Integration of A Lipase Gene into the Bacillus subtilis Chromosome: Recombinant Strains Without Antibiotic Resistance Marker

Document Type: Research Paper

Authors

Institute of Industrial Genetics, Allmandring 31, University of Stuttgart, 70569 Stuttgart, Germany

Abstract

A new system is presented for the generation of recombinant Bacillus subtilis strains without antibiotic markers. This system is based on two plasmids constructed in Escherichia coli. The first plasmid pHM30 contains an incomplete hisI gene, the last gene in the histidine biosynthesis operon of B. subtilis and part of the genes yvcA and yvcB of unkown function flanking hisI at the 3´-end. The spectinomycin resistance gene is inserted between hisI and the downstream yvcAB region. Transformation of B. subtilis with this plasmid pHM30 led to spectinomycin resistant, histidine auxotrophic strains. The integrated parts of pHM30 act like a docking station for the second plasmid pHM31. The plasmid pHM31 contains the same yvcAB region but a complete copy of the hisI gene and no antibiotic resistance marker. Heterologous genes to be expressed in B. subtilis were inserted into a multiple cloning site between hisI and the downstream region. Transformants of B.subtilis/pHM30 with pHM31 derivatives were selected on minimal medium without histidine. By double crossovers during homologous recombination the heterologous genes were integrated, replacing the defect copy of hisI and the spectinomycin resistance gene. The plasmids were also successfully applied in the chromosomal integration of the lipase gene of Bacillus thermocatenulatus under a B. subtilis glucose regulated promotor/antiterminator system.

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 his operon. Downstream of
the his operon in B. subtilis, the yvcA and yvcB genes
of unknown function are located. A 1.3 kb fragment
containing the C-terminal end of yvcA and N-terminal
end of yvcB was amplified by PCR and fused with the
fragment containing the incomplete hisF/I genes.
Finally, a spectinomycin resistance gene was inserted
between the his-yvc region to give pHM30. The plasmid pHM31 contains the same yvc-region and the
same C-terminal end of hisF but has the complete hisI
gene and lacks the antibiotic marker. Therefore, integration of pHM30 into the B. subtilis chromosome by
two crossovers should lead to spectinomycin resistant,
his auxotrophic mutants. The insertion of pHM31 into
ding glcT gene was amplified together with the promotor, the terminator/antiterminator sequence and ribosomal binding site of ptsG, then fused with the lipase
gene and inserted into pHM31 to give pHM67. A
sporulation negative B. subtilis 3NA (genotype spoA3,
Michel and Millet, 1970) was transformed by pHM30
and the resulting transformants were selected by
spectinomycin resistance. The colonies turned out to
be histidine negative as expected. One of the clones
was transformed again with pHM67 and the transformants were selected on minimal glucose medium lacking histidine. Testing of these colonies showed that
most of them were spectinomycin sensitive but positive for lipase. The homologous recombination events
are illustrated in Figure 2. To see that there were no
further DNA rearrangements the chromosomal DNA
of B. subtilis 3NA, 3NA/pMH30 and 3NA/pHM67
were isolated and digested with endoR MunI. The
resulting fragments were then separated on an agarose
gel, blotted on nitrocellulose filter and hybridized with
digoxigenin labeled pHM31 DNA (Figure 3). A 3.58 kb
MunI band was observed in the wild type 3NA, a 4.13
kb band in 3NA/pHM30 and a 5.78 kb in 3NA/pHM67
as expected, which indicates that no other further
rearrangements happened except for the homologous
recombination events illustrated in Figure 2.
To see that the lipase gene was actively expressed
in the recombinant strain, the wild type 3NA and the
recombinant 3NA/pHM67 were grown in minimal
medium with glycerol and the ptsGHI promoter was
induced by addition of glucose. Every two hours samples of the induced cultures were harvested and the
cells were lysed by ultrasonic treatment. Lipase activity was determined by incubation of the crude extracts
with p-nitrophenyl palmitate and measuring the
change in the absorption at 410 nm by a spectrophotometer. Only very low lipase activities were found in
the B. subtilis 3NA wild type strain (0.013 U/mg protein) whereas the recombinant strain with pHM67



upstream of the ptsGHI operon. Transcription of
ptsGHI starts from a constitutive ptsGHI promotor
within the C-terminal end of glcT and ends at a terminator sequence between the glcT and ptsG gene. In the
presence of glucose the GlcT antiterminator protein
binds between the promotor and the ptsG gene at a
transcriptional antiterminator (RAT) sequence leading
to transcription of ptsGHI. Therefore, the correspon

ding glcT gene was amplified together with the promotor, the terminator/antiterminator sequence and ribosomal binding site of ptsG, then fused with the lipase
gene and inserted into pHM31 to give pHM67. A
sporulation negative B. subtilis 3NA (genotype spoA3,
Michel and Millet, 1970) was transformed by pHM30
and the resulting transformants were selected by
spectinomycin resistance. The colonies turned out to
be histidine negative as expected. One of the clones
was transformed again with pHM67 and the transformants were selected on minimal glucose medium lacking histidine. Testing of these colonies showed that
most of them were spectinomycin sensitive but positive for lipase. The homologous recombination events
are illustrated in Figure 2. To see that there were no
further DNA rearrangements the chromosomal DNA
of B. subtilis 3NA, 3NA/pMH30 and 3NA/pHM67
were isolated and digested with endoR MunI. The
resulting fragments were then separated on an agarose
gel, blotted on nitrocellulose filter and hybridized with
digoxigenin labeled pHM31 DNA (Figure 3). A 3.58 kb
MunI band was observed in the wild type 3NA, a 4.13
kb band in 3NA/pHM30 and a 5.78 kb in 3NA/pHM67
as expected, which indicates that no other further
rearrangements happened except for the homologous
recombination events illustrated in Figure 2.
To see that the lipase gene was actively expressed
in the recombinant strain, the wild type 3NA and the
recombinant 3NA/pHM67 were grown in minimal
medium with glycerol and the ptsGHI promoter was
induced by addition of glucose. Every two hours samples of the induced cultures were harvested and the
cells were lysed by ultrasonic treatment. Lipase activity was determined by incubation of the crude extracts
with p-nitrophenyl palmitate and measuring the
change in the absorption at 410 nm by a spectrophotometer. Only very low lipase activities were found in
the B. subtilis 3NA wild type strain (0.013 U/mg protein) whereas the recombinant strain with pHM67

showed 0.25 U/mg of lipase activity. The induction
kinetics of the lipase gene with glucose in B. subtilis
3NA/pHM67 is shown in Figure 4.
DISCUSSION
The successful integration and expression of the lipase
gene into the B. subtilis chromosome and removal of
the antibiotic resistance gene proves that this new system for genetic engineering of B. subtilis is efficient
and easy to handle. With plasmid pHM30, any B. subtilis strain can be made histidine auxotrophic and ready
for integration of any other gene which is inserted into
pHM31. By just one further transformation, one gets
recombinant strains free of antibiotic resistance genes.
The same principle might be used for insertions of
recombinant genes into other biosynthetic or catabolic
operons where inactivation leads to auxotrophic or any
other growth negative mutants, allowing the engineering of B. subtilis with multiple insertions. In addition
this method could be extended to any other bacterial
strain able to take up linear DNA.

Acknowledgements
We are grateful to Silke Weber for technical assistance. We
thank Dr. DR Zeigler from the Bacillus Genetic Stock Center
for providing us with Bacillus strains and plasmids, and Prof.
R. Mattes for support and interest in this work.







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