Beruflich Dokumente
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host for a wide range of metagenomic applications. First, (Jones et al., 2000) by homologous recombination of
A. baylyi ADP1 is naturally competent and can take up plasmid pSalAR_Pu_lux to generate A. baylyi ADPWH-
either naked or plasmid DNA making it a suitable alter- Pu-lux (Fig. 1). Plasmid pSalAR_Pu_lux contains frag-
native to E. coli for genetic engineering (Palmen and ments of the salA and salR genes cloned either side of
Hellingwerf, 1997; Barbe et al., 2004; Metzgar et al., Pu-luxCDABE, and is derived from pGEM-T [Promega,
2004). The DNA transformation efficiency of ADP1 can UK (Huang et al., 2005)], which belongs to a family of
reach 10-2 (de Vries et al., 2003), which is more efficient cloning vectors that have been shown not to replicate in
than calcium chloride-treated E. coli (Palmen and Helling- strain A. baylyi ADP1 (Young and Ornston, 2001). Recom-
werf, 1997; Barbe et al., 2004; Metzgar et al., 2004). bination of pSalAR_Pu_lux with the mutated salA gene of
Second, A. baylyi ADP1 can easily perform multigene A. baylyi ADPW67 results in replacement of the disrupted
trappings which may overcome the in cis transcription salA gene with a functional gene, and transformants could
limitation associated with single trapping in SIGEX. Third, be identified on the basis of their ability to grow on 2.5 mM
A. baylyi ADP1 is able to express a wide range of salicylate as sole carbon source (SAA). The frequency
heterologous genes (Kok et al., 1999; Melnikov and of the gene transformation was 6.1 ⫾ 0.7 ¥ 10-6. Poly-
Youngman, 1999; Huang et al., 2005; Young et al., 2005), merase chain reaction (PCR) amplification and DNA
implying that the gene expression and protein folding in sequencing using the primer pairs salA_flank_for
A. baylyi ADP1 may be highly robust. Finally, it is pro- and luxC_rev, and salAR_rev and luxE_fwd (Fig. 1 and
posed that A. baylyi ADP1 should have similar regulation Table 2), confirmed the presence of the Pu promoter and
machinery to the bacteria present in natural water and luxCDABE cassette in the chromosome of ADPWH-
soils, which makes it an ideal host to reconstruct complex Pu-lux (Fig. 1). We have previously shown that the salA
regulatory processes involving complex regulators and promoter is strong enough to transcribe large inserts
signal chemicals. between salA and salR (Huang et al., 2005), and this was
In this study, we cloned the RpoN (s54)-dependent confirmed here as ADPWH-Pu-lux was able to grow on
Pu–XylR regulatory system into the chromosome of salicylate agar (SAA; data not shown) and displayed
A. baylyi ADP1, as a foundation for future studies involv- salicylate-induced bioluminescence, indicating that salA
ing multigene cloning and acclimatization of exogenous and salR were functional and expressed (Figs S1A
DNA. For this purpose, we have fused the Pu promoter and S2A).
with a bioluminescence reporter operon luxCDABE. The Second, xylR-Km was inserted into the salA gene of
corresponding regulatory gene xylR from the TOL plasmid ADPWH-Pu-lux to create A. baylyi ADPWH-Pu-lux-xylR
of P. putida mt-2 was inserted into an adjacent location in by using plasmid pSalA_Km_xylR as a donor and
the chromosome. We examined the effects of inducers, ADPWH-Pu-lux as a recipient (Fig. 1). Plasmid pSalA_
carbon sources and amino acids on activation of the Pu Km_xylR contains a xylR-terminator-Km cassette flanked
promoter. We confirmed that the Pu promoter–XylR regu- by two homologous fragments of salA (Fig. 1). The trans-
latory system not only functioned in A. baylyi ADP1, but formants were selected on Luria–Bertani (LB) agar with
also reproduced the expression characteristics observed 10 mg ml-1 kanamycin. The transformation frequency was
in its original host – P. putida mt-2. We also revealed that 7.7 ⫾ 0.3 ¥ 10-6. The insertion of the xylR-terminator-Km
the repression of the Pu promoter can be caused by cassette completely prevented read-through from the
aspartic acid or asparagine, but not glutamine, although salA promoter, and A. baylyi ADPWH-Pu-lux-xylR dis-
all these three amino acids are involved in bacterial nitro- played no expression of luxCDABE in the presence of
gen metabolism. Our results support the proposition that salicylate (Fig. S2B). Unlike the eukaryotic luc reporter
A. baylyi ADP1 could be a useful alternative host for func- (Willardson et al., 1998), the bacterial reporter operon
tional metagenomics. luxCDABE provides self-sufficient bioluminescence
expression without addition of the substrate (e.g. luciferin)
for the reaction. The luxCDABE reporter acts as a more
Results and discussion sensitive reporter of gene expression than lacZ in
A. baylyi ADP1, and permits detection of gene expression
Construction of A. baylyi ADPWH-Pu-lux-xylR
over a wider range, which allows the unambiguous detec-
To investigate the behaviour of the Pu promoter–XylR tion of promoter activity (Siehler et al., 2007).
regulatory system in A. baylyi ADP1, we constructed a
bioluminescence reporter mutant strain, ADPWH-Pu-lux-
Pu expression in A. baylyi ADPWH-Pu-lux-xylR is
xylR, in which luxCDABE was fused with the Pu promoter
induced by toluene and xylene
and regulated by XylR expressed in trans.
First, Pu-luxCDABE (6163 bp) was inserted into the Exposure of ADPWH-Pu-lux-xylR colonies grown on LB
salAR operon of the salA mutant A. baylyi ADPW67 agar plates to toluene, m-, p- or o-xylene vapours
Fig. 1. Outline of construction of the A. baylyi ADPWH-Pu-lux-xylR bioluminescence reporter. ADPWH-Pu-lux was produced by integrating the
suicide plasmid pSalAR_Pu_lux containing Pu promoter-luxCDABE flanked on either side by salA and partial salR sequences, into ADPW67
and replacement of the kanamycin (Km) resistance cassette in salA. The resulting Km-sensitive strain ADPWH-Pu-lux can grow on SAA plates
as it now contains an uninterrupted copy of salA. The suicide plasmid pSalA_Km_xylR containing a Km resistance gene, terminator and xylR
gene flanked on either side of partial salA (salA1 and salA2), was then integrated into ADPWH_Pu_lux to give the Km-resistant strain
ADPWH-Pu-lux-xylR. The terminator sequence was included between the xylR and Km genes to prevent transcriptional read-through of
luxCDABE from the salA promoter without activation of the Pu promoter. The correct insertion of each of these chromosomal cassettes was
confirmed by colony-PCR and sequencing. DNA length is not shown to scale.
10000 o-xylene
toluene
Pu promoter activation in A. baylyi ADPWH-Pu-lux-xylR
is growth medium dependent
1000
The Pu promoter–XylR regulatory system is RpoN (s54)
dependent (Cases and de Lorenzo, 2005). The results
100
described above confirm that the RpoN (s54) protein of
A. baylyi ADP1 can substitute for the function of the
P. putida RpoN and allows the XylR and the Pu promoter
10 to function well in ADPWH-Pu-lux-xylR, in a similar way to
0 0.5 5 50 500 its original host P. putida. XylR and growth medium-
Inducer concentration (µM) dependent regulation of the Pu promoter in Pseudo-
monas has been well documented (Marques et al., 1994;
Fig. 2. Sensitivity of A. baylyi ADPWH_Pu_lux_xylR to toluene and
xylenes (P < 0.05). Bioluminescence was measured at OD600 = 0.8
Cases et al., 1996) (for review, please see Cases and
in LB medium. The error bars represent standard error for three de Lorenzo, 2005), so experiments were performed to
bioluminescence readings from three replicate samples. test whether growth medium-dependent regulation of Pu
expression in ADPWH-Pu-lux-xylR was also similar to
regulation in P. putida. As in its original host P. putida
(generated by placing a filter tip dipped in substrate on the
mt-2, the XylR-regulated Pu promoter in A. baylyi
agar plate) resulted in strong bioluminescence while
ADPWH-Pu-lux-xylR was repressed during exponential
un-induced colonies remained dark (Fig. S1B). However,
growth phase when bacteria were grown in rich medium
no bioluminescence was observed after exposure to
such as LB, in spite of the presence of toluene or xylenes,
phenol, benzene, naphthalene, sodium salicylate (sodium
and induced in stationary phase (Fig. 3A). This phenom-
2-hydroxybenzoate), 3-hydroxybenzate, 4-hydroxyben-
enon is classically referred to as ‘exponential silencing’
zate, benzoate or catechol (data not shown), which indi-
(Cases et al., 1996). However, the Pu promoter in
cates that the Pu promoter–XylR regulatory system in
A. baylyi ADPWH-Pu-lux-xylR was immediately activated
A. baylyi ADPWH-Pu-lux-xylR specifically responds to
regardless of growth phase when bacteria were grown
toluene and xylenes, as observed in P. putida mt-2. These
in minimal medium-glucose (MMG) in the presence of
results provide further evidence that salicylate dependent
toluene or xylene (Fig. 3B), which is in good agreement
expression of luxCDABE was blocked by introduction of
with observations of Pu-dependent mRNA expression in
the xylR-terminator-Km construct, and confirm that the
P. putida (Marques et al., 1994). Interestingly, addition of
xylR-terminator-Km construct did not prevent toluene/
5 g l-1 yeast extract to MMG medium repressed the Pu
xylenes dependent expression of the luxCDABE cassette.
promoter in ADPWH-Pu-lux-xylR during the first 7 h of
ADPWH-Pu-lux, which does not contain xylR in the chro-
growth (Fig. 4). Collectively, these experiments show that
mosome, cannot be induced by toluene or xylene (Fig.
the activation of Pu promoter in A. baylyi ADPWH-Pu-lux-
S2), which confirms that induction of lux expression in
xylR can be affected by the growth medium. The Pu
ADPWH-Pu-lux-xylR is mediated by XylR, rather than a
promoter’s immediate activation in minimal medium but
native xylR-like regulator. This is supported by BLASTP
significant delay in the presence of yeast extract suggests
and Pfam domain analyses of the genome sequence of
the repression of Pu promoter could be related to some
A. baylyi ADP1, which show that this strain does not
chemicals in yeast extract.
contain any regulatory proteins that display significant
similarity to XylR.
Aspartic acid and asparagine but not glutamine
ADPWH-Pu-lux-xylR is not only highly specific but also
represses Pu promoter activity in ADPWH-Pu-lux-xylR
highly sensitive, capable of detecting toluene or xylenes
as low as 0.5 mM in liquid cultures grown to late log Rich media such as LB and MMG supplemented with
phase (Fig. 2). The bioluminescence became stronger as yeast extract contain more complex chemical compounds
toluene/xylene concentration increased within the range than minimal medium such as MMG, including high
0.5–500 mM (Fig. 2), showing that ADPWH-Pu-lux-xylR concentrations of amino acids. Therefore, the delay of Pu
can be used to quantify XylR-dependent regulation of Pu promoter activation in LB and MMG with yeast extract
promoter expression. Acinetobacter ADPWH-Pu-lux-xylR may be associated with global regulatory mechanisms
cannot metabolize toluene and toluene more or less has linked to amino acid metabolism, as proposed by
inhibit effect on cells growth. Because of its high specific- Marques and colleagues (1994). As the Pu promoter is
20000 2 20000 2
LB
1.6
LB A1 1.6 LB+m-xylene A2
18000 LB+toluene 18000
OD600
1.2
1.2
OD600
16000 16000 0.8
0.8 0.4
Relative Bioluminescence
14000 14000 0
Relative Bioluminescence
0.4
0 10 20 30
12000 12000 Time (h)
0
0 10 20 30
10000 Time (h)
10000
8000 8000
LB-Biolum
6000 LB-Biolum 6000
LB+m-xylene-Biolum
LB+toluene-Biolum
4000 4000
2000 2000
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (h) Time (h)
20000 MM+glu
2 MM+glu
B1 20000 B2 2 MM+glu+m-xylene
MM+glu+toluene
1.6 1.6
18000
OD600
18000 1.2
OD600
1.2
16000 0.8
0.8 16000
0.4
0.4
Relative Bioluminescence
0
Relative Bioluminescence
14000 14000 0 10 20 30
0 Time (h)
0 10 20 30
12000 12000
Time (h)
10000 10000
8000 8000
6000 6000
4000 4000
MM+glu-Biolum
MM+glu-Biolum MM+glu+m-xylene-Biolum
2000 2000
MM+glu+toluene-Biolum
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (h) Time (h)
Fig. 3. Pu promoter activity in A. baylyi ADPWH-Pu-lux-xylR is dependent on the growth media (P < 0.05). In LB (rich medium), the Pu
promoter was only induced after 10–15 h even in the presence of toluene (A1), m-xylene (A2), p-xylene (A3) or o-xylene (A4). However, in
MMG (minimal medium), Pu promoter was induced immediately in the presence of toluene (B1), m-xylene (B2), p-xylene (B3) or o-xylene
(B4). MM, minimal medium.
RpoN (s54) dependent and rpoN expression is known to estimated individual amino acid concentration in yeast
be repressed by high nitrogen availability in P. putida extract. The addition of aspartic acid (3.76 mM) or aspar-
(Cases and de Lorenzo, 2005), three amino acids: aspar- agine (3.78 mM) to MMG fully repressed the Pu promoter
tic acid, asparagine and glutamine, which are associated for 23 h, the period of observation (Fig. 4). In contrast, the
with bacterial nitrogen metabolism, were tested for their addition of glutamine (3.42 mM) to MMG immediately
effects on Pu promoter repression. Minimal medium- activated and enhanced Pu promoter activity (Fig. 4).
glucose was supplemented with 0.5 g l-1 aspartic acid, However, despite these differences, ADPWH-Pu-lux-xylR
asparagine or glutamine, which is equivalent to the displayed similar growth characteristics in MMG supple-
OD600
LB 1.2
18000 1.6 LB+p-xylene 18000
0.8
OD600
1.2 16000 0.4
16000
Relative Bioluminescence
0.8 0
Relative bioluminescence
14000 14000 0 10 20 30
0.4 Time (h)
12000 12000
0
0 10 20 30
10000 10000
Time (h)
8000 8000
2000 2000
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (h) Time (h)
MM+glu
20000 MM+glu+p-xylene 20000 MM+glu
2
1.6
B4 2 MM+glu+o-xylene
18000 18000 1.6
B3
OD600
OD600
1.2
1.2
16000 0.8 16000
Relative Bioluminescence 0.8
0.4 0.4
14000 0 14000 0
Relative Bioluminescence
0 10 20 30 0 10 20 30
Time (h)
Time (h)
12000 12000
10000 10000
8000 8000
6000 6000
4000 4000
MM+glu-Biolum
MM+glu-Biolum
2000 2000 MM+glu+o-xylene-Biolum
MM+glu+p-xylene
0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time (h) Time (h)
Fig. 3. cont.
mented with glutamine, aspartic acid or asparagine nitrogen status (Arcondeguy et al., 2001). The main
(Fig. 4). Cell densities in MMG supplemented with amino pathway for glutamine assimilation in Acinetobacter is
acids were twice higher than in MMG-only medium, likely to involve conversion of glutamine to glutamate
indicating that ADPWH-Pu-lux-xylR could use glutamine, and then to the TCA cycle intermediate oxoglutarate. In
aspartic acid and asparagine as nitrogen and carbon contrast, asparagine is converted to aspartate and then
sources. This shows that the Pu promoter can be to fumarate, the TCA cycle intermediate immediately
repressed or induced in response to the presence and downstream of succinate. All three amino acids can be
metabolism of a single, specific amino acid, as suggested used to generate energy via the TCA cycle, but only
for the Pu promoter–XylR regulatory system in P. putida glutamine metabolism also acts to signal high nitrogen
(Marques et al., 1994). status, which could help to explain why asparagine and
Glutamine is an important metabolite in bacterial aspartate both act as repressors of Pu expression, but
physiology (Forchhammer, 2007) and an indicator of glutamine does not.
OD600
inducing substrate. Bacteria grew at similar
0.8 rates in media supplemented with YE or
50000 amino acids (inset). The Pu promoter was
0.4
immediately induced and expressed at an
increased level in MMG-glutamine, but
expression was delayed 7 h in YE. Pu
Relative bioluminescence
0
40000 0 10 20 30 promoter activity was significantly inhibited in
Time (h) MMG-aspartate and MMG-asparagine. MM,
minimal medium.
30000
20000
10000
0
0 5 10 15 20 25 30
Time (h)
Carbon source effect on Pu activity of activity in P. putida (Cases et al., 1999). Acinetobacter
ADPWH_Pu_lux_xylR baylyi ADP1 lacks a glucose PTS system and oxidizes
glucose to gluconate in the periplasm (Barbe et al., 2004;
When multiple carbon sources are available, bacteria
Young et al., 2005), and this characteristic of glucose
preferentially use economically favourable substrates
(Holtel et al., 1994). Bacteria achieve this regulation
through carbon catabolite repression (CCR) (Stulke 20000
and Hillen, 1999). In the presence of preferred carbon 18000 Succinate
sources (typically glucose), bacteria will repress the 16000 Glucose
Relative bioluminescence
assimilation might explain the lack of glucose-induced pairs in the presence and absence of inducing sub-
catabolite repression in ADPWH-Pu-lux-xylR. Velazquez strates, thereby isolating novel degradative and reg-
and colleagues (2004) have proposed that the Entner– ulatory genes from both culturable and unculturable
Doudoroff catabolites 6-phosphogluconate (6-PG) and bacteria.
2-dehydro-3-deoxyphosphogluconate (KDPG) are signal
molecules for Pu promoter repression in P. putida. Acine- Experimental procedures
tobacter baylyi ADP1 lacks both a G6P dehydrogenase
Bacterial strains and media
and 6-phosphogluconolactonase required to produce
these catabolites from G6P (Barbe et al., 2004). ADPWH- The bacterial strains and plasmids used in this study are
Pu-lux-xylR was found to be able to grow on 6-PG as the listed in Table 1. Acinetobacter baylyi ADP1 and its
sole carbon source; it indicates 6-PG can be transported mutants were incubated at 28°C or 30°C, and E. coli at
into the cells as carbon source. However, the addition of 37°C. All chemicals were obtained from Sigma-Aldrich
6-PG ranging from 0.1 to 300 mM in LB media had no and were analytical grade reagents. Luria–Bertani
effect on Pu promoter activity (data not shown). It sug- medium or minimal medium (Huang et al., 2005) was
gests that KDPG instead of 6-PG should be a repression used for the cultivation of bacteria as appropriate. Minimal
signal of Pu promoter activity in Acinetobacter baylyi medium-glucose and MMS were prepared by the addition
ADP1, which is in good agreement with the observation of of 30 mM glucose or succinate to minimal medium.
the XylR-regulated Pu promoter in P. putida KT2440 host Salicylate agar was prepared using 2.5 mM salicylate
by del Castillo (del Castillo and Ramos, 2007). as a sole carbon source in minimal medium with 1.4%
Noble agar (Marine BioProduct, Canada). Amino acid-
supplemented media were prepared by adding 5 g l-1
The effect of carbon/nitrogen ratio on Pu
yeast extract (YE), 0.5 g l-1 aspartic acid, 0.5 g l-1 aspar-
promoter activity
agine or 0.5 g l-1 glutamine to MMG. Benzene, benz-
Carbon metabolism is controlled by carbon-derived oates, catechol, naphthalene, phenol, toluene, m-xylene,
signals and the availability of nitrogen and other nutrients p-xylene, o-xylene, 2-hydroxybenzoic acid (salicylic acid),
(Commichau et al., 2006). We examined Pu promoter 3-hyroxybenzoic and 4-hydroxybenzoic were also added
activity in MMG supplemented with varying concentra- to plates or cultures for induction or growth tests. Ampi-
tions of ammonia or nitrate, and containing m-xylene as cillin (Amp) at 100 mg ml-1 and 50 mg ml-1 kanamycin (Km)
an inducing substrate. Expression of the Pu promoter was used for E. coli, and 10 mg ml-1 Km for A. baylyi ADP1
increased in media with a high carbon/nitrogen ratio with or its mutants when required.
nitrate as a nitrogen source, but showed no significant
change in media with ammonia as a nitrogen source
Gene transformation
(Fig. 6). XylR activates Pu expression by activating the
alternate sigma factor RpoN, which also regulates genes Preparation of competent A. baylyi ADP1 cells was per-
involved in nitrogen assimilation. In P. putida, rpoN formed as described previously (Palmen et al., 1993;
expression is modulated by nitrogen availability (Cases Huang et al., 2005). Briefly, ADPW67 or ADP1_Pu_lux
and de Lorenzo, 2005), so it is possible that nitrogen was grown in 5 ml of LB at 30°C with shaking at 200 r.p.m.
modulates Pu expression as a result of the effect of nitro- overnight. Two hundred microlitres of culture was then
gen on the activity and expression of RpoN. diluted into 5 ml of fresh LB medium and incubated for 2 h
In conclusion, this study has shown that the substrate, to make the cells competent. Cells were harvested by
nutrient and growth-dependent properties of the Pu 3000 r.p.m. centrifugation and re-suspended in 0.5 ml of
promoter–XylR system can be reconstituted in a surro- fresh LB. Two micrograms of intact plasmid DNA was
gate host, A. baylyi ADP1, which accurately mimics the added to the 0.5 ml competent cells (109 cells ml-1) and
regulatory and physiological characteristics of bacterial incubated for 2 h at 30°C. The cells were then plated on
species commonly found in natural habitats such as soils selective media to obtain the appropriate transformants.
and groundwater. Acinetobacter baylyi ADP1 is likely to
provide a better genetic host than E. coli, at least in this
Molecular techniques materials
case, for functional metagenomic approaches such as
SIGEX, as E. coli can show non-specific compound Escherichia coli JM109 (Promega, UK) was used for
induction and non-characteristic regulation (Willardson plasmid cloning and preparation according to the manu-
et al., 1998), particularly when expressing plasmid-borne facturer’s instructions. All oligonucleotide primers were
genes. The chromosomal integration strategy described obtained from MWG Biotech (http://www.mwg-biotech.
in this study can easily be adapted to screen libraries of com/) and are listed in Table 2. QIAquick gel extraction kit
DNA fragments containing putative regulator–promoter (Qiagen, UK) was used to purify DNA from agarose
OD600
C/N=6 significant effect on Pu activity in A. baylyi
16000 C/N=8 0.8 ADPWH-Pu-lux-xylR (P > 0.1).
C/N=10 B. Varying the carbon/nitrogen ratio by
0.4 providing nitrate as sole nitrogen source and
Relative Bioluminescence
6000
4000
2000
0
0 5 10 15 20 25 30
Time (h)
B. NO3-
2 C/N=1
20000 C/N=1 C/N=4
1.6 C/N=6
C/N=4 C/N=8
OD600
1.2 C/N=10
18000 C/N=6
C/N=8 0.8
C/N=10 0.4
16000
0
0 10 20 30
14000 Time (h)
Relative Bioluminescence
12000
10000
8000
6000
4000
2000
0
0 5 10 15 20 25 30
Time (h)
gels for cloning or sequencing as the manufacturer’s in- an initial denaturation at 95°C for 5 min, followed by 35
structions. Plasmid construction and chromosomal in- cycles of 94°C for 1 min, 58°C for 1 min and 72°C for
sertions were confirmed by PCR and DNA sequencing. 1 min, and a final extension at 72°C for 10 min. After
Polymerase chain reaction was carried out using a amplification, 10 ml of PCR product was examined by
Sigma PCR kit. For colony-PCR, single-colony material agarose-ethidium bromide gel electrophoresis. The PCR
re-suspended in 50 ml of PCR reaction mixture was used fragments from four clones were purified from the gel
as DNA template material. (QIAquick gel extraction kit, Qiagen) and confirmed by
DNA sequencing. A clone containing a plasmid in which
the Pu promoter sequence was in the same orientation as
Plasmid construction
luxCDABE was selected and designated as pSalAR_Pu_
Constructing pSalAR_Pu_lux. The 320 bp Pu promoter lux (Fig. 1).
fragment was excised from pMC2 by EcoRI and ligated
with partially EcoRI-digested pSalAR_lux (Huang et al.,
Constructing pSalA_Km_xylR
2005) (Fig. 1). Escherichia coli JM109 competent cells
(Promega, UK) were transformed with the ligation mixture, • Overlap extension PCR to create restriction cut sites.
and then spread on LB Amp for selection. Sixteen colo- EcoRI and BamHI restriction sites were created within the
nies were investigated by colony-PCR (Sigma PCR kit) salA gene by overlap extension PCR (OEP) as previously
using primers salA_end_for and luxC_rev (Table 2), with described (Huang et al., 2005). Two salA fragments,
salA1 (907 bp) and salA2 (924 bp), were separately Pu-lux-xylR. The presence of xylR was confirmed by
amplified by PCR using a colony of ADP1 as DNA colony-PCR using the primer pair xylR1_for and xylR_rev
template and the primer pairs salA_flank_for–salA_BE_ (Table 2). Failure to grow on SAA was used to confirm
rev and salA_BE_fwd–salA_revH (Table 2). Polymerase homologous recombination of ADPWH-Pu-lux-xylR.
chain reaction amplifications were performed with an
initial denaturation at 95°C for 5 min, followed by 35
Nucleotide sequencing and sequence analysis
cycles of 94°C for 1 min, 58°C for 1 min and 72°C for
1 min, and a final extension at 72°C for 10 min. The All DNA samples (PCR products or plasmids) were
PCR products were isolated from an agarose gel using sequenced using dye terminator sequencing on an
QIAquick gel extraction kit (Qiagen, UK). To fuse the two Applied Biosystems 3730 DNA analyser. DNA sequences
salA fragments, a PCR amplification (using the same were aligned and edited using BioEdit (Tom Hall, Depart-
reaction conditions as above, except an extension time ment of Microbiology, North Carolina State University) to
at 72°C for 2 min) was carried out using 1 ml of diluted confirm correct insertions. The plasmid pSalAR_Km_xylR
(1:100) salA1 and salA2 fragments as DNA templates has been fully sequenced and submitted to the National
and primers salA_flank_for and salA_revH (Table 2). The Center for Biotechnology Information (NCBI) and the
resultant PCR product of the OEP salA fragment contains accession number is DQ202262.
EcoRI and BamHI restriction sites. It was isolated from an
agarose gel (QIAquick gel extraction kit, Qiagen) and
Acinetobacter baylyi ADPWH_Pu_lux and
then cloned into pGEM-T (Promega) to give pSalA_BE
ADPWH_Pu_lux_xylR induction
(Fig. 1).
All experiments were carried out in triplicate and bio-
• Construction of pSalA_Km_xylR. The kanamycin gene luminescence assay was measured in triplicate, and
(1472 bp) was excised from pRMJ2 (Jones and Williams, means ⫾ standard error are presented. The T-test was
2003) by EcoRI and BamHI and ligated into EcoRI– performed using Excel (Microsoft Software) for statistical
BamHI-digested pSalA_BE to create pSalA_Km (Fig. 1). analysis. Pu promoter activity was monitored by measur-
The xylR gene fragment (2399 bp) was excised from ing relative bioluminescence (bioluminescence divided by
pTS174 by EcoRI and ligated into EcoRI-digested OD600). For each measurement, at each time point 100 ml
pSalA_Km to create pSalA_Km_xylR (Fig. 1). Colony- of samples were analysed in triplicate in clear-bottomed
PCR and sequencing was performed to confirm that xylR black 96-well optical microplates (Nalge Nunc Interna-
had been correctly inserted using primer pairs xylR1_for tional, USA). OD600 and bioluminescence were measured
and xylR_rev (Table 2). The PCR condition was: initial using a Synergy HT Multi-Detection Microplate Reader
denaturation at 95°C for 5 min, followed by 35 cycles of (Bio-Tek, UK).
94°C for 1 min, 50°C for 1 min and 72°C for 2 min, and a
final extension at 72°C for 10 min. Different inducers. A single colony of A. baylyi
ADPWH_Pu_lux_xylR was inoculated in 5 ml of LB liquid
medium placed in a 30 ml glass universal tube, and incu-
Construction of ADP1 mutants
bated at 28°C overnight. The cells were re-inoculated into
The construction of ADPWH-Pu-lux-xylR is outlined in a fresh medium after a 1:25 dilution. Inducers were pre-
Fig. 1. pSalAR_pu_lux was integrated into A. baylyi added into media as appropriate. Toluene, p-, o- or
ADPW67 by homologous recombination and selection m-xylenes, phenol, benzene, naphthalene, 2-hydroxyben-
on SAA to give ADPWH-Pu-lux. Acinetobacter baylyi zoic (salicylic acid), 3-hyroxybenzoic, 4-hydroxybenzoic,
ADPW67 has a kanamycin gene inserted into the salA benzoate or catechol were separately added into LB as
gene and cannot grow on SAA plates with salicylate as a inducer, respectively, with final concentration of 500 mM.
sole carbon source. The integration of pSalAR_pu_lux by Samples were incubated at 28°C with shaking at 150 r.
double-cross-over homologous recombination replaced p.m. OD600 and bioluminescence were measured over a
the disrupted salA gene with a functional copy of the gene period up to 27 h.
and therefore enabled the transformants ADPWH-Pu-lux
to grow on SAA. To confirm the presence of the Pu pro- Induction on solid media. Bacteria were incubated on LB
moter and luxCDABE cassette, colony-PCR reactions agar plates with inducers at 28°C. For salicylate assays
were performed using the primer pairs salA_flank_for and salicylate was added to LB agar to a final concentration
luxC_rev, and salAR_rev and luxE_fwd (Table 2), and the of 2.5 mM. For toluene and xylene assays toluene or
PCR products were sequenced. xylene vapour was used to induce the XylR-regulated
pSalA_Km_xylR was similarly integrated into ADPWH- Pu promoter. Three microlitres of toluene or xylene was
Pu-lux, with selection for Km resistance to give ADPWH- loaded into a filtered tip, which was placed onto LB agar.
Bioluminescent plates were imaged in the dark using a del Castillo, T., and Ramos, J.L. (2007) Simultaneous
Versa-Doc Imaging System (Bio-Rad Laboratories, Herts, catabolite repression between glucose and toluene
UK). Images were captured using the ‘high sensitive metabolism in Pseudomonas putida is channeled through
different signaling pathways. J Bacteriol 189: 6602–6610.
chemiluminescent’ setting for 30 s with a Nikon 50 mm
Commichau, F.M., Forchhammer, K., and Stulke, J. (2006)
lens at f1.4. Regulatory links between carbon and nitrogen metabolism.
Curr Opin Microbiol 9: 167–172.
Carbon and nitrogen source effects. Acinetobacter sp. Dal, S., Steiner, I., and Gerischer, U. (2002) Multiple operons
ADPWH_Pu_lux_xylR was inoculated into MMG, MMS connected with catabolism of aromatic compounds in
and LB to compare the effect of carbon source on Pu Acinetobacter sp. strain ADP1 are under carbon catabolite
activity. Ammonia chloride, sodium nitrate and glucose repression. J Mol Microbiol Biotechnol 4: 389–404.
Duetz, W.A., Marques, S., Wind, B., Ramos, J.L., and
were added into nitrogen-free minimal medium (MMN,
vanAndel, J.G. (1996) Catabolite repression of the toluene
minimal medium without ammonium chloride) to make degradation pathway in Pseudomonas putida harboring
media with carbon/nitrogen ratios of 1, 4, 6, 8 and 10. The pWWO under various conditions of nutrient limitation in
inducers toluene, p-, o- and m-xylene were separately chemostat culture. Appl Environ Microbiol 62: 601–606.
added to a final concentration of 500 mM. Cultures were Forchhammer, K. (2007) Glutamine signalling in bacteria.
incubated at 28°C with 150 r.p.m. shaking. Front Biosci 12: 358–370.
Handelsman, J. (2004) Metagenomics: application of genom-
ics to uncultured microorganisms. Microbiol Mol Biol Rev
Acknowledgements 68: 669–684.
Holtel, A., Marques, S., Mohler, I., Jakubzik, U., and Timmis,
We thank Professor Peter Williams of University of Wales, K.N. (1994) Carbon source-dependent inhibition of
Bangor, for providing A. baylyi ADPW67. Xyl operon expression of the Pseudomonas-Putida Tol
plasmid. J Bacteriol 176: 1773–1776.
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