Transformation...

Bernard Jenni BJenni at datacomm.ch
Thu Sep 10 01:19:05 EST 1998


You can find some answer to your question by reading the papers of 
- Reusch et al.1986. Poly-beta-hydroxybutyrate membrane structure and
its relationship to genetic transformability in E.coli. J.Bacteriol.
168:553-562. and
- Huang & Reusch. 1995. Genetic competence in E.coli requires
PHB/calcium/polyphosphate membranes complexes and certain divalent
cations. J.Bacteriol. 177:486-490.

These authors observed that the development of competence in E.coli is
coincident with de novo synthesis of PHB into the cytoplasmic menbranes.
They reported also the formation of calcium/phosphate/PHB complexes,
forming channels allowing the transfer of DNA.
They also put forward the hypothesis that the competence development of
E.coli is physiologically (i.e., naturally) and not physicochemically
(i.e., by the addition of non-physiological amounts of CaCl2) regulated.

In fact, E.coli is able to develop a natural competence when incubated
in calcareous freshwater which contains only a few mM of calcium.
Contrarily, no transformation is possible in pure LB medium, which
contains only 0.25 mM of free Calcium. In freshwater, no temperature
jump is necessary for having transformants, but temperature jumps
increase their numbers. Of course, I am speaking here from natural,
physiological transformation, which gives less efficiencies as compared
with the methods followed by molecular biologists.

For those who are interested, we have published these results:
B. Baur, K. Hanselmann, W. Schlimme, B.Jenni
Genetic transformation in freshwater: E.coli is able to develop natural
competence.
Applied and Environm. Microbiology, 62(10):3673-3678 (1996)

Bernard Jenni


George Tsimiklis wrote:
> 
> .... that part makes common sense.  However, I still don't understand how
> the MgCl2 and the CaCl2 play a role in this destabilization of the
> membrane.  Is the role of adding MgCl2 or CaCl2 understood, or is it done
> simply because it works ... without a known scientific reason?
> 
> George Tsimiklis
> 
> Elena Sanchez-Heras wrote:
> 
> > Basiccally the mechanism is as follows: when we make competent cells,
> > what we do is to desestabilize the membrane biochemically so we make it
> > more liable to penetration. When we transform those competent cells, the
> > heat shcok literally make holes in that pretreated membrane, so the
> > foreign DNA can penetrate in the cell.
> >
> > Elena Sanchez-Heras
> >
> > On Sat, 5 Sep 1998, George Tsimiklis wrote:
> >
> > > I would like to know the mechanism of transformation of competent
> > > bacteria.  What is the purpose of the heat shock?  Biochemically, how
> > > does MgCl2 and CaCl2 increase the efficiency of the transformation?
> > > Do they have an effect on bacterial transport proteins?



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