Field of Science

R. capsulatus growth curves in RCV medium

My upstairs GTA colleague and I were surprised that the Bioscreen growth curves in the previous post didn't show a dip in OD600 of the GTA-overproducer strain like that seen in manual (non-automated) growth curves.  This dip is thought to be caused by the lysis of GTA-producing cells as GTA production peaks when cells hit stationary phase.

We thought part of the problem might be that I used the standard YPS medium which is based on modest concentrations of yeast extract and peptone.  The clearest/most-recent published demonstration that GTA-producing cultures used RCV, a simpler 'defined' medium based on malate, and showed that the apparent lysis occurred in medium with 0.5 mM PO4 but not in medium with 10 mM PO4.

So I redid the growth curves for all 6 strains, using both high-P and low-P versions of RCV (kindly supplied by my upstairs colleague).  The results are not inconsistent with the Westbye results, but they're not at all compelling.  None of the strains decreases in OD600

The problem is that there's quite a bit of between-strain variation in growth and in the stability of the stationary phase OD.  (Within each strain the replicate wells give very similar results, with one exception.)

The graph below shows growth in the high-phosphate medium.  The main graph shows OD600 on a log scale (appropriate to exponential growth), and all the strains appear to stably reach similar densities.  But the inset shows the same data on a linear scale, which makes the variation look more significant.  The overproducer strain stops growing abruptly at OD600 = 0.7 a lower density than the other strains.

Here's the cells in the low-phosphate medium.  There's an initial drop in OD600, over the first 10 hours, but then all the strains grow steadily except strain YW1, where the individual wells grew at different rates for no apparent reason.  Again the linear-scale inset shows the substantial variation at stationary phase.  The overproducer DE442 again stops growing, this time at OD600 = 0.8, and now its OD falls by about 20% over the next 40 hours.

I really don't feel comfortable drawing any solid conclusions from this one experiment, especially since there's a blip in many of the growth curves at a point where I stopped and restarted the runs to add more time when I realized that 3 days wasn't going to be long enough.  Even though the shaking only stopped for 2-3 minutes, and the trays of cells remained in their holder with the lid closed, most of the strains had an abrupt change in OD600.  (You can see the blips at hour 63.)

Plan:  Do the run again.  This time I'll pre-grow the cells into log phase in high-P and low-P RCV.  medium (the upstairs colleague has offered me enough medium to do this).  And I'll plan on pausing the run at key times to take samples that I can assay for GTA production.

What can we learn from growth curves?

Here's the results of the Bioscreen growth curves I ran for Rhodobacter capsulatus strains:

Each dot is the mean OD600 of 15 replicate wells, each containing 300 ┬Ál of culture, with ODs read every 20 minutes for 45 hours.  The cultures all grew up at about the same times, but I've shifted the X-axes so the curves don't overlap.  OD values below about 0.015 are not significantly above the backround absorption of the culture medium. The Y-axis is a log scale, so when doubling time is constant the dots will fall in a straight line.

I did these runs 'just-in-case', because I'm going to be working with Rhodobacter capsulatus at Memorial University in Newfoundland for the next few months (on sabbatical leave) and thought they probably wouldn't have a convenient Bioscreen that I could use.

Now I need to figure out what we learn from these, and whether I should do any more experiments before I leave UBC.

The simplest expectation is that once the cells have adjusted to the medium (after 'lag phase') they will grow at a constant rate until they run out of nutrients or experience other bad consequences of high cell density (little oxygen, accumulation of toxic byproducts).  But all of these cultures instead exhibit 'diauxy', a mid-growth shift from one resource to another.  We see  this as a brief slowing or even cessation of growth at about OD=0.05 (orange shaded band), after which growth resumes, often at a different rate.  The pause occurs because the cells need time to adjust their metabolism to a change they've caused in the medium, such as exhaustion of one nutrient or new availability of another. 

I don't know enough about R. capsulatus metabolism to speculate about what the change might be, but it might affect production of Gene Transfer Agent particles.  The pause isn't due to lysis of GTA-producing cells, because it's not changed in the ∆∆ strains, which have deletions of the GTA gene cluster and lysis gene.

SB1003, B10 and YW1 are all 'wildtype' strains, I think.  Strain YW1 grows much slower than the others, although it still speeds up after the growth pause, and it reaches a slightly lower final density.

Strain DE442 carries a mutation that causes over-expression of the GTA genes and over-production of GTA particles.  Growth curves in a 2013 paper found that this strain had a substantial drop in OD once growth ceased, thought to be due to lytic release of GTA particles, but no drop is seen in the Bioscreen culture.  That work used a low-phosphate version of a different medium, RCV.  But an earlier paper found strong lysis with the same complex medium I used (YPS), and low lysis with the high-phosphate (10 mM) standard RCV medium.

The lab upstairs has both low-phosphate and high-phosphate versions of the RCV medium, so I'm going to repeat the time course with both.

growth time courses

In a few weeks I'll be headed for the Maritimes, for the final part of my sabbatical work on Gene Transfer Agent.  But before I leave here I want to run some detailed growth time courses on GTA-producing strains, taking advantage of the BioScreen machine belonging to the lab next door.

I'll first do a trial run with all the strains I have,  to check the basic growth kinetics under the Bioscreen growth conditions.  Then I'll see if I can combine the growth measurements with testing for the amounts of GTA produced.