Dynamic Range and Specificity in Bacterial Two-Component Signaling Mark Goulian University of Pennsylvania
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Dynamic Range and Specificity in Bacterial Two-Component Signaling Mark Goulian University of Pennsylvania CpxA NO3-, NO2- EnvZ Cu2+ envelope PO42- osmolarity stress Mg2+ OmpR CpxR TMAO glucose 6P quinones K+ FimZ ~30 two-component signaling systems in E. coli K-12 RssB BaeR QseB BaeS QseC Zn2+ Fe3+ CpxA NO3-, NO2- EnvZ Cu2+ envelope PO42- osmolarity stress Mg2+ OmpR CpxR TMAO glucose 6P quinones K+ FimZ ~30 two-component signaling systems in E. coli K-12 histidine kinase: response regulator: RR P HK QseC Fe3+ HK BaeR QseB BaeS Zn2+ HK RssB P RR cell envelope stress low Mg2+, antimicrobial peptides CpxA osmolarity?, procaine CpxR outer membrane permeability (porins), ... secreted chaperones, proteases .. magnesium transporter, cell envelope modifications ... cell envelope stress low Mg2+, antimicrobial peptides CpxA osmolarity?, procaine CpxR outer membrane permeability (porins), ... secreted chaperones, proteases .. magnesium transporter, cell envelope modifications ... RR P HK HK HK Many histidine kinases are bifunctional – they phosphorylate and dephosphorylate their cognate response regulator P RR The phosphorylation cycle leads to: •Insensitivity to changes in levels of the HK and RR •Dynamic range modulation through autoregulation •Cross-talk suppression EnvZ CpxA OmpR CpxR FimZ HK Some two-component systems are auto-regulated BaeR QseB RssB RR BaeS QseC Why autoregulate? ~30 two-component systems in E. coli Tim Miyashiro low Mg2+, antimicrobial peptides phoP phoQ P PhoP yfp PhoQ/PhoP regulated promoter cfp Reference promoter P PhoP yfp cfp PhoQ/PhoP regulated promoter Reference promoter YFP: The output is graded, not switch-like YFP/CFP Average YFP/CFP (a.u.) 1.4 CFP: P_mgrB 1.2 1 0.8 0.6 0.4 0.2 0 0.01 0.1 1 ++(mM) Mg Mg 30mM Mg++ 100 μM Mg++ (mM) 10 100 phoP phoQ low Mg2+ PhoQ PhoQ PhoQ P PhoP P PhoP PhoP phoP phoQ What happens to the level of PhoP-P when you increase expression of PhoP and PhoQ? HK kk HK HK P RR P RR steady-state [RR-P] KMp , kcat p ( kk /kcat p ) KM p total [RR] Output is insensitive (robust) to variations in total [HK] and total [RR}. Observed experimentally for EnvZ/OmpR and several other systems 1.2 6 1 5 YFP/CFP YFP/CFP PhoP-regulated transcription 0.8 0.6 0.4 4 3 2 0.2 1 0 0 0 3 2 1 PhoP / PhoPwild type inducible promoter: phoP phoQ 4 0 1 2 3 PhoP / PhoPwild type phoP phoQT281R (phosphatase- mutant) 4 30 High signal: autoregulation boosts the output 25 [RR-P] 20 15 10 autoregulation 5 0 0 10 20 30 40 [RR]total 50 For low to intermediate signal the system operates in saturation autoregulation has little effect on the output. + autoregulation: phoP phoQ - autoregulation: phoP phoQ PhoP-P regulated transcription (mgrB transcription) 0.8 YFP/CFP 0.7 0.6 + autoregulation WT - autoregulation 0.5 P1- 0.4 0.3 0.2 0.1 0 0.01 0.1 1 10 100 Mg (mM) Autoregulation has no effect on steady-state behavior down to 10 M Mg++ Very high stimulus (growth-limiting) Mg++ mgrB transcription 1.2 1 YFP/CFP 0.8 WT + autoregulation ΔP1 - autoregulation 0.6 0.4 0.2 0 0 2 4 6 8 10 Tim e (hr) Autoregulation increases the dynamic range low Mg2+ PhoQ PhoQ PhoQ P PhoP P PhoP PhoP What happens when we get rid of the phosphatase activity? phoP phoQ Wild-type PhoQ PhoQT281R (phosphatase-) yfp PhoQ/PhoP regulated promoter (mgrB, … ) cfp Reference promoter Mixed Population PhoQT281R (phosphatase-) Phase CFP “Phenotypic Switching” YFP low Mg2+ PhoQ PhoQ PhoQ P PhoP P PhoP PhoP 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. CpxA NO3-, NO2- EnvZ Cu2+ envelope PO42- osmolarity stress Mg2+ OmpR CpxR TMAO glucose 6P quinones K+ FimZ RssB BaeR QseB BaeS QseC Zn2+ Fe3+ NO3-, NO2- OmpR K+ ? CpxA EnvZ Cu2+ envelope PO42- osmolarity stress Mg2+ Albert Siryaporn RssB BaeR QseB BaeS QseC Zn2+ Fe3+ glucose 6P CpxR Can we evolve cross-regulation between a histidine kinase and a non-cognate response regulator? FimZ TMAO quinones ompC CpxA misfolded proteins in the periplasm, .. EnvZ osmolarity, procaine, ... OmpR CpxR , ... degP HK RR P HK secreted chaperones, proteases .. HK outer membrane permeability (porins), ... , ... P RR EnvZ and CpxA are bifunctional ompC EnvZ CpxA OmpR CpxR , ... outer membrane permeability (porins), ... OmpR P CpxA CpxA CpxA 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. P OmpR Histidine Kinase HK853 fromThermotoga maritima (Marina et al, 2005) Catalytic domain histidine Helix 2 Helix 1 DHp domain Helix 1 CpxA EnvZ Helix 2 SQQRLLSDISHELRTPLTRLQLGTALLRRRSGESKELERIETEAQRLDSMINDLLVM DRTLLMAGVSHDLRTPLTRIRLATEMMSEQDG--YLAESINKDIEECNAIIEQFIDY catalytic Histidine Kinase HK853 fromThermotoga maritima (Marina et al, 2005) Catalytic domain histidine Helix 2 Helix 1 DHp domain Helix 1 CpxA EnvZ Helix 2 catalytic SQQRLLSDISHELRTPLTRLQLGTALLRRRSGESKELERIETEAQRLDSMINDLLVM 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. Acknowledgements Lab members Albert Siryaporn David Chow Melissa Lasaro Elizabeth Libby Andrew Lippa Sri Ram Manuela Roggiani Manan Shah Michele Kim (former) Eric Batchelor Paige Derr Tim Miyashiro Justin Kanoff Ben Kline Sangmin Lee Michael Laub (MIT)
