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Developmental Biology of Bacillus Subtilis: Parts 1, 2 and 3 Lecture Notes

Key words and terms

Bacillus subtilis, spore, sporangium, asymmetric septation, forespore, mother cell, FtsZ, Z-ring, nucleoid, axial filament, RacA, DivIVA, DNA translocase, engulfment, cortex, coat, Spo0A, sF, sE, sG, sK, s factor, reporter gene, anti-s factor, anti-anti-s factor, intercellular communication, pro-protein, regulated intramembrane proteolysis.

Lecture notes

Ferdinand Cohn discovered the bacterium Bacillus subtilis and its spores in 1877.

B. subtilis is related to the anthrax-causing bacterium Bacillus anthracis.

Spore formation by B. subtilis is a powerful model system in which to ask fundamental questions about cellular differentiation and morphogenesis in a bacterium.

1. Spore formation is a tale of two cells


Principal stages of spore formation (sporulation)

  • Sporulation is triggered by nutrient limitation
  • At the onset of sporulation, a single rod-shaped bacterial cell (termed the predivisional sporangium) divides asymmetrically, forming two cells: the small forespore (destined to become the spore) and large mother cell (nurtures the forespore).
  • Initially the two cells lie side-by-side. Subsequently the mother cell “swallows” the forespore in a process called engulfment that is similar to phagocytosis. During engulfment the mother cell membranes migrate around the forespore and fuse, pinching it off as a cell-within-a-cell.


How does asymmetric cell division (septation) occur?

  • Bacteria divide through the action of the tubulin-like protein FtsZ, which forms a cytokinetic ring termed a Z-ring. Interestingly, actin (not tubulin) plays this role in eukaryotic cells.
  • During vegetative growth, the Z-ring forms in the middle of the rod shaped bacterial cell, marking the future site of cell division and resulting in two equal daughter cells.
  • During sporulation, two Z-rings form, one near each pole. Only one Z-ring is converted into a division septum, the other is disassembled. The result is two daughter cells of unequal size.


How are chromosomes segregated during asymmetric septation?

Ordinarily, chromosome segregation occurs prior to cell division. During sporulation, however, chromosome segregation takes place by a unique mechanism before, during, and after asymmetric cell division.

  • First, the two chromosomes (often referred to as nucleoids) present in the predivisional sporangium are rearranged into an elongated structure termed the axial filament, with the two origins of replication anchored at opposite poles.
  • The proteins RacA and DivIVA are important for positioning of the origins at the cell poles. RacA binds to the chromosome, especially near the origin, and in turn interacts with DivIVA, which is anchored at the cell poles.
  • Given this positioning of the chromosomes, only a fraction of the forespore chromosome is present in the forespore itself following asymmetric septation. The remainder of the chromosome is pumped from the mother cell into the forespore through the septum. An ATP-dependent DNA translocase located in the division septum mediates this process.


What are events that occur during the final stages of sporulation?

  • Remodeling of forespore chromosome
  • Formation of a protective layer of cell wall material called the cortex
  • Formation of a thick protein coat
  • Lysis of the mother cell to release the mature spore


The mature spore can survive for years, but is also capable of resuming normal vegetative growth upon the return of favorable environmental conditions.

2. Cell-specific transcription factors drive gene expression


Transcription factors that act in cell-specific manners orchestrate the events of sporulation (including asymmetric septation, forespore engulfment, cortex and coat assembly) and drive the distinct cell fates of the mother cell and forespore.

  • Spo0A = Master regulator of sporulation. Is active in the predivisional sporangium in response to nutrient limitation.
  • ?F = first forespore-specific transcription factor, active following asymmetric septation
  • ?E = first mother cell-specific transcription factor
  • ?G = replaces ?F in the forespore following engulfment
  • ?K = replaces ?E in the mother cell at late times during sporulation


The ?F, ?E, ?G, and ?K factors belong to a family of transcription factors in bacteria known as RNA polymerase sigma (?) factors. ? factors bind to RNA polymerase and direct it to specific promoter sequences on the chromosome.

The activity of transcription factors is often measured and/or visualized by a reporter gene. One popular reporter gene is that encoding the Green Fluorescent Protein (GFP), which can be easily monitored by fluorescence microscopy. When the gene for GFP is fused to a promoter controlled by ?E, green fluorescence is observed exclusively in the mother cell. In contrast, a fusion of this reporter gene to a promoter controlled by ?G results in forespore-specific fluorescence.

A major focus of work in the Losick laboratory has been to understand the mechanisms by which these transcription factors are activated in the correct cell at the correct time during development.

As an example, the known mechanisms of ?F activation are presented:

  • The ?F protein is produced in the predivisional sporangium (under the control of Spo0A), but only becomes active in the forespore following asymmetric septation.
  • An anti-? factor called AB binds and inactivates ?F prior to asymmetric septation and in the mother cell after asymmetric septation.
  • However, ?F escapes AB in the forespore. How? An anti-anti-? factor called AA disrupts the AB•?F interaction, thus releasing active ?F.
  • AA itself is regulated by phosphorylation. AA~P is inactive and unable to disrupt the AB•?F interaction.
  • A phosphatase called E converts AA~P ? AA, thus activating AA, which in turn disrupts the AB•?F interaction, releasing active ?F.
  • How does this happen only in the forespore? We don’t know the full answer to this, but an important clue is that the E phosphatase is situated in the asymmetric septum that separates the mother cell and forespore. It is likely that this localization allows E to act preferentially on the forespore side of the septum to dephosphorylate and activate AA, in turn activating ?F.

 

3. The two cells talk to each other!


Three intercellular signaling pathways link gene expression and development in the forespore and mother cell:

i. First, ?F activity in the forespore initiates a signal transduction pathway that leads to ?E activation in the mother cell (?F ? ?E)

ii. Next, ?E activity in the mother cell causes ?G activation in the forespore (?E ? ?G)

iii. Finally, ?G activity in the forespore initiates a signal transduction pathway that directs ?K activation in the mother cell (?G ? ?K)

These are two-way conversations, moving from the forespore to the mother cell, the mother cell to the forespore, and back again!


Listening in on one conversation: How does forespore ?G direct activation of mother cell ?K?

 

  • ?K is synthesized in the mother cell as an inert pro-protein, called pro-?K. The pro-?K protein has an N-terminal extension (~20 amino acids) that anchors the protein in the mother cell membrane, thus rendering it inactive. To be activated, a protease must cleave pro-?K, liberating a soluble and active form of ?K.
  • Mature ?K is not detected in sporangia lacking ?G, indicating that the forespore sigma factor is required for proteolysis and activation of ?K in the mother cell.
  • How does ?G control pro-?K proteolysis in the adjacent cell? The ?K protease, which is itself a membrane protein, is held inactive by two inhibitory proteins in the mother cell membrane. However, this inhibitory complex can be disrupted by a signaling protein produced in the forespore under the control of ?G. (Secretion of the signaling protein across the forespore membrane allows it to come in contact with the pro-?K protease and its inhibitors in the mother cell membrane.) Upon disruption of this complex, the protease cleaves pro-?K into its mature, active form.


Interestingly, the mode of ?K activation and the pro-?K protease have been highly conserved through evolution. Pro-?K processing is an example of regulated intramembrane proteolysis, given that the active site of the pro-?K protease is located within its membrane-spanning regions of the protein. Studies in diverse cell types have revealed that regulated intramembrane proteolysis is a widely-conserved mechanism for activating membrane-bound regulatory proteins, including the human sterol response element binding protein (SREBP), a key transcription factor that regulates cholesterol metabolism. Moreover, the pro-?K protease is itself a member of a widely conserved protease family that participates in pathways of regulated intramembrane proteolysis in many cell types, including human cells. In fact, one of the proteases responsible for activating SREBP in human cells shares many features, including catalytic residues and mechanism, with the pro-?K protease!


Future research challenge: How do the many hundreds of proteins produced by Spo0A, ?F, ?E, ?G, and ?K collaborate to direct morphogenesis and ultimately produce a mature spore?

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