Lecture 3 - Exam 5

Question 1

What is cellular differentiation?

Answer 1

A fundamental strategy used by cells to generate specialized functions at specific stages of development. It refers to the process in which cells of the same source gradually produce cell types with different morphological structures and functional characteristics.

Question 2

What is the essence of cell differentiation?

Answer 2

Is the selective expression of the genome in time and space. The genome sequence is the same in every cell type, but the pattern of gene expression is different!!

Question 3

What are some examples of cell differentiation?

Answer 3

Myxobacteria fruiting and hunting as a wolfpack and Chlamydia spreads as elementary bodies and differentiates into reticulate bodies within human cells.

Question 4

What is one of the best studied Gram + bacteria?
What is special about it?

Answer 4

Bacillus subtilis.
It is naturally competent so genetic manipulation is made possible.

Question 5

Sporulation by Bacillus subtilis has what kind of cell differentiation?

Answer 5

Temporally-regulated

Question 6

What do endospores contain?

Answer 6

A complete copy of the chromosome, the bare minimum concentrations of essential proteins, and high concentration of calcium bound to dipicolinic acid (taken into the cell and forces all the water out)

Question 7

The spore has what kind of structural components?
What does the structure of the spore do?

Answer 7

An inner membrane, two peptidoglycan layers, and an outer-keratin-like protein coat.
The structure protects the genomic DNA from heat, radiation, and attack by most enzymes and chemical agents. They are so resistant that they can exist for centuries.

Question 8

The resistance makes spores of pathogenic species likely candidates for __________.

Answer 8

bioterrorism

Question 9

Bacillus and Clostridium organisms initiate the sporulation process when ___________ conditions are detected.

Answer 9

Unfavorable

Question 10

The sporulation process is a carefully orchestrated cascade of events at what kind of levels?
What does this involve?

Answer 10

At both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcriptional factors, proteases and phosphatases.

Question 11

What is Spo0A?

Answer 11

The master sporulation regulator. It is a global transcriptional regulator that controls diverse sporulation, virulence and metabolic phenotypes.

Question 12

Describe the first part of the lifecycle of endospore formers.

Answer 12

(A) sporulation: Upon sensing certain environmental conditions, endospore formers activate Spo0A and initiate sporulation.
-The first morphological event is the formation of a polar septum, which creates a larger mother cell and smaller forespore. The mother cell engulfs the forespore, and the two cells work together to assemble the dormant spore.
-Calcium dipicolinic acid (Ca-DPA) is synthesized in the mother cell and transported into the forespore in exchange for water (pushes the water out).
-Cortex is formed between the two membranes, and coat proteins polymerize on the surface of the mother cell-derived membrane.
-Once the spore is mature, the mother cell lyses and releases the dormant spore into the environment.

Question 13

Describe the second part of the lifecycle of endospore formers.

Answer 13

(B) Germination: Upon sensing the appropriate small molecule germinants, the spore initiates a signaling cascade that leads to activation of cortex hydrolases and core hydration, which is necessary for metabolism to resume in the germinating spore.

Question 14

Spore formation occurs in ___ stages, and each stage is controlled by?

Answer 14

7. Controlled by a specific sigma factor(s)

Question 15

Spore formation is triggered in response to an environmental signals, often…?

Answer 15

Low nutrient concentrations

Question 16

In Bacillus subtilis (and many other spore formers), sporulation is controlled, in part, by?

Answer 16

Quorum sensing peptides.

Question 17

B. Subtilis sporulates more efficiently at ______ cell densities. Describe what happens at both low and high cell densities.

Answer 17

High ;
At low cell densities, the phosphorelay system that activates sporulation genes is dephosphorylated by Rap phosphatases.
At high cell densities, signaling pentapeptides enter the cells and inhibit Rap phosphatases, allowing phosphorylation of the sporulation relay system, resulting in expression of sporulation genes.

Question 18

Sporulation B. subtilis is a cascade of….?

Answer 18

gene expression events in both the mother cell and developing spore.
-Gene expression is controlled temporally by a subset of sigma factors in both mother cell and developing spore.

Question 19

Describe the stages of spore formation.

Answer 19

Stage 0: Cell chooses one of the two pole division sites (oH - starvation sigma factor: transcriptional activator of Spo0A)
Stage 1: Chromosome replication and axial filament (oH activates Spo0A, the master regulator of sporulation)
Stage 2: Septation: the cell divides into two unequal compartments (oF in forespore, oE in mother cell)
Stage 3: Engulfment: The mother cell engulfs the forespore (oG in forespore, oE in mother cell). The spore is now encircled by two membranes.
Stage 4: Destruction of mother cell chromosome; Cortex develops (oG in forespore, oK in mother cell).
Stage 5: Layers of coat proteins are deposited on the outer membrane (oG in forespore, oK in mother cell)
Stage 6: Maturation: production in dipicolinic acid by the mother cell and uptake into the spore (oG in forespore, oK in mother cell). Dipicolinic acid uptake promotes spore dehydration.
Stage 7: Release of the spore from the sporangium (mother cell)

Question 20

Describe the vegetative cell.

Answer 20

Normal growth and division into two equal daughter cell (sigma A - housekeeping sigma factor)

Question 21

What is the cortex?

Answer 21

A thick layer of peptidoglycan that forms between the two membranes.

Question 22

The decision to initiate sporulation requires vegetative cells to?

Answer 22

Recognize specific environmental and nutritional signals and assimilate these cues into a robust response.

Question 23

All sporulating Firmicutes use the __________________ regulator, _____, as a key checkpoint for integrating environmental signals.

Answer 23

Conserved transcriptional regulator ; Spo0A

Question 24

The response regulator Spo0A initiates what? How?
What is Spo0A DNA-binding activity is directly controlled by?

Answer 24

Initiates spore formation by activating or repressing the expression of genes encoding early sporulation regulators.
Phosphorylation.

Question 25

Describe (in more detail) the Spo0A master regulator of sporulation.

Answer 25

A key protein that directs the transcriptional regulation of downstream genes, including asymmetric division and the expression of oF and oE, which are special transcription factors of pre-spore and mother cell, respectively. These factors decide the regulation of the development of the spore.

Question 26

Where does Spo0A bind?

Answer 26

Spo0A binds to a DNA sequence containing a ‘0A-box’ where it exerts its role by acting as a transcriptional activator or repressor.

Question 27

Besides being required for the onset of sporulation, Spo0A is also involved in the transcriptional regulation of various other stationary phase processes. What is an important example of this?

Answer 27

Spo0A has a profound effect on the global gene expression pattern of B. subtilis.

Question 28

Spo0A influences the expression of ________ B. subtilis genes.
-Of these genes, how many are under the direct control of Spo0A?
-Several of these encode proteins that themselves are _______ or _______ involved in transcriptional regulation, explaining the global effect of Spo0A on transcription.

Answer 28

520 ;
121 ;
direct or indirect

Question 29

The levels of Spo0A protein and activity increase gradually during what stages of sporulation?

Answer 29

The early stages of sporulation.

Question 30

In B. subtilis, Spo0A phosphorylation is orchestrated by?

Answer 30

A complex regulatory network known as a phosphorelay.

Question 31

What does phosphorelay consist of?

Answer 31

Several orphan sensor histidine kinases (KinA-E) and two phosphotransfer proteins (Spo0f and Spo0B).

Question 32

What does the KINA-E kinase do?

Answer 32

Directly phosphorylates Spo0F, which subsequently transfers the phosphate to Spo0B and finally to Spo0A.

Question 33

What inhibits Spo0A phosphorylation levels? How?

Answer 33

Anti-kinases and two different classes of phosphatases inhibit Spo0A phosphorylation levels by either blocking kinase activity or stripping Spo0A or Spo0F of its phosphate. “Push and pull of phosphorylation”

Question 34

So, why would the cell want half of the population to be vegetative and half of the population to be sporulating?

Answer 34

The sporulating ones are dying, don’t want every single one to die.

Question 35

The complexity of the phosphorelay functions as a ____________, creating?
What does this ensure?

Answer 35

Noise generator, creating heterogeneous levels of Spo0A phosphorylation within a population.
This ensures only a portion of its population commits to sporulation.

Question 36

How does B. subtilis control initiation of sporulation?

Answer 36

-Histidine kinases (e.g. KinA) are activated by different signals (starvation, nitrogen levels) and have a signal detection domain and a kinase domain.
-Ultimately, the transcription factor Spo0A is phosphorylated, and this is a negative regulator of growth, and an inducer of transcription of early sporulation-specific genes.
Having multiple phosphorelay proteins involved provides a series of “checks and balances” in order to tightly regulate sporulation initiation.

Question 37

Control of timing of sporulation by Bacillus subtilis by selective gene expression:
What is it determined by?

Answer 37

Determined by specific, active sigma factors:
-In a B. subtilis vegetative cell under physiological normal conditions, sigma A (o^A) is the housekeeping sigma factor.
-Starvation induces sigma H (o^H)
-Activation of o^H and Spo0A leads to asymmetric division*
-Early compartmentalized gene expression with o^F becoming active in the forespore and o^E in the mother cell.
-When membranes fuse at the pole of the cell, a second round of compartmentalized gene expression occurs, with o^G becoming active in the forespore and o^K in the mother cell.

Question 38

During sporulation, cytokinesis happens ________ to chromosomal partitioning being complete.
-What is the protein SPoIIIE and what kind of domains does it have?
-After completing the translocation of chromosomal DNA, SpoIIIE moves to…?

Answer 38

Prior ;
It is a DNA translocase, with a membrane domain and a motor domain, that pumps the chromosomal DNA across the septum membrane into the forespore ;
SpoIIIE moves to the forespore pole before the completion of engulfment.

Question 39

The resistance of spores to environmental attacks is attributable to the ?

Answer 39

Unique morphology of the spore, which is formed by several layers: the core, the cortex, the coat, the crust, and in some cases, the exosporium. These protect the spore core.

Question 40

What does the spore core contain?

Answer 40

the DNA, RNA, proteins (very few), calcium dipicolinate (DPA or dipicolinic acid), small, acid-soluble spore proteins (SASPs), and calcium. 0

Question 41

What does the DNA in a spore associate with?
Dipicolinic acid (DPA) has an ability to…?
Dipicolinic acid plays a key role in?

Answer 41

The DNA in a spore is associated with DNA binding proteins and calcium dipicolinate (DPA, which replaces water, conferring resistance to heat and UV radiation).
DPA has an ability to bind and chelate Ca2+ ions.
DPA plays a key role in the dehydration, and mineralization of the spore core (dehydration of the spore core is the main factor underlying the resistance of spores to wet heat).

Question 42

Spores of B. subtilis contain several _________________, which comprise up to 20% of total spore core protein.

Answer 42

Small, acid soluble spore proteins (SASP)

Question 43

The multiple alpha/beta SASP have been shown to confer…?

Answer 43

Resistance to UV radiation, heat, peroxides, and other sporicidal treatments.

Question 44

The spore cortex is?

Answer 44

A thick layer of peptidoglycan between an inner and outer spore membrane.

Question 45

The spore coat consists of ?

Answer 45

Proteins deposited by the mother cell arranged in an inner and outer layer in stage 5.

Question 46

Maturation involves?

Answer 46

A thickening of the spore coat and entry into dormancy.
Spore can be dormant for years.

Question 46

Germinant receptors located in the inner spore membrane allows spores to?

Answer 46

Rapidly exit dormancy in response to nutrients, such as amino acids.

Question 47

The extreme resistance and durability of the spore is due to?
What is it?

Answer 47

The spore coat.
The spore coat is a multilayered, proteinaceous covering that provides protection against many stresses and toxic chemicals.

Question 48

The spore coat is assembled from?
In B. subtilis, there are more than ____ coat proteins, which represents 50-80% of the total spore protein.

Answer 48

Components synthesized in the mother cell compartment of the sporulating cell.
70

Question 49

What does the spore coat consist of?

Answer 49

Concentric layers of protein:
Basement layer, inner coat, outer coat, and crust.

Question 50

Coat assembly is regulated at the __________ level by the __________ expression of ?
It is also regulated at the _________ level by a small group of?

Answer 50

Transcriptional ; sequential ; expression of individual coat genes
Protein ; coat morphogenetic proteins that coordinate both the recruitment of coat proteins to specific coat layers and spore encasement.

Question 51

The spore coat confers resistance to ? And acts as?

Answer 51

Lysozyme. Acts as a sieve by interfering with the trajectory of disinfectant substances.

Question 52

Are there differences between the sporulation programs of Bacillus and Clostridium, and different Clostridium species?

Answer 52

Yes.

Question 53

What are the differences between sporulation in Bacillus and Clostridia?

Answer 53

In Bacillus, Spo0A is phosphorylates by a phosphorelay system initiated by orphan HKs, mainly KinA and Kin B . Once activated, the phosphorylated Spo0A initiates the sporulation sigma factor cascade involving four downstream sigma factors (oF, oE, oG, and oK).
In Clostridium, no phosphorelay system is present. Rather, orphan HKs phosphorylate Spo0A directly. Second, the last sigma factor in the Bacillus model, oK, was shown to play a dual role in Clostridium, one early, upstream of Spo0A, and another late, downstream of oG, which is analogous to its role in Bacillus.

Question 54

Clostridiales are significant pathogens of humans and animals. Some examples?

Answer 54

Tetanus, Botulism, Foodborne illness

Question 55

Several pathogenic species directly _______ toxin production to sporulation.
Often, alternative _____________ coupled to sporulation induction control toxin regulons.

Answer 55

couple.
sigma factors

Question 56

Asymmetric division eventually leads to the activation of four sporulation-specific sigma factors: ?
These sigma factors are essential for?
Are these sporulation-specific sigma factors highly conserved?

Answer 56

oF, oE, oG, and oK.
Essential for sporulation because they coordinate the activation of distinct transcriptional programs within the mother cell and forespore, respectively, that culminate in the formation of a metabolically dormant spore.
Yes.

Question 57

For dormancy to be a viable survival strategy, spores must be able to?
Spores continually monitor their surroundings using an array of?

Answer 57

Germinate rapidly when nutrients become available again.
Germination receptors embedded in the inner spore membrane.

Question 58

The transition from spore to vegetative cell occurs in three stages:

Answer 58

Germination:
-Spore germination is induced by environmental germinants, calcium dipicolinate is released from cell, spores lose heat resistance, and protein synthesis begins.
Ripening:
-Transcription and translation machinery are functional, ATP synthase is active, biosynthesis of lipids occurs, and stress response mechanisms functional
Outgrowth:
-TCA cycle functions, respiration via ETC and oxidative phosphorylation, production of cell structure proteins and peptidoglycan, DNA replication, cell division, and protein secretion

Question 59

Sensing of germinants by ______________ triggers a signal transduction cascade.
-During spore formation, the uptake of Ca2+-Dipicolinic acid into the forespore requires 7SpoVA proteins which form a…?

Answer 59

inner membrane germinant receptors (IMGR) ;
Mechanosensitive channel that opens and closes based on membrane tension.
During germination, IMGR sense germinants and send a signal to SpoVA that triggers the channel to open. CaDPA is released through the open SpoVA channel

Question 60

Release of CaDPA triggers…?

Answer 60

Activation of cortex lytic enzymes (CLEs) SleB and CwlJ, resulting in cortex hydrolysis.

Question 61

What do CLEs act on?

Answer 61

Act only on peptidoglycan with the cortex-specific modification muramic acid S-lactam (MAL)***
-The complete release of CaDPA and complete cortex hydrolysis results in the completion of germination.

Question 62

In some Clostridia (Clostridium perfringens and Clostridioides difficile), different CLE proteins are present, which are activated by?

Answer 62

Bile salts (Csp proteins), resulting in rapid spore germination.