrient

4. Catabolism
Chemical changes/set of reaction that carbon or energy source undergo
• Catabolite reactions produce 12 precursor metabolites for synthesis
Precursor metabolites
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
Catabolic pathway that leads to
its synthesis
Glycolysis
Glycolysis
Glycolysis
Glycolysis
Glycolysis
Glycolysis
TCA cycle
TCA cycle
TCA cycle
TCA cycle
Pentose phosphate
Pentose phosphate
Reducing power : redox reaction
ATP : stored energy; compound that stores chemical energy (Adenosine triphosphate)
Energy used during
other steps of metabolism
ATP
ADP
Energy conserved
during catabolism

5. 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)

6. 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.

7. 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.

8. Biosynthesis – metabolic factory uses 3 products of catabolism
• Precursor metabolites
• ATP
• Reducing power
Building blocks for macromolecules
Eg. Biosinthesis pathway :
asparagine
TCA
glycolysis
pentose-phosphate pathway
a
oxaloacetate
pyruvate
lysine
Ribose-5
phosphate
b
methionine
aspartate
threonine
pyruvate
isoleucine
histidine
Driving force that fuels biosynthesis – reducing power stored mostly in the form of
NADPH

9. 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.

10. Polymerisation
Molecular 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,

11. 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

12. Assembly
 Macromolecules assembled into cellular structures
 Assembly 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

13. 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 substratelevel phosphorylation.

14. 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.

15. 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)
- Sulfate reduction
Sulfate (SO42-)
Hydrogen sulfide (H2S)
- Nitrate reduction
Nitrate (NO3-)
Nitrite (NO2-)
- Fumarate reduction
Fumarate (HOOC-CH=CH- Succinate (HOOO-CH2COOH)
CH2-COOH)
-Denitrification
Nitrate (NO3-)
Nitrogen gas (N2)
Trimethylamine oxide
reduction
Trimethylamine oxide
Trimethylamine
Anaerobic Respiration

16. Fermentation
 Fermentation 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.

17. - 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.

18. Nutritional classes of microorganism
Source of energy (ATP)
Source of C atoms
Chemical Rxn
Light Energy
Organic
compounds
Chemoheterotrophs Photoheterotrophs
CO2
Chemoautotrophs
Photoautotrophs
Heterotroph
organic compounds as a source of carbon
Autotroph
uses CO2 as a source of carbon

19. Genetic of microorganism
• DNA structure
• Replication of DNA
• Regulation of gene expression
Genotype / Phenotype
• Genotype
• Phenotype
cell genetic plan
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

20. Incidence of Mutation
• Spontaneous mutation
• Induced mutation
Natural course of microbial growth resulted
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)

21. 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)

22. Genetic Exchange Among Bacteria
Genetic exchange – transfer of genes from one cell to another
In 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

23. • 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

24. Conjugation
Transduction

25. 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 cloned
2. 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 cell
5. Propagating the host cell to produce a clone of cells that carries the
clone of genes

26. 1. Obtaining a piece of DNA that carries
the gene to be cloned
cell
DNA
DNA containing the gene to be cloned
Is purified from intact cells

27. 2. Splicing that DNA into a cloning vector
(a DNA molecule that a host will replicate)
DNA molecules
DNA fragments
• Purified DNA is cut into pieces
and spliced into cuts made in
cloning vector DNA molecules
• Cutting DNA using restriction
endonuclease
cloning vector
molecules
Eg. plasmid
Recombinant DNA

28. 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

29. 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

30. 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

31. 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 longer
DNA 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

32. THE END