Plasmid 49 (2003) 2–8
www.elsevier.com/locate/yplas
Development of a gene expression vector for Thermus
thermophilus based on the promoter of the respiratory
nitrate reductase
Renata Moreno,1 Olga Zafra,1 Felipe Cava, and Jose Berenguer*
Departamento de Biologıa Molecular, Centro de Biologıa Molecular Severo Ochoa, Universidad Aut
onoma de Madrid,
Cantoblanco, Madrid 28049, Spain
Received 12 August 2002, revised 12 September 2002
Abstract
A specific expression system for Thermus spp. is described. Plasmid pMKE1 contains replicative origins for Escherichia coli and Thermus spp., a selection gene encoding a thermostable resistance to kanamycin, and a 720 bp DNA
region containing the promoter (Pnar), and the regulatory sequences of the respiratory nitrate reductase operon of
Thermus thermophilus HB8. Two genes, encoding a thermophilic b-galactosidase and an alkaline phosphatase were
cloned in pMKE1 as cytoplasmic and periplasmic reporters, respectively. The expression of the reporters was specifically induced by the combined action of nitrate and anoxia in facultative anaerobic derivatives of T. thermophilus HB27
to which the gene cluster for nitrate respiration was transferred by conjugation. Overexpressions in the range of 200fold were obtained for the cytoplasmic reporter, whereas that of the periplasmic reporter was limited to 20-fold, with
respect to their intrinsic respective activities.
Ó 2002 Elsevier Science (USA). All rights reserved.
Keywords: Thermophilic; Vector; Expression; Thermus; Reporter
1. Introduction
Production of enzymes from thermophilic organisms is most commonly achieved by heterologous expression on a mesophilic host (Coolbear
et al., 1992; Ishida et al., 1997; Vieille et al., 1996).
However, many complex enzymes, like heterooligomers or those requiring covalently bound co*
Corresponding author. Fax: +34-91-397-8087.
E-mail address: [email protected] (J. Berenguer).
1
Both authors have contributed equally to this work.
factors, can rarely be produced by this method,
requiring the purification from their natural thermophilic source. The search of genetic tools for the
overexpression of such enzymes in an homologous
thermophilic context has been an important goal
for many years (de Grado et al., 1999; Lasa et al.,
1992b; Mather and Fee, 1992).
Thermus spp. are an important source of biotechnologically relevant enzymes because of the
high number of strains available all over the world,
and their usually simple requirements for growth
(Coolbear et al., 1992). This, and the natural
0147-619X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
PII: S 0 1 4 7 - 6 1 9 X ( 0 2 ) 0 0 1 4 6 - 4
R. Moreno et al. / Plasmid 49 (2003) 2–8
competence of many Thermus spp. strains (Friedrich et al., 2001; Koyama et al., 1986), prompted
the development of genetic tools for these organisms that presently allow the isolation of insertional mutants (Lasa et al., 1992a), the cloning of
genes in plasmids (Koyama et al., 1990a; Koyama
et al., 1990b; Lasa et al., 1992b; Mather and Fee,
1992) or integrative vectors (Tamakoshi et al.,
1997; Tamakoshi et al., 1999) and the development
of selection method for the thermostabilization of
proteins from mesophiles (Kotsuka et al., 1996;
Tamakoshi et al., 2001; Tamakoshi et al., 1995).
However, the lack of knowledge on the regulation
of potential transcription promoters (Maseda and
Hoshino, 1995) precludes the development of genetic tools for the controlled expression of proteins. In a recent work, we have used the promoter
region of a respiratory nitrate reductase operon
from Thermus thermophilus HB8 (Ramirez-Arcos
et al., 1998a) to express a thermostable eukaryotic
ribozyme (Vazquez-Tello et al., 2002). In this article, we use an improved version of this promoter
to develop a bifunctional Escherichia coli–Thermus
plasmid, in which the expression of a cloned enzyme gene can be induced by adding nitrate to
anoxic or microaerophilic cultures. To analyze
such an expression, we use for the first time in
Thermus two genes encoding thermophilic cytoplasmic and periplasmic reporters. Finally, we
demonstrate that the presence of transcriptional
activators encoded within the mobilizable element
for the respiration of nitrate (nar) is necessary for
such an induction.
2. Materials and methods
2.1. Bacterial strains
Thermus thermophilus HB8 was obtained from
the American Type Culture Collection (Rockville,
Maryland, USA). T. thermophilus HB27 was a gift
of Dr. Koyama. The strain T. thermophilus
HB27::nar is a derivative of HB27 that contains
the nar cluster (Ramirez-Arcos et al., 1998b). The
E. coli strain DH5a [supE44 flacU169 (/80
lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1
relA1] was used for genetic constructions.
3
2.2. Cell growth, transformation, and Pnar induction
Escherichia coli cells were grown in LB (Lennox,
1955) at 37 °C. Kanamycin (30 mg/L) or/and ampicillin (100 mg/L) were used when needed. Aerobic growth of T. thermophilus was developed at
70 °C under shaking (150 r.p.m.) in TB medium
(Ramirez-Arcos et al., 1998a). For plasmid selection, kanamycin (30 mg/L) was added to TB plates.
T. thermophilus transformation was achieved on
naturally competent cells as described (de Grado
et al., 1999). Transformation of E. coli was carried
out as described (Hanahan, 1983). For the induction of the Pnar promoter, cells were grown aerobically in the absence of nitrate up to an OD550 of
0.2, and transcription was activated by the addition
of KNO3 (40 mM) and the simultaneous arresting
of the shaker(time 0 in induction experiments).
2.3. General methods and plasmid construction
Plasmid purification and restriction analysis
were developed as described (Sambrook et al.,
1989). Automatic methods (Applied Biosystems)
were used for DNA sequencing. Plasmid pMK18r is
a derivative of pMK18 (de Grado et al., 1999) in
which the orientation of the kat gene and the
Thermus replicon were reversed. Plamid pKMPA
was previously described (Castan et al., 2002).
Plasmid pPSbgal is a derivative of pUC119 (Viera
and Messing, 1987) that express a b-galactosidase
from Thermus T2 (Koyama et al., 1990b) under the
control of the promoter of the S-layer gene (PslpA)
(Faraldo et al., 1992). Plasmid pNIT3 is a derivative
of pUC119 that contains a 5.4 kbp DNA fragment
encoding the first (narC), and part of the second
(narG) genes of the respiratory nitrate reductase
operon (Ramirez-Arcos et al., 1998a), and upstream
sequences that includes the Pnar promoter region.
Oligonucleotides O-citCNde (50 CGCATATG
CACCTCCGGCCCCA30 ) and pUCreverse (50 C
AGGGAACAGCTATGAC30 ) were used for the
amplification by PCR from plasmid pNIT3 of a
1.8 kbp DNA region containing the Pnar promoter
and upstream sequences. A 720 KpnI–NdeI DNA
fragment from the PCR product was inserted into
a derivative of pMK18r which contained a modi-
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R. Moreno et al. / Plasmid 49 (2003) 2–8
fied multicloning site sequence (Fig. 1A). The sequence of the Pnar promoter was submitted to the
EMBL GenBank (Accession No. AJ504993).
2.4. Enzyme activities
The nitrate reductase (NR) activity was measured at 80 °C (Ramirez-Arcos et al., 1998a) with
methyl viologen as the electron donor and potassium nitrate (40 mM) as the electron acceptor on
entire cells permeabilized with (0.1% w/v) tetradecyl-trimethyl ammonium bromide (Snell and
Snell, 1949). The activities corresponding to the bgalactosidase and the alkaline phosphatase were
assayed at 70 °C on soluble fractions corresponding to samples of equivalent cell mass obtained
Fig. 1. The pMKE1 plasmid. (A) Restriction map of pMKE1. The genes encoding a thermostable resistance to kanamycin (kat) and
the Thermus replication protein (repA) are indicated. Sequences derived from pUC18 are represented as a thick line, in which the
replicative origin for E. coli (oriE) is labeled. The promoter of the nitrate reductase operon (Pnar) is indicated as a plasmid insert.
Symbols for restriction enzymes: (E) EcoRI; (H) HindIII; (K) KpnI; (N) NdeI; (Nc) NcoI; (Nt) NotI; (P)PstI; (S) SalI; (Sp) SphI; (Xb)
XbaI. (B) Sequence of the Pnar promoter. The transcription start is indicated with an arrow. Restriction sites for BamHI, KpnI, and
NdeI are labeled over the sequence. The putative-35 promoter sequence is labeled by a gray frame, the ribosome binding site is in bold,
and the ATG start codon is underlined. There is no obvious -10 promoter sequence.
R. Moreno et al. / Plasmid 49 (2003) 2–8
after disruption of the cells by sonication. The
b-galactosidase activity was assayed with orthonitrophenyl-galactopyranoside (ONPG) as chromogenic substrate in buffer 20 mM Tris–ClH, pH
8. The phosphatase activity was assayed with paranitrophenyl-phosphate as chromogenic substrate,
in a 100 mM Tris–ClH buffer, pH 8. For both
activities, enzyme units were normalized with respect to the cell mass as described by Miller
(Miller, 1972).
3. Results and discussion
3.1. Cloning of reporter genes in pMKE1
The main features of plasmid pMKE1 are depicted in Fig. 1A. The plasmid includes a minimal
replicon for Thermus spp. (de Grado et al., 1998)
that encodes the replication protein (RepA). It
also has a replicative origin from the high copy
number plasmid pUC18 that allows its amplification and the development of constructions in
E. coli, and the kat gene, encoding a thermostable
kanamycin nucleotidil transferase transcribed
from a bifunctional E. coli–Thermus promoter,
that can be selected both at 37 and 70 °C (Lasa
et al., 1992a). For the expression in Thermus spp.,
a 720 bp DNA region, that contains the Pnar
promoter and downstream sequences encoding the
leader mRNA from the first gene (narC) of the
operon encoding for the respiratory nitrate reductase from T. thermophilus, was included (Fig.
1B). Single restriction sites for NdeI, NcoI, EcoRI,
SalI, HindIII, and NotI are located downstream
Pnar, in such a way that the NdeI site overlaps the
translation start codon (CATATG) located at the
adequate distance from a Thermus ribosome
binding site (GGAGG).
To evaluate the ability of pMKE1 to direct the
controlled expression of desired proteins, we chose
two genes, one encoding a b-galactosidase from a
Thermus spp. (Koyama et al., 1990b) as cytoplasmic reporter, and the other encoding a periplasmic
alkaline phosphatase (AP) from T. thermophilus
(Castan et al., 2002), respectively. In both instances, we used NdeI (50 ) and HindIII (30 ) restriction sites to isolate the coding region of these
5
genes from plasmids pPSbgal and pKMPA. The
resulting plasmids were named pMKEbgal and
pMKEPA, respectively.
3.2. Expression of cytoplasmic and periplasmic
reporters in T. thermophilus
Preliminary work from our laboratory, had
shown that the narCGHJIK1K2 operon, encoding
the respiratory nitrate reductase of T. thermophilus,
was located within a DNA region (‘‘nar element’’)
that could be transferred by conjugation from its
natural host, a facultative anaerobe strain of T.
thermophilus, to the aerobic strain T. thermophilus
HB27, allowing the exconjugant (HB27::nar) to
grow anaerobically (Ramirez-Arcos et al., 1998b).
It was also shown in that work that in both, the
natural host and the exconjugant strains, the expression of the nitrate reductase required the
combined action of anoxia and nitrate (RamirezArcos et al., 1998a). Thus, these data suggested the
existence of two regulatory proteins, one signaling
anoxia and the other nitrate, in a way similar to
that of the expression of the nitrate reductase A in
E. coli. In this system, the reduced FNR protein
(anoxia detection) and the phosphorilated NarL
component of the NarX/L two-component system
(nitrate signaling), bind to specific DNA sequences
upstream the -35 promoter box, leading to the recruitment of the RNA polymerase and the subsequent transcriptional activation of the promoter
(Unden and Bongaerts, 1997).
In order to check if such a regulatory circuitry
was associated with the presence of the ‘‘nar element,’’ the natural host of the element, and the
aerobic (HB27) and its facultative anaerobic
derivative (HB27::nar), were transformed with
pMKE1, pMKEbgal, and pMKEPA, and inductions experiments were developed as described in
Section 2.
In Fig. 2A we show the expression of the bgalactosidase activity along the time in the three
strains. As it can be seen, whereas in the aerobic
HB27 strain the b-galactosidase activity remained
constant along the time (30–40 units), a clear induction was detected after 2 h of static incubation
with nitrate in its HB27::nar derivative, reaching
its maximum (6800 units) after 20 h of incubation.
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R. Moreno et al. / Plasmid 49 (2003) 2–8
Fig. 2. Expression of reporter genes. Parallel aerobic cultures of T. thermophilus strains HB8 (natural host of the ‘‘nar element’’), HB27
and HB27::nar transformed with plasmids pMKE1, pMKEbgal, or pMKEPA were subjected to induction by stopping the shaker and
the simultaneous addition of nitrate. The b-galactosidase (A) or alkaline phosphatase (B) activities were followed along the incubation
time.
Therefore, the expression from the Pnar promoter
responds to positive controls encoded by the ‘‘nar
element.’’ It is also interesting to note that the
presence of plasmid pMKEbgal does not increase
the intrinsic b-galactosidase activity of the
HB27::nar strain in uninduced cultures, which remains at the same level as when the plasmid
pMKE1 was used for transformation, whereas in
the natural host of the ‘‘nar element’’ a 3-4 fold
expression of the b-galactosidase activity (130
units) was evident even before the induction. This
suggests a lower threshold level for the expression
from Pnar in the natural host than in the exconjugant strain. Whether the difference in sensitivity
was at the level of nitrate or oxygen detection can
not be deduced at present, and merits future investigations, but we can conclude that it is not an
effect related to the construction of the plasmid
pMKE1. In any case, and from an applicative
point of view, the fact that Pnar remained completely silent in uninduced cultures of the
HB27::nar strain, makes this promoter/host system
a good candidate for the expression in T. thermophilus of putatively toxic proteins.
The expression of the periplasmic reporter
confirmed the above conclusions about the re-
quirement of transcriptional activators encoded by
the ‘‘nar element’’ for the induction of Pnar (Fig.
2B). However, the overexpression level observed
with this periplasmic reporter was much lower (10to 20-fold the basal level) than with the b-galactosidase, a limitation that could be the additive
consequence of two factors. On one hand, the
basal phosphatase activity of this strain represents
probably an overestimation, as to it can contribute
up to four different phosphatases (Pantazaki et al.,
1998). On the other hand, the AP expression seems
to be saturated after 3 h, probably as consequence
of the existence of a ‘‘bottle neck’’ at the secretion
level. In this sense, the signal peptide of the alkaline phosphatase overexpressed contains two arginines at its N-terminus, which allow its secretion
through the Tat (twin arginine transport) system
of E. coli (Angelini et al., 2001). Therefore, it could
be possible that a similar system was a limiting
step for enzyme overproduction in Thermus.
3.3. Concluding remarks
In conclusion, we have described a plasmid/host
system that can be used for the overexpression of
selected proteins directly in T. thermophilus up to a
R. Moreno et al. / Plasmid 49 (2003) 2–8
level 200-fold higher for a cytoplasmic enzyme,
and, at least, 10-20 for periplasmic ones. This
system is based on a complex promoter which responds to the simultaneous effects of two inexpensive input signals (nitrate and anoxia). It is
likely that these signals interact with specific receptors that, either directly as in the case of FNR,
or through a signal transduction system, like
NarX/L, bind to the promoter and form a nucleoprotein complex that is competent for recruiting
the RNA polymerase to start the transcription. It
is also clear from our experiments that these activators are coded by the ‘‘nar element.’’ In fact, we
have identified recently an homologue of the FNR
family encoded upstream of the narCGHJIK1K2
operon which could be one of the regulators proposed (unpublished results). Nevertheless, the
Pnar promoter is not active in E. coli (not shown),
as it could be expected from the absence of any of
the sequence binding motifs so far described for
FNR and NarL factors, making necessary to
identify and purify such specific factors from
Thermus spp. to analyze their interactions with the
Pnar promoter.
Acknowledgments
This work has been supported by project no.
BIO2001-1267 from the MCYT. An institutional
grant from Fundaci
on Ram
on Areces is also acknowledged. O. Zafra is the holder of a fellowship
from Comunidad de Madrid. F. Cava holds a
fellowship from MCYT. The financial support of
BIOTOOLS B & M to R. Moreno is also acknowledged.
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Communicated by M. Espinosa