Field of Science

GTA competition experiments

I'm in St. John's for the 'summer'*, doing GTA-related experiments in Andrew Lang's lab at Memorial University of Newfoundland ('MUN').

The first experiments I'm going to do are growth competitions between GTA-producing strains and otherwise-identical non-producer strains created by deleting the GTA genes.  Because GTA production requires cell lysis, we predict that the non-producers should outcompete the producers.

While I was still in Vancouver I did detailed growth curves of the various strains.  Preliminary ones are described here, and I'll paste the graph from the latest ones below:

I wanted to check the effect of phosphate concentration in GTA production and culture growth, so I only used two strains, SB1003 (wildtype) and DE442 (a GTA overproducer).  I used two PO4 concentrations; 0.1 mM, which should allow high GTA production and reduced growth, and 10 mM, which should cause low GTA production and better growth.  The growth differences should be detectable only for DE442.  (I also used three different cell densities.  I'm only showing the results for cultures started at the highest density, but the others grew similarly with the expected delays.)

I measured GTA production at two times, by removing cultures from some wells, filtering out the cells, and using the cell-free supernatants to transduce an RifS strain to RifR.

The results are below.  (The upper graph is plotted on a linear scale, and the lower graph is the same data plotted on a log scale, for easier comparison of growth rates.)  The growth curves are very similar to those from a previous experiment (RR#1438) where I didn't measure GTA production.

The GTA production happened as expected.  SB1003 produced no significant GTA in high PO4, and a modest amount (780 transductants per ml) in low PO4.  DE442 produced lots more GTA under all conditions, but about 4-fold more in low PO4 than in high PO4.

On the linear scale the two strains appear to have very similar exponential growth rates, but the log scale reveals that DE442 (the GTA overproducer) is slower in exponential growth.  DE442 also reaches a lower final densities (SB1003, OD ~ 1 - 1.08; DE442 OD ~ 0.7).

The growth differences are unlikely to be directly due to the lysis required by GTA production, because the GTA differences caused by the different PO4 levels do not correlate with OD differences.

DE442 is not isogenic with SB1003; it carries a mutation that blocks synthesis of the red accessory pigment.  Could DE4432’s pigment phenotype be responsible for its poorer growth?  These were aerobic cultures in a dark room, so the growth difference is not a direct consequence of differences in photosynthesis.

I don't think it would be straightforward to transfer the ‘overproducer’ mutation into the SB1003 background, since typical transduction frequencies are less than 1/1000, and we have no way to select for overproducer colonies against the background of normal colonies.  If the pigment difference causes the growth difference, we could transfer the wildtype pigment allele into DE442 or the mutant allele into SB1003.  I wonder how the parent strain of DE442 (Y262, I think) grows.

* It's definitely not summer yet here.  Icy winds anywhere near the coast, and several thin snowfalls in the last few days. I remain hopeful, because most of the trees are finally getting their leaves, and the spring bulbs are blooming.

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