The Bacillus thuringensis strain called “kurstaki” includes an operon on its genome that encodes for the insecticidal toxin, CryB1. The cryB1 gene was the one that was introduced into corn to create the first versions of insect-resistant Bt corn. Another approach for battling insect pests like corn borers and gypsy moth larvae is to simply spray the Bt crystal proteins (or even the B. thuringiensis cells themselves) onto plants. Compared to transgenic crops, these spraying approaches are certainly less ‘high-tech’, but are comparatively cheap and often effective. However, for the lost-cost approaches to be effective, agro-pharma companies need strains of B. thuringiensis that produce a lot of crystal protein. Therefore, they are always looking for Bt strains that produce a lot of toxin, and for ways to genetically manipulate them to enhance toxin production. In some of the Bt strains discovered in the field, the details are modestly different from B. thuringiensis kurstaki: the toxins differ subtly from CryB1 and the architecture of the cry operon(s) differ as well.

A recently isolated ‘champion’ Cry producer is strain YBT2000. It produces an insecticidal toxin protein called Cry6. The gene encoding this protein is part of an operon than can be depicted as shown below. The cry6 gene is 1.8 kbp; orf2 is 1.2 kbp. 

PAK 2.1A

Define the numbered items shown in the figure (lengthy text is not needed; just state what these items are).

State the number of different transcripts and proteins that will be produced from the operon.

YBT2000 produces a lot of Cry6. Could it be improved to produce even more? A research group is trying to complete this by following a genetic approach: they generated random mutations in the chromosome of YBT2000 by growing the cells in the presence of EMS (ethyl methanesulfonate), a chemical mutagen. They then screened a large library of such mutants to identify a rare few mutants that produced higher-than-normal levels of Cry6 (see Figure 2 and Figure 3). These researchers were very interested in understanding how the mutation in Mutant-1 caused the observed increase in Cry6 production. If they could understand that, then maybe they could find a way to make even more Cry toxin in strain YBT2000 and possibly in other Bt strains.

Here is what they learned in their characterization of Mutant-1:

• The mutation responsible for the high level of Cry6 produced in Mutant-1 is located in the
• gene immediately downstream of cry6 (in the same operon). No one knows the function of the protein encoded by this gene, so the gene is currently called orf2 and the corresponding protein is Orf2.
• The relative amount of Cry6 produced by wild-type YBT2000 and Mutant-1 is shown in Figure 2.

• The amount of cry6 operon mRNA levels in wild-type YBT2000 and Mutant-1 is shown in Figure 3. 

The researchers have developed the following working hypothesis: Orf2 regulates Cry6 protein levels by controlling transcription initiation of the cry6 operon.

PAK2.1B Consider the results in Figures 2 and 3.

• Are they consistent with the researchers’ hypothesis? Why?
• Do they provide strong support for their hypothesis or are there other mechanisms by
• which Mutation-1 could give the observed results without affecting transcriptional initiation of the cry6 operon?
• Let’s assume for now that the researchers’ hypothesis is correct: Orf2 can affect transcription initiation of the cry6 operon and that this involves Orf2 protein binding to the DNA located upstream of cry6. Such regulation can be either positive or negative. Which scenario applies here? The next two questions ask you to consider these possibilities.
• PAK2.1C Consider first the possibility that Orf2 mediates positive regulation of cry6 expression. If this were true:
• Would Orf2 be considered an activator or a repressor?
• What would be the effect of mutation-1 on the activity of Orf2? Would it enhance or
• diminish its activity?
• What would be the effect of deleting orf2 from the genome of YBT2000?
• PAK2.1D To help answer this question, the orf2 gene was sequenced. DNA sequencing indicated that Mutation-1 is a one-bp deletion located in codon #10 of the orf2 coding sequence. Does this affect your opinion of whether Orf2 is an activator or a repressor? Briefly explain your answer.