Stringent Response in Myxococcus xanthus What we know… Starve for amino acidsFruiting body formation Starve for amino acidsAccumulation of (p)ppGpp Is there a connection between initiation of fruiting body development and (p)ppGpp accumulation? ??Starve for aa Accumulate (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.) RNA synthesis *RNA synthesis in M. xanthus compared to E. coli *Serine hydroxamate induced starvation…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 to amino acid availability. E. Coli M. xanthus --DK 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 production Fruiting 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 DK527 w/o relA+ No fruiting DK101 w/o relA+ fruiting - light DK101 w/ relA+ DK527 w/ relA+ 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