Field of Science

Thinking about the fraction-competent issues

The post-doc is polishing up has latest manuscript, and on reading it over yesterday I realized that we have data to clarify a very old issue.  Here's a link to an old post on this topic:

In the old days, the best way to estimate the distribution of competence among the cells of a 'competent' culture was to measure the proportion of cells that became transformed by selectable markers that were on two separate DNA fragments (usually markers carried by a single donor strain, but far enough apart on the chromosome that they were never taken up on the same fragment.

Such assays typically find that cells that have been transformed by one marker are enriched for cells transformed by the other marker - the fraction of double transformants is higher than expected from the fractions of either single transformants - and this is used to estimate the fraction of the cells in the culture that are not competent.

Now, from the post-doc's work, we have data telling us what fractions of the cells have acquired one selected marker have also acquired one, two, three or more unselected fragments of donor DNA, and we want to use this data to unpack the relationship above.

1. all parts of the donor chromosome are equally likely to be taken up and recombined into the recipient chromosome,
2. all the cells in the competent culture are equally likely to take up DNA
3. taking up and recombining one DNA fragment does not alter the cell's probability of taking up and recombining another one,
4. we ignore that only a single strand is replaced at a recombination site, and that the donor and recipient strands at this site separate the first time the cell divides
5. we ignore the possibility of mismatch repair at these heteroduplex sites.

the frequency of cells transformed by two unlinked markers should be the product of the frequencies of cells transformed by each marker (under assumption 1 above, equal to the square of the single-marker transformation frequency).

For now I'm mainly interested in what happens if assumptions 2 and 3 are not valid.  (The post-doc is more interested in 4 and 5, and the plan (hope) is that we'll pool our thinking and explain the whole business.)

Most traditional analysis has focused on relaxing assumption 2.  If some cells in the culture are equally competent and the rest not competent at all (and all the other assumptions hold), then the fraction of the cells that are competent is the ratio of the product of the two observed single transformation frequencies to the observed double-transformation frequency.  If the ratio is 1.0, then we'd conclude that all the cells are competent. 

What if we instead relax assumption 3?  What if taking up one DNA fragment uses up cellular resources and so reduces the cells' ability to take up more fragments?  At the extreme, no cell would take up more than one fragment.  The frequency of cotransformation would then be zero, and our ratio would be infinity.  If the resource-depletion was not absolute, then the ratio would be smaller, but it would still be larger than 1.0.  So if we observed  ratio greater than 1.0, we'd conclude that some cells can only take up one DNA fragment.

But deviations from assumptions 2 and 3 push the ratio in opposite directions.  The typical observation of a ratio less than 1.0 means that assumption 2 must be invalid but not that assumption 3 is valid. In principle, how far below 1.0 the ratio is sets a limit on how much of an effect deviations from assumption 3 can be having, but we haven't worked out the math to calculate this.  For example, if the observed ratio were 0.5, could this be because a quarter of the cells were competent but only half of those were able to take up more than one fragment?

Conversely, seeing a ratio greater than 1.0 would mean that assumption 3 was invalid but not that assumption 2 was valid, and we could in principle calculate the range of deviations consistent with a particular observed ratio. 

We could also view relaxing assumptions 2 and 3 a different way, thinking about variation in the levels of expression of competence genes leading to differences in how much machinery/resources cells have available.  What if some cells are more competent than others?  Some cells might not have enough resources to take up even a single fragment, some might have only enough resources to take up a single fragment, and some might have enough resources to take up two or more fragments.  What would our ratio look like then?

So.  For each of two selected markers, the post-doc's data gives us the actual distributions of cells that also took up no, one, two or three additional fragments: 0.35 had none, 0.40 had one, 0.21 had two, and 0.04 had three.  What can we do with these numbers?

The single-segment transformation frequency for the two selected markers was each about 0.03.  I forget the double-transformant frequency, but I remember that it predicted that only about 0.1 of the cells were competent (relaxing assumption 2), so I think it must have been about 0.01.

How far could assumption 3 be relaxed and still give the observed numbers?  Could 0.2 of the cells have been competent if only 0.65 of these were able to take up more than one fragment?

Consistent with this, only two of 20 unselected clones had recombined donor segments. 

If I have lots of KanR colonies from yesterday's hypercompetence-enrichment step...

  1. Pool the colonies (from each of 6 original mutagenized cultures).
  2. Freeze part.
  3. Make chromosomal DNA preps from the rest.  These will be used for sequencing and for the backcrosses (steps 5 & 6)
  4. As controls also make DNA from the pooled NovR colonies from yesterday.
  5. Use the 2 DNAs from the StrR parent and the 2 DNAs from the CmR parent to transform KW20 to StrR and to CmR.
  6. Maybe use the DNAs from the wildtype parent to transform KW20 to KanR.
  7. (Incidentals: make lots more BHI agar, pour plates, use frozen competent KW20, test transformation by NalR fragment, wash and autoclave flasks again.)
Tomorrow (one final round of hypercompetence selection):
  1. Pool the backcross colonies derived from each of 6 original mutagenized cultures.
  2. Freeze part.
  3. Grow part in log phase for at least 2 hr (like yesterday)
  4. Transform with a different marker (NalR fragment, or MAP7 DNA with selection for NalR or SpcR).
  1. Pool the transformant colonies (from each of 6 original mutagenized cultures).
  2. Make chromosomal DNA preps from them for sequencing.

Yet more on-the-fly experiment planning

Hm, yesterday's attempt to do two rounds of enrichment for hypercompetent mutants yielded very few colonies (about 10 total).  That's probably because I didn't let the NovR cells grow to a high enough density before transforming them with KanR DNA.  There were so few cells present with the KanR DNA was added, 5 x 10^5/ml - 6 x 10^6/ml based on the numbers of colonies that grew on the plain plates after the second round transformation).

But I can do the second round enrichment again, by pooling all the NovR colonies that grew on these plain plates.  I have between 1000 and 60,000 colonies to pool, depending on the culture, and the cultures were diluted so much that I don't have to worry that any of these colonies might be KanR. And the colonies are all NovR transformants from the previous round, though many of them are identical descendants of the original mutants.

Plan:  Put some BHI on the plates and scrape them to resuspend all the colonies.  Dilute the pooled cells in sBHI to OD600 = 0.1, then dilute 50-fold in more sBHI.  Grow (37°C, shaking) for 2-3 hr, until OD600 reaches 0.05.  Add KanR chromosomal DNA and let continue growing for another hr.  Dilute and plate on plain, Nov and Kan plates.

Progress:  OK, I've counted the colonies (previously had just estimated them), pooled them (excluding one contaminated plate), and now they're growing in 10 ml sBHI at a starting OD600 of 0.002.  Now I need to pour lots of plates.

AAACCCKKK!!! - I screwed up the selection

I did Day 1 of the big experiment yesterday.  But because the NovR DNA fragment the postdoc gave m hadn't transformed very well in the test, and I didn't have enough of it for the large volumes I was transforming, I used MAP7 DNA for the transformations that select for hypercompetent mutants.  At the time I thought the only disadvantages were a slightly lower overall transformation frequency and the 1-2% risk (for each new mutant) that transformation would also replace the mutation itself.

I forgot that, because the MAP7 DNA carries resistance to all the antibiotics we commonly select for, the pool of NovR cells I'd get would contain 1-2% cells resistant to teach of the other antibiotics.  This means that I can't just reselect the cells in the NovR pool for hypercompetence using DNA with another mutation, because this will just select those cells in the pool that got that mutation in the first round of selection for hypercompetence.

I see several options:

Option 1:  Yesterday I froze lots of mutagenized cells that hadn't yet been incubated with any DNA.  I can thaw these out, grow them for an hour at low density, and transform them to NovR with either the NovR DNA fragment or chromosomal NovR DNA that doesn't carry any other antibiotic resistances.  (I may have an old stock of this DNA.)   Then I can grow the pooled cells in medium with novobiocin for 6 hr, dilute them, and do the second-round transformation (NalR or KanR DNA)) and plate.  If I'm using the NovR fragment I may need to do this transformation on cells that have been concentrated - this will increase background, but the next round of hypercompetence-selection should take care of this.

Option 2 (BAD): I can plate the NovR transformants and manually check single colonies for resistance to the antibiotic I want to use next, by toothpicking them onto other antibiotic plates.  Then I could either test them individually for hypercompetence or pool the ones that are sensitive to whatever antibiotic I plan to use next.  Testing them individually would make the planned pool-sequencing unnecessary, and pooling them would be a lot of work.  But I might as well freeze my pools of NovR cells for later manual screening, just in case we  want to do this.

Option 3 (BAD):  I can make DNA from the pooled NovR colonies from the strain carrying the StrR mutation linked to sxy and the strain carrying the CmR mutation lnked to murE, and use this DNA to transform wildtype cells to StrR and CmR. Then I can do another round of hypercompetence selection, transforming the pooled cells with a marker that's not linked to the one I selected for.  BUT, these new pools will probably (maybe) include cells with the unselected antibiotic resistances from the MAP7 DNA.

Expanded plans for Option 1: 
  1. Thaw 1 vial of each of the 7 cultures (wildtype treated with 0, 0.05 and 0.08 mM EMS, StrR and CmR treated with 0.05 and 0.08 mM EMS).  
  2. Dilute way down in sBHI, to a density equivalaent to that used yesterday.  Grow 1 hr or more at low density.  
  3. Look for NovR chromosomal DNA.  If using the PCR fragment, concentrate the cells by filtration just before adding the DNA.
  4. Incubate cells with DNA for 15 or 30 min, then DNase-I-treat, filter, wash, and resuspend in sBHI + Novobiocin.  
  5. Grow 6 hr or more, keeping OD600 below 0.1.  
  6. Transform a fraction of this culture (no benefit from using it all).  Add KanR chromosomal DNA or NalR PCR fragment.  Incubate 15 min, DNase-treat and plate (if KanR) or grow for 90 min before plating (if NalR).

Timing for the big mutagenesis experiment

In the previous planning post I ended with the following breakdown:
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)
- See more at:

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)

 I'd like to get the whole thing one as quickly as possible, because next week may be our last chance until next April to get the DNAs in for sequencing.

I have the EMS, and I've streaked out the strains, but I didn't inoculate single colonies last night.  Could I still do the big Day 1 mutagenesis, transformation and selection today, or will there not be enough time to have the cells grow up first from single colonies?

What's the actual time commitment for day 1?
  1. Inoculate cells from single colonies into sBHI, grow to OD600 = 0.1.  (time = several hr)
  2. Add equal volume of sBHI containing 2X the desired concentrations of EMS.  Incubate 30 min at 37°C.  Cool cells down quickly. (time = 30 min)
  3. Filter-wash cultures (7 cultures) and resuspend in larger volumes of sBHI. (time = 30 min?)
  4. Grow washed cells for 90 min or until OD is back to 0.1. (time = 90 min)
  5. Dilute part of each culture and grow for 1 hr longer. (time = 1 hr)
  6. While cells are growing, filter-concentrate and freeze the rest of the cultures.
  7. Add NovR DNA fragment to cultures. Incubate 30 min, then DNase I for 5 min. (time = 40 min)
  8. Filter cultures, resuspend in sBHI + novobiocin. (time = 30 min)
  9. Grow (at least 6 hr or) overnight.
  10. The next morning, plate some cells on Nov5 plates, freeze some, and grow some back into log phase for KanR transformation with MAP7 DNA.
OK, I think I can do steps 1-8 today.

How much EMS to use:

In #181 I calculated that adding 79.5 µl of EMS to 15 ml culture gave 0.05 mM, and 127.3 µl gave 0.08 mM.  So for my 10 ml of cells I would use 53 µl and 85 µl of EMS.

What about the volumes of culture to use?
  1. Mutagenize 10 ml at OD600 = 0.1
  2. Resuspend in 80 ml.
  3. Freeze 70 ml after 90 min (concentrate cells first) and dilute 10 ml to 40 ml in fresh sBHI
  4. Grow 1 hr, add DNA etc, filter.
  5. Freeze half the cells.
  6. Resuspend the rest in 100 ml + novobiocin2.5.  Grow 6 hr - overnight
Should I mutagenize a larger volume?  10 ml will be only about 3 x 10^9 cells.  In #181 (the original EMS experiment) I used 15 ml at OD600 of 0.33, which is about 5 times as many cells.

How much EMS do I have?  OK, 5 ml (after brief "Where on earth did I put it???").  It doesn't keep well once opened ("Store under inert gas") so maybe I should double the volumes for the first two steps and freeze more of the mutagenized cells for possible later analysis.  So use 106 µl and 170 µl EMS for 20ml cultures, and dilute to 160 ml.

What about the EMS-contaminated waste culture and tips?

EMS is inactivated in 1.0 M NaOH.  My filter flask will contain about 400 ml of EMS waste (including washes), so I'll add 16 g of solid NaOH to that, and let it sit for an hour before neutralization and disposal.  I'll put the contaminated tips in there too.

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)
- See more at:
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)
- See more at:

Timing of competence development in a new murE749 transformant

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="">murE749
.  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).

  1. Pour lots of plain, Cm and Cm+Nov plates
  2. Thaw out frozen competent Rd cells
  3. Incubate with DNAs, add DNase I for 5 min
  4. Filter to wash away the DNA and (more importantly) the DNase I
  5. Resuspend in sBHI (t = 0)
  6. Grow 1 hr, add chloramphenicol (1 µg/ml I think)
  7. Take aliquots to tubes with MAP7 DNA at t = 1 hr, 1.5 hr, 2 hr, 2.5 hr, 3 hr (longer?).  
  8. Dilute and plate on plain, Cm and Cm+Nov plates.
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

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:
  1. Wildtype H. influenzae Rd
  2. H. influenzae Rd carrying the normal sxy gene and a streptomycin-resistance mutation.  StrR is only about 50 kb from sxy, so we can use it to enrich for hypercompetence mutations in sxy.
  3. H. influenzae Rd carrying the normal murE gene and a chloramphenicol-resistance mutation about 4 kb from murE, which we can use it enrich for hypercompetence mutations in murE.
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?

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:
  1. Start with a mixture of competent cells and log-phase cells, such that I expect a transformation frequency of about 10^-7.  This frequency is higher than we would see in the planned mutant-hunt, because I want to have a predictable and easily measured number of transformants to start with.  
  2. Incubate 5 ml cells at a density of 10^8 (OD600 = 0.03) with 1 µg MAP7 DNA for 15 min,  Plate an aliquot with and without novobiocin.  Dlilute the rest of the culture 100-fold with fresh medium and let grow for 1 hr, and then add novobiocin at 2 µg/ml to prevent further growth of NovS cells.
  3. Plate aliquots again and at hourly intervals to check on the growth and survival of the NovS and NovR cells.
  4. Just in case the dying NovS cells are toxic in the liquid culture, though they'll be much more dilute than on plates, after a couple of hours of novobiocin selection I'll wash the culture by filtration and resuspend the cells in fresh novobiocin medium and let them continue growing.  This will only take a few minutes and will also remove the residual NovR DNA.
  5. After five hours of selection the NovR cells will have doubled about 10 times.  If the NovS cells survived growth in novobiocin but didn't divide I should see a 1000-fold increase in the frequency of NovR colonies on my plates, from about 10^-7 to about 10^-3.  If most or all of the NovS cells died the increase will be even greater.
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...)

  1. Day 1.:Streak out the CmR strain.
  2. Day 2: Grow the CmR strain and mutagenize for 30 min with EMS.  Wash away the EMS, freeze some of the cells for later work. Dilute the rest right away and grow in log phase for 2-3 hr or more.  Add NovR DNA for 30 min and plate on lots of plates.  
  3. Day 3: At this stage each hypercompetent NovR colony is likely to be an independent mutant.  Pool all of them and isolate DNA.
  4. Additional Day 2?: We want many hundreds of transformants, and can scale up the mutagenesis and transformation cultures,  but the selection is limited by the need to not put more than ~5x10^7 NovS cells on each plate.  Maybe we should also try enriching for NovR before plating, by adding novobiocin to the broth and growing for a few hours or overnight.  This would let us screen a lot more cells and thus find more mutants, although we'd sacrifice independence.  Again we'd then isolate DNA from the NovR culture or pooled NovR colonies.
  5. Still Day 3: Transform competent wildtype cells with this DNA, selecting for CmR.   The expected transformation frequency is about 10^-4 to 10^-3, so we can easily select many thousands of independent transformants to pool.  1-5% of these should have the desired hypercompetence mutations.
  6. Day 4: Grow the pooled transformants and transform them in log phase to StrR.  
  7. Day 5: Almost all the transformants should be the desired hypercompetence mutants. Pick some of these for competence testing and pool the rest.
  8. Prep the DNA of a number of individual isolates and of a large pool of colonies.  Sequence these.  Look for specific mutations in the individual cultures and for enrichment of mutations in the pool.
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?

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 ( 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

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.

Conference spam...

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:

Sincerely yours,

Ms. Tina Lee
Organizing Committee of Molmed-2013

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


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.

Does hydroxurea treatment induce competence in H. influenzae?


Here's the data.  Wildtype cells at low density (3.4 x 10^7 cfu ml; no spontaneous competence) were incubated for 100 minutes in medium containing NovR DNA and different concentrations of hydroxyurea.  One set of cultures also contained cAMP to partially induce the competence regulon.  The upper graph shows that 5 and 10 mM hydroxyurea did not inhibit cell division (about 3 doublings), and that 20 and 50 mM allowed only 1-2 doublings.  As expected the effect was independent of cAMP.  The lower graph of transformation frequencies shows that competence was not significantly induced or enhanced by hydroxyurea.

One more hydroxyurea experiment (well, two actually)

I've now shown fairly convincingly that being competent does not enhance H. influenzae's ability to cope with hydroxyurea, which stalls replication forks by blocking the synthesis of the dNTP precursors needed for DNA synthesis.

Experiment 1:  To more completely test my hypothesis that cytoplasmic competence-regulated proteins are induced to mitigate the damage caused by stalled DNA replication, I'm going to test whether cells lacking the proteins DprA or RadC are more sensitive to hydroxyurea than are wildtype cells.
We have all the mutants in the freezer:
  • dprA deletion:
  • radC deletion:
  • rec1 mutation:  This is a very old strain, one of the original H. influenzae transformation mutants.  It should be completely defective in recombinational repair, but I'm not sure how this interacts with hydroxyurea.  Hydroxyurea induces recA in E. coli.
  • recB/recC mutation:  This is another very old strain, not a modern knockout, but its phenotype was well studied.  
I just streaked them all out so I can test them on Saturday.  I'll use the same method I did for the first hydroxyurea experiments, growing them to log phase and then diluting them way down to about 2000 cfu/ml in medium with and without 50 mM hydroxyurea and following them for several hours and overnight by plating 50 µl aliquots.

Later:  Here's a paper that tested the effect of hydroxyurea on a lot of E. coli mutants.  Their assay was the ability to form colonies on agar plates containing 10 mM hydroxyurea.  (To me this seems more likely to select for mutants resistant to the effects of hydroxyurea, but that's what they did.)  They found that a recA mutant formed 1000-fold fewer colonies, and a recB mutant 10,000-fold fewer colonies.

Role for radA/sms in Recombination Intermediate Processing in Escherichia coli Beam Saveson, and Lovett  J. Bacteriol. vol. 184 no. 24 6836-6844

Experiment 2:  While looking for any work on the effects of recA mutations on sensitivity to hydroxyurea, I discovered (i) a paper showing that hydroxyurea induces competence in Legionella pneumophila, and (ii) mention of perhaps-unpublished data showing the same thing for Streptococcus pneumoniae.  The L. pneumophila authors state in their Discussion that "We currently favor the hypothesis that stalling of the DNA replication fork is the primary signal leading to competence development."

So I should definitely also test whether hydroxyurea induces competence in H. influenzae.  This will be easy: grow cells to log phase, dilute into medium containing marked DNA (MAP7, 200 ng/ml) and different concentrations of hydroxyurea (0, 5, 10, 20, 50 mM).  Grow for 1-2 hr and plate ± novobiocin.  

Details added later for experiment 2: I should probably also test cells whose competence has been partially induced with cAMP.  Because these cells have a baseline transformation frequency of 10^-5 - 10^-4, I can use them in a dilute culture (say 10^7/ml).  The cultures without cAMP will need to be at higher density to detect small effects on competence.  Since I've noticed that the concentrations of hydroxyurea that inhibit cell division in dilute cultures are not as inhibitory for denser cultures, I'll include a 100 mM concentration too. (Maybe high concentrations of organic matter partially neutralize or overwhelm the hydroxyurea.)
Charpentier et al.  Antibiotics and UV Radiation Induce Competence for Natural Transformation in Legionella pneumophila  J. Bacteriol. vol. 193 no. 5 1114-1121.

Not the birthday present I would have preferred

I seem to have now thoroughly disproved one of my favourite hypotheses, that cytoplasmic genes in the competence regulon act to help cells survive depletion of pools of deoxyribonucleotides (dNTPs).

Last week's experiment tested whether cultures with higher levels of competence were less affected by hydroxyurea, which inhibits synthesis of dNTPs.  It found no correlation, but the conclusions were weakened by presence of many non-competent cells in the cultures.  So in this new experiment I also measured the numbers of surviving cells that had become transformed to novobiocin resistance by marked DNA I added to the cultures.  Because these cells must have been competent to become transformed, their survival should specifically show how hydroxyurea affects competent cells.

The results show that the frequency of transformed cells was not increased by hydroxyurea treatment, in fact it was lowered in 2 of the 4 cultures and unchanged in the other 2.

I tested 4 cultures, each with and without 50 mM hydroxyurea:

  • 'Kc' is wildtype cells with competence partially induced by 0.2 mM cyclic AMP (1/5 the dose I used previously).  The expected transformation frequency (TF) in log phase is 10^-5 - 10^-4.
  • '5c' is the hypercompetent strain RR563, fully induced by addition of 0.2 mM cAMP.  Expected TF is 10^-3 - 10^-2.
  • '5' is the hypercompetent strain RR563.  Expected TF is 10^-5 - 10^-4 in exponential growth, higher in a dense culture.
  • '7' is the very hypercompetent strain RR749.  Expected TF is ~ 10^-3.

The first graph shows total cells (cfu) over 4 hr of incubation with (open symbols) and without (solid symbols) hydroxyurea.  The cells are at different densities, all more dense than in the previous experiment, because I also needed to plate for transformants.  With no hydroxyurea the cells grew exponentially as expected (RR749 doubling time 25 min, 5c slower because of the cAMP, and Kc and 5 slowing down as they became dense.  Growth of the two relatively dense low-competence cultures (Kc and 5) was only transiently slowed by hydroxyurea, whereas growth of the two low-density maximally competent cultures stopped and cell numbers fell.

The second graph (below) shows the transformation results, which should reflect the growth and survival of the competent cells in each culture. (The dashed line and red arrow indicate an 'upper-limit' data point where no transformants were seen.)  For wildtype cells + cAMP (blue lines) hydroxyurea had identical effects on competent and non-competent cells.  For the fully hypercompetent strain RR749 (purple) the competent cells were slightly more affected.  For the partially hypercompetent strain RR563 (green), transformants were reduced about 5-fold by hydroxyurea, and for RR563 + cAMP (red) transformation was decreased 10-fold at 90 min and undetectable at 230 min (dashed line and red arrow).

I left the cultures shaking overnight and plated them again the next day (graphs below).  I'm only showing this for completeness; it doesn't really add anything to the conclusions.  All the no-hydroxyurea cultures were at about 10^9 cfu/ml, and the hydroxyurea cultures were between 10^4 and 10^7 cfu/ml.  The transformation frequencies of the hydroxyurea cultures were the same as (wildtype + cAMP) or about 10-fold lower than their untreated counterparts.

I can think of some caveats, but they're quite weak. For example, it's possible that hydroxyurea prevents competent cells from becoming transformed, or causes them to become unable to take up DNA.  There may also have been a confounding effect of cell density - the two relatively dense cultures were much less affected by the hydroxyurea.

But overall, the obvious conclusion is that being competent does not help cells survive or grow when dNTP pools are depleted by hydroxyurea treatment.  So I wonder what the cytoplasmic genes in the competence regulon contribute.  It's certainly possible that they've been selected  for their recombination-enhancing effects, as everyone else assumes, but this depends on the assumption that recombination is the funciton of DNA uptake, which I still think very unlikely.

A clearer perspective on CC-BY reuse

Over the weekend I posted and discussed the results of my survey on the editing and re-publication of open access articles in what pretend to be multi-author edited books containing new material.  The articles are published under the original authors' names, but the titles and text have been lightly edited, and the original publications are either not cited or cited in an obscure appendix.  When I composed the survey I thought that this reuse was permitted by the CC-BY license, but now (after a lot of Twitter discussion) I think that this particular form of reuse contravenes the license in at least two ways.  Because the reuse is illegal, the best remedy is legal action by the journals that originally published the papers.

Here's the relevant legalese from the CC-BY license:
  1. If you distribute, publicly display, publicly perform, or publicly digitally perform the Work or any Derivative Works or Collective Works, You must keep intact all copyright notices for the Work and give the Original Author credit reasonable to the medium or means You are utilizing by conveying the name (or pseudonym if applicable) of the Original Author if supplied; the title of the Work if supplied; to the extent reasonably practicable, the Uniform Resource Identifier, if any, that Licensor specifies to be associated with the Work, unless such URI does not refer to the copyright notice or licensing information for the Work; and in the case of a Derivative Work, a credit identifying the use of the Work in the Derivative Work (e.g., "French translation of the Work by Original Author," or "Screenplay based on original Work by Original Author"). Such credit may be implemented in any reasonable manner; provided, however, that in the case of a Derivative Work or Collective Work, at a minimum such credit will appear where any other comparable authorship credit appears and in a manner at least as prominent as such other comparable authorship credit.
Contravention #1:  In the specific case of articles from scientific journals, it's not clear (to me) whether this requires citation of the original publication or just listing the names of the authors.  However, PLOS's description of their CC-BY license explicitly says that any reuse must cite the original article. (I can't find anywhere on the BioMed Central site that explicitly says this; they just quote the standard CC-BY license.)

So, at least for papers from PLOS journals and probably for papers from other OA journals, the book publisher is contravening the license by not conspicuously including a citation to the original publication.

Contravention #2:  The CC-BY license prohibits 'implied endorsement'.  Here's what the Creative Commons wiki says:
"All CC licenses prohibit using the attribution requirement to suggest that the original author or licensor endorses or supports a particular use of a work. This "No Endorsement" provision protects reputation, and its violation constitutes a violation of the license and results in automatic termination."
This means that the book publisher cannot simply list the original authors of the article as authors of the book chapter.  Instead they must say something like "This is an edited version of the paper by the original authors" or otherwise make it clear that these authors are not responsible for this new publication.  Similarly, the book must not list the authors as "Contributors", since this also implies that the authors endorse the new work.

The appropriate response is legal action by the journal agains the book publisher:  My survey of authors found that authors are most concerned about how this reuse could affect their reputations; they want to be sure that their work will be correctly cited and that they are not held responsible for the reuse.  Preventing the two contraventions described above would go a long way to eliminate the authors' concerns.

In principle the individual authors could sue the book publisher, or maybe organize a class action suit.  But in this situation I think legal action should be the responsibility of the journal publishers. The authors have paid substantial fees to the publishers, and legal action to protect their rights should be considered part of the cost of running an open access journal.

I still think that open access journals should give potential authors more information about the risks of the CC-BY license as well as its benefits.  But taking responsibility for defending authors' rights would let journals provide this information in a much more positive way.  For example, they could say:
"The CC-BY license protects the rights of authors to have their publication correctly cited when it is reused, and to not be seen as responsible for any alterations.  This journal will take legal action to defend these rights if they are infringed."

Experimental design good, results discouraging

Well, I tested whether competence helps cells survive treatment with hydroxyurea, and the answer appears to be 'No'.

First a reminder of why I did this experiment: For years I've been hypothesizing that the function of at least some cytoplasmic genes in the H. influenzae competence regulon is to stabilize replication forks that have stalled because of a shortage of nucleotides.  Because the simple chemical hydroxyurea specifically inhibits the enzyme ribonucleotide reductase, which is needed to convert NTPs to dNTPs for DNA synthesis, the most important experimental question is whether competence protects cells from the harmful effects of hydroxyurea, with or without DNA uptake. - See more in this post.

What I did:  Cells with different levels of competence, in exponential growth in rich medium, were transferred to the same medium with and without 50 mM hydroxyurea, and growth and survival were followed by plating and by measurement of OD600.

What I observed:  Over a 3 hr period where DNA replication was arrested by hydroxyurea, cultures that were constitutively or partially competent did not exhibit increased growth or survival.

 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

The cells:
  1. K: Wildtype strain KW20.  Not competent in exponential growth but inducible by starvation.
  2. KC: Wildtype strain KW20 with 1 mM cAMP added 45 min earlier to induce moderate competence
  3. 5: Mutant strain RR563.  Has a hypercompetence mutation in sxy so is moderaately competent in exponential growth.  Similar transformation frequency to KC.
  4. 6: Mutant strain RR648.  Has a knockout of sxy so cannot become competent at all.
  5. 7: Mutant strain RR749.  Has a hypercompetence mutation in murE.  Competence is fully induced in exponential growth.
The results:  

Cell growth:  These cells were diluted at t = 0 into medium ± hydroxyurea, and their growth was followed by measuring the turbidity of the culture. Cells with arrested replication are expected to continue growing but to cease division (the cells form filaments), and that's what these cells did - growth was slowed only slightly by 50 mM hydroxyurea.

Cell division:  The same initial cultures were diluted 1:50,000 into medium ± hydroxyurea and their numbers were followed by plating and counting colonies.  Now we see that hydroxyurea did arrest cell division; the cells with hydroxyurea doubled only once or twice in the time that the control cells doubled more than seven times. 

Cell survival:  This is the same data as the above graph, with the addition of cfu counts after the very dilute cultures continued incubating overnight.  Ignore the '300 min' label on the X-axis; this was really after another 16 hr of incubation.  Cells in some of the hydroxyurea cultures divided a few more times, one culture kept the same cfu, and the cfu of the cells with cAMP decreased about 10-fold, probably due to the cAMP's general perturbation of gene expression.  There's no correlation with level of competence - the most competent cells increased only a bit more than the cells unable to become competent.  (The cells in the control cultures grew overnight to the expected 10^9 cfu/ml.)

Complications and plans:  One weakness of this experiment is that many (perhaps most) of the cells in a competent culture are not transformable, so many may not be expressing the cytoplasmic proteins that I hypothesize are protective.  This could reduce the sensitivity of the experiment by a lot.  

One way to clarify this would be to also assess survival of the transformable cells, by adding novR transforming DNA to the cultures and plating cells on novobiocin plates as well as plain plates.  This will make the experiment more complicated, largely because I'd have to work with less dilute cultures and do some dilutions for all the cells on plain plates.On the other hand, having the results of the experiment I've just done will let me streamline the plating, partly making up for the extra work and the uncertainty of survival and transformation frequency on the nov plates.  This strategy won't work for cultures that aren't competent at all (there will be no novR transformants), so I'd leave out the KW20 and RR648 cultures.  But it should work nicely for KW20+cAMP, and for RR563 and RR749. 

Could I also leave out most or all of the no-hydroxyurea controls?  Do I expect the number of transformants to parallel the total numbers of cellsIn the absence of hydroxyurea?  Perhaps not, since new competent cells will continue to become transformed over the time of the experiment.  So I'd better retain these controls.

Unexpected discovery: Cells grow faster when they're very very dilute.  The control cells in the top graph (blue lines) appear to be growing exponentially as expected; the log-scale lines are straight until the second-last time point.  The doubling time is about 35 minutes, which is typical for our cultures.  But when the cells were at a very low density in the same medium (second graph), they grew with a doubling time of about 24 minutes, faster than I've ever seen!  So I should do a separate experiment, following change in cfu of wildtype cells from from very dilute to more dense.  

Survey results by author's number of publications

A Twitter discussion got us wondering whether author's opinions about CC-BY editing and republication would depend on the seniority of the author.  Perhaps authors who had spent decades building their reputations would feel they had more at risk.  Or maybe authors just starting out would feel that their reputations were more vulnerable.

My $25 one-month Survey Monkey upgrade lets me filter the data for graphing, but doesn't make it easy to export the numbers to Excel.  So here are the charts for each publication category.

I haven't bothered labelling the answer choices because the different publication categories gave very similar results.  Respondents who have yet to publish their first paper feel the same as those with more than 20 publications.

I also tried filtering by whether or not the respondent had published any open-access papers.  This didn't affect the results either.

Later:  I figured out how to get the data into Excel, so here's a graph that allows more direct comparison:

And here are the questions again:

Survey results: what should be done about CC-BY reuse?

(As indicated in places below, I've later added points to this post as a result of ongoing Twitter discussions.)
 My first posts on this new problem reported that a for-profit publisher is editing and republishing open access articles as if they were new contributions to special-topic books (here), and described concerns raised by authors I had spoken with (here).  These concerns were largely dismissed by some advocates of open access, who commented that (i) authors should have realized that this is permitted by the obligatory CC-BY license, and (ii) authors should not complain since this is additional exposure for their work and ideas.

I felt that it's unreasonable to expect authors to have anticipated this particular form of reuse, especially since there's no evidence that open access advocates anticipated it.  And I thought most of the concerns authors raised in discussion with me were very reasonable (here).  So I circulated a short survey to get solid data on how authors feel about this new practice.

The survey responses (here) make it clear that authors are seriously concerned about the ways this reuse could harm their reputations.  This is to be expected - I think most scientists see their scientific reputation as even more important than their funding.  The many comments also make it clear that most authors had no idea this republication was happening, even though most of them had published open access articles.

More than 40% of authors in the survey said that they would not have accepted the CC-BY license if they had known this republication could happen.  If nothing is done, these concerns will seriously hinder the spread of open access publishing.

What should be done?  Open access advocates and publishers (the honourable ones, not the predatory ones) could just keep quiet and hope that the problem doesn't become generally known.  That probably won't work out well.  The present problem may be limited to one publisher (Apple Academic Press) but the explosive increase in predatory publishers of open-access journals suggests that it will grow; see the more than 300 publishers (not just journals) on Jeffrey Beall's list.  And awareness of the problem will spread each time authors discuss where to send their next paper.

Open access publishers could also work behind the scenes to ensure that CC-BY articles republished under the authors names are conspicuously labeled as having been previously published and, if appropriate, as having been edited without the authors' participation.  If this effort was successful I think it would eliminate most of the authors' concerns about their reputations.  Enforcing it would probably require expensive and ongoing legal actions, but (added later) I think any journal that requires CC-BY should accept the responsibility of legally protecting their authors' interests in this license.

(Added later)  Although CC-BY doesn't explicitly specify that the journal citation must be included along with the authors' names (not being designed for journal articles), K. Fortino (@kennypeanuts) pointed me to PLOS's very clear statement that full citation of the article is the required form of attribution.  All CC licenses prohibit 'implied endorsement'; that is, the reuse must not imply that the original source approves the reuse.  The offending books I've looked at typically describe all of the article authors as 'contributors' in a list at the beginning of the book; this is clearly a form of implied endorsement.

Because open access articles are a major user of CC licenses, OA advocates and publishers could also work together to develop a specific CC license that better meets the needs of authors and publishers.  It might allow everything that CC-BY does, but also require (i) prominent listing of the journal citation with the authors' names and (i) if the article had been edited from the original publication, whether the authors have approved this editing.  Maybe call it 'CC-OA'.

Finally, open access publishers could actively inform authors about these issues and their efforts to control them.  There are many ways to do this, but the strongest point of contact is when the author agrees to the CC-BY license.  Open access publishers already use this access point to provide authors with information about the benefits of this license.  Now that this problem and the reasonable author concerns have been identified, I think it would be disingenuous of publishers to not also give authors this information.

Open access publishers and advocates have enthusiastically promoted the benefits of CC-BY publication to authors (the BMC text is typical).  In a previous post I drew an analogy with informed consent in clinical trials, suggesting that OA publishers would be negligent if, in promoting the common good of CC-BY licensing, they did not inform authors of the personal risks as well as the personal benefits.

Remember, I'm an advocate of open access, not an enemy. In the short term, increasing awareness of this problem may scare off authors who might otherwise remain ignorant of it.  But if we do nothing about it, in the long term we risk losing many authors who would otherwise invest their limited grant funds to make their articles open.

Survey results

Here are the results of my survey on concerns about republication of CC-BY articles.  I've put a screenshot of the survey questions below the graphs of the results.  I discuss the implications in a separate post.

The survey link was emailed to the Evoldir and Ecolog-L mailing lists and posted to the SSE and CSEE facebook sites (reaching ~20,000 evolutionary biologists and ecologists), and emailed to four UBC departmental faculty lists.  It was also posted on this blog.  The text of the email is provided at the end of this post.

Survey participants:

Survey opinions:
I would be happy to have the quality of my paper recognized. I would be happy that my scientific contribution is being widely disseminated. I would welcome this as another entry in my publication list. I would want to have received a share of the profits. I would want the collection to be freely available. I would want the collection to be a high-quality contribution to the field. I would want the paper's original publication to be conspicuously credited. I would want the paper to be unaltered. I would worry about editing errors. I would worry that the editing has changed my interpretations. I would worry that the book's goals may conflict with mine. I would worry that my citation record will be confused. I would worry that colleagues will think I've self-plagiarized by publishing the same article twice. I would want to learn more about copyright options. I would not have accepted the CC-BY license if I'd known this could happen. In future I would not publish in journals that require the CC-BY license. In future I would not publish in open-access journals. - See more at:
I would be happy to have the quality of my paper recognized. I would be happy that my scientific contribution is being widely disseminated. I would welcome this as another entry in my publication list. I would want to have received a share of the profits. I would want the collection to be freely available. I would want the collection to be a high-quality contribution to the field. I would want the paper's original publication to be conspicuously credited. I would want the paper to be unaltered. I would worry about editing errors. I would worry that the editing has changed my interpretations. I would worry that the book's goals may conflict with mine. I would worry that my citation record will be confused. I would worry that colleagues will think I've self-plagiarized by publishing the same article twice. I would want to learn more about copyright options. I would not have accepted the CC-BY license if I'd known this could happen. In future I would not publish in journals that require the CC-BY license. In future I would not publish in open-access journals. - See more at:

The survey choices:
(A thread on Twitter claimed that this survey was very biased.  However, all of the negative choices were ones authors had raised with me; the positive ones I added to give balance.  Perhaps the tweeters mistakenly thought this was a general survey about open access instead of a very narrowly focused survey about one particular form of CC-BY reuse.)

Comments/concerns from survey participants:
  • In future it will be obligatory in the UK to publish in an open access format (Green or Gold)
  • Frustrating but an acceptable by product of the shifting landscape of publishing
  • I would want to be contacted before publication and have the right to decline
  • I would want to be consulted first and appropriately recognized for the original contribution and if being sold for profit the authors should receive a share of profits
  • Editing of published manuscripts should be illegal.
  • In addition to accurate citation of my original publication, I would want the citation to clearly indicate if the paper was copied exactly or adapted.
  • always assumed OA was so people could read freely only
  • I got my book!! republished this way! I guess that a intermediate (no commercial use) CC license should be required.
  • I would be infuriated that I was not notified that they stole my work. I have published in PLOS and this would make me seriously reconsider doing so ever again - especially since I paid $1350 to PLoS so the work would be freely available to everyone and anyone having to pay $100 for this book is being ripped off
  • is there a different license, still making freely available / disseminable, but restricting this kind of republishing?
  • I would want a disclaimer on the book that clearly states that the individual articles are free on the web.
  • The whole idea offends me
  • I believe authors are bound by the agreements they make.
  • I know these are not mutually compatible responses but its a complicated problem!
  • Some of the above seem to conflict with the explicit wording of the CC-BY. Seems like if you agree to CC-BY, you can't really argue some of these.
  • I would like to be asked and it should be after a number of years
  • I should be able to expect nothing less than ethical conduct by publishers, regardless of what is legal
  • I would like the licensing to be more transparent
  • Who is making money off of this? It is creepy that they don't have to ask.
  • This is truly frightening. Surely profiting from this is in violation, no?
  • I don't think it would put me off publishing entirely using CC-BY, but it has certainly made me think twice!
  • The CC-BY license should proscribe for-profit use by individuals other than the author(s).
  • I would want them to let me know before they do any changes and ask me if these changes are correct!
  • I think this would be fine, if proper attribution were clearly given for original work
  • It is not clear from the situation description whether the original authors are listed as authors of the paper in the volume. If so, I would also have checked 1-3
  • While I know that the CC BY requires only attribution, I think this scenario presents some outcomes that are potentially unintended by many parties who publish in OA journals. I worry that this scenario is essentially duplication of the original work in a way that is anathema in academia. I'm very happy to allow someone to profit by using the CC BY licenses as long as such reuse was actually transformative. As it is, it appears that the CC BY license essentially allows plagiarism to be carried out for profit. In my opinion, such works should not be treated as citable literature in academics, and perhaps the open access licenses should be modified to reflect this goal.
  • I would want them to make clear the origins of the work and original context
  • I would be absolutely furious if my work was used without my explicit permission. How dare someone attempt to profit off my without doing anything.
  • The point is we pay extra to make an article open to the public. No one should be profiting from the information later. Also the book will be somewhat out of date by publication time and may not be that cohesive since it wouldn't include subscription articles. In other words I would worry about the overall quality of the book.
  • I do not want people to make money from my work which is published OA. The idea behind OA is to disseminate science freely for everybody!
  • I would be angry that others are making a profit of my work while I paid extra to have it FREELY available
  • This practice is clearly unethical and violates normative publication standards of the scientific community
  • In the future I would publish with the CC-BY-NC-ND license, which would legally prevent abuse by publishers:
  • I would like to have at least been contacted, even though probably not required under the cc license terms
  • I would prefer to be contacted about this before they edit my paper, as a courtesy.
  • I'd prefer the CC non-commercial share alike license.
  • I don't care because my colleagues are smarter than that.
  • Either the paper should be unaltered, or it should be marked clearly as a derivative work with a different set of authors, citing the original from which it was derived conspicuously.
  • Regarding the answers to the last two I will try not to publish in these type of journal unless absolutely necessary
  • I would not be happy to have the work reprinted for profit. Science should be not for profit: the main objection is that others are making profit from something that they ethically should not be selleing even though it is currently legal.
  • I'd be furious!
  • I would be surprised and annoyed I was never told that my work would be re-published somewhere else (I should have the option to decline).
  • My main concern is that it is scientific plagiarism, whether it is legal or not. It would be inappropriate to publish the same data twice without clearly referencing it, therefore it is inappropriate that anyone can do this to our work.
  • If I agreed to the copyright contract, then this is acceptable. An author has an obligation to understand these things.
  • It certainly makes me think twice about publishing in OA journals, but I will probably continue to do so.
  • You are doing massive harm to the OA cause by trying to scare the children
  • Above all, they need to actually notify authors when this happens
  • authors should definitely be notified of this possibility and preferable be given an option to opt in or out
  • Once someone alters text in the paper- the editors should get approval for that. I have no problem with someone bundling papers (similar to Amazon reading lists) and selling that "value added service". However, they shouldn't change the underlying articles. Perhaps provide an introduction to the volume and then introductions for each paper and why it is significant / ties into the theme of the volume. It should also be made perfectly clear in these volumes what the original citation should be. Also, authors of the original papers could be offered a page or two for commentary - how their paper fits into the theme of the volume and relates to other research.
  • I would have liked to be asked for permission
  • Except for reediting, which is unacceptable, this practice is entirely legal, and not necessarily directly against the spirit of open access. Two points are important: (1) insisting that a book chapter is clearly labeled as available for free, and (2) educating researches about these kinds of phenomena so that they know they don't have to pay for access.
  • if you have research council funding in the UK, then you must publish open access.
  • I would want notification my paper was being reprinted
  • There are several aspects that seem highly unethical, but the least ambiguous is the publication under my name of words that are not my own.
  • I would want to be warned that my paper should be used
  • I don't find the major difference from people just printing my papers. Then money goes to the manufacturer of the printer, ink and paper.
  • I believe altering the text and putting my name on it would be against the license, so I would demand a copy of the chapter to check if it is altered in any way. If not I would accept it because I accept the license agreement I have signed (and checked before signing).
  • I don't think distribution per se is a problem at all, as long as the source is cited and correctly reproduced. Actually, I would like being asked for an "update" of a previous paper
  • I would want authors to contact primary authors as a professional courtesy; however, I still support open access publishing
  • I would opt for CC-BY-SA as I do for open source code
  • I would worry that the work may no longer be up to date, and may be incorrect in light of more recent findings.
  • I'd prefer a CC-BY-NC license to stop this
  • In the future I'd license under the Creative Commons Attribution-NoDerivs 3.0 United States License (CC BY-ND 3.0 US)
  • I wish I knew before this was done
  • I would like to be asked if I agree with the re-publication and the alterations by the publisher of the multi-author book!
  • I feel that this violates the spirit of open-access publication and may discourage other authors from publishing open-access
  • Maybe shifting to CC-BY-ND in future would help to avoid these issues...
  • If the original paper isn't being cited, it's breaking the terms of CC-BY and should be disallowed
  • For the first two... it would really depend on the book. What if it's a creationist book that's re-using my work, with or without editing, to trash evolution? I wouldn't be happy!
  • My feelings are mixed. Communication with the authors should be required and authors should have a say in the editorial process
  • I would want to have given permission or been made aware of the book
  • I would be angry that this practice might confuse and weaken the peer-review and merit-based foundation of research science publication.

Here's the full text of the email:
Dear Colleagues,

I've recently discovered that some commercial publishers are re-editing articles from open-access journals and publishing them as multi-author books, without the authors' knowledge (example here).  Although most authors I've spoken with find this objectionable it's quite legal, since open-access articles are usually published under Creative Commons attribution-only (CC-BY) licenses. 

Before pressing for any changes I'd like to get a broad set of researchers' opinions on this, so I've prepared a short (3 question) survey.  Here's the link:

Please feel free to pass this survey link on to other researchers or scientific email lists (I've already sent it to Evoldir and Ecolog-L).



p.s.  If you'd like more information I've also discussed this issue on my blog:
Dr. Rosemary J. Redfield  

  Professor, Dept. of Zoology    Univ. of British Columbia                             
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