1. The Bacillus subtilis sporulation
A bacterial model of differentiation Or
How to survive

2. Spore formation in Bacillus subtilis and other gram-positive bacteria
B. subtilis is a gram-positive soil-dwelling bacterium that has the
ability to differentiate into endospores when severely starved for nutrients.
(carbon, nitrogen, and/or phosphorus source).
Spores are metabolically dormant and highly resistant to a variety of
environmental insults, including UV and gamma radiation, reactive
oxygen, high and low temperature, acid and alkali conditions, hydrolytic
enzymes, and organic solvents.
Spores can remain dormant for long periods of time (perhaps up to 50 years),
but will germinate when they encounter a nutrient-rich environment containing
one or more of two specific ‘germinants’ (L-alanine and/or a mixture of
L-asparagine, fructose, glucose, and potassium ions).
Spore formation in B. subtilis is a simple developmental
event that involves the generation of two different cell types (forespore
and mother cell) with different programs of gene expression and different
developmental fates. Thus, in addition to being of interest to microbiologists,
spore formation in Bacillus is also of considerable interest to developmental
biologists.
In addition to B. subtilis, several other species of gram-positive bacteria
make endospores. Bacillus anthracis (anthrax), Clostridium botulinum (botulism),
Clostridium tetani (tetanus), Clostridium perfringens (gas gangrene, food poisoning),
Clostridium difficile (antibiotic-associated and pseudomembranous colitis).

5. Bacillus subtilis growth and sporulation
Relatives: B. cereus, B. anthracis
time
Log(N)
inoculate
4 Sporulate, asymmetric cell div 4
3 Gain genetic competence 3
2 Gain motility 2
1 log growth, symmetric cell division 1
Quorum
sensing
~7 hours VII stages
Genetics:
>200 genes/proteins

6. Life cycle of Bacillus subtilis
B. subtilis can sporulate when the environmental conditions become unfavorable ?
division cycle
sporulation-germination cycle
metabolic and environmental signals
Start with a schematic overview of the life cycle of B. subtilis. Use this slide to draw attention to the question mark. This is the important developmental decision.

7. Microscopy of sporulation

8. Endospores of Bacillus have been the primary model system
DNA complexed with
SASPs
Dipicolinic acid
Brock Biology of Microorganisms, vol. 9, Chapter 3

9. Stage of sporulation I II III IV to VI (vegetative growth)
(vegetative growth) I
(axial filament formation) II
(polar division)
III
(engulfment)
IV to VI
(cortex and coat formation)
Mother cell lysis
and release of free spore
Mutants defective in sporulation that are blocked at each of the above
morphological stages have been identified.
Nomenclature of mutants: spo; stage of block; locus; allele
Example: spo0K141

11. Sporulation Stages in Bacillus
I Replication
0 Growth
II Assymetric Septum
Sporulation Stages in Bacillus
VII Free Spore
III Engulfment
VI Mother Cell Lysis
IV Cortex
V Coat

12. Stages of Sporulation in Bacillus subtilis
Germination
Stage 0
Stage II1
Stage II3
Stage III
Stage IV
Stage V
Stage VI
Stage VII
Normal vegetative growth
Engulfment
Cortex formation
Maturation
Asymmetric septation
Prespore protoplast
Coat formation
Release

13. Spore germination
In response to nutrients, the spore will exit its dormant state and enter back into vegetative growth
free
cell
spore

16. B. subtilis sporulation stages
2 chromosomes, axial fibers I
2 cells, cell wall M D II
Engulfment
III
Double membrane IV
Cortex
V-VI
Coat
VII
Spore maturation
Mother cell lysis (DNA)

17. Stages of Sporulation in Bacillus subtilis
How is the process regulated?
Stage 0
Stage II1
Stage II3
Stage III
Stage IV
Stage V
Stage VI
Stage VII
spo0
spoII
spoIII
spoIV
spoV
spoVI

18. Stages of Sporulation in Bacillus subtilis
Golden age of Bacillus genetics
Identify spo genes
Clone spo genes
Determine their interdependencies
Stage 0
Stage II1
Stage II3
Stage III
Stage IV
Stage V
Stage VI
Stage VII
spo0
spoII
spoIII
spoIV
spoV
spoVI

19. Deciding to sporulate s s sporulate 3 3 Chromosomes: ? 2 Cell density:1
1 Starvation: ? s H
sporulate
What is monitored? [GTP] = (C, N, P)
Not ppGpp
“quorum sensing” peptides
Food low, [cell] low
Ok ?
Food low, [cell] high
Trouble ahead !
: undamaged, complete, and polar placement.

20. How is the septum placed asymmetrically?
Polar Division
How is the septum placed asymmetrically?

21. FtsZ:
Ring at septum

22. A Downward Spiral

23. Asymmetric Cell Division in B
Asymmetric Cell Division in B. subtilis Involves a Spiral-like Intermediate of the Cytokinetic Protein FtsZ Cell 109:

25. (from Daniel and Errington, 2003. Cell. 113: 767)

30. Spo0J and Soj spoIIA Spo0J Soj spoIIE spoIIG
Spo0J (a ParB homolog) is required for efficient chromosome
segregation during vegetative growth in B. subtilis. Spo0J
binds to a series of DNA sites (parS sites) in the origin-proximal
20% of the chromosome, and these sites are believed
to be involved in partitioning.
In addition to its role in partitioning, Spo0J also is needed
for efficient spore formation. Spo0J regulates the initiation
of sporulation by antagonizing the function of Soj.
SoJ (a ParA homolog), a DNA-binding protein present
in an operon with Spo0J, inhibits the initiation of sporulation
in the absence of Spo0J. Soj inhibits the onset of sporulation
by binding and inhibiting transcription of promoters of sporulation
genes that are normally activated by Spo0A~P
(spoIIA, spoIIE, spoIIG).
Spo0J Soj spoIIE
spoIIA
spoIIG

31. Roles of Spo0A-P and the alternative s factor sH
in polar cell division
Spo0A~P,
Spo0H
(from Margolin, Curr. Biol. 12: R ).
Early in sporulation, FtsZ localization switches from a midcell
position to a bipolar localization.
Spo0A and the phosphorelay proteins Spo0F and Spo0B are needed for
the midcell to bipolar switch of FtsZ rings.
A constitutively active form of Spo0A is sufficient to cause
polar septa formation during exponential growth. These results indicate
that Spo0A~P drives transcription of one or more genes that promote
formation of polar septa.
The alternative s factor sH (encoded by spo0H) is also needed for the
switch in FtsZ localization and for polar septation.

32. Formation of polar septa during sporulation
appears to proceed through an intermediate
spiral-like FtsZ structure
Spo0A~P: expression of SpoIIE
Spo0H: increased FtsZ expression
(from Margolin, Curr. Biol. 12: ).

33. FtsZ spiral formation and polar septation
is mediated by increased transcription
of SpoIIE and FtsZ during sporulation
Early during sporulation, Spo0A~P drives expression of spoIIE, a gene encoding a phosphatase needed for later events in development. SpoIIE co-localizes with FtsZ, first in a spiral-like structure, and then in polar rings.SpoIIE has also been shown to bind directly to FtsZ.
Early in sporulation, transcription of FtsZ increases. Increased transcriptionis mediated primarily by a sH-dependent promoter (P2).
Bacterial single mutants in which either spoIIE or the P2 promoter are inactivated make asymmetric septa during sporulation. However,double mutants in which both the spoIIE gene and the P2 promoter of ftsZare deleted are unable to make FtsZ spirals or polar septa .In addition, simultaneous overexpression of both spoIIE and ftsZ result in formation of FtsZ spirals followed by polar septa in growing cells in the absence of sporulation stimuli (e.g. nutrient deprivation). These results indicate that ‘polar switching’ of FtsZ localization is mediated by increased transcription of spoIIE and ftsZ early in development.
wt (spoIIE+;
P2+-ftsZ)
spoIIE+;
P2∆-ftsZ
spoIIE∆;
P2+-ftsZ
Three hours after the induction of sporulation, cells were fixed, stained with
the membrane dye FM4-64, and examined by fluorescence microscopy
(from Ben-Yehuda and Losick, Cell. 109: 257).

34. Role of sE in polar septum formation
sE (the product of the spoIIGB gene) is synthesized as an inactive precursor
before formation of the asymmetric septum. Active
(processed) sE is generated only in the mother cell where it directs
gene expression in that cell type.
Null mutations in spoIIGB or mutations that prevent activation of
sE result in formation of ‘disporic’ cells. These are cells that contain
two asymmetric septa, one at each pole. Both of the forespore-like
compartments in disporic cells contain a chromosome, but the larger
mother cell is anucleate. Of course, viable spores do not develop from
a disporic cell.
The disporic phenotype of spoIIGB mutants indicates that sE must
be needed for transcription of a gene or genes whose product(s)
somehow act to inhibit formation of a second polar septum.
A recent report indicates that at least three of the sE -controlled
genes that inhibit the second polar septum are spoIID, spoIIM, and
spoIIP.
spoIID,
spoIIM,
spoIIP
sE -
minus
sE -
positive

35. Chromosome segregation during sporulation in B. subtilis
(from Pogliano et al Curr.Opin. Cell Biol. 6: 586)

36. Role of RacA in chromosome segregation during sporulation
In exponentially growing cells, chromosomes are segregated prior to
cell division.
In sporulating cells, The chromosome becomes organized into an
elongated structure (axial filament). Chromosome segregation occurs after
most of the asymmetric septum has formed. Initially, only about 35%
of one of the chromosomes is located within the forespore. After septation,
The remainder of the chromosome is transferred from the mother cell
into the forespore.
Recent results indicate that the DNA-binding protein RacA is needed
for axial filament formation and for efficient translocation of DNA into
the forespore. RacA is localized to the cell poles in sporulating cells,
suggesting that it might act to anchor the chromosome to this region.
Consistent with this idea, expression of RacA is not expressed in vegetatively
growing cells and artificial expression of RacA during exponential growth
results in movement of nucleoids towards the cell poles (not shown).
racA+ strain
racA- strain
DAPI
(DNA)
DAPI
+ FM4-64
(membrane)
DAPI
(DNA)
DAPI
+ FM4-64
(membrane)
sporulating cells
(from Ben-Yehuda et al Science. 299: 532)

37. RacA may cooperate with Spo0J and Soj to
anchor chromosomes during sporulation
(from Errington et al Mol. Microbiol. 49: 1463)

38. Role of SpoIIIE in chromosome translocation
Mutations in spoIIIE cause a defect in chromosome translocation
into the forespore, indicating that SpoIIIE is needed for chromosome
transport into the forespore. SpoIIIE localizes to the asymmetric (polar)
division septum.
The carboxyl-terminal region of SpoIIIE is critical for the ability of this
protein to promote import of the forespore chromosome. Point mutations in
the carboxyl-terminal region cause a defect in chromosome translocation.
This region in SpoIIIE is homologous a region in DNA transfer (Tra) proteins involved in conjugation-mediated plasmid transfer in Streptomyces.
(from Levin and Losick.
p In Prokaryotic
Development ASM
Press. Washington, DC).

39. Promoter recognition by sigma factors   '
RNA polymerase core enzyme
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

40. Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

41. Promoter recognition by sigma factors
Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

42. Sigma factors in sporulation
Old gene names New gene names Protein names Location
rpoD sigA 37,A vegetative
spo0H sigH H vegetative
spoIIG sigE 37,E mother cell
spoIIA sigF F forespore
spoIIIC sigK K mother cell
spoIIIG sigG G forespore

43. Sigma factors in sporulationH H A A
Starvation (and other signals) Stage 0

44. Sigma factors in sporulationE H F A E F A H A
Starvation (and other signals) Stage II/III

45. Sigma factors in sporulationE H H F A A
Starvation (and other signals) Stage 0

46. Sigma factors in sporulationE E H F F F A E A H A
Starvation (and other signals) Stage II/III

47. Sigma factors in sporulationK G E F A K E G F A
Starvation (and other signals) Stage IV
Starvation (and other signals) Stage III

48. Sigma factors in sporulation
Sporulation: regulated sigma factor cascade
… but what regulates the sigma factors?
Not transcriptional control
What kind of post-transcriptional control?
How are events coordinated? Checkpoints?

72. Genes required for the initiation of sporulation (spo0 genes)
spo0A: Encodes a response regulator and key transcription factor.
Spo0A~P binds and activates transcription of promoters of genes required
for later events in sporulation. Spo0A-P also represses transcription of
the transcriptional repressor AbrB, resulting in increased expression
of genes involved in alternative stationary-phase responses (degradative
enzyme biosynthesis, motility, antibiotic production, competence
development).
kinA, kinB, kinC: Code for histidine protein kinases that serve
as sources of phosphate for Spo0A.
spo0F: Encodes a response regulator that transfers phosphate from
KinA, KinB, and KinC to the histidine-phosphoprotein Spo0B.
spo0B: Encodes a phospho-transfer protein that serves as a
direct phosphate donor to Spo0A.

73. Biochemical analysis of the phospho-relay
(from Burbuyls et al
Cell. 64: 545)