Basic Medical Microbiology(genetics) Medical Microbiology(genetics) 2015 Begumisa MG lecture notes series Page 1 Microbial Genetics I. THE BASIS OF HEREDITY All information necessary

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



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).



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



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.



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



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.



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



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.



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!



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



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



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.

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Basic Medical Microbiology(genetics) Medical Microbiology(genetics) 2015 Begumisa MG lecture notes series Page 1 Microbial Genetics I. THE BASIS OF HEREDITY All information necessary