Stringent Response in Myxococcus xanthus

1
Stringent Response in Myxococcus xanthus
What we know
Starve for amino acids
Fruiting body formation
Starve for amino acids
Accumulation of (p)ppGpp
Accumulate (p)ppGpp
??Starve for aa
Fruiting bodies
Is this a causal relationship?
Is there a connection between initiation of
fruiting body development and (p)ppGpp
accumulation?
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Manoil and Kaiser (1980)In M.xanthus

• (p)ppGpp accumulates rapidly when starved (aa)
• All known conditions that initiate fruiting body
  formation also elicit an increase of (p)ppGpp
  concentration
• Mutant DK527 fails to accumulate (p)ppGpp after
  starvation and it does not differentiate
• Can this mutant be used to distinguish if there
  is a causal relationship?

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Is the mutant DK527 like the E. coli relA- mutant?
M. xanthus
--DK527
RNA synthesis in M. xanthus compared to E. coli
RNA synthesis
-- DK101
Serine hydroxamate induced starvationalso
increases (p)ppGpp
Time after aa starvation w/ serine hydroxamate
(min.)
If DK527 is a relA-, then RNA synthesis should
be uncoupled to amino acid availability.
E. Coli
--relA(wild type)
RNA synthesis
wild-typesstable RNA synthesis/ tRNA
availability relA- and DK527uncoupled
-- relA-
Time after aa starvation serine hydroxamate (min.)
DK527 parallels relA- mutant in E. coliBoth
fail to accumulate (p)ppGpp after starvation, and
subsequently do not form fruiting bodies or spores
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Therefore, DK527 is hypothesized to be a relA-
mutant.
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How do we test to see if DK527is really a relA-
mutant?
Complementation!
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Complementation
E. coli relA
E. coli relA
DK527 relA
relA
(p)ppGpp production Fruiting body formation
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How can we integrate something so that it
replicates?
Specific Integration
DK101 and DK527 transformed with pMS132
Negative control DK101 and DK527 transformed
with pMS1321, lacks E. coli relA gene
E. coli relA
Mx8
Presence of plasmids were confirmed by Southern
blot
pMS132
To control gene expression, the light-inducible
carQRS promoter was used
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Examination of fruiting body development in
plasmid-carrying derivatives when starved
light
- light
DK101 w/ relA
DK101 w/ relA
fruiting
fruiting
DK101 w/o relA
DK101 w/o relA
fruiting
fruiting
DK527 w/ relA
DK527 w/ relA
fruiting
fruiting
DK527 w/o relA
DK527 w/o relA
No fruiting
No fruiting
relA expression is controlled by lightso, why
did we get fruiting?
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Expression of E. coli relA protein before
exposure to light was measured by Western blot
(see gel)
Control gene expression w/ light-inducible
promoter
Still shows sufficient amt. of relA protein to
regain (p)ppGpp accumulation and rescue fruiting
in dark
Is recovery of development the result of a
second-site supressor?
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Transduction and homologous recombination
M. xanthus relA
10 out of 10 transductants lost the E.coli relA
gene (screen with probe in S. blot) 7 our of 10
kept DK527 phenotype associated with
relA not a second-site supressor relA rescues
the DK527 mutant
Tet resistance
Mx8
pMS133
Plasmid w/ DK527, but lacks E.coli relA
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The moral of the story is

• DK527 mutant is lacking the relA gene
• Rescue of the DK527 can be attained by
  complementaion with relA
• Fruiting occurs after (p)ppGpp accumalatestherefo
  re, there is a causal relationship b/w
  starvation, (p)ppGpp accumulation and fruiting
• (p)ppGpp is necessary for differentiation