Biomedical Engineering Reference
In-Depth Information
There are several ways in which DNA can be
introduced into streptomycetes, including transfor-
mation, transfection and conjugation. Transforma-
tion is achieved by using protoplasts, rather than
competent cells, and high frequencies of plasmid DNA
uptake can be achieved in the presence of poly-
ethylene glycol (Bibb
et al
. 1978). Plasmid mono-
mers that are covalently closed will yield 10
6
-10
7
transformants/
Table 8.4
Streptomyces
plasmids that have been used
in the development of vectors.
Mode of Copy Host
Plasmid Size replication number range
pIJ101
8.8 kbRolling circle 300
pJV1
11.1 kbRolling circle
Broad
pSG5
12.2 kbRolling circle
20-50
Broad
g of DNA, even with plasmids up to
60 kb in size. Open circular and linearized molecules
with sticky ends transform with 10 -100-fold lower
efficiency (Bibb
et al
. 1980). The number of trans-
formants obtained with non-replicating plasmids
that integrate by homologous recombination into
the recipient chromosome is greatly stimulated by
simple denaturation of the donor DNA (Oh & Chater
1997). This stimulation reflects an increased fre-
quency of recombination rather than an increased
frequency of DNA uptake. Electroporation has been
used to transform streptomycetes, since it bypasses
the need to develop protoplast regeneration pro-
cedures (Pigac & Schrempf 1995, Tyurin
et al
.
1995). For electroporation, limited non-protoplasting
lysozyme treatment is used to weaken the cell wall
and improve DNA uptake. Intergeneric conjugation
of mobilizable plasmids from
E. coli
into strepto-
mycetes (see p. 142) is increasingly being used,
because the required constructs can be made easily
in
E. coli
and the conjugation protocols are simple.
For intergeneric conjugation to occur, the vectors
have to carry the
oriT
locus from RP4 and the
E. coli
strain needs to supply the transfer functions
in trans
(Mazodier
et al
. 1989).
Transformants are generally identified by the
selection of appropriate phenotypes. However, anti-
biotic resistance has much less utility than in other
organisms, because many streptomycetes produce
antibiotics and hence have innate resistance to
them. One particularly useful phenomenon is that
clones harbouring conjugative plasmids can be
detected by the visualization of pocks. The property
of pock formation, also known as
lethal zygosis
, is
exhibited if a strain containing a conjugative plasmid
is replica-plated on to a lawn of the corresponding
plasmid-free strain. Under these conditions, clones
containing plasmids are surrounded by a narrow
zone in which the growth of the plasmid-free strain
is retarded (Chater & Hopwood 1983).
ยต
SCP2*
31 kbTheta
1-4
SLP1
17.2 kbRolling circle Integrating Limited
pSAM2
10.9 kbRolling circle Integrating Broad
Vectors for streptomycetes
With the exception of RSF1010 (see p. 145), no plas-
mid from any other organism has been found to
replicate in
Streptomyces
. For this reason, all the
cloning vectors used in streptomycetes are derived
from plasmids and phages that occur naturally in
them. The different replicons that have been sub-
jugated as vectors are listed in Table 8.4. Nearly all
Streptomyces
plasmids carry transfer functions that
permit conjugative plasmid transfer and provide
different levels of chromosome-mobilizing activity.
These transfer functions are very simple, consisting
of a single transfer gene and a repressor of gene
function.
Plasmid SCP2* is a deriviative (Lydiate
et al
. 1985)
of the sex plasmid SCP2. Both plasmids have a size
of 31.4 kb and are physically indistinguishable,
although SCP2* exhibits a much more pronounced
lethal zygosis reaction. SCP2* is important because
it is the progenitor of many very low-copy-number,
stable vectors. High-copy-number derivatives have
also been isolated with the exact copy number (10 or
1000) being dependent on the sequences from the
replication region that are present. SLP1 and pSAM2
are examples of
Streptomyces
plasmids that normally
reside integrated into a specific highly conserved
chromosomal transfer RNA (tRNA) sequence (Kieser
& Hopwood 1991). Many different specialist vectors
have been derived from these plasmids, including
cosmids, expression vectors, vectors with promo-
terless reporter genes, positive-selection vectors,
temperature-sensitive vectors, etc., and full details
can be found in Kieser
et al
. (2000).
