The Bacillus subtilis sporulation

A bacterial model of differentiationOr

How to survive

Spore formation in Bacillus subtilis andother 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, reactiveoxygen, high and low temperature, acid and alkali conditions, hydrolyticenzymes, 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 containingone or more of two specific ‘germinants’ (L-alanine and/or a mixture ofL-asparagine, fructose, glucose, and potassium ions).

• Spore formation in B. subtilis is a simple developmentalevent that involves the generation of two different cell types (foresporeand mother cell) with different programs of gene expression and differentdevelopmental fates. Thus, in addition to being of interest to microbiologists,spore formation in Bacillus is also of considerable interest to developmentalbiologists.

• In addition to B. subtilis, several other species of gram-positive bacteriamake endospores. Bacillus anthracis (anthrax), Clostridium botulinum (botulism),Clostridium tetani (tetanus), Clostridium perfringens (gas gangrene, food poisoning), Clostridium difficile (antibiotic-associated and pseudomembranous colitis).

Bacillus subtilis growth and sporulation

time

Log(N)

inoculate

1 log growth, symmetric cell division

1

2 Gain motility

2

3 Gain genetic competence

3

4 Sporulate, asymmetric cell div

4

Relatives: B. cereus, B. anthracis

~7 hours VII stages

Genetics: >200 genes/proteins

Quorumsensing

Life cycle of Bacillus subtilis

• B. subtilis can sporulate when the environmental conditions become unfavorable

?division cycle

sporulation-germination

cycle

metabolic and environmental signals

Microscopy of sporulation

Dipicolinic acid

DNA complexed withSASPs

Endospores of Bacillus have been the primary model system

Brock Biology of Microorganisms, vol. 9, Chapter 3

Stageof sporulation

0(vegetative growth)

I(axial filament formation)

II(polar division)

III(engulfment)

IV to VI(cortex and coat formation)

Mother cell lysisand 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; alleleExample: spo0K141

0 Growth

I Replication

II Assymetric Septum

III Engulfment

IV Cortex

V Coat

VI Mother Cell Lysis

VII Free Spore Sporulation Stages in Bacillus

Stages of Sporulation in Bacillus subtilis

Stage 0

Normal vegetative growth

Stage II1

Asymmetric septation

Stage II3

Engulfment

Stage III

Prespore protoplast

Stage IV

Cortexformation

Stage V

Coatformation

Stage VI

Maturation

Stage VII

Release

Germination

spore freecell

Spore germination

In response to nutrients, the spore will exit its dormant state and enter back into vegetative growth

0

B. subtilis sporulation stages

M D

2 chromosomes, axial fibers

I 2 cells, cell wall

II Engulfment

III Double membrane

IV Cortex

V-VI Coat

VII Spore maturationMother cell lysis (DNA)

Stages of Sporulation in Bacillus subtilis

Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII

How is the process regulated?

spo0 spoIII spoVIspoIV spoVspoII

Stages of Sporulation in Bacillus subtilis

Stage 0 Stage II1 Stage II3 Stage III Stage IV Stage V Stage VI Stage VII

spo0 spoIII spoVIspoIV spoVspoII

Golden age of Bacillus genetics• Identify spo genes• Clone spo genes• Determine their interdependencies

Deciding to sporulate

A

H

?

sporulate

1

1 Starvation:What is monitored? [GTP] = (C, N, P)

2 Cell density:

2?

“quorum sensing” peptides

Food low, [cell] low

Food low, [cell] high

Ok ?

Trouble ahead !

3

3 Chromosomes: : undamaged, complete, and polar placement.

Not ppGpp

How is the septum placed asymmetrically?

Polar Division

FtsZ:

Ring at septum

A Downward Spiral

Asymmetric Cell Division in B. subtilis Involves a Spiral-like Intermediate of the Cytokinetic Protein FtsZ Cell 109:257-266.

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

Spo0J and Soj

• Spo0J (a ParB homolog) is required for efficient chromosome segregation during vegetative growth in B. subtilis. Spo0Jbinds to a series of DNA sites (parS sites) in the origin-proximal20% of the chromosome, and these sites are believedto be involved in partitioning.

• In addition to its role in partitioning, Spo0J also is neededfor efficient spore formation. Spo0J regulates the initiationof sporulation by antagonizing the function of Soj.

• SoJ (a ParA homolog), a DNA-binding protein presentin an operon with Spo0J, inhibits the initiation of sporulationin the absence of Spo0J. Soj inhibits the onset of sporulationby binding and inhibiting transcription of promoters of sporulationgenes that are normally activated by Spo0A~P (spoIIA, spoIIE, spoIIG).

Spo0J Soj spoIIE spoIIA

spoIIG

Roles of Spo0A-P and the alternative factor H

in polar cell division

• Early in sporulation, FtsZ localization switches from a midcell position to a bipolar localization.

• Spo0A and the phosphorelay proteins Spo0F and Spo0B are needed forthe midcell to bipolar switch of FtsZ rings. A constitutively active form of Spo0A is sufficient to causepolar septa formation during exponential growth. These results indicate that Spo0A~P drives transcription of one or more genes that promoteformation of polar septa.

• The alternative factor H (encoded by spo0H) is also needed for theswitch in FtsZ localization and for polar septation.

(from Margolin, 2002. Curr. Biol. 12: R391-392).

Spo0A~P,Spo0H

Formation of polar septa during sporulationappears to proceed through an intermediate

spiral-like FtsZ structure

Spo0A~P: expression of SpoIIESpo0H: increased FtsZ expression

(from Margolin, 2002. Curr. Biol. 12: 391-392).

FtsZ spiral formation and polar septationis mediated by increased transcriptionof 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 H-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

spoIIE∆;P2∆-ftsZ

Three hours after the induction of sporulation, cells were fixed, stained withthe membrane dye FM4-64, and examined by fluorescence microscopy

(from Ben-Yehuda and Losick, 2002. Cell. 109: 257).

Role of E in polar septum formation• E (the product of the spoIIGB gene) is synthesized as an inactive precursor •before formation of the asymmetric septum. Active (processed) E is generated only in the mother cell where it directsgene expression in that cell type.

• Null mutations in spoIIGB or mutations that prevent activation of E result in formation of ‘disporic’ cells. These are cells that containtwo asymmetric septa, one at each pole. Both of the forespore-likecompartments in disporic cells contain a chromosome, but the largermother cell is anucleate. Of course, viable spores do not develop froma disporic cell.

• The disporic phenotype of spoIIGB mutants indicates that E mustbe 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 E -controlledgenes that inhibit the second polar septum are spoIID, spoIIM, and spoIIP.

E -positive

E -minus

spoIID,spoIIM,spoIIP

Chromosome segregation duringsporulation in B. subtilis

(from Pogliano et al. 2003. Curr.Opin. Cell Biol. 6: 586)

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 aftermost 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 neededfor 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 vegetativelygrowing cells and artificial expression of RacA during exponential growthresults in movement of nucleoids towards the cell poles (not shown).

racA+ strain racA- strain

DAPI(DNA)

DAPI(DNA)

DAPI+ FM4-64

(membrane)

DAPI+ FM4-64

(membrane)sporulating cells (from Ben-Yehuda et al. 2003. Science. 299: 532)

RacA may cooperate with Spo0J and Soj to anchor chromosomes during sporulation

(from Errington et al. 2003. Mol. Microbiol. 49: 1463)

Role of SpoIIIE in chromosome translocation

•Mutations in spoIIIE cause a defect in chromosome translocationinto 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 thisprotein to promote import of the forespore chromosome. Point mutations inthe 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. 167-189. In ProkaryoticDevelopment. 2000. ASMPress. Washington, DC).

Promoter recognition by sigma factors

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

'

RNA polymerase core enzyme

Sigma factor

Promoter recognition by sigma factors

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

Promoter recognition by sigma factors

Figure from Griffiths et al (1996) Introduction to Genetic Analysis, 6th ed., WH Freeman and Co.

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

Sigma factors in sporulation

A A

AA H

H

H

H

Starvation (and other signals)Stage 0

Sigma factors in sporulation

A

A

AA

H

H

E

E F

F

Starvation (and other signals)Stage II/III

Sigma factors in sporulation

A A

AA H

H

H

HE

E

F

F

Starvation (and other signals)Stage 0

F

F

F

F

E

E

Sigma factors in sporulation

A

A

AA

H

H

E

E F

F

Starvation (and other signals)Stage II/III

Sigma factors in sporulation

A

A

E

E F

F

K

K

G

G

Starvation (and other signals)Stage III

Starvation (and other signals)Stage IV

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?

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 requiredfor later events in sporulation. Spo0A-P also represses transcription ofthe transcriptional repressor AbrB, resulting in increased expressionof genes involved in alternative stationary-phase responses (degradativeenzyme biosynthesis, motility, antibiotic production, competencedevelopment).• kinA, kinB, kinC: Code for histidine protein kinases that serveas 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 adirect phosphate donor to Spo0A.

Biochemical analysis of the phospho-relay

(from Burbuyls et al. 1991.Cell. 64: 545)