Document Type : Research Paper
Authors
Institute of Industrial Genetics, Allmandring 31, University of Stuttgart, 70569 Stuttgart, Germany
Abstract
Keywords
INTRODUCTION
The introduction of heterologous genes in organisms
by plasmids, viruses or integration into the chromosomes is usually selected by antibiotic resistance
markers. The occurrence of multiple antibiotic resistances in pathogenic organisms is a growing problem
and there are concerns that antibiotic resistance genes
in transgenic plants and microorganisms used for food
production might cause a further spread of this problem. Today the main method to remove an antibiotic
resistance gene from a chromosome is to use antibiotic resistance gene cassettes flanked by recognition
sites for site-specific recombinases, for instance the
Cre or FLP recombinases. When the recombinase
genes are transiently expressed in the recombinant
organism, the antibiotic resistance genes are excised.
This has been done successfully in plants as well as in
bacteria (Marx and Lindstrom, 2004; Kopertekh et al.,
2004). There are only a few other methods reported
that generate recombinant strains free of antibiotic
resistance genes. One method was described by Brans
et al. (2004). They brought the lysine biosynthesis
gene lysA of Bacillus subtilis under the control of a β-
lactamase promotor. Then they introduced the β-lactamase repressor gene blaI together with an antibiotic
resistance marker and the gene of interest into the
chromosome of this strain which made the cells conditionally auxotrophic for lysine. The blaI gene and the
resistance marker were flanked by long direct repeats
allowing the loss of the cassette by a single crossover.
The eviction of the blaI and antibiotic resistance genes
were identified simply by selecting lysine prototrophic strains. In another method previously described by
Fabret et al. (2002), a gene cassette with an antibiotic
resistance marker and a gene for counterselection were
integrated into the chromosome. As before, the cassette was flanked by direct repeats which led to spontaneous eviction of the cassette via homologous
recombination. The antibiotic sensitive strains were
selected by the counterselection marker upp (uracil
phosphoribosyl-transferase) which make cells sensitive to 5-fluorouracil. Fabret et al. (2002) used this
method for generating chromosomal deletions and
chromosomal point mutations, but in principle it can
also be used to introduce foreign genes into a chromosome too. This was shown by Zhang et al. (2006) who
used the same strategy, but with a different counterselection gene (an IPTG inducible E. coli mazF gene) to
integrate a gene of interest into the chromosome or to
generate point mutations in chromosomal genes. All
three methods were developed for B. subtilis.
Bacillus subtilis is classified as a food-grade organism, i. e. it produces no endotoxins and other toxic
substances and can be safely used for production of
enzymes for food processing or for pharmaceuticals.
Besides E. coli, it is the most deeply investigated bacterium. The genome is completely sequenced (Kunst et
al., 1997) and all except 271 essential genes were
knocked out recently (Kobayashi et al., 2003). A major
advantage of B. subtilis is its genetic ability to take up
any double-stranded DNA from the environment. This
type of DNA is degraded to a single strand during
uptake and efficiently integrated into the chromosome
via homologous recombination (Dubnau, 1991). Such
natural competence for transformation was exploited
in the methods described above for generating recombinant strains without antibiotic resistance markers.
The disadvantage of all three methods is the need for
tight regulation systems or well functioning counterselection markers. In the following study, we describe a
new way for generating recombinant B. subtilis strains
without antibiotic resistance marker which, in contrast
to the known methods does not need any sophisticated
regulatory system or counterselection.
MATERIALS AND METHODS
Strains and culture conditions: Escherichia coli
JM109 was used as host for transformation (YanischPerron et al., 1984). Bacillus subtilis 3NA was transformed by taking advantage of its natural competence.
Competent cells were prepared and transformed
according to protocol No 3.8 (Bron, 1990) with plasmid DNA from E. coli linearized with SacI. E. coli and
B. subtilis cells were grown at 37ºC on LB agar plates
and in LB liquid medium or minimal liquid medium
and agar plates [2 g/l (NH4)SO2, 14.8 g/l K2HPO4, 5.4
g/l KH2PO4, 1.9 g/l trisodium citrate, 0.2 g/l
Mg2SO4.2H2O, 0.02 % (w/v) casamino acids, 15 g/l
agar; protocol No 3.8] with 0.5 % w/v glucose and 0.5
% glycerol, respectively. The media were supplemented with spectinomycin (100 µg/ml) and histidine (20
µg/ml) when necessary.
Molecular techniques: All standard molecular techniques such as restriction enzyme analysis, ligation,
PCR, transformation of E. coli were carried out as
described in Ausubel et al. (1994). For Southern blots
the DNA was blotted onto nitrocellulose and
hybridized with pHM31 DNA and λ DNA labeled with
digoxigenin, according to the manufacturer’s instructions (Roche, Germany).
Plasmid constructions: The plasmids pHM30 and
pHM31 were constructed from pIC20HE
(Altenbuchner et al., 1992). A DNA fragment from B.
subtilis containing the C-terminal part of hisF, the
complete downstream hisI gene, which are the last two
genes in the his operon (http://genolist.pasteur.fr/
SubtiList/), were amplified with the primers S3597
(5´-CGC GGA TCT CGA AGC TC-3´) and S3598 (5´-
AAA AAA GCT AGC ACC CAA TAT AAA TCT AAA
TAC-3´), cleaved with endoR MluI and NheI and
inserted between the MluI/NheI sites of pIC20HE to
give pJOE4476.1. From same PCR fragment a
MluI/BsaAI fragment was inserted into pIC20HE cut
with MluI and BsaAI to give pJOE4475.2. Hereby the
C-terminal end of hisI was deleted. A 1.3 kb fragment
containing the C-terminal end of yvcA and N-terminal
end of yvcB downstream of hisI was amplified by PCR
using the primers S3599 (5´-GGA TGC AGT ATG AAT
GAC AA-3´) and S3600 (5´-AAA AAA GCA TGC GCG
GGT CAT CTT TTG AGA T-3´), cleaved with endoR
BamHI and SphI and inserted into pIC20HE to give
pJOE4482.1. From pJOE4482.1 the cloned PCR fragment was isolated again together with vector DNA as
a BamHI/ScaI fragment to replace the corresponding
restriction fragment in pJOE4476.1 to give pHM31
and in pJOE4475.2 leading to pJOE4519.1. Finally, an
EcoRI/EcoRV fragment from plasmid pDG1730
(Guérout-Fleury et al., 1996) encoding a spectinomycin resistance gene was inserted between the two
SacII sites of pJOE4519.1 leading to formation of the
plasmid pMH30. Hereby a 108 bp SacII fragment was
deleted from pJOE4519.1. The lipase gene (lip) without signal sequence for export was isolated from plasmid pT-BTL-2 (Rua et al., 1998) as a NdeI/BamHI
fragment and first inserted into the E. coli expression
vector pJOE4042.1 giving plasmid pJOE4615.1. For
regulated expression of the lipase gene in Bacillus, the
B. subtilis ptsGHI promotor (Stülke et al., 1997)
together with the corresponding antiterminator gene
glcT, the terminator/antiterminator sequence and ribosomal binding site of ptsG were amplified with the
primers S4163 (5´-AAA AAA CAA TGG CCC GGG
AAG GAC AGC CGA TTG AAA-3´) and S4164 (5´-
AAA AAA CAT ATG AAT TGA CCT CCT CTT TTT-3´).
The resulting PCR fragment was inserted between the
MfeI/NdeI sites of pJOE4615.1. Finally, the glcT-lip
fusion fragment was isolated again as a XmaI-fragment and inserted into pHM31 giving the pHM67 plasmid.
Induction kinetics of the Bacillus thermocatenulatus
lipase: Lipase activity in the strains 3NA and
3NA/pHM67 was determined as follows. Cells were
grown at 37ºC in minimal medium with 0.5% w/v
glycerol to an optical density (OD550) of 0.4. Glucose
was added to a final concentration of 0.5% w/v and the
cells were further incubated. Samples were taken
immediately and after 2, 4 and 6 h, the cells were
washed in 0.05 M sodium phosphate puffer, pH 7.0 and
lysed by ultrasonication. The crude extract was cleared
by centrifugation and the lipase activity was determined by adding 10 µl of crude extract to 990 µl of
reaction buffer (0.05 mM NaPO4, 5 mM Na-desoxycholate, 0.8 mM p-nitrophenyl palmitate). The resulting change in absorption was measured in a spectrophotometer at 410 nm for 1 min at 65ºC. One unit of
lipase corresponds to the release of 1 µM p-nitrophenol
(molar extinction coefficient: 15.200 mol-1 cm-1) per
min (Kaufmann and Schmitt-Dannert, 2001). Protein
concentrations were determined according to Bradford
(1970), using bovine serum albumin as standard.
RESULTS
Basically, to obtain marker-free recombinant strains
DNA must be integrated into the B. subtilis chromosome by homologous recombination with an incomplete B. subtilis gene from the histidine biosynthesis
operon which leads to auxotrophy for histidine. This
first event is selected by an antibiotic resistance marker. These auxotrophic strains can then be used for integration of the target genes by a second plasmid. This
plasmid has now the complete his gene but lacks the
antibiotic resistance marker. This allows a selection of
the his prototrophic strains, and by homologous
recombination the target genes are integrated and the
antibiotic resistance marker replaced. For this purpose
we constructed two plasmids which are shown in
Figure 1. Both plasmids pHM30 and pHM31 were
constructed from the E. coli plasmid pIC20HE
(Altenbuchner et al., 1992) which can not in
B. subtilis. The plasmid pHM30 contains the C-terminal part of hisF and the N-terminal part of hisI gene,
the last two genes in the