Field of Science

Why GTA genes can't be maintained by 'selfish' transmission

Below is the line of reasoning showing that the genes responsible for producing GTA particles cannot maintain themselves or spread into new populations by GTA-mediated transfer of themselves into new cells.  I initially worked this out with a rigorous set of mathematical equations, but then realized that the problem was so glaringly obvious that math isn't needed.

The main GTA gene cluster is too big to fit inside a single GTA particle, so GTA particles can't transmit DNA that converts a GTA- cell into a GTA+ cell.  Some genes outside the main cluster are also required for GTA production.

But GTA particles can (and do) contain one or more individual GTA genes.  If a fragment containing a particular GTA gene is injected into a formerly-GTA+ cell that is now GTA- because it has a mutated version of this gene, the resulting recombination can restore the cell's original GTA+ genotype.

But these transfer events would not allow GTA+ cells to invade a GTA- population, or to maintain themselves in the face of loss of GTA function by mutation.  That's true for all known GTA systems, even in the simplest (imaginary) case where production of GTA particles requires only a single gene that could easily fit into a GTA particle, as illustrated below.  

Why?  Three factors together require that production of GTA particles reduces the total number of GTA+ cells in the population:

Problem 1:  GTA particles can only be released to the environment if the GTA+ producer cell lyses.  So each production event removes one GTA+ cell from the population.

Problem 2:  The GTA genes in the producer cell are not over-replicated as a phage genome would be, so each production event can produce at most one G+ particle (containing the GTA gene or cluster).  

If all steps occurred with 100% efficiency, problems 1 and 2 would allow, at best, replacement of the lost GTA+ cell with a new one created by GTA-mediated recombination.  But this would not maintain the numbers of GTA+ cells in the face of occasional loss of GTA genes by mutation or deletion.  Nor would it allow GTA+ cells to invade a GTA- population.

Problem 3:   Production of GTA particle production, transmission of their DNA to recipient cells, and recombination with the recipient genome are all likely to be at least moderately inefficient.  Here's a partial list of expected inefficiencies:
  1. Burst size:  Actual burst sizes are unknown, but packaging all the DNA in a R.capsulatus. genome would need 841 particles, which is much larger than typical burst sizes for DNA phages.  Capsid proteins may be limiting, since they would be produced from single-copy GTA genes rather than replicated phage genomes.
  2. Dispersion:  The GTA particles will disperse in the environment, and many will probably not find cells to attach to.
  3. Stability:  Lab preps of GTA particles are unstable in non-optimal storage conditions, so many particles will likely fall apart.
  4. Recombination efficiency:  Only one DNA strand enters the cytoplasm, and some DNA degradation is likely.  The highest observed transduction frequency is only ~4^-4, (theor. max: 1.2^-3) so recombination efficiency is probably only ~0.3.  Recombining in a novel gene will be less efficient than simple strand replacement
  5. Self-conversion:  Some G+ particles may attach to cells that are already GTA+.

Might GTA be a vaccination system for infecting phages?

My work at Dartmouth (to be described in upcoming posts) showed conclusively that genes encoding Gene Transfer Agents (such as the GTA system of Rhodobacter capsulatus) cannot be maintained by 'selfish' transfer of either whole GTA gene clusters or single GTA genes into GA- recipients.  Neither can the GTA genes be maintained by general recombination benefits that can arise when fragments of chromosomal DNA are transferred into new cells.  So, although 'gene transfer agent' does accurately describe one activity of these genes, it cannot be the activity for which they are selected.

The main obstacle to the maintenance of GTA genes, which applies to all the benefits is that any GTA+ cell that actively produces GTA particles cells must die, since cell lysis is needed to release their particles into the environment.  Another obstacle, applying to selfish transfer, is that GTA genes are not over-replicated during GTA production (and are not preferentially packaged), so each cell death can produce only one GTA+ particle. 

I presented these results at the Analytical Genetics conference last week, and asked the other participants if they could think of alternative benefits of producing GTA particles.  Sanna Koskiniemi from Uppsala University made the very interesting suggestion that GTA particles could serve as a syringe, packaging DNA fragments from a phage that's infecting the producer cell and transferring these fragments into other as-yet-uninfected cells, where they could trigger development of CRISPR immunity.

I love this idea and want to test it.  It doesn't overcome the cell-death obstacle, but it does overcome the selfish-transfer obstacle since a single producer cell could produce many particles of phage DNA from a single phage genome, and more if the phage genome is replicated.

One way to see if this could provide sufficient benefits to maintain the GTA genes is by simulation modeling like that I used to examine the recombination benefits.  This could clairfy the important factors that would need to be examined.

Here I want to start considering experimental tests of this hypothesis.

The ideal test would be to infect the GTA-producing strain with a phage, preferably under low-growth conditions where phage infections are often abortive.  (Luckily R. capsulatus produces most of its GTA under such conditions.)  Then some recipient cultures would be exposed to the GTA-containing culture medium (and some not, as controls), and then all exposed to a lysate of the phage.

"But wait!", you say.  "Won't the GTA-containing culture medium also contain some phage?"  Yes, probably.  I don't think there's any way to inactivate the phage particles without also inactivating the GTA particles, or vice versa.  We might be able to come up with either perfectly-abortive infection conditions (where infected cells don't produce any phage), or a cellular mutation that prevents phage production.  If not, we might have to combine the GTA-exposure and phage-infection steps.

"And won't any phage lysate also contain some GTA particles?"  Yes, probably.  But we could use a GTA- mutant as the host for lysate production.  Not the mutant that can't lyse, but the one with the main GTA gene cluster completely deleted.

What resources are available for this project?  First I checked with my GTA colleagues, who confirm that R. capsulatus does have a CRISPR-Cas9 system.  Then I asked if there were any well-characterized phage systems able to infect R. capsulatus.  Until quite recently the answer would have been 'No', but a recent paper reported the isolation and sequences of 4 R. capsulatus phages.  A Mu-like phage of R. capsulatus has also been characterized, but it did not form plaques on SB1003.

The report about the 4 new phages used a different host strain (YW1-derived, not SB1003), so the first thing I'll need to do is check whether they form plaques on SB1003.  Then I'll need to play around with infection and plating conditions...  My idea of fun!