Microbial Genetics – Microbiology Outline Notes

Microbiology Lecture Notes
Microbial Genetics


I. Deoxyribonucleic acid (DNA)
A. 2 Strands, Double helix

B. Composed of Nucleotides
1. Phosphate, deoxyribose sugar, nitrogen base
2. Nitrogen Bases
a. Adenine (A), Guanine (G), Thymine (T), Cytosine (C)
3. A–T and C—G are complementary bases, hydrogen bonded to each other

C. DNA Replication
1. Parental DNA strands unwind at origin
2. Complementary nucleotides are added to each exposed parental strand
3. Replication occurs at replication forks in both directions
4. Each new DNA strand contains one original parent strand and one new strand

 

II. Bacterial Genome – all genetic material (DNA) in a cell

A. Chromosomes – one circular DNA molecule attached to plasma membrane
1. Genes – segments of DNA along chromosome that code for proteins & RNA

B. Plasmids
1. Self-replicating circular pieces of DNA; about 1-5 % size of chromosome
2. Contain non-essential genes for cell metabolism
3. Plasmids may contain genes to:
a. Allow for survival under certain conditions, i.e. presence of antibiotics
1) Gene codes for enzyme that degrades antibiotic, i.e. penicillin
a) R factor plasmids —– Antibiotic Resistant Bacteria
b. Increase pathogenicity
1) Genes code for toxins and attachment to intestinal cells in strain
of E. coli that causes diarrhea
4. Shared between bacteria

III. Protein Synthesis

A. DNA (gene) —–transcription—— mRNA ——translation—— Protein

B. Transcription
1. A segment of DNA (gene) is transcribed to produce mRNA
2. mRNA is a single stranded
3. mRNA provides the directions for how to make a protein
4. RNA polymerase binds to DNA site called promoter to initiate transcription

C. Translation
1. mRNA is read at ribosomes
2. tRNA’s bring amino acids to the ribosomes
3. Peptide bonds form between amino acids and the protein is produced
IV. Control of Gene Expression

A. Induction and Repression regulate transcription of mRNA
1. i.e. controls proteins (enzymes) that cell produces

B. Induction
1. Activates (turns on) the transcription of a gene
2. Inducer – substance that induces transcription and leads to the production of enzymes
3. The enzymes that are produced are called inducible enzymes
4. This occurs when these enzymes are needed by the cell
C. Repression
1. Inhibits gene expression, decreases synthesis of enzymes
2. Repressor – a protein that blocks transcription
3. This stops the production of products that the cell doesn’t currently need

V. Inducible Gene Regulation (Example – Lac Operon Model)
A. ß-galactosidase is an enzyme that breaks down lactose to glucose and galactose
B. When E. coli is in a medium without lactose, there is no ß-galactosidase in the cell
C. When lactose is added to the medium (and there is no available glucose), E. coli produces
large amounts of ß-galactosidase so it can use lactose as food.
D. Operon genes (segments of DNA on bacterial chromosome) regulate this process
1. Structural genes
a. Enzymes required for lactose uptake and use in E. coli
1) ß-galactosidase, lac permease and transacetylase
2. Control genes
a. Promotor – site where RNA polymerase binds (necessary for transcription)
b. Operator – stop and go signal to initiate transcription for structural genes
1) In “OFF” mode until turned on by presence of inducer (lactose)

E. Lac Operon
1. Lactose is the Inducer.
2. Lactose binds to repressor protein which causes it to detach from the operator that it
was blocking.
3. Now RNA polymerase can bind to promoter allowing transcription and translation of
the structural genes. *Note: Lack of glucose also enhances binding of DNA Polymerase
4. E. coli can now digest and use lactose for energy.

VI. Repressible Gene Regulation (Example – Arginine production)
A. This operon is always in the “ON” mode and is shut off when product (arginine) is no longer
needed because it is in excess in the cell.
B. When it is in excess in the cell it acts as a corepressor and binds to and activates the repressor
protein
C. The repressor protein then binds to the operator and blocks transcription
D. This stops further production of arginine
VII. Mutations

A. Driving force of evolution; adds variation to the population

B. Change (mistake) in the base sequence (gene) of DNA
1. May change the protein that is coded for by that gene which could alter the structure or
physiology of that organism
2. Could have no effect, lethal effect, or beneficial effect on the organism
3. Most mutations are deleterious, i.e. harmful and lethal
4. Beneficial mutations create antibiotic resistance bacteria
5. Mutations are also responsible for increased pathogenicity of microbes
a. E. coli – certain strains produce toxins and increased intestinal cell adherence
b. Salmonella enterica – mutation of protein in outer membrane helps it resist
phagocytosis

C. Spontaneous vs. Induced Mutations
1. Spontaneous Mutations – random changes in DNA due to errors in DNA replication
a. No known mutagen caused mistake
2. Induced Mutations – result from exposure to known mutagen
a. Mutagen – increases frequency of mutations
1. Chemical Mutagen – ex) benzo[a]pyrene in smoke
2. Physical Mutagen – ex) U.V. radiation
3. Mutation Rate – probability the at gene will mutate when a cell divides
a. Spontaneous rate = 10-6 (one in a million replicated genes)
b. With Mutagen = 10-4 – 10-6 (one in a 1000 or 10,000)

D. Carcinogens
1. Mutagens that cause cancer in animals, including human
2. Carcinogenic – cancer causing
VII. Transmission of Genetic Material in Bacteria

A. DNA transfer from one bacterium to another
1. Plasmid
2. Chromosomal fragment

B. Modes of Transfer
1. Transformation
2. Conjugation
3. Transduction

 

C. Transformation
1. Genes are transferred as “naked” DNA
2. Griffith Experiment – (1928)
a. Showed DNA could be transferred between bacteria
b. Genetic Recombination
1) One bacterium donates DNA (genes) to another bacterium which is
then incorporated into the recipients chromosome

Griffith Experiment

Used two strains of Streptococcus pneumoniae
1. Virulent (pathogenic) – has capsule to escape phagocytosis
2. Avirulent – no capsule

Step 1 – Injected living encapsulated (w/ capsule) strains into mouse
-Mouse died
-Colonies of encapsulated bacteria were isolated from dead mouse

Step 2 – Injected living non-encapsulated (w/out capsule) strain into mouse
-Mouse remained healthy
-A few colonies of non-encapsulated bacteria were isolated from mouse
*most were killed by phagocytosis in mouse

Step 3 – Injected heat-killed encapsulated strains into mouse
-Mouse remained healthy
-No colonies could be isolated from mouse (bacteria already dead before injection)

Step 4 – Injected living non-encapsulated strains and heat-killed strains into mouse
-Mouse died
-Colonies of encapsulated bacteria were isolated from dead mouse
*In nature (perhaps your GI tract), when bacteria die and are lysed, they release their DNA to the environment. Other bacteria may be able to take up the released DNA fragments and incorporate them into their own chromosomes by recombination. In this case, pathogenic genes could be passed to an otherwise non-pathogenic bacterium.

 

D. Conjugation
1. Involves transfer of a Plasmid
2. Requires cell to cell contact
a. Gram – cells use sex pili for contact
b. Gram+ cells use sticky substance for contact

Conjugation in E. Coli

1. Plasmid in known as F factor (f for fertility)
a. Carries genes for sex pili and transfer of plasmid to another cell
2. Donors carrying F factor plasmids are known as F+ cells
3. Recipients who don’t have F factor plasmids are F- cells
4. After donors transferred a copy of the F factor plasmid, recipient become F+ cell
Resistance factors (R factors)
1. Plasmids with antibiotic resistant genes
a. Code for enzymes that inactivate antibiotics
2. Resistant genes can accumulate in one plasmid
a. Ex) Plasmid R100 contains resistance genes for sulfonamides, streptomycin,
chloramphenicol and tetracycline
3. Can be transferred through conjugation between different species, such as E. coli and
Salmonella
E. Transduction
1. DNA is transferred from one bacterium to another by a virus that infects bacteria
a. Bacteriophage (phage) – virus that infects bacteria