Download Conjugative plasmids are circular pieces of DNA that not only

Conjugative plasmids are circular pieces of DNA that not only replicate in host
bacteria, but also encode machinery to transfer itself into bacteria that do not
contain the plasmid. This process is termed conjugation. Nickase is an enzyme
that initiates the transfer of the plasmid from “donor” bacteria into “recipients”,
but is not required for replication in the donor.
1. In Staphylococcus epidermidis, the first CRISPR spacer region is identical to a
region of the Nickase gene (nes) from the conjugative plasmid named pG0400.
In Figure 1B, why is it important that the authors chose to make silent mutations
in the region of nes? Why do the authors focus exclusively on spacer region 1,
what do you think is in the other two spacers?
2. In figure 1, conjugation experiments were performed by mixing an S.
epidermidis donor strain carrying the pG0400 plasmid (RN4220) with recipient
strains that lacked the plasmid, and conjugation was allowed to occur during
growth on a plate overnight. The conjugation efficiency was determined by
dividing the number of transconjugants (the recipient cells that incorporated the
plasmid) by the total number of recipient cells. How was the number of
transconjugates determined? How do you think these cells were distinguished
from recipient (no plasmid) and donor cells (which have the plasmid)?
BTW, there is a mistake in the legend to figure 1. The conjugation efficiency is
calculated as transconjugants/recipients.
3. What was the evidence that suggested to the authors that nes DNA was the
target rather than mRNA? How did the authors test this – what trick did they
use to change the DNA but not the RNA?
4. Why did the authors also test for interference using transformation? What is
the important difference between the plasmids they used for the transformation
experiments and the ones they used for the conjucation assay?
5. If the sequences in the CRISPR spacer regions specify the DNA target to be
interfered with, how does the cell discriminate the sequence on the plasmid from
the DNA sequence that is present in the chromosomal CRISPR locus? I.e., how
do you think CRISPR discriminates self from non-self?