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

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 and characterize spo genes• Determine their interdependencies

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

Errington and Mandelstam (1986)• Care in using reporter gene (lacZ)• How is regulation exerted?• Dependence of spoIIA expression on other genes

spoIIA

Goals of Errington and MandelstamMeasure spoIIA expression: wt vs spo mutants

spoIIA

spoIIAA spoIIAB spoIIAC

Psp

lacZ

Psp

ß-galactosidase

spoIIA::lacZ

Goals of Errington and Mandelstam

Psp spoIIA::lacZB. subtilis wild type spoIIA::lacZB. subtilis spo0A43B. subtilis spo0B136B. subtilis spo0D8B. subtilis spoIIAA562B. subtilis spoIIB131B. subtilis spoIID298B. subtilis spoIIIA65B. subtilis spoIIIB2B. subtilis spoIIIC94B. subtilis spoIVA67B. subtilis spoIVB165B. subtilis spoIVC23B. subtilis spoVA89B. subtilis spoVB91B. subtilis spoVC134B. subtilis spoVIA513B. subtilis spoVIB520B. subtilis spoVIC610B. subtilis gerE36

Need a way to move around spoIIA::lacZ

Measure spoIIA expression: wt vs spo mutants

. . .

Goals of Errington and Mandelstam

Psp spoIIA::lacZ

Make spoIIA::lacZ transportable

105J19 105J19cat

105J19: temperate phage

Infection

Lysis Lysogeny

Goals of Errington and MandelstamMake spoIIA::lacZ transportable

Psp spoIIA::lacZ105J19 105J19cat

Wt (105J19 spoIIAA::lac-cat)Wt (105J19)lysogen

recombination

spo- lysogen spo- lysogen spo- lysogen spo- lysogen

Transducing phage

. . .

Select for Cmr

Goals of Errington and MandelstamMeasure expression from spoIIA::lacZ

OH

OH

OH

OH OO

NO2

HO

OH

OH

OH

OH

OH

O

NO2

Starve cells Break cells

+ ONPG

ß-gal Conclusion?

Goals of Errington and MandelstamMeasure expression from spoIIA::lacZ

Starve cells Break cells

+ MUG

ß-gal

OH

OH

OH

OH

OH

O

OH

OH

OH

OH OOMUG

Moral Beware of equating what is measured with what is sought

Induction of spoIIA::lacZ during sporulation

ß gal activity(ONPG)

Time after move to starvation medium 0 h 6 h

(Figure 2a)

wt spoIIA::lacZ

wt

Induction of spoIIA::lacZ during sporulation

ß gal activity(MUG)

Time after move to starvation medium 0 h 6 h

(Figure 2b)

wt spoIIA::lacZ

wt spoIIA::lacZ spoIIA+

spoIIA-

wt

Moral Beware of side effects. Context is important.

Why does spoIIA::lacZ expression fall?

Psp

Transcriptionalregulators

transcription translation

RNAdegradation

proteindegradation

RNA secondary structure

Not well understood

Presence of proteases

Cm

Why does spoIIA::lacZ expression fall?

ß gal activity(MUG)

Time after move to starvation medium 0 h 3 h

(Figure 3)

Rate of protein degradation constant

+Cm

+Cm+Cm

+Cm

Why does spoIIA::lacZ expression fall?

Psp

transcription translation

RNAdegradation

proteindegradation

MUG

RNA

Time after starvation

Why controls spoIIA::lacZ expression?

Table 2

Spo0proteins

SpoIIproteins

SpoIIIproteins

SpoIVproteins

SpoVproteins

Why controls spoIIA::lacZ expression?

Table 2

Spo0proteins

SpoIIproteins

SpoIIIproteins

SpoIVproteins

SpoVproteins

Nonregulatory protein

Nonregulatory protein

Nonregulatory protein

Nonregulatory protein

Nonregulatory protein

1. How does this work?

Hint: Spo0AA is a sigma factor

Why controls spoIIA::lacZ expression?

Table 2

Spo0proteins

SpoIIproteins

SpoIIIproteins

SpoIVproteins

SpoVproteins

1. How does it get started?

Environmental sensing + signal transduction