1. Fig. S1 Cytoplasm V c Periplasm V pp Microenvironment V e D pp AiAi A pp AeAe k IMOM * * * D

2. A Type IIa Type Ia AiAi k AeAe D VcVc dada dada * VeVe * B k D VcVc dada dada * VeVe * AiAi AeAe D imp Fig. S2

3. Percent effector activation 0 40 60 20 80 Basal Induction level Fig. S3 10 2 10 6 V e /V c 10 3 10 4 10 5

4. E nM hr -1 0 Shutoff Induction 200 400 500 E opt OFF R S Q E max P Fig. S4 V e /V c 10 2 10 5 10 4 10 3

5. E opt E max Cost Benefit f max Activation E Cost or Benefit Fig. S5

6. A B v Low Benefit v opt = 0 10 0 10 1 10 3 10 5 Cell density n T cells/ml 10 2 10 4 ×10 6 2.2 1 0.8 1.4 1.8 10 5 10 6 10 7 10 8 Cell density n T cells/ml 10 4 10 6 10 8 High Benefit v opt = inf v Fig. S6

7. Ab e P Ab i Sensor E VeVe P + * Fig. S7

8. C OFF ON v d a hr -1 10 -1 10 0 10 1 10 2 10 0 10 1 10 2 10 3 10 -1 Type I Type II Fig. S8

9. Fig. S9 AiAi k AiAi k AiAi k AeAe D V V B D D S P N + sensor S P N + S P N + A

10. Log 10 ( v ) v calculated v observed Log 10 ( v ) v calculated v observed Fig. S10 A B

11. Log 10 ( v calculated ) 1 23 4 5 6 7 Log 10 ( v observed ) 1 2 3 4 5 6 7 sin CSF1 cin lux rhl las tra CSF2 luxS car ain HAI1 agr comX CAI-1 CSP* CSP luxS* Fig. S11 cep