1. CLONING IN GRAM POSITIVE
BACTERIA
E.g.: Bacillus Subtilis

2. Cloning in Gram-positive bacteria
1.Characterization of the base composition of
Gram-positive bacteria: <30% GC~>70%
2. Preferred codons: Differ greatly from one
organism to another.

3. Vectors for cloning in Bacillus subtilis
and other low-GC organisms
1. Characters of vectors:
plasmids from S. aureus can be transformed
into B. subtilis, but segregative stability are
greatly reduced following insertion of
exogenous DNA.
2. Construction of vectors: Shuttle vectors for
E.coli and another host cell

4. Many of the cloning vectors used with Bacillus subtilis and other low-
GC bacteria are derived from plasmids found in Staphylococcus aureus
• Development of B.subtilis with the
observation (Ehrlich 1977) that plasmids from
S.aureus can be transformed into
B.subtilis, where they replicate and express
antibiotic resistance normally.

5. Properties of some S.aureus plasmids used vectors in
B.subtilis
PLASMID Phenotype Size Copy no. Comments
conferred
on host cell
pC194 Chloramphe 2906 bp 15 High mol wt
nicol res. DNA
pE194 Erthromycin 3728 bp 10 Temp
res. sensitive for
replication
pUB110 Kanamycin 4548bp 50 Conjugal
resistance transfer

6. • Because of the difficulties in direct cloning in
B.subtilis hybrid plasmids were constructed
which can replicate in both E.coli and B.subtilis
• Most of the plasmid were constructed
between Pbr322 and pC194,with such
plasmids E.coli can be used as an efficient
intermediate host for cloning.
• Plasmid preparations extracted from E.coli
clones are used to transform B.subtilis cells.
Such preparations contain MCS.

7. 1. Structural instability of recombinant DNA in
B.subtilis: Longer DNA fragments often
undergo rearrangements.
2. Major reasons for structural instability :
Mode of replication
DNA fragments in plasmids replicating by a
rolling-circle mechanism are likely to be
unstable. While DNA fragments in plasmids
replicating by theta mechanism tend to be
stable

9. Transcription and translation
1. The composition of the core RNA polymerase
in B.subtilis and other low-GC hosts resemble
that of E.coli and main sigma factor is sigma70,
but the number is different.
2. Many promoters contain TGTG motif at –16
region.

10. 3. The ribosome of B.subtilis differ from that of
E.coli in that B.subtilis ribosome's lack a
counterpart of the largest E.coli ribosome's
protein, S1.
4. E.coli ribosome's can support the mRNA from
other organisms, but B.subtilis ribosome's
only support the homologous mRNA.

11. Controlled expression in B. subtilis and
other low-GC hosts
1. Controlled expression in B. subtilis :
Plasmids contain T7 promoter and Lac
operator as well as LacI, T7 RNA polymerase
gene is inserted into B. subtilis chromosome
DNA under the control of xylose-inducible
promoter

12. 2. Controlled expression in L.lactis :
Low-copy plasmids contain a phage middle
promoter and phage ori, the gene of interest
is inserted into the downstream of the
promoter. Following infection of φ31 phage,
the plasmid number rapidly increases and the
gene can be expressed in this way

13. Secretion vectors for low-GC bacteria
1. The difference of signals between the low-GC
bacteria and other organisms
1) The N-termini are more positively charged.
2) There are also larger and the extra length
distributed among the three regions of the
signal peptide.

14. Vectors for systematic gene
inactivation
1.Vector used for insertional mutagenesis in
B.subtilis: pMUTIN
2. Properties of pMUTIN
1) an ability to replicate in B.subtilis
2) a report gene to facilitate the measurement
of the target gene
3) the inducible Pspac promoter

16. Properties of pMUTIN
• An inability to replicate in B.subtilis, which
allows insertional mutagenesis
• A reporter lacZ gene to facilitate the
measurement of expression of the target gene
• The inducible Pspac promoter to allow
controlled expression of genes downstream of
and found in the same operon as the target
gene