Growth and Cell division Flashcards
What are the 3 most important elements that bacteria need for growth?
Carbon, Nitrogen, oxygen
What are growth factors?
Growth factors are additional things added into the media in order for the bacteria to grow properly
-not every organisms requires the same growth factors
Examples: amino acids, cholesterol, NADH, Heme…
Do all bacteria require the same growth factors?
No
Which bacteria can synthesize all 20 amino acids from glucose?
E-coli
What are photoautotrophs?
Get their carbon from CO2 and use light energy
ex: plants, algae and cyanobacteria
What are photoheterotrophs
Get their carbon from carbon containing compounds but get their energy from light
ex: green and purple nonsulfur bacteria
What are chemoautotrops
get their carbon from CO2 and their energy from chemical compounds
ex: hydrogen, sulfur, and nitrifying bacteria
Chemoheterotrophs
Get their carbon from carbon containing compounds and their energy from chemical compounds
ex: Lots of examples. includes organisms that undergo aerobic & anaerobic respiration as well as fermentation
How can microbes be organized based on their oxygen requirement
1: aerobes: need air
2. anaerobes: cannot be in the presence of air
3. Facultative anaerobes: don’t need oxygen but can be around it
Why is O2 necessary for obligate aerobes?
It is the final electron acceptor of the ETC
Why is O2 deadly for obligate anaerobes?
Because they lack the 2 enzymes to eliminate toxic forms of oxygen (superoxide, hydroxyl radicals, and hydrogen peroxide) formed by oxygen reducing enzymes
What are the 2 enzymes that aerobic and facultative anaerobic bacteria use to eliminate toxic oxygen forms?
- Superoxide dismutase
2. Catalase
What does the catalase test differentiate between?
two types of gram positive bacteria
- streptococci
- staphylococci
How does the catalase test differentiate between the 2 bacteria?
Streptococcus lacks the catalase enzyme so will not bubble
Staphylococcus has the enzyme and will bubble (positive test result)
What are Psychrophiles
Bacteria that grow at very low temperatures
-like 10 ºC
What are Mesophiles
Bacteria that grow around body temperature (37ºC)
What are thermophiles and hyperthermophiles
Bacteria that like hot and very hot temperatures
-65ºC ish and close to 100 ºC
What are the 3 kinds of bacterial groupings based on optimal pH for growth?
Acidophiles (0-5.5)
Neutrophiles (5.5-8.5)
Alkaliphiles (8.5-14)
What are Halophiles?
Bacteria that require high salt concentrations to grow
What are Barophiles
Bacteria that require high hydrostatic pressure to grow
-found in the bottom of the ocean
What occurs during the Lag phase of typical bacterial growth?
- No net growth, little or no cell division
- Bacteria take this time to adapt to new surroundings, or recover from toxic products
- Synthesis of new machinery/enzymes/coenzymes to produces the necessary growth products
What can result in an extended lag phase for the bacteria?
if they are transferred from a rich growth medium to one that is lacking in readily utilizable nutrients and growth factors.
-need to take time to synthesize enzymes
What occurs during the log phase
- Rapid exponential growth of the population
- Cellular enzymes working at full capacity
- Mass and volume increase proportionally to production of new cellular components like DNA and protein
What is steady state growth
Cells in which the mass and volume are increasing proportionally to the production of new cellular components
In what stage are the cells most healthy
In the log stage
-this is where you want to sample cells from
What happens during the stationary phase?
- Maintenance metabolism state during which there is little to no net replication
- Cells stop growing
- Depletion of key nutrients and oxygen, accumulation of toxic wastes
Which stage is most representative of bacteria in nature ?
The stationary phase
What happens in the death phase?
Depletion of cellular energy
Cell lysis due to autolytic enzymes
What are 6 physiological changes for the bacteria that occur during the stationary phase?
- Become smaller and rounder
- Changes in surface properties: become more hydrophobic and adhesive
- Changes in the membrane phospholipid composition
- methylation of the DB in unsaturated FA to give cyclopropyl derivatives - Increase in the protein and RNA turnover rate
- Synthesis of new proteins (like PhoE in phosphate limiting situations)
- Increase in resistance to environmental stress
The synthesis of which compound helps in the resistance to environmental stress?
starvation sigma factor, σs (KatF) encoded by the rpoS or katF gene.
σs (KatF) is required for…?
The synthesis of at least 30 proteins induced by carbon, nitrogen, and phosphate starvation
What is the definition of a sigma factor in general?
bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters.
What are 2 other regulators for stationary phase gene expression
- Cyclic AMP-cAMP receptor protein complex (cAMP-CRP)
- Guanosine tetraphosphate (ppGpp)
- also called alarmone
How does ppGpp work ?
Inhibits RNA and tRNA synthesis during amino acid starvation
-leads to an inhibition of ribosomal protein synthesis
When can you define a growth yield constant Y?
When a single nutrient is the sole source of energy and carbon
What is Y?
amount of dry weight of cells produced per weight of nutrient.
-ex: if Y = 0.5, then 50% of the nutrient is converted to cellular material and the other half is oxidized to CO2
What conditions mean that a culture is in steady state?
when all its components double with each division
Under what 2 conditions can steady state be achieved?
- Maintaining the cell culture by subculturing
2. while the cells are in continuous growth
How can the growth rate be controlled by the flow rate?
The concentration of a nutrient is kept low so it is the limiting nutrient for growth. Thus, when a drop of medium enters the growth chamber, the growth-limiting nutrient is used up immediately and the cells cannot continue to grow until the next drop of medium enters
What are No, Nt, and n?
No = the initial number of cells Nt = the number of cells at time t n = the number of generations in time t
Nt = ?
Nt = No x 2^n
log Nt = log No x n log2
n = (log Nt - log No) /log2
n= (log Nt - log No) / 0.301
what is k?
The growth rate constant
k = ?
k = n/t
also = (log Nt - log No) / 0.301t
AND = 1/g
what is g?
generation time or doubling time
-time taken for Nt = 2No
g = ?
1/k
What is D?
the dilution rate
-also happens to be equal to the growth rate constant k
D = ?
F/V
f= flow rae
v= culture volume
2 important points about the measurement of growth
- Growth is defined as an increase in mass.
2 Growth can be measured by any growth parameter provided it increases proportionally to mass.
How does turbidity measure growth?
- Turbidity is measuring the amount of light that can pass through a cell culture
- the more dense the culture (i.e more growth), the less light will pass through
*the amount of light scattered by a bacterial cell suspension is proportional to its mass. (to an extent)
What is the Beer-Lambert law?
Log (I/Io) = -xl
can also be expressed as Log(Io/I) = xl
What is Io, I, x, and, l
Io = incident light (light that arrives at the receptor) I = transmitted light x = cell density l = cell path (standard value in cm)
What is I/Io and what is its relationship to x?
I/I0 is the fraction of light transmitted and decreases exponentially with x
what is Log(Io/I)?
Log(Io/I) is referred to as the optical density OR turbidity
-it is the opposite of light passing through (?)
What is OD
Optical density
= xl
OD is directly proportional to ____ if the mass per cell remains constant
cell density
What is a limitation to using a spectrophotometer to measure cell growth?
If the cell culture is too dense you can get light bouncing off multiple cells and hitting the detector
-thus lowering the turbidity reading
What are some limitations of using total cell count?
- Does not distinguish live or dead cells
2. Cannot be performed on low density cultures
What are 5 methods of growth measurement?
- Turbidity
- total cell count
- viable cell counts (using serial dilutions and colony formation)
- Dry weight
- Total proteins
What is a limitation of viable cell counts
Will give an underestimation if cells are clumped or in chains because each clump or chain will give rise to a single colony.
What are 2 examples of total protein methods of growth measurement?
- Coomassie blue stain assay and BSA standard curve
2. Total nitrogen content using the Kjeldahl method
What is cell division technically
The division of a mother cell into two daughter cells separated by a septum
What is cell separation ?
Comes after cell division and results in two distinct daughter cells
What are 4 events that occur before septum formation?
- DNA synthesis initiated when the cell reaches a critical cell mass.
- Two copies of chromosome are formed.
- Chromosomes separate - sister chromosomes move to opposite cell poles
- Replication origin anchored to cell poles; remaining chromosomes continue to migrate to daughter cells.
In Gram negative bacteria, cell separation occurs ____ septation
With
In gram positives, why does cell separation not occur with septation?
once septation is complete the cells will still be connected by the cell wall
6 steps in septum formation
- MinE forms a membrane-associated ring at cell center relieving the inhibition of FtsZ ring formation by MinCD .
- FtsZ recruited to the cell center and begins forming the FtsZ ring (probably with help from ZipA)
- Other proteins (FtsA, FtsW) recruited to the ring to from the septal ring.
- Further recruitment of other proteins (e.g. FtsK, FtsI, FtsN) and the septal ring constricts and MinE ring disassembles.
- Constriction is completed.
- MinE forms a new ring at the centers of the daughter cells.
What is the role of MinCD in the formation of the septum?
it inhibits the FtsZ ring formation from occuring when it is not time for the cell to divide
-prevents septation
What is the role of MinE
To relieve the action of MinCD and allow the Fts Z ring to form
What are the 2 systems that determine the location of septum formation?
- the Min system : MinCD prevent FtsZ ring formation. The location of MinCD is determined by MinE (which keeps it away from the midcell region)
- The nucleoid occlusion system : The nucleoid mass interferes with FtsZ ring formation
What is the 3 for 1 model
Describes the way new peptidoglycan is inserted into the cell wall during cell division
- involves breaking existing bonds (by autolysins), synthesizing new peptidoglycan, and joining new peptidoglycan to the cell wall.
- 3 units of peptidoglycan are inserted and 1 old one removed
