Question

3. (5 points) Researchers wanted to investigate how the bacterial species Colwellia degrades oil. They found an inducible operon consisting of promoter (P), operator (O), repressor (R), and structural genes for metabolic oil-degrading enzyme-complex (C). Based on the genotypes below, explain which mutant(s) can make functionally active enzyme in presence and absence of oil? Explain. Wild-type: R+P+o+c+ (make active enzymes only in presence of oil) Mutant 1: R+pto+c-/F'R+P o+c+ Mutant 3: R-Pto+C/F' R+p+o+c+ Mutant 2: R-Pto+c+/F' Ripto+c+ Mutant 4: R+ptoʻct/F R+p+o+c+

Answer 1

Consider an oil-inducible operon comprising of a promoter (P), an operator (O) and a cassette of genes that work by metabolizing the oil and converting it into a usable form for the bacteria. The operon is under the control of the repressor (R). When the oil is not there, the repressor can bind to the operator and stop the transcription of the downstream cassette. On the other hand, when the oil is present, the repressor is bound by the oil, which acts as an inducer, thereby preventing the repressor from binding to the operator. As a result, the transcription is initiated.

Tra ption Absence of Oil P 0 Cassette B oil Transcription Presence of Oil P. 0 Cassette

The above figure depicts the scenario for the wild-type R+P+O+C+ scenario. The cassette is active only in the presence of the oil and hence the proteins are produced only when the oil is present.

1. Mutant 1 is R+P+O+C-/F'R+P+O+C+. Thus the bacteria is mutant for the cassette proteins. However, it has an F plasmid in its chromosome which contains the Cassette. Thus the bacteria acts like the wild-type and can produce the enzymes only in the presence of the induce oil.

2. Mutant 2 is R-P+O+C+/F'R+P+O+C+. Thus the bacteria lacks R and hence should be able to produce the cassette proteins irrespective of the presence of oil. However, it has an F plasmid incorporated into its genome which does have R. If the concentration of R is high enough, the bacteria would act like wild-type and produce the cassette enzymes only in the presence of oil. However, if the R concentration produced from the F plasmid is pretty low, it will not be able to repress the native cassette. And a minimal amount of cassette proteins will be produced even in the absence of oil. However, upon the addition of oil to the culture, the cassette expression would increase due to the activation of the F' cassette.

3. Mutant 3 is R-P+O+C-/F'R+P+O+C+. This bacteria completely lacks the cassette in its genome but has a cassette supplemented by the F'. Hence the bacteria would act like wild-type bacteria and produce the cassette enzymes only in the presence of the oil.

4. Mutant 4 is R+P+O-C+/F'R+P+O+C+. The bacteria lacks the genomic Operator. Thus the cassette expression becomes independent of the repressor and the bacteria will be able to produce the cassette enzymes in the presence as well as the absence of oil.

Thus each of the mutants can in practice make the enzyme.