Biomedical Engineering Reference
In-Depth Information
subtilis
are more likely if endogenous plasmids are
used, Bron and colleagues have developed the cryp-
tic
Bacillus
plasmid pTA1060 as a vector (Haima
et al
. 1987, Bron
et al
. 1989).
Because of the difficulties experienced in direct
cloning in
B. subtilis
, hybrid plasmids were con-
structed which can replicate in both
E. coli
and
B. subtilis
. Originally most of these were constructed
as fusions between pBR322 and pC194 or pUB110.
With such plasmids,
E. coli
can be used as an efficient
intermediate host for cloning. Plasmid preparations
extracted from
E. coli
clones are subsequently used
to transform competent
B. subtilis
cells. Such pre-
parations contain sufficient amounts of multimeric
plasmid molecules to be efficient in
B. subtilis
-
competent cell transformation (see p. 140).
Table 8.2 lists some of the commonly used shuttle
plasmids. Note that some of them carry some of the
features described earlier for
E. coli
plasmids, e.g. the
E. coli lacZ
Cloning in Gram-positive bacteria
In Gram-positive bacteria, the base composition of
the different genomes ranges from
<
30% GC to
>
70% GC. Given this disparity in GC content, the
preferred codons and regulatory signals used by
organisms at one end of the % GC spectrum will not
be recognized by organisms at the other end. This
in turn means that there are no universal cloning
vehicles for use with all Gram-positive bacteria.
Rather, one set of systems has been developed for
high-GC organisms (e.g. streptomycetes) and another
for low-GC organisms. This latter group comprises
bacteria from the unrelated genera
Bacillus, Clostrid-
ium and Staphylococcus
and the lactic acid bacteria
Streptococcus, Lactococcus
and
Lactobacillus
.
Vectors for cloning in
Bacillus subtilis
and other low-GC organisms
-complementation fragment, multiple
cloning sites (MCS) (see p. 53) and the phage f1
origin for subsequent production of single-stranded
DNA in a suitable
E. coli
host (see p. 70).
α
The development of
B. subtilis
vectors began with
the observation (Ehrlich 1977) that plasmids from
S. aureus
(Table 8.1) can be transformed into
B.
subtilis
, where they replicate and express antibiotic
resistance normally.
As can be seen from Table 8.1, none of the natural
S. aureus
plasmids carries more than one selectable
marker and so improved vectors have been con-
structed by gene manipulation, e.g. pHV11 is pC194
carrying the
Tc
R
gene of pT127 (Ehrlich 1978). In
general, these plasmids are stable in
B. subtilis
, but
segregative stability is greatly reduced following
insertion of exogenous DNA (Bron & Luxen 1985).
Reasoning that stable host-vector systems in
B.
The influence of mode of replication:
vectors derived from pAM
β
1
Early in the development of
B. subtilis
cloning vectors,
it was noted that only short DNA fragments could be
efficiently cloned (Michel
et al
. 1980) and that longer
DNA segments often undergo rearrangements (Ehrlich
et al
. 1986). This structural instability is independ-
ent of the host recombination systems, for it still
occurs in Rec
−
strains (Peijnenburg
et al
. 1987).
Table 8.1
Properties of some
S. aureus
plasmids used as vectors in
B. subtilis.
Phenotype conferred
Plasmid
on host cell
Size Copy no.
Other comments
pC194
Chloramphenicol
2906 bp
15
Generates large amount of high-molecular-weight DNA when carrying
resistance
heterologous inserts
pE194
Erythromycin
3728 bp
10
cop
-6 derivative has copy number of 100.
resistance
Plasmid is naturally temperature-sensitive for replication
pUB110
Kanamycin
4548 bp
50
Virtually the complete sequence is involved in replication maintenance,
resistance
site-specific plasmid recombination or conjugal transfer
