In E. Coli, the Malk Operon Encodes Three Genes: Malk, Lamb, and Malm. It Is Activated

6). (14 Points)

In E. coli, the malK operon encodes three genes: malK, lamB, and malM. It is activated by the MalT protein which is transcribed from the malT gene. The malT gene is unlinked to the MalK operon. The operon is transcribed from a regulatory region as shown below.

The malK gene is required for the cell to use the disaccharide maltose as a C-source. The lamB gene encodes the LamB protein. It is a trimeric outer membrane protein that allows diffusion of maltose (a glucose-glucose disaccharide) and maltodextrins (long polymers of glucose) into the periplasm. LamB is required for diffusion of the lengthy maltodextrins across the outer membrane. However, maltose is small enough to cross using another porin called OmpF. In addition to forming a pore, LamB is also required for adsorption of phage lambda to the cell surface. The function of malM gene product is unknown (and not relevant to this problem).

If one selects mutants that are resistant to infection by lambda, but still able to use maltose as a C-source, all of the mutations map in the lamB gene. Thus LamB mutants cannot grow when maltodextrins as the only C-source but they can grow with maltose as the sole C-source because it diffuses into the cell via OmpF.

A). Starting with a wild-type E. coli strain, you isolate 5-BrU induced mutants that are resistant to infection by phage lambda. (5-BrU causes GC to AT and AT to GC substitutions).

What are 6 different types of 5-BU induced mutants would you expect to find? Would any be suppressed by an amber suppressor? If so, which ones. Would any be highly likely to be dominant? If so which ones? (6 Points).

[1 point for each of 6 out of 8.

malT region- missense and amber in malT gene. Some ambers suppressed missense not suppresed. Promoter down mutation in promoter of malT not ever suppressible and always dominant.

MalK operon-

Mutations in the activator or promoter sites of the malK operon causing lower expression of MalK and thus lower expression of the structural genes. These would also be dominant. None suppressible. Polar mutation in malK, some suppressible. Missense and nonsense in lamB. None of the missense suppressed, some nonsense suppressed.]

B). Assume your lamB mutant contains a nonsense mutation. How would you isolate lamB revertants? (2 Points).

[Demand growth on maltodextrins as sole C-source].

C). What are three different types of revertants you would be likely to obtain?

Which ones would be resistant or sensitive to lambda infection? (6 Points).

[(2 Points for each type and explanation).

True revertants -lambda sensitive, second site mutations in the nonsense codon that make a missense substitution that inserts an acceptable amino acid residue into LamB –probably also lambda sensitive, unlinked amber suppressor – lambda sensitive,

Bypass suppressor – lambda resistant].