In the previous post I realized that I don't know how quickly competence will develop in cells that have just acquired a murE hypercompetence mutation.  In that post this was a practical problem (how long should the mutagenized cells be incubated for), but it also has research-grade implications.  That's because we still have no idea how these mutations turn on the competence regulon.  If they take a long time to have an effect, I would suspect some sort of gradually developing imbalance that eventually changes Sxy expression, whereas if they act quickly I'd suspect a more direct effect.

How could I find this out?  I would need to introduce the murE749 hypercompetence mutation into a murE+ cell, presumably by transformation, and then follow the development of competence.  I can do this because we have strains with a CmR cassette that's about 70% linked to murE in transformations. Strain RR797 has the cassette and the murE749 mutation; strain RR805 has the cassette and murE+. So if I transform competent wildtype cells with RR797 DNA and select for chloramphenicol resistance, about 70% of the CmR cells should have the murE749 mutation.

If I do this CmR selection in broth (plating on the side just to check that the transformation worked), I can check how long it takes for the culture to increase its log-phase competence from a transformation frequency of <10 10="" 70="" a="" about="" expected="" i="" mixture="" of="" s="" that="" the="" to="">murE74910>
.  In parallel, as a control that will also provide useful information, I'll transform with RR805 DNA and measure how long the CmR cells and unselected cells take to completely lose competence and resume exponential growth in sBHI at low density.  (I think I've measured loss of competence before but not carefully.)
How long will this take?  I think it's a one-day experiment after I make the DNAs (or if I find old stocks of the DNAs in my fridge (later - found RR805 DNA but not RR797 DNA so I'll plan on remaking them both).

Would we learn anything?  Yes if competence development after transformation is very fast (appears as quickly as chloramphenicol resistance) or very slow, no otherwise.

The EMS mutagenesis mutant hunt - progress and plans

By Rosie Redfield on Sunday, September 22, 2013 I wrote about this general experimental plan here, and described the preliminary test of the NovR selection here.  The EMS mutagen has been ordered and should arrive within the next few days.  The experiment isn't as urgent as we originally thought, because the DNA sequencing plans have changed, but it still needs to be done soon, so I'd better be ready to get started.  Here's the plan:

Do the initial mutagenesis in three different strains:

To minimize spontaneous competence the cells will be pre-grown in sBHI at low density for at least 2 hr.

Each strain will be incubated with 0.0, 0.05 and 0.08 mM EMS for 30 min at an initial OD600 of 0.05. Maybe the volume will be 10 ml.

After the 30 min mutagenesis each culture will be thoroughly washed by filtration to remove the EMS and resuspended in fresh sBHI (50 ml? 100 ml?).

First plan: The cultures will be grown for 2 hr - that should be enough for any mutational changes in sxy to cause elevated competence.  The density will need to be kept low - ideally below OD600 = 0.1.  Will it be enough time for expression of the murE-mutant hypercompetence phenotype?  Hard to say, since we don't know how the mutations cause their phenotype.  Ideally I would do an experiment to find this out.  (How? Not easily, it seems...   I could transform competent wildtype cells with DNA from a CmR murE749 mutant, selecting for CmR after 1 hr expression time, and then at intervals (every hour?)  transforming aliquots of the culture to NovR with MAP7 DNA.  But the cells would initially have to be competent, so I'd have to grow the culture for at least a couple of hours at low density to grow-out this competence before I would be able to detect any log-phase competence caused by the murE mutation...)  Instead I think I'll just hope that 2 hr of log-phase growth is enough time for a new murE mutation (or any other mutation) to cause hypercompetence.  Wait, is there any compelling reason not to grow them longer?  The volume will keep getting larger with the repeated dilutions, but I could just freeze some of the cells after 1 or 2 hr and continue growing the rest for another hour or two.  The frequency of the mutants I'm looking for shouldn't change with growth, since none of the hypercompetence mutations we know of slow growth.

So new plan:  Grow the mutagenized cultures for 90 minutes.  The OD should be back up to about 0.1.  Freeze 3/4 of the cells (concentrate by filtration before freezing).  Dilute the rest back to OD 0.025, grow for another hour (OD back to 0.1), and then add transforming DNA.
The postdoc is making me a prep of NovR PCR fragment that I can use for these transformations.  I'll want to do a test transformation first, using this DNA with normal competent cells, to determine how much DNA to use.  We don't want to use MAP7 chromosomal DNA because (1) the efficiency is low because most of the DNA is from other parts of the chromosome, and (2) it will transform other parts of the competent cells' chromosomes, potentially removing the mutation we want to isolate.

Incubate the cells with DNA for 30 min, add DNaseI and wash the cells by filtration to remove both the DNA and dead cells.  Resuspend in medium with novobiocin and grow for at least 6 hr (or overnight) before plating on nov5 plates. (We usually use nov at 2.5 µg/ml, but NovR transformants grow just as well on plates with 5 µg/ml, and most spontaneous NovR mutants don't.)  Freeze some of the cells instead of plating them all.

If the cultures are grown overnight with novobiocin,  I should probably increase the novobiocin to 5 µg/ml after the first few hours.  In the morning I could plate some of the cells for NovR colonies, and just grow some in log phase for 2-3 hr before transforming them with KanR DNA to select the ones that are genetically hypercompetent (eliminating the ones that were accidentally competent for the first selection).  Alternatively I can wait and pool the NovR colonies that grow up on the plates.  Because the culture should be already enriched for hypercompetent mutants I won't need to worry about 'bald spot' effects but can plate relatively dilute cultures on the kan plates.  (I won't assume this but check by plating different concentrations of course.)

Next I pool all the transformants from each StrR or CmR culture (KanR if I've done the second round of selection, NovR if I haven't) and prep DNA from them.  I use the DNA to transform competent wildtype cells to StrR or CmR, grow the pooled cells into log phase, and select for hypercompetent cells with NovR DNA.  (This could be a PCR fragment or chromosomal DNA (wait, do I have a NalR strain in the freezer?).)

For the wildtype culture, I can just do one more round of hypercompetence selection, with NalR, or I can do an unselected transformation (or select for NovR as an unlinked marker) and then do the NalR hypercompetence selection.

Pool the NalR transformants from each culture, extract DNA and sequence.  If there are hypercompetent mutants we expect to see peaks of novel alleles at and around the sites of the mutations.

So, the whole experiment:  First streak out the cells.  Then dilute and grow, mutagenize, wash, dilute and grow, freeze, dilute and grow, transform with NovR, wash, grow with nov, freeze, grow with nov, plate.  (Pool), dilute and grow, transform with KanR, plate.  Pool KanR, make DNA, transform to CmR or StrR, plate.  Pool, dilute and grow, transform to NalR, plate, pool, make DNA, sequence.

AAACCKKK!  Maybe it will be clearer if I partition it into days?

Day -1: Streak out the cells.  (3 strains)
Day 0:  Inoculate single colonies overnight.  (3 strains)
Day 1: Dilute and grow, mutagenize, wash, dilute and grow,
freeze, dilute and grow, transform with NovR, wash,
grow with nov, freeze, grow with nov, maybe plate.   (9 cultures)
Day 2: (Pool), dilute and grow, transform with KanR, plate.
(6 cultures (not the controls))
Day 3: Pool KanR, make DNA, transform Rd to NovR, CmR or StrR,
plate. (6 cultures)
Day 4: Pool, dilute and grow, transform to NalR, plate.  (6 cultures)
Day 5: Pool, make DNA, ready for sequencing. (6 DNA preps)

Well, at least I now know what I'll be getting into.

Selecting for rare NovR cells by enrichment in broth - would it work?

By Rosie Redfield on Saturday, September 14, 2013 In the previous post about searching for new hypercompetent mutants I mentioned that our ability to find rare NovR transformants in a log-phase culture is limited by the need to put relatively small numbers of NovS cells (less than 5 x 10^7) on each plate.  If we use more cells we see large 'bald spots' where the NovR cells are unable to form colonies; we speculate that this is because of toxic effects of too many NovS cells dying around them.  When the problem is severe we see no NovR colonies at all even though hundreds were plated. One solution is just to distribute the cells over more and bigger plates.  Scaling up by ten-fold is easy, but scaling up by 100-fold is a lot more work and more expense for plates and medium.  In the experiment we're now considering, we expect the NovS cells to outnumber the NovR cells by about 10^8 to 10^9, so we'd like to scale up by a thousandfold.

An alternative that we've never tried is to add novobiocin to the liquid culture for a few hours before plating the cells on agar medium containing novobiocin.  This scaling up would let us amplify the rare NovR cells by letting them double repeatedly while the NovS cells stalled or died.  Ten doublings (about 5 hr of growth) would bring the NovR density up from 10^-9 to 10^-6, so that plating on a moderate number of plates would capture the full diversity of the initial transformant population.  We would no longer be able to assume that separate NovR colonies descended from independent transformations, but for this experiment that's not important.

I think I'll try this today, using a normal log-phase culture rather that the EMS-mutagenized cells we'd use in the planned mutant-hunt:
If this works as expected, we'll be able to start our big experiment with a very large mutagenized culture and pool many more independent transformants.  This will give us a much more diverse pool of hypercompetent mutants for our sequencing.

OK, the first try didn't work because I appear to have added bacteria to the sBHI agar instead of novobiocin (!!!) so what should have been novobiocin plates were plates with about 10^4 tiny colonies of H. influenzae embedded in the agar!

But the second try worked very nicely.  The red lines are the planned experiment, with a small number of competent cells added to a 100-fold excess of non-competent cells; the green lines are just the competent cells by themselves.  Each cell prep was incubated with NovR transforming DNA for 15 minutes, and then novobiocin was immediately added to the culture.  The non-competent cells (open red squares) continued growing a bit and then stalled (the competent cells had already stopped growing because they had been starved to induce competence).  After two hours the transformed NovR cells (solid red and green squares) started doubling and the non-transformed NovS cells (open red and green squares) started dying, just as they should.  By the next morning the cultures were thick and consisted of only NovR cells. This result means that we can incorporate selection for NovR transformants in broth in the mutagenesis experiment and so won't  have to distribute the transformation mixture over hundreds of Nov plates.

A better strategy for finding new hypercompetence mutations in murE If we did decide to buy some EMS (it's cheap and readily available from Sigma) and repeat the mutagenesis, I think we should use a different strategy to select for hypercometent mutants.  Specifically, we should focus on getting more  strains whose hypercompetence is caused by mutations in murE (see this old post for a description of the murE results).  We have four such mutants now, which change two different amino acids, and we don't understand how they work.  Having more independent mutants would help clarify the situation, whether we get new mutations or just more of the same mutants. How to do this?  We would start by mutagenizing cells with EMS, but instead of using wildtype cells we'd use cells carrying a neutral chloramphenicol-resistance insertion (CmR) that's within a few kb of murE.  The mutagenized cells would be incubated in log phase with a NovR DNA fragment, and the rare transformants wuld be selected by plating on novobiocin and pooled.  Since the transformation frequency of wildtype cells in log phase is less than 10^-9, many of these should be hypercompetent mutants.  DNA from the pooled transformants would be used to transform wildtype cells, selecting for CmR to enrich for mutations in and near murE, and these 'backcrossed' transformants would again be selected for hypercompetence by transformation in log phase, this time using MAP7 DNA and selecting for streptomycin resistance.  DNA from the pooled transformants would be sequenced, as would individual hypercompetent isolates.

We need to think about the numbers.  Say 1-5% of the original NovR transformants are hypercompetent strains with murE mutations (in the original experiment we found 4/150).  If we pool their DNA and backcross, selecting first for CmR, 1-5% of the colonies will have murE hypercompetence mutations.  Even if these mutants were only 100-fold more competent in log phase than normal cells, selecting for these by transformation to StrR would give mostly colonies with the desired mutations.

(The murE mutants we have are about 10^6-fold more competent, so we might miss mutations giving weaker hypercompetence phenotypes...)

Should we also do an unfocused search for any hypercompetence mutations?  We could do this in parallel, replacing the selection for CmR on Day 3 with selection for NovR.  We might still get mainly mutations in murE, because these give such a strong phenotype.  If we wanted to target mutations in sxy, we could first do a second round of selection for transformation using StrR MAP7 DNA and then select for StrR in the backcross.  Since StrR is not very close to sxy we'd want to be gentle with our Day 3 DNA prep so the fragments were long.

A new way to make money from researchers?

By Rosie Redfield on Friday, September 13, 2013 Basically, you give World Biomedical Frontiers $38 and they list your paper's Abstract on their website along with whatever supplementary explanation of the work you provide.  I gather that "cutting-edge biomedical research" means "research by people who gave us $38". A bit of surfung suggests that you might then add a note like this to your publication list:

"This paper has been selected to be featured in World Biomedical Frontiers (http://biomedfrontiers.org/cancer-2013-may-2-5/) because of its innovation and potential for significant impact. World Biomedical Frontiers [ISSN: 2328-0166] focuses on cutting-edge biomedical research from around the globe."

(We don't work on influenza.)

Dear Dr. Redfield:

Your recent paper about influenza-“Defining the DNA uptake specificity of naturally competent Haemophilus influenzae cells” (published in “Nucleic acids research.2012 Sep;40(17):8536-8549.”) has been selected to be featured in our next issue of World Biomedical Frontiers, because of its innovation and potential for significant impact.

Research results with significant potential to improve health – or to treat or prevent disease – often deserve an immediate leap onto the “front page”. However, scientific breakthroughs don't always make the front page – and some don't make any page! We are the platform for you to stand out from among ~100,000 papers published each month, in order to attract more attention from the public and potential investors.

World Biomedical Frontiers [ISSN: 2328-0166] focuses on cutting-edge biomedical research from around the globe. Our website receives more than 11,000 visits per month from an international audience of academic and industrial researchers and developers, providing greater opportunity for your results to be recognized and appreciated.

If you accept our invitation to feature your paper on our website, a $38 processing fee will be charged. We will then post the abstract/summary of your paper in the latest section of Infection and Immunity, with additional information from you highly recommended to further explain your novel findings and concepts in plain language; photos and/or figures are welcomed. Here are two examples (1 and 2).

In order to report breaking publications in a timely fashion, we ask that you contact us within 2 weeks if you wish your paper to be featured in our next issue.

Michael S. Yang, M.D & Ph.D.
World Biomedical Frontiers, LLC
New York, USA
Phone: 1-(917) 426-1571
E-mails: email@example.com
Website: http://biomedfrontiers.org/

A new role for next-gen sequencing in our research

By Rosie Redfield on Wednesday, September 11, 2013 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.

Conference spam...

By Rosie Redfield on Sunday, September 08, 2013 The 3rd Annual World Congress of MolMed-2013
November 13-16, 2013        Haikou, China

Dear Rosemary J. Redfield,

This is Tina. How are you?

I'm writing to follow-up my last invitation letter as below, would you please give a tentative reply? Thank you very much.

We are pleased to announce that the 3rd World Congress of Molecular Medicine (MolMed-2013) will be held during November 13th to 16th, 2013 in Haikou, China. On behalf of the organizing committee, we sincerely hope you could attend our congress and give a speech at the session, with your special contribution on your research area.

We believe MolMed-2013, which has been endorsed by Haikou Convention Bureau, will be the perfect opportunity and platform to highlight the high profile permanent biotech convention!

Haikou is world-famous for its unique and charming tropical views, relaxed and happy natural environment. As a capital of Hainan Province, Haikou gathers all what are famous in Hainan. Especially in the old Haikou, the building’s style mixed with Portuguese, French and Southeast Asian style. These streets used to be divided into areas for medicine, silk and clothing, agricultural products, stationery, etc. Hainan boasts for its rich and souvenirs as a paradise for tourists.

Would you please indicate if your schedule is available during that time?
More details about our congress, please kindly log on: http://www.bitlifesciences.com/Molmed2013/

Ms. Tina Lee
Organizing Committee of Molmed-2013

Do any H. influenzae competence genes protect against replication fork stalling?

By Rosie Redfield on Tuesday, September 03, 2013 The top graph shows two mutant strains with near-normal resistance to hydroxyurea: radC- and rec1-.Growth and division wasn't affected by 10 mM hydroxyurea but was stalled by 50 mM. This  effect was also seen when the cultures were allowed to grow overnight from their initial very low density (~ 4 x 10^3 cells/ml).  Cultures with 10 mM hydroxyurea grew dense (turbid), and those with 50 mM were still transparent.

In E. coli the competence-induced RadC protein has been implicated in replication fork stabilization; Rec1 is the H. influenzae homolog of the recombinational repair protein RecA. These strains' lower density in 50 mM hydroxyurea at 220 minutes may or may not be significant. And here's the really sensitive strains. The recBC mutant was the positive control; in E. coli recBC mutants are known to be very sensitive to hydroxyurea because they cannot cope with stalled replication forks, and here we see that the most of the H. influenzae mutant cells were killed by 50 mM hydroxyurea.  The 10 mM culture also failed to grow up overnight.

The big result:  Cells with a deletion of the competence gene dprA were even more sensitive to hydroxyurea than the recBC mutant.  This is important for two reasons.  First, dprA homologs are ubiquitous in bacteria but no function outside of competence has been identified. In particular, in E. coli a dprA mutation had no effect when tested in various combinations with known 'recombination' mutants.  Second, dprA is the poster child for genes thought to be induced in competence specifically to promote recombination.  My new result suggests that other effects should also be considered.
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