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Dynamic Range and Specificity in Bacterial Two-Component Signaling

Dynamic Range and Specificity in Bacterial Two-Component Signaling. Mark Goulian University of Pennsylvania. RssB. FimZ. RstB. KdpD. UhpB. CusS. PhoQ. TorS. DpiA. ArcB. NarQ. BasS. PhoR. DcuS. CpxA. YedV. EnvZ. QseC. BarA. YehU. YfhK. HydD. YpdA. NarX. RcsC. RcsD.

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Dynamic Range and Specificity in Bacterial Two-Component Signaling

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  1. Dynamic Range and Specificity in Bacterial Two-Component Signaling Mark Goulian University of Pennsylvania

  2. RssB FimZ RstB KdpD UhpB CusS PhoQ TorS DpiA ArcB NarQ BasS PhoR DcuS CpxA YedV EnvZ QseC BarA YehU YfhK HydD YpdA NarX RcsC RcsD NtrB TorR BasR RcsB KdpE DpiB NarL CheB CheY RstA PhoB PhoP DcuR CreB CusR UhpC NarP YedW YfJR UvrY YehT YpdB QseB OmpR CpxR HydG ArcA NtrC CheA CreC BaeR AtoC BaeS AtoS envelope stress PO42- osmolarity Mg2+ Cu2+ TMAO glucose 6P NO3-,NO2- quin-ones K+ ~30 two-component signaling systems in E. coli K-12 EvgA EvgS Zn2+ Fe3+

  3. RssB FimZ RstB UhpB KdpD CusS PhoQ TorS DpiA NarQ ArcB DcuS BasS PhoR CpxA QseC YedV BarA HydD YehU YfhK YpdA EnvZ NarX RcsC RcsD NtrB TorR BasR RcsB KdpE CheY CheB DpiB NarL RstA DcuR PhoP CreB PhoB CusR UhpC NarP YehT CpxR OmpR HydG YfJR UvrY ArcA YedW QseB YpdB NtrC P P CheA CreC BaeR AtoC BaeS AtoS envelope stress PO42- osmolarity Mg2+ Cu2+ TMAO glucose 6P NO3-,NO2- quin-ones K+ ~30 two-component signaling systems in E. coli K-12 HK HK HK EvgA RR RR EvgS Zn2+ Fe3+ histidine kinase: response regulator:

  4. PhoQ CpxA EnvZ PhoP OmpR CpxR cell envelope stress osmolarity?, procaine low Mg2+, antimicrobial peptides outer membrane permeability (porins), ... secreted chaperones, proteases .. magnesium transporter, cell envelope modifications ...

  5. PhoQ CpxA EnvZ PhoP OmpR CpxR P P cell envelope stress osmolarity?, procaine low Mg2+, antimicrobial peptides outer membrane permeability (porins), ... secreted chaperones, proteases .. magnesium transporter, cell envelope modifications ... Many histidine kinases are bifunctional – they phosphorylate and dephosphorylate their cognate response regulator HK HK HK RR RR • The phosphorylation cycle leads to: • Insensitivity to changes in levels of the HK and RR • Dynamic range modulation through autoregulation • Cross-talk suppression

  6. RssB FimZ RstB KdpD UhpB CusS TorS PhoQ DpiA ArcB NarQ DcuS BasS PhoR CpxA QseC YedV EnvZ YfhK YehU YpdA HydD BarA NarX RcsC RcsD NtrB TorR BasR RcsB KdpE CheY NarL CheB DpiB RstA DcuR CreB PhoP PhoB CusR UhpC NarP YpdB ArcA YedW CpxR OmpR QseB YfJR YehT UvrY HydG NtrC CheA CreC BaeR AtoC BaeS AtoS RR HK Some two-component systems are auto-regulated EvgA EvgS Why autoregulate? ~30 two-component systems in E. coli Tim Miyashiro

  7. PhoQ PhoP low Mg2+, antimicrobial peptides phoP phoQ

  8. cfp Reference promoter PhoQ/PhoP regulated promoter yfp P PhoP

  9. cfp Reference promoter PhoQ/PhoP regulated promoter yfp Average YFP/CFP Mg++ (mM) P PhoP YFP: The output is graded, not switch-like CFP: 30mM Mg++ 100 μM Mg++

  10. phoP phoQ

  11. PhoQ PhoP PhoP PhoP P P low Mg2+ PhoQ PhoQ phoP phoQ What happens to the level of PhoP-P when you increase expression of PhoP and PhoQ?

  12. HK HK HK RR P P kk RR KMp, kcat p Output is insensitive (robust) to variations in total [HK] and total [RR}. Observed experimentally for EnvZ/OmpR and several other systems ( kk /kcat p ) KM p steady-state [RR-P] total [RR]

  13. phoP phoQ phoP phoQT281R PhoP-regulated transcription YFP/CFP YFP/CFP PhoP / PhoPwild type PhoP / PhoPwild type inducible promoter: (phosphatase- mutant)

  14. autoregulation High signal:  autoregulation boosts the output [RR-P] For low to intermediate signal the system operates in saturation  autoregulation has little effect on the output. [RR]total

  15. + autoregulation: phoP phoQ phoP phoQ - autoregulation: PhoP-P regulated transcription (mgrB transcription) + autoregulation - autoregulation  Autoregulation has no effect on steady-state behavior down to 10 M Mg++

  16. Very high stimulus (growth-limiting) Mg++ mgrB transcription + autoregulation - autoregulation Autoregulation increases the dynamic range

  17. PhoQ PhoP PhoP PhoP P P low Mg2+ PhoQ PhoQ What happens when we get rid of the phosphatase activity? phoP phoQ

  18. cfp Reference promoter PhoQ/PhoP regulated promoter (mgrB, … ) yfp Wild-type PhoQ PhoQT281R (phosphatase-) Mixed Population

  19. PhoQT281R (phosphatase-) Phase CFP YFP “Phenotypic Switching”

  20. PhoQ PhoP PhoP PhoP P P low Mg2+ PhoQ PhoQ phoP phoQ For a simple two-component systems containing a bifunctional histidine kinase, positive autoregulation boosts the response range at high stimulus without affecting the response to low and intermediate stimulus.  provides a stable form of positive feedback that prevents the steady-state output from rolling off at high stimulus.

  21. RssB FimZ RstB UhpB KdpD CusS TorS PhoQ DpiA ArcB NarQ DcuS BasS PhoR CpxA BarA QseC YedV YehU YfhK YpdA EnvZ HydD NarX RcsC RcsD NtrB TorR BasR RcsB KdpE DpiB NarL CheY CheB RstA PhoP PhoB CreB DcuR CusR UhpC NarP CpxR OmpR QseB HydG YedW UvrY YfJR YehT YpdB ArcA NtrC CheA CreC BaeR AtoC BaeS AtoS envelope stress PO42- osmolarity Mg2+ Cu2+ TMAO glucose 6P NO3-,NO2- quin-ones K+ EvgA EvgS Zn2+ Fe3+

  22. RssB FimZ RstB CusS UhpB KdpD PhoQ TorS DpiA NarQ ArcB PhoR BasS DcuS CpxA YehU EnvZ YfhK YpdA YedV HydD QseC BarA NarX RcsC RcsD NtrB TorR RcsB BasR KdpE NarL CheB CheY DpiB RstA CreB DcuR PhoP PhoB CusR UhpC NarP YpdB YehT YfJR YedW CpxR UvrY QseB OmpR ArcA HydG NtrC CheA CreC BaeR AtoC BaeS AtoS envelope stress PO42- osmolarity Mg2+ Cu2+ TMAO ? glucose 6P NO3-,NO2- quin-ones K+ Can we evolve cross-regulation between a histidine kinase and a non-cognate response regulator? Albert Siryaporn EvgA EvgS Zn2+ Fe3+

  23. CpxA EnvZ CpxR OmpR P P degP ompC osmolarity, procaine, ... misfolded proteins in the periplasm, .. , ... , ... outer membrane permeability (porins), ... secreted chaperones, proteases .. HK HK HK RR RR EnvZ and CpxA are bifunctional

  24. CpxA EnvZ CpxR OmpR P P ompC , ... outer membrane permeability (porins), ... CpxA CpxA CpxA OmpR Cross-talk can be detected in the absence of EnvZ and CpxR, but it is blind to signal and shows no evidence of phosphatase activity. OmpR

  25. Helix 2 Helix 1 catalytic Histidine Kinase HK853 fromThermotoga maritima (Marina et al, 2005) Catalytic domain histidine Helix 2 Helix 1 DHp domain CpxA SQQRLLSDISHELRTPLTRLQLGTALLRRRSGESKELERIETEAQRLDSMINDLLVM EnvZ DRTLLMAGVSHDLRTPLTRIRLATEMMSEQDG--YLAESINKDIEECNAIIEQFIDY

  26. Helix 2 Helix 1 catalytic Histidine Kinase HK853 fromThermotoga maritima (Marina et al, 2005) Catalytic domain histidine Helix 2 Helix 1 DHp domain CpxA SQQRLLSDISHELRTPLTRLQLGTALLRRRSGESKELERIETEAQRLDSMINDLLVM EnvZ DRTLLMAGVSHDLRTPLTRIRLATEMMSEQDG--YLAESINKDIEECNAIIEQFIDY Residues important for specificity of interaction with response regulator Skerker et al. Cell 2008. (Laub lab) Change of specificity mutants are blind to signal and show no evidence of phosphatase activity in vivo.

  27. Acknowledgements Lab members • Albert Siryaporn • David Chow • Melissa Lasaro • Elizabeth Libby • Andrew Lippa • Sri Ram • Manuela Roggiani • Manan Shah • Michele Kim Michael Laub (MIT) • (former) • Eric Batchelor • Paige Derr • Tim Miyashiro • Justin Kanoff • Ben Kline • Sangmin Lee

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