Sporulation

Vegetative cell divides asymmetrically:septum (new cell wall) produced near one end

Sporulation

Larger cell (“mother cell”)engulfs smaller (“forespore”)

mother cell

forespore

Sporulation

The forespore now has two membrane layers

Sporulation

Mother cell DNA is breaking down--this cellis “programmed” to die

Sporulation

Mother cell and forespore both produce sporecoat layers of protein and other materials

core

cortex

coat

Sporulation

Mother cell lyses and releases spore

Spore resistance

Heat (many species’ spores can be boiled) Drying Organic solvents Salts Lysozyme and other degradative enzymes UV

Sporulation

Spore structure

Core• DNA• SASPs protect against heat, UV, oxidation• Dehydration protects against heat, oxidation, chemical reactions

Cortex• Peptidoglycan; protects against organic solvents

Coat• Protein layers; protects against lysozyme and many small molecules

Exosporium• Protein and polysaccharide• Functions in sieving, attachment, enzymes, ??

Assembly of the spore coat

B. subtilis spore

500 nm

How do we know what proteins are in the spore coat?

Lai et al., Proteomic analysis of the spore coats of B. subtilis and B. anthracis, J. Bacteriol. 185:1443 (2003)

Spore protein extract SDS-PAGE Coomassie-stained

proteins

Density-gradient centrifugation

Bacillussporulatedin DSM

50%Renografin

(1.2 g/ml)

20%Renografin(1.08 g/ml)

12,000 × g

diatrizoate

celldebris

cells andphase-darkspores

phase-brightspores

MALDI-MS identification of proteins

protein

fragment(e.g., with protease)

ionize

determine masswith mass spectrometer

2D Gel electrophoresis

2D Gel electrophoresis

high m.w.

low m.w.

+ –mostnegative

mostpositive

How did we learn about the roles of morphogenetic proteins?

Little and Driks, Functional analysis of the B. subtilis morphogenetic spore coat protein CotE, Mol. Microbiol. 42:1107 (2001)

Spore protein extract SDS-PAGE Western Blot

mutations in cotE

How did we learn about the roles of morphogenetic proteins?

Ozin et al., Morphogenetic proteins SpoVID and SafA form a complex during assembly of the Bacillus subtilis spore coat, J. Bacteriol. 182:1828 (2000)

Spore protein extract immunoprecipitation SDS-PAGE Western Blot (anti-SpoIVD)

wt spoV

ID m

utan

t

wt spoV

ID m

utan

tsafA

mutant

How did we learn about the roles of morphogenetic proteins?

Bauer et al., Functional regions of the Bacillus subtilis spore coat morphogenetic protein CotE, J. Bacteriol. 181:7043 (1999)

in situ immunofluorescence anti-CotE antibody

wt cotE mutant

spoIVA mutant

What about the exosporium?

Little known about function 20+ known proteins and glycoproteins in B. anthracis

• BclA is major surface protein• ExsFA and ExsFB exosporium proteins not found in B. subtilis

Contains enzymes that may modify germinants• Alanine racemase• Inosine hydrolase