Chapter 15, Problem 30P

The E.coli MalT protein is a positive regulator of several mal operons, which are induced in the presence of the sugar maltose. The gene that encodes MalT was identified in a screen for mutants causing constitutive expression of mal operons; the operons were transcribed even in the absence of maltose. The screen involved a lacZ transcriptional fusion reporter gene in which the regulatory region of a maltose-inducible operon was fused to the coding sequences of lacZ.

a. Bacteria with a lacZ- mutation are transformed with the reporter gene and spread on petri plates containing the β-galactosidase substrate X-gal. What color would the colonies be if the plates also contained maltose? What if the plates had X-gal but no maltose?

b. In the screen, scientists mutagenized the lacZ- bacteria before transforming them with the reporter gene, and then spread the transformed bacteria on plates with X-gal and no maltose. All of the colonies were white except for one colony that was blue. At this stage of the analysis, researchers could not establish whether the gene mutant in the blue colony encoded a positive or a negative regulator of mal operons. Suppose first that the gene encoded a positive regulator. (i) How could the wild-type protein respond to maltose? (ii) How would the mutation affect protein function? (iii) Describe the likely nature of the mutation in the gene at the molecular level. Now answer these same three questions for the hypothesis in which the gene encoded a negative regulator (a repressor) of mal operon expression.

c. How do you think the scientists figured out that MalT was a positive regulator and not a repressor? (Hint: Think about what would happen in each case if the researchers attempted to identify the malT mutant using a plasmid library made from the genome of a wild-type strain versus a plasmid library made from the genome of the mutant strain.)