A new role for next-gen sequencing in our research

Last night the post-doc told me that he expects to have leftover sequencing capacity in the next big run, and asked if I know of any old material that should be sequenced.  After a bit of discussion we realized that some very old experiments should be reinvestigated.

Way back 23 years ago, when I first started my lab, I mutagenized and froze some wildtype cells with EMS, planning to use selection for log-phase competence to isolate regulatory mutants from the mutagenized cells.  This plan succeeded - my initial selection identified a series of 'hypercompetent' mutants with mutations in the sxy gene.  We now know that these mutations destabilize the base-paired stem in sxy mRNA and allow its translation under what are usually non-inducing conditions.  Several years later I thawed out two more vials of these cells and repeated the selection.  This identified nine new hypercompetent mutants, four with sxy mutations identical to ones I had originally found (sxy-1, sxy-2 and two of sxy-5), four extremely hypercompetent mutants with mutations that were eventually mapped to the murE gene (we still don't know how these cause their extreme hypercompetence), and one that remains unmapped.

We now plan to use next-gen sequencing to (1) identify the cause of the unmapped mutation, and (2) identify additional hypercompetence mutations in the two remaining vials of frozen mutagenized cells (treated with 0.05 and 0.08 mM EMS).

1.  The unmapped mutation:  This strain (RR735) has a phenotype like the sxy mutants, moderately competent in log phase (transformation frequency 10^-6 - 10^-5) and fully competent at high cell density.  There were hints from the original experiments that the mutation might be in or near sxy, as there was some evidence of linkage to the StrR locus, but sequencing of the ~400 bp around the known sxy mutations did not find any change.  We did create a 'backcrossed' mutant (RR753) by transforming wild-type cells with RR735 DNA and selecting and screening for hypercompetence (RR expts #804 & #805 and CM expts #690).

The solid circles in the upper graph below show its transformation time course.  (This is a scan of a notebook figure, since the 1995 MacDraw files can't be opened.)   The open circles are the wildtype strain KW20, with the two earliest points giving no transformants (expected TF for wildtype cells at this density is less than 10^-9).  The upper lines are the highly hypercompetent murE
mutants that were also being investigated.

Strategy 1:  We'll sequence both the original mutant and the backcrossed strain.  This should identify one or more segments of RR735 DNA in RR753, and this may be sufficient to identify a candidate hypercompetence mutation if the background frequency of mutations is low enough.  But the background frequency of mutations may be too high.  The EMS treatment caused about 50% mortality, but we don't know how much of this was a direct consequence of EMS damage and how much due to lethal mutations.  In two other strains sequencing of ~400 bp of sxy found additional mutations that we concluded were unrelated to hypercompetence (but we never directly tested this).

Strategy 2:  We'll repeat the backcross, again pooling transformants (selected for StrR?) and selecting for hypercompetence by transforming in log phase with a NovR DNA fragment.  Based on the mutant phenotype we expect a substantial fraction (maybe half?) of the NovR transformants to carry the hypercompetence mutation.  (The math:  RR753 has a log-phase TF about 1000-fold higher than KW20, and we expect a point mutation to transform at a frequency of about 3x10^-3.)  At this point we could pool all the NovR colonies and sequence the pool, or we could either test individual clones, or we could do a second round of selection by transforming the round 1 pool to another marker, again in log phase.

2. Selecting for a pool of new transformants with hypercompetence mutations***: I first need to check the viability of the old frozen cells, since they've been through a partial freezer meltdown.  I'll do this by thawing both vials and plating to check the cfu/ml.  So as not to waste the cells, I'll dilute them into sBHI, let them grow for a couple of generations at low cell density, and then re-freeze them.  Before refreezing I'll do two things. (1) Plate again for cfu/ml to check that they are growing.  (2) Concentrate the cells by collecting them on a filter and resuspending them in a smaller volume.  This will both make freezing more convenient and wash away any DNA released by all the cells that were killed by the EMS treatment.

Round 1 selection for hypercompetence:  The thawed cells will be diluted, checked for cfu/ml, , and resuspended at a OD600 of about 0.01.  Cells will be grown to OD600 = 0.1, incubated with a NovR DNA fragment for 20 min, and plated on nov plates.  (To eliminate background due to new novR mutations the novobiocin will be at 5 µg/ml rather than the usual 2.5 µg/ml.)  This will take a lot of plates because we want to plate all the cells and we don't want to put more than 5x10^7 cfu on each 90 mm plate.  We'll include no-DNA controls and the control vial of non-mutagenized cells.  The novR colonies from this experiment will be pooled (maybe one pool for each original vial, depending on how many colonies there are.  If the experiment is well done there shouldn't be enormous numbers of colonies.  In the previous best experiment, 1 ml of cells at an OD600 of ~0.08 (~ 4 x 10^8 cfu/ml) gave about 5-10 transformants.  Depending on how many viable cells we start with, we could have thousands...

Round 2 selection for hypercompetence:  The novR transformants will be pooled, diluted and grown into log phase (at least 2 hr at OD600 less than 0.05).  They'll then be incubated with DNA carrying a different genetic marker (a NalR PCR fragment?) so we can again select for transfomation in log phase.  Because the modest number of NovR colonies will have created a bottleneck, here we won't need to worry about maintaining a large population.  The NalR transformant colonies will then be pooled and the pool's DNA sequenced.  We'll then examine the pooled sequences for strong overrepresentation of particular mutations, especially in sxy and murE.

The only big concern is the need to get these experiments done quickly because of the time frame for the other sequencing they need to mesh with.

***Later:  The vials of frozen mutagenized cells turned out to contain very few viable cells.  If we can easily get some more EMS we can redo the experiment from scratch (the mutagenesis is fast and easy, since I worked the details out 20 years ago), but otherwise we'll have to abandon Part 2.