Download - Sporulation Vegetative cell divides asymmetrically: septum (new cell wall) produced near one end
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Sporulation
Vegetative cell divides asymmetrically:septum (new cell wall) produced near one end
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Sporulation
Larger cell (“mother cell”)engulfs smaller (“forespore”)
mother cell
forespore
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Sporulation
The forespore now has two membrane layers
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Sporulation
Mother cell DNA is breaking down--this cellis “programmed” to die
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Sporulation
Mother cell and forespore both produce sporecoat layers of protein and other materials
core
cortex
coat
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Sporulation
Mother cell lyses and releases spore
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Spore resistance
Heat (many species’ spores can be boiled) Drying Organic solvents Salts Lysozyme and other degradative enzymes UV
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Sporulation
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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, ??
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Assembly of the spore coat
B. subtilis spore
500 nm
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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
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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
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MALDI-MS identification of proteins
protein
fragment(e.g., with protease)
ionize
determine masswith mass spectrometer
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2D Gel electrophoresis
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2D Gel electrophoresis
high m.w.
low m.w.
+ –mostnegative
mostpositive
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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
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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
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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
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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