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Sporulation. Vegetative cell divides asymmetrically : septum (new cell wall) produced near one end. Sporulation. mother cell. Larger cell (“mother cell”) engulfs smaller (“forespore”). forespore. Sporulation. The forespore now has two membrane layers. Sporulation. - PowerPoint PPT Presentation
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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
