Mico Genetics

Why do we study microbial genetics?

To know & prevent diseases

To know & study Antibiotic Resistance

Biotechnology

• Evolution and emerging threats of diseases

DNA Replication

Based on template and base-pairing

• Double Helix

• Base Pairs

• DNA ( A-T & G-C) pair

• RNA ( A- U & G- C) pair

Flow of Information

DNA —> RNA —> Protein

DNA Replication Structure/ Steps

1) Helicase -Separating parent DNA apart

• Enzyme

  2) Single Strand binding protein → Stabilizes DNA strand so they STAY APART

  3) Primase - Makes short RNA Primer on DNA template

  4) Topoisomerase - Relieves tension ahead of Fork (Enzyme)

  5) DNA Polymerase iii - Synthesize new DNA strands on both templates

  6) DNA Polymerase I - Digest RNA primers and replace with DNA

  7) DNA Ligase - Connects and bonds DNA fragments together

Directions of DNA

DNA only synthesized in 5 → 3 Direction

Leading Strand (Top)

• Continuous synthesis towards the fork

Lagging Strand (Bottom)

• Discontinuous synthesis away from fork

What is the replication of bacteria?

Bacteria Replication chromosomes is bidrectional

• Vertical Gene Transfer (for prokaryotic)

• Asexual reproduction (one parent → two daughters)

Binary Fission

is the vertical gene transfer for bacteria

“It’s the cell division for bacteria” ** KNOW THE STEPS

1. Chromosome replicates and divides into 2 nascent cells.

2. Septum grows between 2 nascent cells.

   • Cell wall division in the middle

3. Cells physically separate, producing 2 new daughter cells.

Gene Expression for Prokaryotic

Genetic information is used within a cell to produce the proteins needed for the cell function

Gene Expression on Humans vs. Bacteria

Humans - Diploid 2 Sets of Chromosomes

Prokaryotes - Haploid 1 Set of chromosomes ( 1 chromosome)

Genotype

Genetic makeup

Phenotype

Physical/Observable apperance

RNA (Ribonucleic Acid)

Single Stranded macromolecule

Ribose Sugar

4 Nitrogenous Base

• Adenine (A)

• Guanine (G)

• Cytosine (C)

• Uracil (U)

DNA ( Deoxyribonucleic Acid)

Double- Stranded macromolecule

Deoxyribose Sugar

4 Nitrogenous Base

Adenine ( A)

Guanine (G)

Cytosine( C)

Thymine ( T)

Transcription

DNA is read by RNA polymerase which synthesize RNA transcription in 5→ 3 direction

It adds RNA nucleotides (A, U, C, G) that are complementary to the DNA template (A pairs with U, T with A, C with G, G with C).

*DNA → RNA
(Think of it as copying a recipe from a book—DNA is the book, RNA is your copy.)

Where does transcription begin?

in : Promoter

a special DNA sequence ahead of Gene

**Transcription and Translation are coupled in prokaryotes (Happens Simultaneously)

mRna

DNA is the template for mRNA

During elongation of mRNA, one strand of DNA acts as the template strand for RNA synthesis

RNA Polymerase creates a mRNA strand that is complementary to the DNA template strand

Translation RNA

Types of RNA involved in Translation:

Ribosomal RNA

Makes up ribosome which will synthesize proteins ( forms peptide bonds between amino acids)

Transfer RNA (tRNA)

RNA that brings correct amino acids to ribosomes during translation

Messenger RNA (mRNA)

RNA that carries codon info to be read by ribosome for translation

Multiple Ribosomes

Can translate one mRNA at the same time (lots of protein made in short time)

Translation

is the process where the RNA molecule is used to synthesize a specific sequence of amino acids, which then fold into a functional polypeptide (protein) from the mRNA sequence

Genetic Code / Codons

is the set of three nucleotide sequences ( called CODONS) that translate genetic information (DNA/RNA) into proteins.

• Multiple codons correspond to a single amino acid, result the code is redundant or degenerated

tRNA

Translation

tRNA helps link the “languages” of nucleotides and amino acids

Genetic Code Read by

Read by Ribosomes

the mRNA nucleotide sequence is “read” as a sequence of codons by Ribosomes

in Prokaryotes 70s ribosomes is a large protein complex

Translation overview

Ribosomes

transfer RNA (tRNA)

Codon

Amino Acid

Genetic Code

mRNA read 5 → 3

Significance of start and stop codons

Regulation of Genes

Gene Expression in Eukaryotic cells is regulated which is particularly important for multicellular organisms in making tissue/ cells/ organs despite having same DNA

Regulation of genes in single cell prokaryotic organism is also important

Why important for Bacteria?

Controls gene expression to conserve energy ( Be more efficient)

Turning Genes on/off

ON : making mRNA transcription then translating into protein ( active)

OFF: Very little protein made (repressed)

Influence of ON/OFF Gene

Influenced Factors cause by internal and external cues

• Feedback within the cell

• Environmental changes ( Temp, pH, osmotic, pressure )

• Chemical signals ( proteins/ molecules from other cells)

• Chemical Tags on DNA

Genes are Regulated by a stimulus

Quorum - Sensing regulates Gene

Quorum sensing is the ability of bacteria to communicate and coordinate group behavior through signaling molecules that turn ON and OFF genes

Bacteria can control gene expression base on cell concentration/density

Operator

a segment of DNA that a repressor protein can bind to

controls whether transcription occurs

Regulatory Gene

Encodes the repressor protein that regulates the operon

** not technically part of the operon

Repressible Operon

Repressible operon is one that is normally active (transcribing genes) but can be turned OFF ( repressed) when a certain molecule is present

It’s repressible because the cell needs these genes to be "on" most of the time, but shuts them off when their product is in excess.

A corepressor, usually the end product of the operon’s pathway, binds to the repressor protein, activating it.

The activated repressor then binds to the operator, blocking RNA polymerase from transcribing the genes.

Repressor : guard that blocks the gene

Corepressor - key that activates the guard

Tryptophan - Actual thing that cell is making (product) of the operon

2 Types of Operons

Repressible - Default stage : On ( needs to be turned OFF when the product is already available)

Inducible - Default stage : Off ( needs to be turned ON by something)

Repressor Operon in E.coli ( TrP) Protein

• synthesize tryptophan using enzymes that are encoded by five structural genes (E,D,C,B,A) located next to each other in the trp operon

• When environmental tryptophan is low, the operon is turned on. This means that transcription is initiated, the genes are expressed, and tryptophan is synthesized. However, if tryptophan is present in the environment, the trp operon is turned off. Transcription does not occur and tryptophan is not synthesized.

When tRP not present

When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase from transcribing the structural genes, stopping expression of the operon.

Inducible Operon

An inducible operon is a group of genes that is normally turned OFF, but it can be turned ON (induced) when a specific substance (called an inducer) is present.

Step 1: Operon is OFF by default

• A repressor protein is attached to the operator (a part of the operon).

• This blocks RNA polymerase from reading the genes.

• So the genes are not being used — no proteins are made.

🟩 Step 2: Inducer appears

• A small molecule (the inducer) shows up — often the thing the cell wants to break down.

• The inducer binds to the repressor.

• This changes the shape of the repressor, so it falls off the DNA.

• Now RNA polymerase can read the genes and make the needed proteins.

Inducer - Lac Operon example

Found in E.coli

• The lac operon encodes three structural genes necessary to acquire and process the disaccharide lactose from the environment

• lactose must be present. This makes sense for the cell because it would be energetically wasteful to create the enzymes to process lactose if lactose was not available.

• In the absence of lactose, the lac repressor is bound to the operator region of the lac operon, physically preventing RNA polymerase from transcribing the structural genes.

• when lactose is present, the lactose inside the cell is converted to allolactose. Allolactose serves as an inducer molecule, binding to the repressor and changing its shape so that it is no longer able to bind to the operator DNA.

• Removal of the repressor in the presence of lactose allows RNA polymerase to move through the operator region and begin transcription of the lac structural genes.

Lac Operon activation depends on glycose levels

Glycose is the preferred sugar by Bacteria

Bacteria grows quickly on glucose

When no glucose is present it is that bacteria will began to consume other sugars like lactose

Positive regulation by cAMP

• cAMP (cyclic adenosine monophosphate) is a small signaling molecule in cells.

• In bacteria, it helps cells respond to low levels of glucose, the cell’s preferred energy source.

When cell consumes all glucose, concentrations of cyclic AMP (cAMP) builds up in the cell

CAP proteins bound by cAMP which allows CAP to bind the promoter to help RNA polymerase to bind and transcribe

When glucose is low:

1. cAMP levels go up.

2. cAMP binds to CAP, forming the cAMP-CAP complex.

3. This complex binds near the lac promoter.

4. It helps RNA polymerase bind better, so transcription of the lac genes increases.

5. If lactose is also present, the repressor is removed → lac operon turns ON fully.

🛑 When glucose is high:

• cAMP levels are low.

• No cAMP → CAP can't bind the DNA.

• RNA polymerase doesn’t bind well → lac operon stays mostly OFF, even if lactose is there.

cAMP

Signal glucose availability

High [Glucose = Low [cAMP]

Low [Glucose] = High [cAMP]

CAP = Catabolite (Activator Protein)

• Helps RNA polymerase transcribe needs [cAMP]

• CAP is inactive without [cAMP]

Bacteria acquire new genes?

Mutations - Alterations of genetic material already present in cell

Genetic Recombination - Gain new genetic material from another through horizontal gene transfer

Mutations

Can alter genes

• Mutations- are permanent changes in the base sequence of DNA this even could alter a genes function

Point Mutations - are mutations that change one ( or a few) nucleotides base within the DNA sequence

• One (or two) nucleotides are
  incorrectly inserted or
  deleted from the DNA
  sequence, which leads to a
  shift in the reading frame
  (frameshift).

Spontaneous Mutations - occur in the absence of mutations causing agents

• DNA Replication

Mutagens ( things that directly or indirectly cause mutations)

-Chemicals (nitrous acid)

-Radiation ( ultraviolet)

Eukaryotes vs. Bacteria

Most Eukaryotes : Forming new organisms and genetic recombination are linked together ( sexual reproduction and meiosis)

Bacteria: Forming new organisms and genetic recombination are NOT linked ( asexual reproduction and occasional genetic recombination)

Recombination

Genetic information can be transferred horizontally between cells of the same generation

Vertical Gene Transfer | in Bacteria

Binary fission

mother cell passes genetic information to two daughter cells

Different generations

Horizontal Gene Transfer | in Bacteria

Exchange of genes between existing bacteria

same generation

3 Majors of Horizontal Gene Transfer

• Transformation

• Transduction

• Conjugation

Transformation

Cells take up exogenous (outside) DNA from it’s environment

-Genetic recombination (exchange of DNA to form new combination of genes) occur to incorporate new DNA.

** transfers DNA from the outside

not many can do this

happens naturally

• Recipient cell must be competent - its in a special physiological state to uptake DNA

Transduction is transfer of DNA by viruses

Transduction is the viral transfer of genetic material from one bacterium to another

DNA transfer to host bacterium via Phage- accidentally packaged piece of previous host chromosome

Phages ( Bacteriophages)

Phages normally infect bacteria to replicate and produce more phages

Sometimes the phages package the wrong, DNA ( fragments of the bacterial host chromosome) which result in transduction

Conjugation “mating” bacteria

Conjugation is the transfer of genetic material in the form of a plasmid from one bacterium to another through sex pilus

The sex pilus is a physical connection between the two bacteria
“ Cell - to - cell”

Donor Bacteria cell will transfer plasma recipient bacteria cell via sex pilus conjugation bridge.

a plasmid is a small, circular DNA molecule that can be transferred from one bacterium to another self-replicating, gene-containing piece of DNA
(about 1-5% of the size of a chromosome) that exists outside
of the chromosome.

Plasmid Transfers

Plasmid transfers from donor to recipient

Cells are the opposite mating type

Donor ( F+) carries plasmid and recipient (F-) does not

** the recipient will always be the one that doesn’t carry the plasmid

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats

• Adaptive immune system within prokaryotes that defend them against phages

• Bacteria gain immunity against phages using CRISPR

• Biologist took the system and used it for foundation for an entirely new gene editing tool potential to revolutionize the world and future

How to gene edit using CRISPR

is a system like a precise molecular scissors that cuts exactly where you want it to so you can change the gene you want.

“ you can do it your way’

Growth of Microbes

Growth refers to an increase in the numbers of cells not an increase in cell size

Prokaryotes Grow

Grow by Binary Fission

grows and increases its cellular components

Temp range

Minimum temperature of growth = lowest temperature at
which there is growth (below this temperature, there is no
growth).
• Maximum temperature of growth = highest temperature at
which there is growth (above this temperature, there is no
growth).
• Optimum temperature of growth = temperature at which
there is the most/best growth (allows the highest rate of cell
division to occur).

Classification for Temp.

Psychrophiles – Microbes that can grow from below 0°C to a maximum of
20°C. Optimal temperature = 15°C.

Psychrotrophs – Microbes that grow at temperatures of 4°C to 25°C.
Responsible for spoiling food in the fridge.

Mesophiles – Microbes that are adapted to temperatures of 15°C - 45°C
(like the human body).

Thermophiles – Microbes growing at 50°C - 80°C (like hot springs)

Extreme Thermophiles (Hyperthermophiles)– Microbes that can survive at
temperatures from 80°C - 110°C (like deep ocean thermal vents).

Thermoduric – Microbes that are generally mesophilic and are able to
survive at high temperatures (70°C or higher) for short periods of time.
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Microbes are limited by temp.

At high temperature, enzymes denature and membranes
become more fluid (likely to rupture).

At freezing temperatures, growth is inhibited by reduced
enzyme activity (or denaturation), increase in fluid viscosity,
and membrane rigidity

Microbes tolerate osmotic stresses

Having enough water is important for any cell.

Bacteria maintain relatively high cytoplasmic solute
concentrations which promotes the inward diffusion of water
into the cell by osmosis.
Osmosis is the movement of water across a semi-permeable
membrane, and it occurs as a result of the different solute
concentrations on either side of this membrane.

The result is a more equal solute
concentration on both sides of the
membrane

Hypotonic & Hypertonic Tolerate osmotic stress

Hypotonic Environment

• Not Harmful to bacteria

Hypertonic Environment

• Most bacteria shrivel ( due to plasmolysis) and stop dividing

• inhibit bacteria growth

Hypertonic Environment Growth

Bacteria can survive high osmotic pressure by increasing the solute concentration of their cytoplasm

Halotolerant: Bacteria are those that can withstand high osmotic pressure exerted by salt

Halophilic : Bacteria are those that REQUIRE high concentrations of salt to survive

Microorganisms Classification for Osmotic Conditions

Halophiles – Microbes adapted to live and grow only in high salinity
(hypertonic), about 5 – 15%. Most are Archaea, but some are bacteria.

Extreme Halophiles – Microbes that can only grow in 15% - 30% saline
environments (like the Dead Sea and the Great Salt Lake).

Halotolerant – Microbes that can survive and grow in slightly higher saline
environments, from 0 - 11% salinity

Osmophiles – Microbes that can tolerate very high sugar concentrations
(high osmotic pressures). Water activity is not always determined by salt.
Water activity only relates to the solute concentration (and solutes can be
salt, sugars, amino acids, or other molecules).
Microorganism classification for osmotic conditions

*Halotolerant bacteria like S. aureus and B. cereus can grow in salty foods, produce
toxins, and cause food poisoning.*

pH and microbial growth

-measurement of the concentration of H+ in a solution

The HIGHER the concentration of H+ the lower the pH

Acidophiles | classification of pH

optimal growth at less than pH of 5.5

Neutrophiles

Optimal growth at range of pH 6-8

Alkaliphiles | classification of pH

optimal growth at pH of 8-10.5

Oxygen Requirements for Microbes

1.  Strict Aerobes – Microbes that rely solely on oxygen as the final electron acceptor for cellular respiration to produce ATP.

2. Facultative Anaerobes – Microbes that can use oxygen but do not require it and are capable of growing in the absence of oxygen.

3. Strict Anaerobes – Microbes that cannot grow in the presence of oxygen at all. Rely on fermentation and/or anaerobic respiration to make ATP.

4. Aerotolerant – Microbes that can tolerate the presence of oxygen but do not use oxygen to make ATP.

5. Microaerophilic – Microbes that require oxygen to grow but cannot tolerate high concentrations of oxygen.

ROS ( Reactive Oxygen Species)

"These are very reactive ions and molecules formed when oxygen is only partly reduced. They can damage almost any large molecule or structure they touch."

Oxygen (O₂) reacts with other molecules in the cell to produce toxic forms of oxygen called reactive oxygen species (ROS).

• Toxic and Harmful to cells - the result of the oxygen not complete

• Cell need enzymes to inactivate break down

Eliminating the harm of ROS

Enzymes and vitamins can detoxify ROS molecules in the cell

Superoxide dismutase

Catalase

Peroxidase

Vitamin C

Vitamin E

4 Groups of Marcos

Nitrogen

Carbon

Hydrogen

Oxygen

Phosphorus

Sulfur

CHONPS **

Trace Elements

Cell Requires very little of them

Sodium, Potassium Chlorine, Magnesium , Calcium Iron

4 STAGES of bacteria Growth

1 Lag Phase

• No increase in number of living bacteria cells

2 Log Phase

• Exponential increase in number of living bacteria cells

3 Stationary Phase

• Plateau in number of living bacteria cells, rate of cell division and death roughly equal

4 Death or decline Phase

• Exponential decrease in number of living bacteria cells

Growth of Bacteria FORMULA

N = number of cells at any generation

N0 = is the starting of number cells

Exponent n is the number of generations