State of the Grad

It’s been absolutely beautiful in Vancouver. I’ve been living the nomadic life in kits and having a blast the last few days.   I’ve got my coffee, just took a Ritalin,  and want to clear out some of the ideas I’ve been entertaining for a little while.

1) Antibiotics inducing transformation by gene transfer agent

I’ve been social and hanging out with the Beatty lab. It started because I saw a grad student, R, giving a talk during the 3 minute thesis competition about the effect of low dose antibiotics on gene transfer agent(GTA) last week. Low doses of certain antibiotics greatly increase the rate of of gene transfer by the phage like GTA of Rhodobacter capsulatusRosies blog has some great background and insight on GTA, and we talked about it last week. Heres the lowdown:

Gene transfer agent is likely the remnants of a degenerate bacteriophage, although grad student C(more on him later) suggested the alternative possibility that it could be an ancestor of phages I haven’t looked at phylogenies to see which is more likely. What makes GTA different from a bacteriophage is that GTA doesn’t transport itself, it moves (assumably)random pieces of host genome. In fact it can’t transport itself as GTA requires several gene components to work that are too large to be moved by GTA. So the big question is how can GTA be selected? It’s conserved so it appears to be under selection, but the evolutionist in me has theoretical problems with it. The problem is that GTA confers seemingly no benefits to the bacteria that have the GTA phenotype. GTA may be benefiting the population as whole because it allows for bacterial sex under certain conditions/stresses which is always good, but the cells that have the GTA phenotype are dead – cell lysing may be required for GTA release, no one knows yet but it’s likely based on how related phages exit bacteria. So the donor GTA genes aren’t being selected for because we assume the host is dead, and the recipient shouldn’t need GTA to be a recipient. Selection can’t see GTA.  A simple simile to explain this problem is that GTA being selected for has the same theoretical difficulties that a gene that only has a phenotype in post-menopausal grandmothers would. Both require group selection theory, they rely completely on group selection theory to exist.

Some questions about gene transfer agent:

a) Is GTA bacteriophage derived?

b) Do all cells that release GTA lyse?

c) Does GTA has any other function that could benefit the donor?

d) What proportion of the donor genome is packaged into GTA?(If it’s really big and efficient, maybe GTA could be maintained in a population just by sheer brute force of transformation)

e) Are GTA rhodobacter that have GTA more likely to be transformed by GTA than non-GTA bacteria?

f) Under what conditions do GTA transformations occur most frequently?

Me and R talked about it over some beers. Her basic assay is growing two bacteria strains that have two different mutations in a photsynthetic pathway together, and selecting for photosynthetically competent colonies that should be the result of gene transfer one way or the other.  She sees the effect with certain gyrase inhibitors, aminocoumarins and quinolones. That’s critical,  C informs me that low levels of gyrase inhibitors have aknown effect of increasing phage production in microflora, and one of R’s strains has a phage that the the other strain is sensitive to, making interpretation of R’s results difficult. On the other hand there may be something to her results as gyrase inhibitors cause DNA damage. There’s a paper(Charpentier et al 2011) that found that these same antibiotics as well as UV induce competence in legionella pneumophila, presumably as a response to DNA damage in the absence of SOS-response.  I suggested she use GTA to make her non-phage containing strain rif and kanamycin resistant and use those as markers to get rid of any phage effect, as well as test for congression.

2) Quorum sensing molecules inducing gene transfer agent

C’s is also in the Beatty lab, working on quorum sensing, r recommended I look into his research. He found that quorum sensing molecules induce gene transfer agent, maybe we would find the same result for competence in HI. Quorum sensing is one of the first things Rosie explained to me her views on when I joined the lab. Her thinking is that people are too quick to jump to the conclusion that bacteria are using them to communicate but rather the simplest explanation and the one that best takes into account how bacteria outside the lab live is that the bacteria use quorum sensing molecules to get a sense of their environment. Bacteria feed by releasing enzymes to break down nutrients and then eat the digested products, but these enzymes are expensive to make and if the cell is in the open ocean or a river they will just diffuse away and be a waste of resources. Instead, the cell could first release the comparatively inexpensive quorum sensing molecules, get a tally of what percentage stick around and then get an idea if the they are in a little nook or in a turbid environment.

So if bacteria use quorum sensing molecules to detect if they are in a good place to eat, and high quorum sensing environments could mean there are other bacteria around and the area is potentially nutrient/DNA rich, it might be a good idea to become competent to be able take up the DNA that could be in the area. This test would not be able to differentiate between the food or sex function of competence, unless maybe a cell can differentiate between quorum sensing molecules sent out by itself and those dispersed by others.

It could be easy to test, as C pointed out that gram negative bacteria use pretty universal sensing molecules so it shouldn’t be too difficult to get experiments going.

3) Could antibiotics increase competence in human microflora?

Given R’s result I’ve been thinking about the effect of antibiotics on transformation, antibiotics increase the amount of bacteria resistant to it by selection, but could they also be increase the basal rate of horizontal gene transfer?

Here’s how I could test this:

Find subjects who have a bacterial infection they are going to be treat with antibiotic (B). At the time of treatment introduce antibiotic(A) sensitive DNA to their microflora that is resistant to both (A) and (B). Does the rate of resistance to (A) decrease when compared to controls that aren’t treated with antibiotics?
Note: I originally wanted to give resistant DNA to sensitive microflora and check for transformants that gained resistance, which would be technically easier and involve less counting counting, but maybe this way someone would be willing to be a study participant.  Almost certainly to test this I’d have to use an animal model which would also be easier if introducing DNA involves directly injecting into the gut or nasopharynx where our competent HI lives,  but maybe I could find a little gel capsule of sometype that could protect DNA from degradation until it reached the target microflora. Maybe something for the Finlay lab. I’ll talk to my new roomie who works for Prof Finlay about it and see if we could get some collaboration going if the idea is sound.
4) A test for the Noisiness of a pathway

I’m not sure about this one but maybe it could work. Some pathways are governed by noise. Noise can be intrinsic or extrinsic. Intrinsic noise is the stochastic effect of biochemicals on transcription or translation, and since the responsible chemicals are found in small amounts only infrequently do they meet and interact with these transcription/translation.
  Pick a pathway affected by noise and have a way to test for it’s composition of a culture. Take two aliquots, vortex one briefly. Test the two aliquots at different time points for phenotype of interest.
Assuming that vortexing mixes the cells intracellular contents and makes them different than the control this test could give a quick and dirty answer to two things:  A) What type of difference does vortexing has on the phenotype and B) when do these differences arise?
A) Does vortexing makes the phenotype go up or down, and by how much, could be indicative of how much the noise is ruled intrinsic/extrinsic factors and how rare the responsible molecules are, and whether the responsible molecules are for the on or off phenotype. Lot of compounding factors.
B) This is the more solid aspect. If the phenotype % changes, how long did it take? If it’s a long time, that means the noise is far away from the phenotype/the phenotype is long lasting, and if its’ instantaneous that means it’s shortlived and directly tied to noise regulation.

5) Are competent and persistant states related?

Give antibiotic(not low dose, lethal) and MAp7 DNA to HI at the same time in late log phase. The antibiotic should kill all cells that aren’t in a persister state. Is the rate of transformation changed from a non antibiotic control? If transformation frequency goes up it means persisters are responsible for more than their share of competent cells, if it goes down it means that persisters aren’t very competent. The post doc helped me with this one

The post doc also shut down phenomenon I was excited about, the observation that colonies on antibiotic plates appear at variable delayed times compared to plain plates. I was thinking this could be due to different growth of competent cells. Here’s the likely culprit and some  homework

That’s it for now! I borked my leg this morning longboarding to Calhouns so I’m gimpy and have to hobble around. I’m picking up game of thrones, going to play some settlers and then heading to the suburbs to see the family.

Random science fact of the day: The universe couldn’t have been able to expand the way it did after the big bang given the speed of light, we know nothing can ever travel faster than the speed of light. So maybe the speed of light was different then. See the variable speed of light hypothesis and John Barrows  for more info

7 Comments (+add yours?)

  1. Rosie Redfield
    Apr 24, 2011 @ 06:58:28

    “…which is always good…” ????


  2. Rosie Redfield
    Apr 24, 2011 @ 07:01:09

    “…presumably as a means as to repair DNA.” ???


    • dnogas
      Apr 24, 2011 @ 15:58:26

      My mistake and sloppy writing, edited. It’s the authors conclusion of the paper I cited that the induction of competence under those conditions in SOS-deficient bacteria is to respond to DNA damage, using competence as a substitute for error-prone SOS to increase genetic diversity. The authors do discuss that “DNA import for repair” is controversial and they found a negative result for a prediction of the theory, transformation deficient mutants should be more sensitive to UV.

      Interestingly they think that replication fork stalling is the mechanism that induces competence, and found that hydroxyurea, a drug that depletes dNTP pools induces competence


  3. Rosie Redfield
    Apr 24, 2011 @ 07:05:17

    The scale of a cell (especially a bacterial cell) is much too small to be affected by the macroscopic mixing caused by vortexing.


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