Stringent Response in Myxococcus xanthus

What we know…

Starve for amino acids Fruiting body formation

Starve for amino acids Accumulation of (p)ppGpp

Is there a connection between initiation of fruiting body development and (p)ppGpp accumulation?

??Starve for aaAccumulate (p)ppGpp

Fruiting bodies

Is this a causal relationship?

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?

Is the mutant DK527 like the E. coli relA- mutant?

Time after aa starvation w/ serine hydroxamate (min.)

Time after aa starvation serine hydroxamate (min.)

RN

A s

ynth

esis

RN

A s

ynth

esis

*RNA synthesis in M. xanthus compared to E. coli

*Serine hydroxamate inducedstarvation…also increases (p)ppGpp

*wild-types=stable RNA synthesis/tRNA availability

relA- and DK527=uncoupled

*If DK527 is a relA-, then RNA synthesis should be uncoupled toamino acid availability.

E. Coli

M. xanthus --DK527

-- DK101

--relA+(wild type)

-- relA-

**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!

DK527 relA

E. coli relA+

Complementation

(p)ppGpp productionFruiting body formation

relA

E. coli relA+

Specific Integration

E. coli relA Mx8

DK101 and DK527 transformed with pMS132

pMS132

Negative control: DK101 and DK527 transformed with

pMS1321, lacks E. coli relA gene

Presence of plasmids were confirmed by Southern blot

To control gene expression, the light-inducible carQRS promoter was used

How can we integrate something so that it replicates?

+ light

DK101 w/ relA+

DK527 w/ relA+

fruiting

fruiting

DK527 w/o relA+

No fruiting

DK101 w/o relA+

fruiting

- light

DK101 w/ relA+

DK527 w/ relA+

fruiting

fruiting

DK527 w/o relA+

No fruiting

DK101 w/o relA+

fruiting

Examination of fruiting body development in plasmid-carrying derivatives when starved

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

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?

Transduction and homologous recombination

Tet resistance Mx8

pMS133

Plasmid w/ DK527, but lacks E.coli relA+

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

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