Assay and characterization of an osmolarity inducible promoter

newly isolated from Bacillus subtilis

Wei-Wei Zhang

Qiu-Rong Gao

Ming-Ming Yang

Hui Liu

Dun Wang
Received: 26 March 2011 / Accepted: 25 January 2012 / Published online: 11 February 2012
Springer Science+Business Media B.V. 2012
Abstract An osmolarity-sensitive promoter fragment,
P23423, isolated from Bacillus subtilis was characterized.
The expression of b-galactosidase (b-Gal) driven by
P23423 was regulated by osmolarity both in Escherichia
coli and B. subtilis. The classical conserved region of this
prokaryotic promoter was found within the sequence of the
cloned fragment, and the putative promoter was identied
as the control element of RNA not coding for protein (a
RNA molecule that is not translated into a protein). The
efciency and benet of this promoter was further dem-
onstrated via osmolarity-induced expression of three other
heterologous proteins in E. coli. Thus, this approach pro-
vided a simple and inexpensive inducible promoter element
for the expression of cloned genes.
Keywords Bacillus subtilis Expression vector
b-Galactosidase Inducible promoter Osmolarity
Introduction
Bacillus subtilis is an important organism from both the
basic and applied research perspectives [1, 2]. With
the completion of the sequencing and annotation of the
B. subtilis 168 strain genome [3], systematic functional
studies can be started. Inducible promoters are very pow-
erful tools in genetic engineering because the expression of
operable genes linked them can be turned on or off at a
given time or condition [46].
Activities of inducible promoters are often regulated by
particular chemical or physical factors. The commonly
used chemically-regulated promoters include Pspac [7],
Pxyl [5], and Pglv [8], which are induced by IPTG, xylose
and maltose, respectively. However, the toxicity of IPTG,
the cost of IPTG and xylose, and the catabolism repression
of glucose [8, 9] restrict the usage of these three promoter
systems, especially in industry. So it is in the interest of the
biotechnological industry to seek new physical-regulated
promoters that are non-patented, cheap and simple to
regulate.
The stress-inducible promoters are suitable for large-
scale protein production such as the temperature, pH,
glucose starvation and anaerobiosis [1012]. Promoters
that respond to osmolarity can be readily used for the
efcient and sensitive regulation of target protein
expression levels [1317]. These promoters can be
induced by simply varying the osmolarity of the growth
medium, without being dependent on carbon or nitrogen
sources, and while having a minimal effect on physio-
logical conditions. Therefore, the promoters are potential
inducible elements for gene expression and industrial
applications.
In this study, an osmolarity-regulated promoter from
B. subtilis was cloned and characterized. The putative
promoter was further identied and predicated via
sequence analysis. Moreover, the efciency and strength
of this promoter was demonstrated via the high-level
expression of homologous and heterologous proteins in
E. coli.
W.-W. Zhang Q.-R. Gao D. Wang (&)
College of Life Sciences, Northwest A&F University,
Yangling 712100, Peoples Republic of China
e-mail: wanghande@nwsuaf.edu.cn
M.-M. Yang H. Liu
College of Animal Sciences, Yangling 712100,
Peoples Republic of China
1 3
Mol Biol Rep (2012) 39:73477353
DOI 10.1007/s11033-012-1566-3
Materials and methods
Bacterial strains, plasmids and growth conditions
The bacterial strains and plasmids used in this study are
listed in Table 1. All organisms were grown in Luria-
Bertani medium (LB) at 37C. When required, the organ-
isms were grown in LBON medium (LB medium without
NaCl) or high osmolarity medium (LB medium with 2.5%
NaCl). The nal concentrations of antibiotics in medium
were 100 lg/ml ampicillin and 5 lg/ml chloramphenicol.
Transformation of E. coli and B. subtilis
Transformation of E. coli cells were performed as descri-
bed by Sambrook et al. [18]. B. subtilis cells were trans-
formed by electroporation [19].
Sub-clone of the promoter
In our previous work, a clone pSI23423 driven osmolarity-
induced expression was screened from B. subtilis promoter
library [10]. Using primers pairs P23up/P23down (5
0
-
TTGGGCCCATATGAACGAATGACC-3
0
/5
0
-TTGGATC
CTGATGAACTGGCTGTCG-3
0
) and P24up/P24down (5
0
-
TTGGGCCCAAAAACCTTAACATCTAAG-3
0
/5
0
-TTGG
ATCCGCTGATTCATAGAATTTGG-3
0
), the putative
promoters P23S and P24S were PCR amplied from
plasmid pSI23423, respectively. Both the resultant pro-
moter fragments were anked by ApaI and BamHI at 5
0
and
3
0
end, respectively. Following enzyme digestion, the P23S
and P24S fragments were cloned into the corresponding
sites of the promoter probe vector pShuttleI, yielding pSI23
and pSI24, respectively.
Construction of expression vectors in E. coli
The P23S promoter (excised from the pSI23 with the ApaI
and BamHI) and the annealed synthesis Shine-Dalgarno
(SD) sequence (with a BglII and sacI cohesive end) were
ligated into the sites of ApaI and SacI in the pBluskm. Then
the promoter with the SD sequence was excised with ApaI
and EcoRI and ligated into the corresponding sites of
pGJ103, resulting in pOMH. The bgaB gene [20] coding
for b-galactosidase of B. stearothermophilus was excised
from the pLJ-7 with EcoRI and SacI, and then cloned into
the corresponding sites of pOMH, resulting in pOMH-
bgaB. The vgb gene [21] was excised from pYG-vgb with
EcoRI and SacI and cloned into the corresponding sites of
pOMH, resulting in pOMH-vgb. Using BioB-1 (5
0
-TT
GAATTCATGGCTCACCGCCCAC-3
0
) and BI-2 (5
0
-TTG
AGCTCCAGCTCATAATGCTGCCG-3
0
) as primers, we
PCR amplied bioB gene [22] from E. coli DH5a genomic
DNA, digested the obtained fragment with the EcoRI and
SacI, and then cloned it into the pOMH vector, resulting in
pOMH-bioB.
b-Galactosidase activity assay
The b-Gal activity assay was carried out as described
previously [20, 23]. The activity was given as Miller units
per ml sample (Mill U/ml).
Table 1 Strains and plasmids
used in this study
Spec
R
spectinomycin-resistance,
Cm
R
chloramphenicol-
resistance, BGSC Bacillus
Genetic Stock Center, NCU new
construction that was
constructed in our lab,
unpublished data, NCT new
construction that was
constructed in this work
Strain or plasmid Description Source
E. coli
DH5a F
-
u80dlacZDM15D (lacZYA-argF) U169 endA1 recA1 hsdR17
(r
k
-
, m
k
?
) DeoR thi-1 phoA supE44 k
-
gyrA96 relA1
Commercial strain
B. subtilis
PY79 B. subtilis 168 prototroph SPb
-
; Cat. no. 1A747 BGSC
Plasmids
pSI23423 Cm
R
, Spec
R
, harboring promoter fragment NCU
pShuttleI Cm
R
, Spec
R,
promoter probe vector NCU
pSI23 pShuttleI with amplied promoter P23S NCT
pSI24 pShuttleI with amplied promoter P24S NCT
pGJ103 clone vector [3]
pLJ-7 bgaB gene donor [3]
pYG-vgb vgb gene donor [18]
pOMH pGJ103 with amplied promoter P23S NCT
pOMH-bgaB Expression vector of bgaB NCT
pOMH-vgb Expression vector of vgb NCT
pOMH-bioB Expression vector of bioB NCT
7348 Mol Biol Rep (2012) 39:73477353
1 3
SDS-PAGE assay
Sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) was performed as previously described [18].
Reverse transcription analysis
The total RNA was prepared with a Total RNA Extract Kit
(Promega, Cat.No.Z3100) from the B. subtilis PY79. The
reverse transcription analysis was performed with a Primer
Extension PCR Kit (Promega, Cat.No.Z3500). The primer
(5
0
-GAAGTTCAGATGAAAATTCAAACGGAATC-3
0
)
that is homologous with the complement strand of the gene
under control of the P23S and 229 bp from the putative
transcription origin site was labeled with (c
32
P) ATP at the
5
0
end, according to the manufacturers instructions.
Nucleotide sequence analysis
The promoter fragments were sequenced by Aoke corp
(Beijing, China). Analysis of DNA sequence of the pro-
moter-active fragment was carried out online with NCBI
blast 2.0 (http: www.ncbi.nih.gov). The promoter was
predicted by softberry (http: www.softberry.com).
Results and discussion
Characterization of an osmolarity-inducible promoter
In our previous work, the promoter library of B. subtilis
was created (Li et al. 2007). By screening the library, we
isolated a clone pSI23423 that demonstrated osmolarity-
induced expression of b-Gal (Fig. 1). In this study, we
further characterized the cloned promoter by quantitatively
determining pSI23423-driven b-Gal production in E. coli
and B. subtilis (Table 2). In both E. coli and B. subtilis,
the b-Gal productions from pSI23423 were higher in
LBON medium supplemented with 2.5% NaCl than that in
LBON medium. The b-Gal production driven by the
pSI23423 cultivated in LBON medium with 2.5% NaCl
reached maximum (2525.36 mill U) at 36 h in E. coli,
which was almost 4.12 times of that in LBON medium.
These results further conrmed that the cloned promoter in
pSI23423 is osmolarity-sensitive in driving expression of
b-Gal in E. coli. The b-Gal production from the pSI23423
in B. subtilis was lower than that in E. coli. However, the
b-Gal production from pSI23423 cultivated in LBON
medium with 2.5% NaCl was 4.05 fold higher than that in
the medium without NaCl in B. subtilis. This implied that
the cloned promoter in pSI23423 might be a potential
osmoregulated element in the genetic engineering of E.
coli and B. subtilis.
Sequence analysis and predication of the putative
promoter
To further characterize the cloned promoter fragment,
sequence analysis was carried out. Sequence analysis
(Fig. 2a) indicates that the inserted fragment upstream of
the reporter gene bgaB in pSI23423 is about 898 bp and
100% identical to the reported B. subtilis genome DNA
(GenBank accession number NC 0009642).
Two conserved prokaryotic promoter regions were
found in the cloned fragment (Fig. 2b). The rst putative
promoter, P23S, was located 127 bp upstream the bgaB,
and the second, P24S, located 647 bp upstream the bgaB.
To nd the authentic promoter, the two probable promoters
were sub-cloned into the promoter probe vector, in which
the bgaB with its native ribosome binding site (RBS) was
used as the reporter, yielding pSI23 and pSI24, respec-
tively. Figure 3a demonstrates that the trend of b-Gal
production from the pSI23 is similar with that from
pSI23423 both in E. coli and B. subtilis. The b-Gal pro-
duction from the pSI23 reached 3122.44 and 294.25
mill U/ml when supplemented with 2.5% NaCl in E. coli
and B. subtilis, respectively, which was higher than that
from the pSI23423. Whereas, no b-Gal activity was
detected from the pSI24 either induced or not induced by
salt. Therefore, the promoter driven inducible expression of
b-Gal in this study is achieved using the P23S.
To examine which gene was under the control of P23S,
we analyzed the sequence downstream of the P23S pro-
moter in B. subtilis genome (Fig. 2b). Partial sequence of
yddE, ygaB, and ygaC was contained in this cloned
Fig. 1 Relevant features of pSI23423. The bgaB, b-Gal encoding
sequences of B. stearothermophilus; The promoter upstream the
reporter gene represents the cloned promoter-activity fragment from
B. subtilis chromosome DNA; ColE1, E. coli ColE1 replicon. Cm
chloramphenicol-resistance marker, Spec spectinomycin-resistance
marker, Rep replicon in B. subtilis. The unique restriction enzyme
sites are marked on the outside of the map
Mol Biol Rep (2012) 39:73477353 7349
1 3
fragment, but no known gene or open reading frame (ORF)
was found in the contiguous region downstream of the
P23S (in the same orientation as the promoter). This sug-
gested that the P23S might be the control element of RNA
not coding for protein. To test the hypothesis, we rst
analyzed the fragment for the conserved sequence of RBS
(Shine-Dalgarno box, SD). The results showed that there
was no typical SD sequence detected downstream of the
P23S in B. subtilis genome, suggesting that the P23S most
likely is a control element of RNA not coding for protein.
A reverse transcription analysis was conducted to deter-
mine if there is any transcript generated by the P23S pro-
moter. As shown in Fig. 3b, the promoter P23S generated
the RNA transcript as expected. Thus, these observations
suggested that that the P23S is a putative promoter driving
expression of RNA.
Table 2 The b-Gal production from pSI23423
Strain Salt
a
(%) OD595 b-Gal activity (U/ml)
12 h 24 h 36 h 48 h 12 h 24 h 36 h 48 h
DH5a (pSI23423) 0 4.41 4.54 4.52 4.39 299.59 564.24 612.95 528.12
DH5a (pSI23423) 2.5 4.37 4.29 4.32 4.35 1033.68 1407.81 2525.36 1610.49
PY79 (pSI23423) 0 4.91 5.01 4.89 4.80 23.46 38.57 54.07 49.82
PY79 (pSI23423) 2.5 4.76 4.88 4.90 4.75 80.57 159.19 218.98 197.14
a
Supplemented salt in LB medium
With or without the salt induction, the b-Gal production from pShuttleI is not detected both in DH5a and PY79
Fig. 2 a Sequence of the cloned putative promoters in pSI23423. -
10 and -35 of putative promoters and transcription origin (black
triangle) were indicated. b Map of the cloned fragment relative to the
genome DNA. The sequence from 2 to 734 comes from the ygaB and
ygaC genes, and the sequence from 735 to 938 comes from yddE
gene. The orientations of genes were indicated
7350 Mol Biol Rep (2012) 39:73477353
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Detection of the expression strength of P23S promoter
To further examine the activity of the promoter, the P23S
fused with an engineered RBS was exploited as a control
element and used to generate expression in the pOMH
vector. Since sufcient unique restriction sites are located
downstream of the promoter, it is convenient to insert the
target gene downstream of P23S in the expression vector
pOMH.
To examine the expression efciency of the recon-
structed promoter system for cloned genes, the coding
region of bgaB was used as a reporter gene and cloned into
the expression vector, pOMH. As shown in Fig. 4a, b-Gal
from the pOMH-bgaB in E. coli under the 2.5% salt
inducible conditions revealed a distinct band with a
molecular mass of approximately 66 kDa, corresponding to
the molecular weight of b-Gal, while a relatively weak
band was visible from that under no salt inducible condi-
tions. These results conrmed that the b-Gal was efciently
expressed under salt induction.
To further demonstrate the efciency of the P23S pro-
moter system, the bioB gene, which is involved in biotin
biosynthesis of E. coli, and the vgb gene coding for Vit-
reoscilla haemoglobin, were inserted downstream of P23S
Fig. 3 a b-Gal production from pSI23 both in E. coli and B. subtilis.
The open square and the solid square represent the b-Gal production
from pOMH-bgaB in E. coli with and without 2.5% salt induction,
respectively; the solid triangle and the open triangle represent the b-
Gal production from pOMH-bgaB in B. subtilis with and without
2.5% salt induction, respectively. b The reverse transcription analysis
of transcript directed by P23S. Lane 1 represents the reverse transcript
(labeled with c
32
P) from E. coli total RNA (this served as negative
control), Lane 2, A9174/HinfI marker, Lane 3 represents the reverse
transcript (labeled with c
32
P) from B. subtilis PY79 total RNA, Lane 4
represents the reverse transcript (labeled with c
32
P) from B. subtilis
PY79 total RNA with 2.5% salt induction
Fig. 4 a SDS-PAGE analysis of bgaB directed by the P23S promoter
system in E. coli harvested after 36 h culture. Lane 1 molecular mass
markers (top to bottom: 116, 66, 45, 35, and 25 kDa); Lanes 2 and 3
expression of bgaB directed by the P23S without and with 2.5% salt
induction, respectively. b SDS-PAGE analysis of bioB directed by the
P23S promoter system in E. coli harvested at 36 h. Lane 1 molecular
mass markers (top to bottom: 116, 66, 45, 35, and 25 kDa), Lane 2
expression of bioB directed by the P23S with 2.5% salt induction,
Lane 3 expression of bioB directed by the P23S without salt
induction. c SDS-PAGE analysis of vgb directed by the P23S
promoter system in E. coli harvested at 36 h. Lane 1 expression of
vgb directed by the P23S without salt induction, Lane 2 molecular
mass markers (top to bottom: 116, 66, 45, 35, and 25 kDa), Lanes 3
and 4, expression of vgb directed by the P23S with 2.5% salt
induction
Mol Biol Rep (2012) 39:73477353 7351
1 3
in the pOMH, respectively. As expected, these two genes
were successfully expressed by this promoter system in E.
coli under 2.5% salt inducible conditions. Only relatively
weak target protein bands were visible under no salt
inducible conditions (Fig. 4b, c), further demonstrating the
efciency of the osmolarity-inducible promoter system
developed in this study.
As Gram-positive soil bacteria, B. subtilis is exposed to
various stress conditions from the natural environment. To
adapt to environmental stress conditions, some mecha-
nisms have been evolved in B. subtilis, such as regulation
of gene expression in response to osmolarity change in a
timely and efcient manner [17, 24]. The elements that are
involved in the regulation of gene expression in response to
environmental stress are valuable for helping us understand
the corresponding mechanisms of bacteria. Moreover, these
elements can also provide potentially convenient expres-
sion elements in the genetic engineering eld, by offering a
safe induction approach that is also cost-efcient. Here, an
osmolarity-sensitive promoter was isolated and character-
ized. The expression of the cloned genes driven by this
promoter was induced by salt. There have been some
osmolarity-sensitive promoters reported, such as the proU
operon of E. coli., which mediate the uptake of glycine
betaine from the environment [14]; The control element of
the major outer membrane proteins, OmpF and OmpC, of
E. coli K-12 [25], and the promoter of the gpdA gene [15]
isolated from NaCl-adapted Aspergillus nidulans
(FGSC359); The operon of B. subtilis pbpE gene induced
by increased salinity [6].
As we know, the response to environmental stress is
usually governed by the sigma factors of RNA polymerase
in bacteria, i.e., r
B
, r
H
, r
S
[24, 2631]. These factors can
activate the specic expression when cells are submitted to
the specic environment such as heat, low pH and high
salt. Amongst, high salt will lead to the increasing of
osmolarity, this activate the sigma factor response to stress.
Sequence analysis revealed that no ORF or typical SD is
found downstream of the P23S promoter and, thereby,
P23S may be a control element of non-coding protein
RNA. With respect to the core elements, the promoter
P23S has a sequence (GTTTGC-N12-CTGATT) similar to
the consensus sequence of B. subtilis r
B
-dependent pro-
moters (GTTTAA-N12-14-GGGTAT). r
B
is a sigma factor
that directs RNA polymerase to promoters of B. subtilis
stress regulon. The expression driven by r
B
will be acti-
vated when bacterium is exposured to environmental stress
described above [31]. This is probably related to the
observation of the expression driven by the cloned pro-
moter under osmotic induction.
P23S indicated the characteristic of expression under
salt induction either in E. coli or in B. subtilis. The suc-
cessful expression of the cloned genes further demonstrated
the efciency of this promoter in E. coli. However, the
expression level in B. subtilis was relatively low. B. subtilis
is a Gram-positive sporulating bacterium, and its regulation
system was more complex than that of E. coli. Possibly
there were some unidentied specic regulators in B.
subtilis, such as some repressive factors or specic
enzymes. These factors might be the reason that affected
the expression strength of the promoter P23S in B. subtilis.
In conclusion, we cloned and characterized an osmoti-
cally induced promoter from B. subtilis, in which high
expression strength of promoter was demonstrated in E.
coli. The efciency and strength of this promoter was
demonstrated via the over-production of homologous and
heterologous proteins. Thus, we found a new osmolarity-
inducible promoter and provided a potential expression
element in biotechnology applications.
Acknowledgment The authors gratefully acknowledge the Bacillus
Genetic Stock Centre of Ohio State University for generously pro-
viding the study materials. And this study was supported by the grants
of NSFC (30872033, 31170609).
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