Question

1. Osmoregulation in E. coli

Like most things we’ve talked about this semester, if eukaryotes do it, bacteria do it too. Thus the existence of signal transduction pathways in bacteria should not surprise you. One of the best-studied signal transduction pathways controls how E. coli responds to changes in osmotic concentrations. EnvZ spans the cytoplasmic membrane and senses changes in osmolarity. OmpR is a transcription factor that works with EnvZ to adapt to these changing conditions. The signal passed between these two proteins is (of course) mediated by phosphorylation. Interestingly, EnvZ possess both kinase AND phosphatase activities.

In low osmolarity conditions, EnvZ displays low kinase/high phosphatase activity. This results in very little OmpR-phosphate (OmpR-P). In high osmolarity conditions, EnvZ displays high kinase/low phosphatase activity. Thus the level of OmpR-P accumulates.

OmpR-P binds to DNA. There are two outer membrane proteins (OMPs) controlled by OmpR: OmpC and OmpF. Under low osmolarity conditions, OmpF is more abundant than OmpC. Under high osmolarity conditions, OmpC is more abundant than OmpF.

Now that you know the background of this system, answer the following questions.

A. Draw a diagram to illustrate this signal transduction pathway.

B. If you created an EnvZ mutant that was always a phosphatase but never a kinase, predict which OMP would be most abundant under high AND low osmolarity conditions.

C. If you created an EnvZ mutant that was always a kinase but never a phosphatase, predict which OMP would be most abundant under high AND low osmolarity conditions.

D. If you created an OmpR mutant that could never be phosphorylated, predict which OMP would be most abundant under high AND low osmolarity conditions.

E. Would the mutant in #2 be dominant or recessive to wild-type EnvZ?

F. Would the mutant in #3 be dominant or recessive to wild-type EnvZ?

G. Would the mutant in #4 be dominant or recessive to wild-type OmpR?

H. Under high osmolarity conditions, OmpR-P acts as an activator of ompC expression and as repressor of ompF expression. Propose a mechanism for how this type of gene regulation occurs.

Answer 1