Biomedical Engineering Reference
In-Depth Information
Box 8.1 Transforming
Bacillus subtilis
with plasmid DNA
Although it is very easy to transform
B. subtilis
with
fragments of chromosomal DNA, there are problems
associated with transformation by plasmid molecules.
Ehrlich (1977) first reported that competent cultures
of
B. subtilis
can be transformed with covalently closed
circular (CCC) plasmid DNA from
Staphylococcus aureus
and that this plasmid DNA is capable of autonomous
replication and expression in its new host. The
development of competence for transformation by
plasmid and chromosomal DNA follows a similar time
course and in both cases transformation is first-order
with respect to DNA concentration, suggesting that
a single DNA molecule is sufficient for successful
transformation (Contente & Dubnau 1979). However,
transformation of
B. subtilis
with plasmid DNA is
very inefficient in comparison with chromosomal
transformation, for only one transformant is obtained
per 10
3
-10
4
plasmid molecules.
An explanation for the poor transformability of
plasmid DNA molecules was provided by Canosi
et al
.
(1978). They found that the specific activity of plasmid
DNA in transformation of
B. subtilis
was dependent on
the degree of oligomerization of the plasmid genome.
Purified monomeric CCC forms of plasmids transform
B. subtilis
several orders of magnitude less efficiently
than do unfractionated plasmid preparations or
multimers. Furthermore, the low residual transforming
activity of monomeric CCC DNA molecules can be
attributed to low-level contamination with multimers
(Mottes
et al
. 1979). Using a recombinant plasmid
capable of replication in both
E. coli
and
B. subtilis
(pHV14) (see p. 149), Mottes
et al
. (1979) were
able to show that plasmid transformation of
E. coli
occurs regardless of the degree of oligomerization,
in contrast to the situation with
B. subtilis
.
Oligomerization of linearized plasmid DNA by DNA
ligase resulted in a substantial increase of specific
transforming activity when assayed with
B. subtilis
and caused a decrease when used to transform
E. coli
. An explanation of the molecular events in
transformation which generate the requirement for
oligomers has been presented by De Vos
et al
.
(1981). Basically, the plasmids are cleaved into linear
molecules upon contact with competent cells, just as
chromosomal DNA is cleaved during transformation of
Bacillus
. Once the linear single-stranded form of the
plasmid enters the cell, it is not reproduced unless it
can circularize; hence the need for multimers to provide
regions of homology that can recombine. Michel
et al
.
(1982) have shown that multimers, or even dimers,
are not required, provided that part of the plasmid
genome is duplicated. They constructed plasmids
carrying direct internal repeats 260-2000 bp long
and found that circular or linear monomers of such
plasmids were active in transformation.
Canosi
et al
. (1981) have shown that plasmid
monomers will transform recombination-proficient
B. subtilis
if they contain an insert of
B. subtilis
DNA.
However, the transformation efficiency of such
monomers is still considerably less than that of
oligomers. One consequence of the requirement for
plasmid oligomers for efficient transformation of
B. subtilis
is that there have been very few successes
in obtaining large numbers of clones in
B. subtilis
recipients (Keggins
et al
. 1978, Michel
et al
. 1980).
The potential for generating multimers during
ligation of vector and foreign DNA is limited.
Transformation by plasmid rescue
An alternative strategy for transforming
B. subtilis
has
been suggested by Gryczan
et al
. (1980). If plasmid
DNA is linearized by restriction-endonuclease cleavage,
no transformation of
B. subtilis
results. However, if
the recipient carries a homologous plasmid and if the
restriction cut occurs within a homologous marker,
then this same marker transforms efficiently. Since
this rescue of donor plasmid markers by a homologous
resident plasmid requires the
B. subtilis recE
gene
product, it must be due to recombination between
the linear donor DNA and the resident plasmid.
Since DNA linearized by restriction-endonuclease
cleavage at a unique site is monomeric, this rescue
system (
plasmid rescue
) bypasses the requirement
for a multimeric vector. The model presented by
De Vos
et al
. (1981) to explain the requirement for
oligomers (see above) can be adapted to account for
transformation by monomers by means of plasmid
continued
