Download Stringent Response in Myxococcus xanthus

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