Biomedical Engineering Reference
In-Depth Information
respectively. Replication of these plasmids does not
lead to accumulation of detectable amounts of single-
stranded DNA, whereas the rolling-circle mode
of replication does. Also, the replication regions of
these two large plasmids share no sequence homo-
logy with the corresponding highly conserved regions
of the rolling-circle-type plasmids. It is worth noting
that the classical
E. coli
vectors, which are derived
from plasmid ColE1, all replicate via theta-like
structures.
Renault
et al
. (1996) have developed a series of
cloning vectors from pAM
Vellanoweth 1993). This is demonstrated by the
observation that
E. coli
ribosomes can support pro-
tein synthesis when directed by mRNA from a range
of Gram-positive and Gram-negative organisms,
whereas ribosomes from
B. subtilis
recognize only
homologous mRNA (Stallcup
et al
. 1974). The
explanation for the selectivity of
B. subtilis
ribosomes
is that they lack a counterpart of the largest
E. coli
ribosomal protein, S1 (Higo
et al
. 1982, Roberts &
Rabinowitz 1989). Other Gram-positive bacteria,
such as
Staphylococcus, Streptococcus, Clostridium
and
Lactobacillus
, also lack an S1-equivalent protein and
they too exhibit mRNA selectivity. The role of S1 is
believed to be to bind RNA non-specifically and bring
it to the decoding site of the 30S subunit, where proper
positioning of the Shine-Dalgarno (S-D) sequence
and initiation codon signals can take place. This
is reflected in a more extensive complementarity
between the S-D sequences and the 3
1. All the vectors carry a
gene essential for replication,
repE
, and its regulator,
copF
. The latter gene can be inactivated by inserting
a linker into its unique
Kpn
I site. Since
copF
down-
regulates the expression of
repE
, its inactivation
leads to an increase in the plasmid copy number
per cell. The original low-copy-number state can be
restored by removal of the linker by cleavage and
religation. This new replicon has been used to build
vectors for making transcriptional and translational
fusions and for expression of native proteins. Poyart
and Trieu-Cuot (1997) have constructed a shuttle
vector based on pAM
β
end of the
16S ribosomal RNA (rRNA) than found in bacteria
which have ribosomal protein S1.
The additional sequence requirements for efficient
transcription and translation in
B
.
subtilis
and other
low-GC organisms probably explain why many
E. coli
genes are not expressed in these hosts.
′
1 for the construction of tran-
scriptional fusions; it can be conjugally transferred
between
E. coli
and a wide range of Gram-positive
bacteria.
β
Controlled expression in
B. subtilis
and
other low-GC hosts
Transcription and translation
The first controlled expression system to be used in
B. subtilis
was the
Spac
system (Yansura & Henner
1984). This consists of the
E. coli lacI
gene and the
promoter of phage SPO-1 coupled to the
lac
operator.
More recently, the
E. coli
T7 system (see p. 74) has
been successfully implemented in
B. subtilis
(Conrad
et al
. 1996). This was achieved by inserting the T7
RNA polymerase gene (
rpoT7
) into the chromosome
under the control of a xylose-inducible promoter
and cloning the gene of interest, coupled to a T7 pro-
moter, on a
B. subtilis
vector. Of course, expression
of the heterologous gene can be made simpler by
putting it directly under the control of the xylose-
inducible promoter (Kim
et al
. 1996). A similar
xylose-inducible system has been developed in
staphylococci (Sizemore
et al
. 1991, Peschel
et al
.
1996) and
Lactobacillus
(Lokman
et al
. 1997). Many
different controllable promoters are available in
Lactococcus lactis
(for reviews see Kuipers
et al
. 1995,
The composition of the core RNA polymerase in
B. subtilis
and other low-GC hosts resembles that of
E. coli
. The number of sigma factors is different in each
of the various genera but the principal sigma factor
is sigma A. Analysis of many sigma A-dependent
Bacillus
promoters shows that they contain the
canonical
10 sequences found in
E. coli
promoters. In
B. subtilis
, at least, many promoters con-
tain an essential TGTG motif (
−
35 and
−
−
16 region) upstream
of the
10 region. Mutations of this region signific-
antly reduce promoter strength (Helmann 1995,
Voskuil & Chambliss 1998). The promoters also have
conserved polyA and polyT tracts upstream of the
−
−
16 region is found in some
E. coli
promoters, such promoters often lack the
35 region. Although the
−
−
35
region, whereas this never occurs in
B. subtilis
.
The translation apparatus of
B. subtilis
differs
significantly from that of
E. coli
(for review, see
