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?
To specifically alter the DNA without changing the protein, which is
required for conjugation.
Why do the authors focus exclusively on spacer region 1, what do you think is in
the other two spacers?
They focus on region 1 because they found that it is identical to a
region of the nes gene of the conjugative plasmid, thus they have a
target to test experimentally. The other two regions didn’t identify a
match in a search of the NCBI database. Because the spacers contain
sequence information from previously encountered infectious agents,
they are likely derived from unknown plasmids or phages.
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?
The plasmid contains an antibiotic resistance gene that allowed for
selection on plates containing mupirocin, and the recipient cells did not
have this gene and thus were unable to grow in the presence of the
antibiotic unless they obtained the plasmid.
How do you think these cells were distinguished from recipient (no plasmid) and
donor cells (which have the plasmid)?
The recipient cells also had a different antibiotic resistance gene that
allowed them to grow in the presence of neomycin. Thus, the
transconjugants had both antibiotic resistance markers. The
experiment then was performed by plating on plates that contained
both antibiotics - donors were selected against due to neomycin and
recipients were selected for by growth on mupirocin.
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?
They showed that CRISPR was required in the recipient cell to limit
conjugation, but nickase is required only in the donor cell and the
protein does not get transferred to the recipient.
How did the authors test this – what trick did they use to change the DNA but
not the RNA?
They disrupted the target sequence in nes by introducing a self-splicing
intron. The DNA is altered but the intron is removed in the RNA after
transcription, regenerating the target sequence specifically in 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 conjugation assay?
Conjugation requires a number of plasmid-encoded factors for
establishing a mating pair, creating a portal between two bacteria, and
allowing for replicative transfer of the plasmid into the recipient (they
are “infectious” plasmids). Nes is one of these factors required for
conjugation, making it difficult to manipulate the target sequence in
the plasmid (eg. change the orientation, Figure 3) without affecting
Nes expression. The use of the transformation assay simply removes
any requirement for Nes function as the assay does not require
conjugation. The plasmids used for the transformation experiments
are not conjugative plasmids, they only have an origin of replication
and a selectable marker.
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?
There are many plausible answers to this question, I think its fun for
the students to explore the possibilities. The answer is that the
CRISPR repeats that flank the spacers in the locus dictate self/non-self
discrimination. The entire CRISPR locus is transcribed in one long
RNA, and the individual crRNAs are generated by cleavage within the
repeat sequences. Therefore, the crRNAs generated from the CRISPR
locus not only contain the spacer sequences in the center, but have
both 5’ and 3’ flanking RNA that are derived from the repeats. If a
crRNA hybridizes to the CRISPR locus, it matches completely along the
length of the crRNA. If a crRNA hybridizes to a target on a phage or
plasmid, there will be non-complementarity at the 5’ and 3’ ends
because these sequences are derived from the repeats, not the target.
The endonuclease complex of CRISPR can recognize the difference and
only cleaves when there is non-complementarity at the ends.