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Microbial Physiology > Lecture 4 - Exam 1 > Flashcards

Lecture 4 - Exam 1 Flashcards

1
Q

Describe the growth of a cell.

A

Increases in mass of the cell

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2
Q

Describe the growth of a population.

A

Increase in number of cells.

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3
Q

What are the factors that affect microbial population growth?

A

Acquisition of nutrients, catabolism of nutrients, anabolism of vital cell compartments, replication of genetic material, division of a cell into two daughter cells and start over.

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4
Q

What are the many ways to measure cell population growth?

A

Turbidity, viable plate counts, total cell counts (microscopic counts), electronic cell counts, membrane filtration, dry weight, protein measurements, most probable number, measures of metabolic activity.

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5
Q

Regarding the ways to measure cell population growth, what do we have to do in order to get very precise numbers?

A

Multiple measurements.

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6
Q

When measuring turbidity, what is one of the most widely-used methods to estimate population numbers?

A

A spectrophotometer.

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7
Q

(Turbidity measurement) Within limits, the __________ by a bacterial cell is proportional to ___________.

A

Within limits, the amount of light scattered by a bacterial cell is proportional to its mass.
So, light scattering is proportional to total mass.

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8
Q

(Turbidity measurement) If average mass per cell remains a constant, light scattering can be used to measure _______?

A

Changes in cell number (i.e. growth).

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9
Q

What is actually being measured when using a spectrophotometer in a turbidity measurement?

A

The fraction of light transmitted through the culture, not the scattered light.

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10
Q

In mathematical terms, what does spectrophotometer provide?

A

The log of the reciprocal of the fraction of transmitted light.

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11
Q

What is the Beer-Lambert Law?

A

States that the absorption of light is directly correlated to the concentration of a solute (or cells, in this case) in a solution.

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12
Q

(Turbidity measurement) The fraction of light does what when the density of the culture increases?

A

Decreases exponentially.

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13
Q

Why aren’t OD readings negative?

A

We actually obtain a negative number, but since we are human and do not like a decreasing, negative number with increasing cell density, we take the reciprocal log to give a positive number.

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14
Q

What are the advantages of using a spectrophotometer to measure turbidity to estimate population numbers?

A

Simple, fast and inexpensive

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15
Q

What are the disadvantages of using a spectrophotometer to measure turbidity to estimate population numbers?

A

Limited linear range, need standard curve of optical density vs cell count, wavelength can be different for different bacteria (normally at OD600)

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16
Q

What are the advantages of direct counts?

A

Accurately measures total number of cells

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17
Q

What are the disadvantages of direct counts?

A

No distinction between live and dead cells (unless live/dead stain used), not accurate for low density cultures, manual, very tedious

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18
Q

We don’t usually use the method of direct counts unless we have?

A

An electronic cell counter (very expensive, but more accurate and can determine cell count and cell size).

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19
Q

What is the second most commonly used technique for estimating population growth?

A

Cultured counts

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20
Q

What are the advantages for cultured counts?

A

Counts only living cells that can form colonies, to determine colony forming units per mL.

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21
Q

What are the limitations of using culture counts?

A

Only gives counts for culturable species, counts only living cells that can form colonies, easy to underestimate the number of cells due to clumping, assumes that every live cell forms a viable colony, need to know appropriate growth conditions (medium, pH, temp), several days to acquire results.

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22
Q

The vast majority of bacterial species cannot currently be ________.

A

Cultured in vivo

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23
Q

What is the “Great Plate Count Anomaly?

A

When every environmental sample has a big difference between the number of cells observed by direct counting compared to the number of colonies counted on a plate. Many factors that can cause this, a few being: choice of growth medium and requirement for nutrition from other microbes.

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24
Q

Uncultivated microbial species are “as-yet-uncultured,” NOT ______

A

Unculturable microbes.
Note: A majority of microbes do not grow on synthetic media.

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25
What is a dry weight measurement?
In dry weight methods, cells are harvested, dried, and weighed. It is fairly accurate, takes time, and there is a need to generate a standard curve of cell/weight for given growth condition.
26
What is a protein measurement?
Measure total protein concentration of cell using Bradford colorimetric assay. It takes times, and there is a need to generate a standard curve of total protein concentration/cell for given growth condition.
27
What are the four phases of cell growth?
Lag phase, log phase, stationary phase, and death or decline phases. Each phase can be very different for different prokaryotic species.
28
Do laboratory conditions reflect actual environments in which microorganisms have evolved?
No
29
What is the lag phase in bacterial cell growth?
There is no increase in number of living bacterial cells. It is a time of adjustment in a new environment.
30
What are the specifics of what goes on in lag phase of bacterial cell growth?
Recovery of cells from toxic metabolites in the previous environment, synthesis of new enzymes or cofactors, many dead cells in the inoculum rather than live cells (causes the lag in the curve), and the size of cells (mass) increases before replication starts.
31
What is the log/exponential phase in bacterial cell growth?
Exponential increase in number of living bacterial cells. Cell mass increases exponentially over time.
32
What are the specifics of what goes on in log phase of bacterial cell growth?
Fastest growing phases due to maximal utilization of available nutrients and optimal environment conditions for cell division, the number of ribosomes in actively growing E. coli cell can be 70,000, and primary metabolites are produced, meaning waste compounds are associated with rapid growth.
33
What are the different variables for exponential/log phase?
G = t/n G = generation time t = time per generation n = number of generations
34
What is generation time?
The rate of exponential growth of a bacterial culture.
35
During exponential growth, what happens to the mass in the culture?
It doubles each generation.
36
How can growth kinetics actually be useful?
When trying to determine starting culture concentration.
37
What is the stationary phase of bacterial cell growth?
Plateau in number of living bacterial cells ; rate of cell division and death roughly equal. When conditions for logarithmic growth have diminished, there is a slowdown and eventual cessation of population growth.
38
What are the specifics for the stationary phase of bacterial cell growth?
Cultures enter stationary phase when: Essential nutrients have been exhausted, accumulation of toxic waste products, changes in environment that affects the cell (pH fluctuation, depletion of O2 for facultative anaerobes).
39
What is the primary sigma factor for bacteria during stationary phase?
RpoS
40
What is RpoS (stationary phase)?
Is a master regulatory factor for stationary phase and stress and starvation gene expression.
41
Does metabolism stop in stationary phase?
NO! Secondary metabolites are produced that help microorganisms compete with on another, like antibiotics.
42
What are the changes associated with stationary phase?
Metabolic activity changes: There is an increased metabolic breakdown and resynthesis of protein and RNA. Developmental changes: reduction in cell size and metabolic activity, morphological changes (from rod to cocci), changes in surface properties (increased adhesive properties), changes in protein composition. Spore production: spores are metabolically dormant cell type. This is a differentiated cell type produced in response to stressful environmental stimuli (prolonged starvation, pH fluctuation, osmotic fluctuation, oxidative stress). Stringent response
43
What is a stringent response?
A temporary inhibition in synthesis of ribosomal RNA (rRNA) and transfer RNA (tRNA). Occurs when cells are starved for an amino acids. This caused "uncharged" tRNA to accumulate and the uncharged tRNA activated an enzyme called RelA.
44
What does RelA do?
Synthesizes ppGpp (alarmone), which slows transcription of ribosomal RNA, resulting in fewer ribosomes.
45
Cell entering stationary phase also become more resistant to __________.
Environmental stresses, such as high temperature, osmotic stress, and certain chemicals.
46
(Stationary Phase gene expression) Cells entering stationary phase become more resistant to environmental stresses. In Gram negative cells, what are these resistance properties due to?
The synthesis of a starvation sigma factor, RpoS. This master regulator regulates expression of at least 60 genes induced by carbon starvation.
47
RpoS is also a regulator for stress response in _________. Is it tightly controlled/regulated?
Exponentially growing cells. Yes, tightly controlled/regulated and needs to be turned on and off depending on environmental conditions.
48
(Stationary Phase Gene Regulation: Alarmones) What does an efficient control of metabolism do? What are the compounds that can accomplish all this?
Senses stress, adjusts growth accordingly, mandates gene expression, and ultimately provides a fitness advantage over poorly-adapted microbial competitors. The second messenger molecules (p)ppGpp are compounds that can accomplish all this. (p)ppGpp are referred to as alarmones.
49
When bacteria experience growth-limiting environmental conditions, the synthesis of alarmones is induced by which enzymes?
RelA and SpoT
50
High levels of (p)ppGpp induce what?
The stringent response. The stringent response allows bacteria to quickly reprogram transcription in response to changes in nutrient availability.
51
What specifically happens during the stringent response?
Ribosomal RNA and transfer RNA synthesis is downregulated, stress-related genes are upregulated, messenger RNA stability and translation altered (decreases), and allocation of scarce resources optimized.
52
How well is rRNA synthesis understood? Ribosome synthesis is coupled with what?
Not well. Coupled to growth rate.
53
Amino acid starvation controls...?
The rate of cell growth, but also inhibits ribosome synthesis.
54
The stringent response is _____-dependent synthesis of ppGpp.
RelA
55
What is the SpoT enzyme?
A bifunctional enzyme synthesized in carbon- and energy-limited environments. Synthesizes and degrades ppGpp.
56
What is the death phase of bacterial cell growth?
Exponential decrease in number of living bacterial cells. Complete depletion of energy occurs -> programmed cell death.
57
What are Viable But Not Culturable (VBNC) bacteria?
Bacteria have reduced metabolic activity and may not grow on nutrient rich media, but may live on less nutrient rich media, as they are adapted to live in less nutrient rich environments.
58
What is the long-term stationary phase?
Mutations in RpoS present a phenotype that allows the mutant to survive in extended media for an extended time (up to 5 years).
59
What is the phenotype called that allows for a long-term stationary phase? What do these cells do? What else is required to allow long-term survival of the culture?
GASP - growth advantage in stationary phase The GASP cells continue to grow and actually replace each other. Programmed cell death, initiated by a stress-response, RpoE, is required to allowed long-term survival of the culture.
60
What is Diauxic Growth?
Preferentially growing on one carbon source before using the second carbon source. Most bacteria (r-strategists) need to grow as quickly as possible to try to outcompete other bacteria in the same environment, so they want to use the most energy-efficient carbon source first.
61
Many bacteria grow preferentially on _____.
Glucose when presented with media containing glucose and other carbon sources (such as lactose).
62
Between glucose and lactose, which is more energy-efficient?
Glucose... no extra enzymes to break it down, goes directly into glycolysis. In order to use lactose, the bacteria must produce the enzyme lactase; then lactase breaks lactose into glucose and galactose. This is longer and less efficient.
63
How many lag phases are in Diauxic growth?
There are two lag phases. In the second lag phase, the enzymes needed to utilize the second carbon source are produced.
64
When does catabolite repression occur?
Catabolite repression occurs when one molecule (usually glucose) represses the synthesis of enzymes needed to grow on the second carbon source.
65
Diauxic growth can occur with what kinds of sources?
Can occur with other C sources, as well as with glucose.
66
What is a chemostat (bioreactor)? What's the concept?
"Steady state growth" Allows continuous growth of cells at controlled rate and density. Concept: At low nutrient concentration, growth rate is a function of nutrient uptake rate and utilization.
67
For dilution rate, what are the variables?
D = dilution rate F = flow rate V = volume
68
What is cell division?
Cell division is the splitting of a mother cell into two daughter cells separated by a septum.
69
When is DNA synthesis initiated?
When a growing cell reaches a critical mass.
70
Describe the process of cell division.
During replication, sister chromosomes move toward opposite ends of the cell. Soon after chromosome replication is complete, inward growth of the cell membrane and peptidoglycan for a septum between the two newly replicated chromosomes. Soon after replication, cell separation occurs (in bacteria that grow as single cells).
71
What is the B period in cell division?
Interval between cell division and initiation of DNA
72
What is "synthesis?"
Time between rounds of DNA replication initiation
73
What is the C period in cell division?
Period of DNA synthesis (genome replication)
74
What is the D period in cell division?
Period between the end of genome replication and the end of cell division.
75
What is the T period in cell division?
Total cell cycle = B + C +D
76
What are the two major events in cell division?
DNA replication: DNA replication & Chromosome partitioning Cell Division: D period
77
What are the proteins that are associated with cell septation and are essential for cell division?
Fts proteins. The role of these proteins in bacterial cell division is very complex.
78
What is FtsZ?
FtsZ is a tubulin homolog involved in bacterial cell division.
79
What is the septal ring?
The FtsZ and its associated proteins are called the septal ring.
80
FtsZ is present in which organisms?
All bacteria and archaea (homolog of tubulin). The septal ring is not present in a newborn cell or at the end of the cells. If you removed FtsZ, then there would be no septal ring, just a line of cells. ftsZ mutants form filaments without constrictions.
81
What is ZipA?
Essential cytoplasmic cell division protein. Stabilizes the FtsZ protofilaments by cross-linking them. Serves as a cytoplasmic membrane anchor for the Z ring. zipA mutant form filaments without constrictions.
82
What is FtsA?
Probably ATPase - interacts with FtsZ to promote continuation of septum formation. ftsA mutants form indented filaments but don't form complete septum.
83
It is imperative that FtsZ rings does what?
It is imperative that FtsZ ring forms at mid cell.
84
FtsZ forms at mid cell. How do cells ensure this happens?
The Min System
85
What does the Min System do?
The Min System regulates central location of FtsZ. The Min System ensures that the mother cell divides into two identically-sized daughter cells. Nucleoid occlusion and the Min System work together to ensure the Z-ring forms at mid cell.
86
________ genetic locus in G- bacteria has three genes, what are they?
Minicell genetic locus in G- bacteria has three genes: minC, minD, and minE, whose protein products are involved in cell division.
87
Describe the MinD protein.
MinD forms a cytoplasmic membrane-associated cup at one cell pole that extends toward midcell. It is drawn to the curvature of the cell and binds at one of the two poles.
88
Describe the MinC protein.
Division inhibitor, inhibits FtsZ from assembly into a Z ring, binds to MinD (MinC cannot directly bind to membrane, must bind to MinD)... MinD has ATPase activity that is required for MinC function.
89
Describe the MinE protein.
Responsible for topological specificity (ensuring the cell divides at midcell), displaces MinCD complex from one pole & forms a ring that initiates displacement of the MinD cup, MinE will chase MinCD complex from one pole to the other. MinCD oscillates back and forth to each pole, preventing FtsZ ring from forming. The only place that FtsZ formation is not being inhibited is at the center of the cell, ensuring you have two equally sized daughter cells.
90
Describe G- bacterial cell division and the different Min proteins involved.
MinE localizes to a ring-like structure in the middle of the cell. The MinCD complex accumulates alternately at the membrane periphery on either side of the MinE ring. -The MinE ring displaces the MinCD complex at the pole... This oscillation of MinD at each pole occurs very rapidly (seconds) ensures that no FtsZ ring is assembled at either the 1/4 or 3/4 sites of the cell halves. -The presence of MinE at midpoint prevents MinD inhibitory activity at this site, and allows FtsZ ring assembly in the center. -The MinE ring disassembles before completion of constriction.

Microbial Physiology (22 decks)

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