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Basic Medical Microbiology(genetics) 2015
Begumisa MG lecture notes series Page 1
Microbial Genetics
I. THE BASIS OF HEREDITY
All information necessary for life is stored in an organism’s chromosomes, which are made up of
DNA (exception: some viruses only have RNA). A chromosome is circular (prokaryotes) or linear
(eukaryotes). Nucleic acids (DNA & RNA) are made up of building blocks called nucleotides. In DNA,
nucleotides are arranged in a twisted double chain called a helix. The particular nucleotide sequence
spells out the “genetic code” that provides information for the synthesis of new DNA (DNA
replication necessary for cell division) and for the synthesis of proteins.
A typical prokaryotic cell contains a single circular chromosome. Bacteria may also have a small,
circular piece of extrachromosomal DNA called a plasmid. Human cells have 46 linear chromosomes.
A gene, the basic unit of heredity, is a liner sequence of DNA nucleotides that form a functional unit
of the chromosome or plasmid. All information for the structure and function of an organism is
coded in its genes. The information in specific gene is not always the same; different versions of the
same gene are called alleles. Using humans as an example, the hair color gene is always found at
the same location on a chromosome, but the different versions or alleles that can exist for hair color
are blond, brunette, red, black, etc. Because prokaryotes only have one chromosome, so they
generally only have one allele for a particular gene. Many eukaryotes have 2 sets of chromosomes
and thus 2 alleles of each gene, which may be the same or different. For example in humans, we
have 46 chromosomes or 23 pair. In each pair, you get one chromosome from your mom and one
for your dad. You may have a blonde hair allele from your mom and a dark hair allele from your dad
(so you get dark hair since the dark hair allele is dominant).
II. DNA STRUCTURE - THE WATSON-CRICK MODEL [DNA = deoxyribonucleic acid]
In the Watson - Crick Model, the DNA molecule is a double-stranded helix, shaped like a twisted
"ladder." Remember that nucleic acids (DNA & RNA) are made up of building blocks called
nucleotides. Each nucleotide is made up of a sugar, a phosphate, and a nitrogenous base. When we
put these nucleotides together to build a DNA ladder, the sides of the ladder are composed of
alternating phosphate groups & sugar molecules. The rungs of the ladder are made up of paired
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nitrogenous bases joined in the middle by hydrogen bonds. The nitrogenous bases are adenine,
thymine, guanine, & cytosine; adenine always pairs with thymine (A-T or T-A) & guanine always
pairs with cytosine (G-C or C-G) [This is called complementary base pairing]. These 4 bases spell out
the genetic message or code!
DNA enters into 2 kinds of reactions:
Replication - replicates the DNA before cell division, so that each new daughter cell will
receive a copy.
Protein Synthesis (Gene Expression); 2 steps: transcription & translation
III. DNA REPLICATION IN PROKARYOTES
Replication begins by an enzyme (topoisomerase e.g. DNA gyrase) breaking the hydrogen bonds
between the nitrogenous bases in the DNA molecule; the double stranded DNA molecule "unzips"
down the middle, with the paired bases separating. As the 2 strands separate, they act as
templates, each one directing the synthesis of a new complementary strand along its length.
If a nucleotide with thymine is present on the old strand, only a nucleotide with adenine can fit into
place in the new strand; if a nucleotide with guanine is present on the old strand, only a nucleotide
with cytosine can fit into place in the new strand, & so on. This is called complementary base
pairing. DNA replication is called semiconservative replication since half of the original DNA
molecule is conserved in each new DNA molecule. Like other biochemical reactions, DNA replication
requires a number of different enzymes, each catalyzing a particular step in the process.
IV. GENE EXPRESSION - PROTEIN SYNTHESIS
A. FROM DNA TO PROTEIN: THE ROLE OF RNA
By the 1940's biologists realized that all biochemical activities of the cell depend on specific
enzymes; even the synthesis of enzymes depends on enzymes! Remember that the DNA
molecule is a code that contains instructions for biological function & structure. Proteins
(enzymes) carry out these instructions. The linear sequence of amino acids in a protein
determines its 3-D structure & it is this 3-D structure that determines the protein's function.
The big question was: How does the sequence of bases in DNA specify the sequence of
amino acids in proteins? The search for the answer to this question led to the discovery of
RNA (ribonucleic acid), which is similar in structure to DNA (deoxyribonucleic acid).
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Three types of RNA:
i. messenger RNA (mRNA) - single stranded; contains codons (3 base codes); mRNA is
constructed to copy or transcribe DNA sequences.
ii. ribosomal RNA (rRNA) - ribosomes "read" the code on the mRNA molecule & send
for the tRNA molecule carrying the appropriate amino acid.
iii. transfer RNA (tRNA) - clover leaf shaped; at least one kind for each of the 20 amino
acids (a. a) found in proteins; each tRNA molecule has 2 binding sites - one end, the
anticodon (also a 3 base code), binds to the codon on the mRNA molecule; the
other end of the tRNA molecule binds to a specific amino acid; each tRNA & its
anticodon are specific for an a. a.!!
Differences between RNA & DNA:
i. RNA nucleotides contain a sugar called ribose while DNA nucleotides contain a
different sugar called deoxyribose.
ii. RNA is single stranded while DNA is double stranded.
iii. In RNA, uracil replaces thymine. There is no thyamine in RNA!!! But, there is
adenine.
B. TWO MAJOR EVENTS IN PROTEIN SYNTHESIS:
i. Transcription [mRNA copies or transcribes DNA sequences]
This process is similar to what occurs in DNA replication. A segment of DNA uncoils
unzips. Free RNA nucleotides, are then added one at a time to one end of the
growing RNA chain. Cytosine in DNA dictates guanine in mRNA, guanine in DNA
dictates cytosine in mRNA, adenine in DNA dictates uracil in mRNA, thymine in DNA
dictates adenine in RNA. This complementary base pairing is just like what occurs in
DNA replication. An enzyme (RNA polymerase) catalyzes this process. After
transcription the mRNA goes out in search of a ribosome. This mRNA molecule will
now dictate the sequence of amino acid in a protein in the next step called
translation.
ii. Translation - actual synthesis of polypeptides or proteins; translate information
from one language (nucleic acid base code) into another language (amino acids);
remember, the sequence of amino acids (the protein's primary structure)
determines what the protein's 3-D globular structure is going to be & structure
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determines function. Translation involves the following stages;
a. Initiation - Begins when the ribosome attaches to the mRNA molecule,
reading its first or START codon. The first tRNA comes into place to pair with
the initiator codon of mRNA (it occupies the peptide site (P) in the
ribosome). The START codon is AUG, which specifies the amino acid
methionine. All newly synthesized polypeptides start with methionine.
b. Elongation - The second codon of the mRNA molecule is then read and a
tRNA with an anticodon complementary to the second mRNA codon
attaches to the mRNA molecule; with its a. a. this second tRNA molecule
occupies the aminoacyl site (A) of the ribosome. When both the P & A sites
are occupied, an enzyme forges a peptide bond between the 2 a. a. & the
first tRNA is released. The first tRNA cannot be released until this peptide
bond is formed, as it will take its a. a. with it!! The second tRNA is then
transferred from the A site to the P site & a third tRNA is brought into the A
site. The ribosome continues to move down the mRNA molecule in this
fashion, "reading" the codons on the mRNA molecule & adding amino acids
to the growing polypeptide chain.
c. Termination - Toward the end of the coding sequence on the mRNA
molecule is a codon that serves as a termination signal (UAG, UAA, UGA).
There are no tRNA anticodons to complementary base pair with this codon.
Translation stops and the polypeptide chain is freed from the ribosome.
Enzymes in the cell then degrade the mRNA strand.
iii. In the prokaryotic cell, no organelles exist, therefore modification/processing of the
polypeptide into a protein occurs in the cytoplasm.
C. THE GENETIC CODE. The mRNA codons for the 20 universal amino acids.
See the table below of mRNA codons for the 20 amino acids. The 3-base codons are written
to the left and the abbreviations of the amino acids they correspond to are written to the
right.
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The amino acid abbreviations in the table are: Ala - alanine; Arg - arginine; Asn -
apararagine; Asp - aspartamine; Cys - cysteine; Glu - glutamic acid; Gln - glutamine; Gly -
glycine; His - histidine; Ile - isoleucine; Leu - leucine; Lys - lysine; Met - methionine; Phe -
phenylalanine; Pro - proline; Ser - serine; Thr - threonine; Trp - tryptophan; Tyr - tyrosine;
Val - valine.
The code has been proven to be the same for all organisms from humans to bacteria - it's
known as the universal genetic code. Notice that most of the amino acids have more than
one code (ex. Arg has 6 codes!). However, each code is specific for an amino acid (ex. UUU
only codes for the amino acid Phe).
Three of the 64 codons do not specify amino acids. Instead they indicate STOP or
termination of the translation process (they say "This is the end of the polypeptide.")
The START codon is AUG, which specifies the amino acid methionine. All newly synthesized
polypeptides have to start with methionine. Since AUG is the only codon for methionine,
when it occurs in the middle of a message, it is ignored as a START codon and is simply read
as a methionine-specifying codon.
V. MUTATIONS
A. A mutation is any chemical change in a cell's genotype (genes) that may or may not lead to
changes in a cell's phenotype (specific characteristics displayed by the organism). Many
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different kinds of changes can occur (a single base pair can be changed, a segment of DNA
can be removed, a segment can be moved to a different position, the order of a segment
can be reversed, etc.). Mutations account for evolutionary changes in microorganisms and
for alterations that produce different strains within species. Mutations often make an
organism unable to synthesize one or more proteins. The absence of a protein often leads
to changes in the organism'’ structure or in its ability to metabolize a particular substance.
B. Spontaneous mutations – occur by chance, usually during DNA replication. Only about one
cell in a hundred million (108) has a mutation in any particular gene. Since full-grown
cultures contain about 109 cells per milliliter, each milliliter contains about 10 cells with
mutations in any particular gene. Because the bacterial chromosome contains about 3,500
genes, each ml of culture contains about 35,000 mutations that weren't present when the
culture started growing. When you think about it that’s a lot of mutations in just one ml!
C. Induced mutations are caused by chemical, physical, or biological agents called mutagens.
i. Chemical Mutagens – ex. Nitrates and nitrites are added to foods such as hot dogs,
sausage, and lunch meats for antibacterial action. Unfortunately these same
compounds have been proved to cause similar mutations and cancer in lab animals
ii. Physical Mutagens - Include UV light, X-rays, gamma radiation, & decay of
radioactive elements; heat is slightly mutagenic.
D. Consequences of Mutations - Most mutations do not change the cell's phenotype. If the
mutation changes the codon to another that encodes the same amino acid, the protein
remains the same. For example if the DNA code is changed from AGA to AGG, the mRNA
codon would change from UCU to UCC. Check your table! The amino acid would not
change. The amino acid would stay serine. In this case the genotype is altered, but the
phenotype stays the same. Having more than one codon for each amino acid allows for
some mutations to occur, without affecting an organism’s phenotype. A mutation that
changes a codon to one that encodes a different a. a. may alter the protein only slightly if
the new a. a. is similar to the original one. However, if a mutation changes an a. a. to a very
different one, there may be a drastic change in the structure of the protein, causing major
complications for the cell. For example, if the structure of an enzyme called DNA
polymerase was greatly altered; the cell would not be able to replicate its DNA and thus
would not be able to multiply.
E. Repair of DNA Damage – Organisms (Bacteria) have enzymes that repair some mutations.
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VI. GENETIC TRANSFER
Gene transfer refers to the movement of genetic information between organisms. In most
eukaryotes, it is an essential part of the organism’s life cycle and usually occurs by sexual
reproduction. Male and female parents produce sperm and egg which fuse to form a zygote, the
first cell of a new individual. Of course, sexual reproduction does not occur in bacteria, but even
they have mechanisms of genetic transfer. Gene transfer is significant because it greatly increases
the genetic diversity of organisms. We’ve already discussed how mutation account for some genetic
diversity, but gene transfer between organisms accounts for even more. In recombinant DNA
technology, genes from one species of organism are introduced into the genetic material of another
species of organism. For example, human genes can be inserted into the bacterial chromosome.
A. BACTERIAL PLASMIDS & CONJUGATION
Most bacteria carry additional DNA molecules known as plasmids:
i. Plasmids are circular DNA molecules, much smaller than the bacterial chromosome.
ii. Plasmids can move in and out of the bacterial chromosome.
iii. Two important plasmids are fertility (F) plasmids and drug resistant (R) plasmids.
a. The F Plasmid - This plasmid contains about 25 genes, many of which
control the production of F pili. F pili are long, rod-shaped protein
structures that extend from the surface of cells containing the F plasmid.
Cells that lack the F plasmid are known as female (recipient) or F (-) cells.
Cells that possess the F plasmid are known as male (donor) or F (+) cells. F
(+) cells attach themselves to F (-) cells by their pili and transfer a copy of an
F plasmid to the F (-) cells through a pilus. The once F (-) cells are now F (+)
and will now produce pili, because they now have the F plasmid that
contains the plasmid genes that code for these pili. This transfer of DNA
from one cell to another by cell-to-cell contact is known as conjugation and
is a form of genetic recombination because new genetic material is
introduced into the cell. This is as close to sex as bacteria get!
b. The R Plasmid –A group of Japanese scientists (1959) discovered that
resistance to certain antibiotics and other antibacterial drugs can be
transferred from one bacterial cell to another. It was subsequently found
that genes conveying drug resistance are often carried on plasmids. Over
the last few decades, R factors have proliferated to the point that some
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infections are difficult to cure with antibiotics.
iv. Note: Plasmids are very important to scientists involved in recombinant DNA
research. Genes of interest can be inserted into plasmids. The plasmids are
introduced to bacteria and the bacteria take them up by endocytosis. As the
bacteria reproduce themselves by binary fission, they replicate the plasmid and pass
it to their daughter cells. The plasmids can then be isolated from all of these
bacterial cells and the gene of interest can be excised (cut out). In this way a large
quantity of a gene of interest can be produced.
B. TRANSFORMATION - A genetic change in which DNA leaves one cell, exists for a time in the
aqueous extracellular environment, & then is taken into another cell where it may become
incorporated into the genome. E.g. Extracts from killed, encapsulated, virulent (disease
causing) bacteria, when added to living, harmless, unencapsulated bacteria, can convert the
latter to the virulent type. By endocytosis, the living, non-virulent bacteria pick up the DNA
from the dead, virulent bacteria and incorporate the DNA into their own DNA. The non-
virulent bacteria now have the genes that code for proteins that transform them into
virulent bacteria. (Read about Griff’s experiment )
C. TRANSDUCTION – Is the injection of foreign DNA by a bacteriophage virus into the host
bacterium. Viruses that infect bacteria are called bacteriophages, which literally translates
to 'bacteria-eater.' Viruses are notorious for their ability to invade a host, hijack the host
cellular machinery, and force it to build millions of copies of the virus. These copies are then
released and go on to attack new hosts, spreading through populations.
Sometimes, instead of just infecting and hijacking the host, the virus picks up and transfers
some of the host cell's DNA. This process is transduction.
VII. GENETIC ENGINEERING
Genetic engineering refers to the purposeful manipulation of genetic material to alter the
characteristics of an organism in a desired way. One of the most useful of all techniques of genetic
engineering is the production of recombinant DNA (DNA that contains information from two
different species of organisms).
A. PROCESS:
For example a particular human gene can be removed from a human chromosome.
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Recombinant DNA is then constructed by inserting that gene into a bacterial plasmid, which
serves as a carrier. The recombinant DNA is then introduced into host bacterium, which
takes up the plasmid. The host bacterial cell then divides and its daughter cells divide,
producing millions of cells that all contain a copy of the human gene of interest. This
process serves at least 2 purposes:
i. Large quantities of the human gene of interest are produced.
ii. The bacteria can read the human gene of interest, producing the protein coded for
on the gene by protein synthesis. The genetic code is universal! We can obtain
large quantities of a particular protein using bacteria.
B. MEDICAL APPLICATIONS
i. Products such as insulin to treat diabetes, human growth hormone to treat
dwarfism, blood-clotting proteins to treat hemophilia, antibiotics, and vaccines have
all been produced by bacteria using recombinant DNA technology.
ii. Using "genetically engineered viruses:" Recently, genetic engineering &
recombinant DNA technology has allowed us to use bacteria to produce the protein
antigens found in the protein capsids of certain viruses (remember, viruses don't
have phospholipid cell membranes - they have proteins coats or capsids). Scientists
determine the genetic code for these proteins & insert the gene into the
chromosome of bacterial cells. The bacteria produce the proteins coded for on the
inserted genes when they go through their regular process of protein synthesis.
These proteins can then be injected as a vaccine (your body doesn't care if the
proteins are in the real viral capsid or if they were made by a bacterium; they are
the same proteins & your body's immune system will respond to these antigens in
the same way). "Genetically engineered viruses" (e.g. hepatitis B, influenza, rabies)
do not pose the same risks as inactivated and attenuated viruses!
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Gene regulation in Bacteria
Transcriptional Activators Turn Genes On
The Tryptophan Operon
The idea that genes could be switched on & off came originally from a study of how bacteria adapt to
changes in the composition of their growth medium. The sequence of bases coding for one or more
polypeptides, together with the operator controlling its expression is called an operon. This
arrangement is of great advantage to the bacterium because coordinated control of the synthesis of
several metabolically related enzymes/ proteins can be achieved. The chromosome of the bacterium
E.coli, a single celled organism, consists of a single circular DNA molecule of about 5 x 106 nucleotide
pairs. This DNA is in principle sufficient to encode about 4000 proteins, although only a fraction of these
are made at any one time. E.coli regulates the expression of many of its genes according to the food
sources that are available in the environment. Five E.coli genes code for the enzymes that manufacture
amino acid tryptophan. The tryptophan repressor is a simple switch that turns genes on and off in
bacteria. These genes are arranged in a cluster on the chromosome & are transcribed from a single
promoter as one long mRNA molecule, a feature that allows their expression to be coordinately
controlled. The promoter is the specific DNA sequence that directs RNA polymerase to bind to DNA, to
open the DNA double helix, and to begin synthesizing an RNA molecule.
When, however, tryptophan is present in the growth medium & enters the cell (when the bacterium is in
the gut of a mammal that has just eaten a meal of a protein, for example), these enzymes are no longer
needed & their production is shut off. This is the molecular basis for this switch: Within the promoter
that directs transcription of tryptophan biosynthetic genes lies an operator. This operator is a short
region of regulatory DNA of defined nucleotide sequence that is recognized by a helix-turn-helix gene
regulatory protein called the tryptophan repressor. The promoter & operator are arranged so that
occupancy of the operator by the tryptophan repressor blocks access to the promoter by RNA
polymerase, thereby preventing expression of the tryptophan-producing enzymes. This block is
regulated in an ingenious way: the repressor protein can bind to its operator DNA only if the repressor
has also bound 2 molecules of amino acid tryptophan. This tryptophan binding tilts the helix-turn-helix
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motif of the repressor so that so that it’s presented properly to the DNA major groove. Without
tryptophan, the motif swings inward & the protein is unable to bind the operator. Thus the tryptophan
repressor is a simple device that switches production of the tryptophan biosynthetic enzymes on & off
according to the availability of free tryptophan.
Because the active, DNA binding form of the protein serves to turn genes off, this mode of gene
regulation is called negative control, and the gene regulatory proteins that function in this way are
called transcriptional repressors or gene repressor proteins. The highlight of major events is as below;
If the level of tryptophan inside the cell is low, RNA polymerase binds to the promoter and
transcribes the five genes of tryptophan Operon.
If the level of tryptophan is high, however the tryptophan repressor is activated to bind the
operator, where it blocks the binding of RNA polymerase to the promoter.
Whenever the level of intracellular tryptophan drops, the repressor releases its tryptophan and
becomes inactive, allowing the polymerase to begin transcribing these genes.
Transcriptional Activators Turn Genes On
A transcriptional activator can operate as a simple on-off genetic switch. Because the active, DNA-
binding form of such a protein turns genes on, this mode of gene regulation is called positive control,
and the gene regulatory proteins that function in this manner are known as transcriptional activators or
gene activator proteins. The bacterial activator protein CAP (catabolic activator protein), activates
genes that enable E.coli to use alternative carbon sources when glucose, its preferred carbon source is
not available. Falling levels of glucose induce an increase in the intracellular signaling molecule cyclic
AMP, which binds to CAP protein, enabling it to bind to its specific sequence near target promoters and
there by turn on the appropriate genes. In this way, the expression of the target gene is switched on or
off, depending on whether cyclic AMP levels in the cell is high or low, respectively.
The Lactose Operon (lac operon)
More complicated genetic switches can be constructed by combining positive and negative controls. The
lac operon in E.coli is under both negative and positive transcriptional control by the lac repressor
protein and CAP, respectively. The lac operon codes for three proteins required to transport
disaccharide lactose into the cell and break it down. One gene codes for enzyme β-galactosidase, the
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second gene directs the synthesis of β-galactoside permease, the protein responsible for lactose
uptake, while the third gene codes for β-galactoside transacetylase whose function is still uncertain.
When E.coli is growing in the absence of lactose, it often lacks mRNA molecules coding for the synthesis
of β-galactosidase. However, in the presence of lactose, each cell has 35 – 50 β-galactosidase mRNA
molecules.
CAP enables bacteria to use alternative carbon sources such as lactose in the absence of glucose. It
would be wasteful however, for CAP to induce the expression of lac operon if lactose is not present, &
the lac repressor ensures that the lac operon is shut off in the absence of lactose. This arrangement
enables the lac operon to respond to and integrate two different signals, so that it’s expressed only
when two conditions are met: lactose must be present and glucose must be absent. Any of the other
three possible signal combinations maintain the gene in the off state, as shown below;
When both glucose and lactose are present, the lac operon is off because CAP is not bound
When glucose is present but lactose is absent, the lac operon is off because the lac repressor is
bound to operator while CAP is not bound
When both glucose and lactose are absent, the lac operon is off because the lac repressor is
bound to operator
Glucose and lactose levels control the initiation of transcription of the lac operon through their effects
on the lac repressor protein and CAP. Lactose addition increases the concentration of allolactose, which
binds to repressor protein and removes it from the DNA. Glucose addition decreases the concentration
of cyclic AMP; because cyclic AMP no longer binds to CAP, this gene activator protein dissociates from
DNA, turning off the operon.