Marc Solioz's 1975 PhD thesis on GTA

PhD students, don't assume that your thesis will moulder unread in the library.  More than 40 years after he submitted it, I'm reading Marc Solioz's PhD thesis (The Gene Transfer Agent of Rhodopseudomonas capsulata).  I want to understand the kinetics of GTA production, and his is the only good data I can find.

Here's what he reported:

A. Stability of and transduction by GTA in various solutions:  He tested a wide range of solutions.  In these studies he didn't try to distinguish between conditions that stabilize GTA for storage and conditions that maximize its ability to attach to cells and inject its DNA.  It's happiest in 1mM each of Na+, Mfg+ and Ca++.  This can be buffered with 10 mM Tris, with or without gelatin or BSA (no effect).  It's destabilized by 10% gycerol, even for freezing.  GTA preps made by filtering culture supernatants should be diluted at least 10-fold to reduce the destabilizing effect of the medium constituents.

B. Inactivation by other factors:  GTA's stability is not affected by temperatures up to 50°C.  Keeping it on ice is not better than room temperature, and there was no difference between partially purified and purified stocks.  It's inactivated by proteases but not RNase or DNase.  It's not inactivated by ether or chloroform, or by phospholipases, consistent with the absence of any membrane.

C. Inactivation by UV:  UV damages DNA so it is expected to inactivate the transducing activity of GTA particles.  To control for experimental variation (a big concern with UV experiments), he compared GTA inactivation to inactivation of phage T2 UV'd together in the same solution.  The action spectra are the same for GTA and T2, but GTA inactivation requires much higher doses, consistent with the small amount of DNA in each particle.

D. Conditions and kinetics of GTA production:   1. Production kinetics: This is the same surprising result (Solioz's term) I showed in the previous post. Cells were grown photosynthetically/anaerobically in a yeast extract + peptone medium.  The dashed line approximates the combined growth curves seen in the four independent experiments, but it's in 'arbitrary units' (I think on a log scale) so I have to infer the cell densities from how my cells grow.



He reports that the initial peak and drop were consistently seen across all his experiments, but that sometimes the drop was not followed by the final high-GTA stage.  He saw a similar pattern using a strain that does not absorb GTA (strain H9), so the changes in GTA titre are not due to changes in the removal of GTA particles from the medium.   However this conclusion is weakened by the description of strain H9 in the methods, which just says 'does not act as a recipient of GTA, with no reference'.)  Other tests he did could not rule out effects of transient inhibitory/inactivating factors in the culture supernatant.

2. Effects of growth conditions on production.  Defined medium RCV gave low titres of GTA.  Yields with different concentrations of yeast extract and/or peptone were variuable, apparently depending even on the batch no. of ingredient used.  Variation sin culture growth rate and final density did not correlate with GTA titres.

3. Isolation of mutants:  He attempted to isolate an overproducer mutant but failed.  The original producer strain B10 carried two phages, so he made a derivative strain, SB1003, that was cured of the phages and carried the convenient RifR point mutation.   This new strain is the one I have been using as the standard donor; it's good to know its provenance.

4. Radiolabelling:  He put in a lot of work to find a way to radioactively label GTA.  This was used to guide the purification studies.

E. Purification of GTA particles:  This is a long section that's not of much interest to me.  He tested a wide range fo the available biochemical techniques used for purification of organelles, phage and molecules.

F. Characterization of the nucleic acid:  He used the single-strand-specific nuclease S1 to show that the DNA in GTA particles is double-stranded.  He used CsCl ultracentrifugation to estimate its base composition as 65% G+C, the same as that of the R. capsulatus genome.  Repeating this analysis with heat-denatured DNA confirmed that the DNA is linear, not closed-circular like plasmid DNA.  Banding in a CsSO4 gradient showed that it is not extensively modified.  In sucrose gradients it co-sedimented with SV40 DNA, suggesting a size of 3.6 x 10^6 Daltons.  How big is this in base pairs or kb, you ask - about 5.5 kb.  He says it would be better to run the DNA in an agarose gel, but this emerging technology wasn't available to him yet.

G. Examination of GTA with the Electron Microscope:  He saw lots of tails, and empty heads, some with tails.  Apparently-full heads came in different sizes, from 150-600 Angstroms  in diameter (15-60 nm).  But he thinks much of this may be artefacts of the purification and EM-preparation procedures.