Stringent Response in Myxococcus xanthus

1. Stringent Response inMyxococcus 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?

2. 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?

3. 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 starvation…also 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-types=stable RNA synthesis/ tRNA availability relA- and DK527=uncoupled -- relA- Time after aa starvation serine hydroxamate (min.) **DK527 parallels relA- mutant in E. coli**Both fail to accumulate (p)ppGpp after starvation, and subsequently do not form fruiting bodies or spores

4. Therefore, DK527 is hypothesized to be a relA- mutant.

5. How do we test to see if DK527is really a relA- mutant? Complementation!

6. Complementation E. coli relA+ E. coli relA+ DK527 relA relA (p)ppGpp production Fruiting body formation

7. 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 relAgene E. coli relA Mx8 Presence of plasmids were confirmed by Southern blot pMS132 To control gene expression, the light-inducible carQRS promoter was used

8. 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 light…so, why did we get fruiting?

9. Expression of E. colirelA 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?

10. Transduction and homologous recombination M. xanthus relA *10 out of 10 transductants lost the E.colirelA 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.colirelA+

11. 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