Physical factors affecting bacterial growth

Environmental Effects on Microbial Growth:

Temperature

- Growth stops to occur at a temperature above the minimum and maximum

- The optimum is closer to the maximum
- When the temperature is low/minimum, the membrane loses its fluidity
- This is called membrane gelling
- Functions involving fluidity will be affected such as transport
- Enzymes will not function very well
- When the temperature is at its optimum
- The enzymatic reactions will be occurring at maximal possible rate
- When the temperature is at its maximum/hot temperature
- Protein denaturation will occur
- Cytoplasmic membrane will be too fluid which will collapse
- Thermal lysis will occur
Classification of Microbes based on the temp of their optimal growth
Molecular Adaptations of psychrophiles
Legend:
> = more
< = Less
Enzymes
- > alpha-helix; < beta-sheet - this will allow greater flexibility of the molecule
- > polar amino acids, lesser hydrophobic amino acids
- < weak bonds: hydrogen and ionic bonds

Cytoplasmic membrane fatty acids:

- The fatty acids need to have means to make up for loss fluidity
- > Cis double bonds
- Will introduce bends bends which allow greater fluidity
- Will have shorter fatty acids

Molecular chaperones cold shock proteins

- Maintain cold-sensitive proteins in an active forms
- Bind specific mRNAs 0 facilitate translation under cold conditions

Cryoprotectants:
- Antifreeze proteins or specific solutes - glycerol or certain sugars, to prevent formation of
ice crystals
- Abundant levels of exopolysaccharide cell surface slime
Molecular Adaptations of Thermophiles/Hyerthermophiles

Enzymes
- > ionic bonding between basic and acidic amino acids
- Highly hydroponic interiors
- This would stabilize the molecule at high temperatures

Cytoplasmic membrane phospholipids:

- Saturated fatty acids
- Long fatty acid chains
- Phospholipid monolayer in some Archaea

Solutes that help stabilize proteins against thermal denaturation

- Di-inositol phosphate, diglycerol phosphate, and mannosyglycerate in certain
hyperthermophiles

Chromosomal DNA positive supercoils

- Reverse DNA gyrase
pH, Osmolarity, and Oxygen

Most organisms:
- Grow best between pH 6 and 8
- The internal pH of a cell must stay relatively close to neutral even though the external pH
is highly acidic or basic
Osmotic Effects on Microbial Growth

- What are:
- Halotolerant microorganisms
- Halophile
- Osmophile
- Xerophile
 - Compatible solutes
- Not toxic to the cell
- Synthesized or pumped into the cell when cell is in a hypertonic environment
- The environment becomes an isotonic solution and can withstand the
high-salt environment
- Highly water-soluble organic molecules
- Include sugars, alcohols, and amino acid derivatives
- NOTE: When the amount of solute increases, then the medium will be hypotonic,
there will be a tendency for the water to leave the cell. The organism can
synthesize more compatible solutes so it can equal the amount of cells that left
the cell

Oxygen and Microbial Growth

- Aerobes
- obligate/strict aerobs:
- Require free oxygen (if oxygen is not present, the molecule cant grow)
- Type of metabolism: aerobic respiration
- Falcutative aerobs
- More versatile
- Can grow with or without oxygen (prefers to grow with oxygen)
- Type of metabolism: aerobic respiration or anaerobic respiration or
fermentation
- Microaerophilic
- Required but at levels lower than atmospheric
- If there is no oxygen they will not grow; if there is too much
oxygen, they will not grow
- Tyep pf metabolism: aerobic respiration
- Anaerobs
- Aerotolerant:
- Not required, and growth no better when O2 present
- Type of metabolis: fermentation
- Obligate
- Oxygen is harmful or lethal
- Type of metabolism: fermentation or anaerobic respiration

- Oxic zone: has oxygen

- Anoxic zone: has no oxygen
a. Test tube A is:
b. Test Tube B is:
c. Test Tube C is Facultative
d. Test Tube D is Microaerophile
e. Test Tube E is: Aerotolerant
 - Why can O2 harm the molecule?
- When oxygen is present, ther are toxic metabolic products such as
- Superoxide
- Hydrogen peroxide
- Hydroxyl radical
- These are oxidizing agents which can extract electrons from other molecules

- Why are there organisms that are not killed from the lack of oxygen?

Catalase: Catalyzed to break down to non-toxic water and oxygen

Peroxidase: NADH will be oxidezed and water is produced
- Catalyzes the oxidation of NADH by hydrogen peroxide so it will be a non-toxic water
Superoxide dismutase: the reduced oxygen will be converted it into hydrogen peroxide
Superoxide Dismutase/Catalase in combination: oxygen is converted into not just hydrogen
peroxide but into non-toxic water and oxygen
Superoxide Reductase: The substrate is super oxide but there is the presence of rubredoxin
and it can be reduced which can create hydrogen peroxide and an oxidezed rubredoxin

Bacterial Nutrition
Classification of microorganisms based on:
1. Carbon source
a. Heterotroph
i. Requires an organic carbon source
1. An organic carbon has both carbon and hydrogen
b. Autotroph
i. Can live off an inorganic carbon source
1. Can live off CO2
2. Electron (hydrogen) source:
a. Organotroph
i. Would require an organic electron source (ex. Glucose, amino acids, fatty
acids)
b. Lithotroph
i. Would require an electron or hydrogen from inorganic sources
3. Energy source
a. Chemotroph
i. Can get energy from oxidation, reduction and other chemical reactions
b. Phototroph
i. Can make energy through light
- All these elements are essential in a cell

- They are needed in bigger quantity

- Trace elements are needed in small amounts

- Can chelate iron from the environment

Bacterial Growth and Reproduction

Overview of Binary Fission

1. The binding of DNAA to OriC initiated replication

2. SeqA will block oriC which will cause the cell to elongate
 3. The cell will then segregate
4. It woll cause a z-ring formation
5. It will then have a cell division

Fts proteins:
- FTS: Filamentous temperative sensitive
- Archaea and Bacteria
- Interact to form division apparatus: divisome

FtsZ:
- Structurally similar to eukaryotic tubulin
- Similar proteins found in mitochondria and chloroplasts
- Defines division plane in the cell
- FtsZ ring forms after DNA replication

- FtsZ proteins
- FtsZ ring: forms the divisor
- ZipA and FtsA
- Anchors the FtsZ to the cytoiplasmic membrane
- FtsA
- Also has ATPase activity
- GTP is also another soure of energy
- FtsI
- Penicillin binding protein
- Peptidoglycan synthesis
- FtsK
- Separates chromosome
MreB:
- Major shape-determining protein in rods, spirullum spirochetes
- Absent in cocci
- Forms actin-like cytoskeleton
Peptidoglycan Synthesis and Cell Division

- FtsZ ring is localized at one point

The peptidoglycan needs to increase in size so it needs to go through these processes:

- Autolysin: introduces breaks in the glycosidic binds in the peptidoglycan layer
- Transglycosylase: forms glycosidic bonds
- Transpeptidase: forms crosslinking of tetrapeptide chains

New peptidoglycan is synthesized in the cytoplasm of the cell

Summary of the peptidoglycan synthesis
 - Bactoprenol will insert itself into the cytoplasmic membrane due to its hydrophobic
properties
- Autolysin breaks the glycolytic bonds in the preexisting peptidoglycan, while
transglycosylase synthesizes them, linking old peptidoglycan with new
- The transpeptidation reaction that leads to the final cross linking of 2 peptidoglycan
chains

Exponential Growth or Logarithmic:

- Population doubles every generation
- 100% increase in population every generation
Example:

Bacterial Growth Curve

- Bacterial growth in a batch culture
- Batch culture: you prepare the setup with the appropriate medium and you will
inoculate it with a known number of bacterial cells and let the organisms grow

- There are 4 phases

- Lag Phase
- There is a lag in the increase of the population
 - There is no increase in the number of cells but they are actively
metabolizing and growing in size
- Can vary in length/time (may be shorter or longer)
- If the cell has insufficient nutrients to prepare itself for exponential
phase, it will need more time
- If it has enough nutrients, it can directly go to the exponential
phase
- Exponential Phase
- Where the cells undergoing binary fission
- Doubling in the number fo cells per generation
- Stationary Phase
- There is no increase of the population of the cells
- Even though this is the are where there are the most viable cells, it is the
point where there are cells that are both dividing and dying
- Death Phase
- Population of cells decrease
- Most cells are dying
- It can also plateu out and it will just remain the same amount

Measurements of Microbial Growth

A. Direct

- You count both the dead and living cells

- Total Counts
1. You can have the total count by staining the cell
 a. This is called Microscopic Count (con’t)
i. Examples
1. DAPI-4’, 6-diamidophenylindole-DNA
2. Acridine orange- DNA
ii. Flow cytometer

- PI will only penetrate those without intact cytoplasmic membrane (dead cells)
- SYTO 9 will penetrate all cells, but since PI is already in the dead cells, it has a stronger
affinity for NA than SYTO9, so the dead cells will remain red while the, live cells will
become green

- Plate Count
 1. There may be many microorganisms that may be to close to each other that they may be
too many to count
a. Due to this, dilution is done
2. You perform a series of dilution
a. Serial dilution
3. A colony is a clone of cells that arose from a single parent cell
4. Constraints in the plate count
a. The medium you used might take longer times to make colonies
b. There may be colonies that may grow below under larger colonies
How to inoculate:
Measurements of Microbial Growth

B. Direct

- The more turbid the molecule is, the higher the optimal density adn this shows more
growth in the medium
- Gives the total count

- Continuous culture: The Chemostat

- Maintains culture in exponential phase for long periods, days and weeks
Stages of Biofilm development
1. Initial Adhesion
a. Presence of planktonic cells
b. Adherence and attachment
c. Microorganism surface interaction
2. Early Developmenr
a. EPS production
b. Cell Division
3. Mature Biofilms
a. EPS matrix
b. Heterogeneity
c. microenvironments
d. Tolerant cells
e. Social interactions
4. Late Stafe Disprsal
a. EPS Matrix remodelling
b. Activate dispersal mechanisms