Download Expression of the six chromate ion transporter

Microbiology (2014), 160, 287–295
DOI 10.1099/mic.0.073783-0
Expression of the six chromate ion transporter
homologues of Burkholderia xenovorans LB400
Yaned M. Acosta-Navarrete,1 Yhoana L. León-Márquez,1
Karina Salinas-Herrera,1 Irvin E. Jácome-Galarza,2 Vı́ctor Meza-Carmen,1
Martha I. Ramı́rez-Dı́az1 and Carlos Cervantes1
1
Correspondence
Instituto de Investigaciones Quı́mico-Biológicas, Universidad Michoacana, Morelia, Michoacán,
Mexico
Carlos Cervantes
[email protected]
2
Laboratorio Estatal de Salud Pública, Secretarı́a de Salud de Michoacán, Morelia, Michoacán,
Mexico
Received 2 October 2013
Accepted 20 November 2013
The chromate ion transporter (CHR) superfamily comprises transporters that confer chromate
resistance by extruding toxic chromate ions from cytoplasm. Burkholderia xenovorans strain
LB400 has been reported to encode six CHR homologues in its multireplicon genome. We found
that strain LB400 displays chromate-inducible resistance to chromate. Susceptibility tests of
Escherichia coli strains transformed with cloned B. xenovorans chr genes indicated that the six
genes confer chromate resistance, although under different growth conditions, and suggested
that expression of chr genes is regulated by sulfate. Expression of chr genes was measured by
quantitative reverse transcription-PCR (RT-qPCR) from total RNA of B. xenovorans LB400 grown
under different concentrations of sulfate and exposed or not to chromate. The chr homologues
displayed distinct expression levels, but showed no significant differences in transcription under
the various sulfate concentrations tested, indicating that sulfate does not regulate chr gene
expression in B. xenovorans. The chrA2 gene, encoded in the megaplasmid, was the only chr
gene whose expression was induced by chromate and it was shown to constitute the chromateresponsive chrBACF operon. These data suggest that this determinant is mainly responsible for
the B. xenovorans LB400 chromate resistance phenotype.
INTRODUCTION
The presence of high concentrations of chromate (hexavalent chromium) in the environment has selected microorganisms possessing mechanisms that allow them to
tolerate the toxic oxyanion (reviewed by Cervantes et al.,
2001). Bacterial resistance to chromate has been documented widely and may be conferred by chromosomal or
plasmid genes (reviewed by Ramı́rez-Dı́az et al., 2008). The
best-studied bacterial chromate resistance system is that of
the Pseudomonas aeruginosa ChrA membrane protein,
which functions as a chemiosmotic pump that expels
chromate from cell cytoplasm using the proton motive
force (Alvarez et al., 1999). ChrA belongs to the chromate
ion transporter (CHR) superfamily (Nies, 2003) that
includes hundreds of homologues from all three domains
of life (Dı́az-Pérez et al., 2007; Henne et al., 2009). The
The authors dedicate this paper to the memory of Professor Jesús
Caballero-Mellado who passed away in October 2010.
Abbreviations: CHR, chromate ion transporter; L, long; RT, reverse
transcription; S, short; q, quantitative.
Two supplementary tables and two supplementary figures are available
with the online version of this paper.
073783 G 2014 SGM
CHR superfamily is composed of two sequence families: (i)
the short-chain monodomain (SCHR) family, made up of
protein pairs of ~200 aa each and (ii) the long-chain
bidomain (LCHR) family with proteins of ~400 aa (Dı́azPérez et al., 2007). Proteins of both the LCHR and SCHR
families have been shown to confer chromate resistance by
a chromate efflux mechanism (Ramı́rez-Dı́az et al., 2008).
The genomes of species of the betaproteobacteria genus
Burkholderia commonly encode multiple CHR homologues
of different subfamilies (Chain et al., 2006; Dı́az-Pérez
et al., 2007). We reported previously that Burkholderia
vietnamiensis TVV75 and Burkholderia xenovorans LB400
possess seven and six CHR homologues, respectively (Dı́azPérez et al., 2007). Thus, to obtain insights on the possible
biological significance of chr gene redundancy, two
approaches were employed in this work. (i) The six chr
homologues from B. xenovorans LB400 were cloned,
transferred to Escherichia coli and the chromate resistance
phenotype was determined in the transformants. (ii)
Expression of the chr genes was tested directly by
quantitative reverse transcription-PCR (RT-qPCR) assays
in B. xenovorans LB400. The six CHR homologues from B.
xenovorans LB400 conferred chromate resistance in E. coli,
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287
Y. M. Acosta-Navarrete and others
depending on the sulfate concentrations of the culture
medium. However, expression of the chr genes in B.
xenovorans was not affected by the sulfate levels. Moreover,
expression of the chrA2 gene, encoded by the megaplasmid,
was induced by chromate exposure.
METHODS
Bacterial strains, culture media and plasmids. B. xenovorans
strain LB400 (Goris et al., 2004) was a gift of J. Caballero-Mellado and
was utilized as the source of chr genes. E. coli W3110 (Hayashi et al.,
2006) and P. aeruginosa strain PAO1 (Holloway et al., 1979) were
used as chromate-sensitive controls in susceptibility tests. E. coli
strains JM101 (Yanisch-Perron et al., 1985) and W3110 were
employed as recipient strains for recombinant plasmids. P. aeruginosa
PAO1 and E. coli CC118 (Martı́nez-Valencia et al., 2012) were used as
hosts for transcriptional fusions.
Culture media employed were: nutrient broth (NB), Luria–Bertani
broth (LB; 1.5 % agar for solid medium), and M9 salts minimal
medium (Sigma) supplemented with 20 mM glucose and 0.1 mM
CaCl2; sulfate levels in M9 were adjusted by varying MgSO4
concentrations. B. xenovorans LB400 was grown at 30 uC in K1
medium (Goris et al., 2004) or in K1 modified (K1m) medium in
which all of the sulfate salts, except MgSO4, were substituted for the
corresponding chloride salts at the same concentrations as in K1.
The pGEM-T vector (Promega) was utilized to recover PCR fragments, which were subcloned into vector pACYC184 (Fermentas) or
into the E. coli/Pseudomonas binary vector pUCP20 (West et al.,
1994). pLP170 (Preston et al., 1997), a binary vector with a
promoterless lacZ reporter gene, was employed for construction of
transcriptional fusions.
DNA sequencing and sequence analysis. DNA sequencing was
carried out at the Department of Genetics, CINVESTAV-IPN,
Irapuato, Mexico. Amino acid sequence similarities were calculated
with Clustal W. Putative promoter sequences were identified
employing PromScan software (http://molbiol-tools.ca/promscan/).
Rho-independent bacterial terminators were searched using the
program FindTerm (Softberry).
Bacterial growth and susceptibility tests. Bacteria were grown
routinely by diluting 1 : 50 overnight cultures in tubes with 4 ml fresh
medium to OD590 ~0.05 as monitored with a spectrophotometer.
After incubating for 18 h with shaking in the indicated medium
and temperature, growth was measured as OD590. For chromate
susceptibility assays, increasing concentrations of K2CrO4 were added
at zero time and incubation continued as described previously.
For induction assays, cultures were diluted and distributed in three
flasks with fresh medium. Control cultures received no additions. To
induced cultures, chromate was added to 2 mM at zero time; after a
2 h incubation, chromate was added at a final concentration of
20 mM to both induced and uninduced cultures. Incubation
proceeded and samples were taken at intervals.
For determination of the minimal sulfate requirement of B.
xenovorans, overnight cultures grown in K1 medium were diluted
in fresh K1m medium with various MgSO4 concentrations.
Subinhibitory chromate levels were determined by growing B.
xenovorans as noted above, except that increasing amounts of
chromate were added from the start of incubation.
Cloning of chr genes. General molecular genetic techniques were
used according to standard protocols (Sambrook et al., 1989). B.
xenovorans LB400 genomic DNA was isolated from NB-grown
288
overnight cultures as described previously (Ausubel et al., 1995).
The B. xenovorans chr genes, including their 59 putative regulatory
regions, were amplified by PCR from genomic DNA utilizing the
oligonucleotides listed in Table S1 (available in Microbiology Online).
PCR conditions were as follows: first denaturing step 95 uC, 2 min; 30
cycles of denaturation 95 uC, 40 s; primer annealing 54 uC, 30 s;
extension 72 uC, 2 min; final extension 5 min, 72 uC. Amplified
fragments were purified using the Wizard SV Gel and PCR Clean-Up
System (Promega) and ligated into the pGEM-T vector. Recombinant
plasmids were transferred by electroporation to E. coli JM101,
selecting transformants on LB agar plates with 100 mg ampicillin
ml21. The cloning process was verified by restriction endonuclease
digestions and by sequencing inserts using M13 forward/reverse
universal primers. DNA fragments containing the chr genes were
obtained by digestions with HindIII/XbaI or HindIII/EcoRI endonucleases and subcloned into the corresponding sites of pACYC184 or
pUCP20 vectors. E. coli W3110 cells were transformed by electroporation with recombinant plasmids and transformants were selected
on LB agar plates with 35 mg chloramphenicol ml21 (for pACYC184)
or 100 mg ampicillin ml21 (for pUCP20).
RT-qPCR. For expression measurements, B. xenovorans cells were
grown at OD590 0.6 (mid-exponential phase) or 1.1 (stationary phase)
at 30 uC with shaking in K1m medium containing various
concentrations of sulfate in the absence or presence of chromate.
Total RNA was isolated by using the TRI Reagent (Molecular
Research Center) and stored at 280 uC. DNA was removed with RQ1
RNase-free DNase (Promega). RNA was quantified by spectrophotometric analysis at 260 nm and RNA integrity was verified on
agarose gels. Oligonucleotide primers and hydrolysis probes for RTqPCR of chr and 16S rRNA reference genes (listed in Table S1) were
designed using the Biosearch Technologies software (https://www.
biosearchtech.com/support/applications/realtimedesign-software),
and were purchased from Biosearch Technologies. Amplification
of chr and 16S rRNA genes was performed in a single tube using
the 59 exonuclease probe RT-qPCR method. RT-qPCR was
performed with total RNA samples (50 ng) and the SuperScript
III Platinum One-Step RT-qPCR Reagent Kit (Invitrogen) on the
LightCycler 480 II System (Roche Molecular Diagnostics). The
amplification signal curves were analysed at absorption wavelengths of 530 nm. Appropriate positive and non-template
controls were included in every test run. Relative expression of
chr genes was normalized with expression values obtained from
the 16S rRNA gene. Estimation of relative gene expression was
performed via the classical calibration dilution curve and slope
calculation. A fivefold dilution series (500–0.05 ng total RNA)
was prepared and used as sample in the RT-qPCR. The efficiency
(E) was obtained from standard curves using the formula
E5(1021/slope–1)6100. Relative expression levels were determined with the efficiency correction method, which takes into
account amplification efficiencies between target and reference
genes (Pfaffl, 2001).
Transcriptional fusions. The putative regulatory regions of B.
xenovorans LB400 chrA2 and chrB genes were amplified by PCR using
specific primers (Table S1) purchased from Integrated DNA
Technologies. To ensure that a putative regulatory region was
included, primers were designed 640 bp upstream and 60 bp
downstream of the corresponding gene’s start codon. Amplified
fragments were purified and cloned into the EcoRI/BamHI sites of the
promoterless lacZ pLP170 vector, and recombinant plasmids were
transferred into P. aeruginosa PAO1 and E. coli CC118 by
electroporation. LacZ activities were determined utilizing the
chromogenic substrate ONPG (Sigma) in permeabilized cells as
described previously (Martı́nez-Valencia et al., 2012). Enzyme
activities [expressed as Miller units (MU)] of control cells containing
only the vectors were subtracted from the values determined in the
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Microbiology 160
Burkholderia xenovorans CHR homologues
fusions. The assays were performed in duplicate in three independent
experiments.
RT-PCR. B. xenovorans cells were grown in K1m medium with 50 mM
sulfate in the absence or presence of 2 mM chromate at 30 uC to an
OD590 0.6. Total RNA (50 ng) was isolated as described previously
and was employed for RT-PCR using the one-step Access RT-PCR
System Kit (Promega). Pairs of primers (Table S1) were used to
amplify intergenic regions from the chrBACF gene cluster. Positive
and negative controls were performed with PCR master mix
(Promega) and with the pair of primers described previously using
genomic DNA or total RNA as templates, respectively.
RESULTS
promoters s70, sN and s54. Further sequence analysis
predicted putative promoters controlled by the typical s70
transcription factor in three chr genes, whereas the two
remaining genes, both from chromosome 1, displayed
promoter sequences related to the s54 transcriptional
regulator (Fig. S1). The chrA2 gene, from the megaplasmid,
has no promoter at all, but appears to be expressed from
the putative s70-dependent promoter of the adjacent chrB
gene (Fig. S1). Rho-independent transcription termination
sequences were not found, but consensus putative
ribosome-binding sites were identified for the five
chromosomal chr genes and for the chrB gene from the
chrBCAF gene cluster (data not shown).
B. xenovorans chr genes
Chromate susceptibility of B. xenovorans LB400
The B. xenovorans LB400 genome contains six genes
encoding proteins from the CHR superfamily distributed
in its three replicons (Table 1). These include four
members of the LCHR family of long proteins: two of
subfamily LCHR1 (ChrA1a and ChrA1b), and one each of
subfamilies LCHR6 (ChrA6) and LCHR2 (ChrA2); and
two members of the SCHR family of paired short proteins,
both of subfamily SCHR1 (Chr1NCa and Chr1NCb). CHR
homologues share 26–50 % amino acid sequence identity
and 28–55 % sequence similarity among them (percentages
for each homologous pair are presented in Table S2).
Analysis of the genomic context of B. xenovorans chr genes
showed that the chrA2 homologue, encoded on the
megaplasmid replicon, forms part of a cluster constituted
of chrBACF genes (Dı́az-Pérez et al., 2007).
Chromate susceptibility tests were performed using NB
medium and incubating cultures at 30 uC to achieve similar
growth of the compared strains. Results showed that B.
xenovorans LB400 is able to grow at higher chromate
concentrations than the E. coli W3110 and P. aeruginosa
PAO1 standard strains (Fig. 1a), indicating that the former
bacterium possesses a functional chromate resistance
determinant(s). Previous exposure of B. xenovorans LB400
cultures, grown in K1 medium, to a subinhibitory chromate
concentration and then challenged with a toxic 20 mM
chromate treatment clearly protected cells, which grew
similarly to the untreated control; in contrast, the growth of
an uninduced culture was inhibited completely (Fig. 1b).
These data suggest that a resistance determinant(s) in B.
xenovorans LB400 is induced by chromate.
Initial inspection of the 59 regions upstream of the chr
genes from the two B. xenovorans chromosomes revealed
no high degree of similarity with common prokaryotic
Expression of B. xenovorans chr genes in E. coli
Table 1. Homologues of the CHR superfamily identified in the
genome of B. xenovorans LB400
Replicon
Homologue*
Chromosome 1
ChrA1a
ChrA6
Chr1NCa
Chromosome 2
ChrA1b
Chr1NCb
Megaplasmid
ChrA2
GenBank
accession no.
Size
(aa)
Q13XT1
YP_559160
Q142U2
YP_557699
Q13YC2/C3
YP_558969/68D
Q13JA2
YP_555187
Q13RY0/Y1
YP_552509/08D
Q13FS3
YP_556416
430
402
190/178d
412
189/176d
398
*Nomenclature according to Dı́az-Pérez et al. (2007) and Dı́azMagaña et al. (2009).
DPaired genes encoding amino/carboxyl domains.
dSizes of amino/carboxyl proteins encoded by paired genes.
http://mic.sgmjournals.org
As its genome does not contain chr homologues (Dı́azPérez et al., 2007), E. coli was used as a chromate-sensitive
heterologous host to test whether Burkholderia chr genes
confer chromate resistance. For this purpose, fragments
containing each chr gene, including their own putative
promoters, were PCR-amplified from B. xenovorans
genomic DNA and subcloned into vector pACYC184. As
the chrA2 gene lacks a promoter, its coding region was
cloned into the pUCP20 vector, which provides an inframe constitutive lac promoter. Recombinant plasmids
with cloned chr genes were transferred individually to the
E. coli strain W3110 and chromate susceptibility tests were
conducted (Fig. 2). In M9 standard medium (containing
excess sulfate at 2 mM), E. coli transformants bearing genes
encoding B. xenovorans homologues ChrA1a, ChrA1b (Fig.
2a) and Chr1NCa (Fig. 2b) displayed a clear chromate
resistance phenotype, whereas transformants with each of
the two remaining CHR homologues behaved as moderately resistant (Chr1NCb) or sensitive (ChrA6) as compared with the vector-only E. coli strain (Fig. 2a, b). To
determine the possible effect of sulfate levels on the
chromate resistance phenotype conferred by chr genes,
susceptibility tests were conducted in M9 medium with 10fold lower (0.2 mM) sulfate. In low-sulfate medium,
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Y. M. Acosta-Navarrete and others
(a) 1.2
(b)
1.0
OD590
0.8
0.6
0.4
0.2
0.0
0
1
2
Chromate (mM)
3
0
3
6
9
Time (h)
Fig. 1. Chromate susceptibility and induction of B. xenovorans LB400. (a) Cultures were grown in NB with the indicated
concentrations of K2CrO4 for 18 h at 30 6C with shaking and OD590 was recorded. E. coli W3110 (#), P. aeruginosa PAO1
(&), B. xenovorans LB400 (m). (b) B. xenovorans LB400 cultures were grown in K1m medium (50 mM sulfate) at 30 6C with
shaking. Control with no additions (#), uninduced (&) or induced (m) with 2 mM chromate at time 0; after 2 h of incubation
(arrow), chromate at a 20 mM final concentration was added to the last two cultures and incubation continued for the indicated
times. Note that different chromate concentrations were used in the assays shown in (a) and (b) because of the distinct culture
media employed in each case. Data shown are means from duplicates of three independent assays. Bars, SE.
transformants with homologues ChrA1a and ChrA1b
behaved similarly to the control strain (Fig. 2c), but
ChrA6 (Fig. 2c), Chr1NCa and Chr1NCb (Fig. 2d)
conferred chromate resistance on E. coli. The chrA2
homologue, cloned in the vector pUCP20, conferred clear
chromate resistance on E. coli under both high- and lowsulfate conditions (data not shown). Thus, under at least
one of the growth conditions tested, all CHR homologues
conferred resistance to chromate on E. coli.
Expression of chr genes in B. xenovorans
To determine the expression patterns of chr homologous
genes in their native host, RT-qPCR assays were carried out
using total RNA from B. xenovorans LB400 grown to the
mid-exponential phase. Relative expression was evaluated
according to the expression of the 16S rRNA gene from B.
xenovorans. To determine whether expression of the chr
genes was related to sulfate levels, transcription was
measured under sulfate concentrations ranging from the
minimal level still allowing optimal growth (quantified as
50 mM; data not shown) up to 2000 mM sulfate. Expression
levels for the 16S rRNA gene were maintained at relatively
constant values among the different growth conditions and
treatments of the B. xenovorans cultures (Fig. S2), thus
validating the use of this constitutive gene as a normalizing
control for expression of the chr homologues.
Relative expression values for the chr genes from the two B.
xenovorans chromosomes varied several hundred-fold,
from 0.2 (chrA1a) to ~100 (chr1NCb), but no significant
differences were observed among these under the distinct
290
sulfate concentrations tested (Table 2). Expression of
the chrA2 gene, encoded on the megaplasmid, displayed
a moderate dual effect of sulfate, with lower relative
expression under medium sulfate concentrations (200 and
800 mM) as compared with threefold higher values shown
at both 50 and 2000 mM sulfate (Table 2). As no significant
differences in expression were found under the distinct
sulfate levels tested in the exponential phase, assays were
conducted using cultures grown in medium with 50 mM
sulfate, with or without chromate exposure, to the
stationary phase. Under this condition, the relative
expression values among the different chr genes showed a
pattern similar to that displayed in the exponential-phase
assays, with homologues from chromosome 2 showing the
highest values (data not shown). However, decreased
transcription of chr genes was observed in the stationary
phase, with expression values 2.3–6.7 times higher in
the cultures grown to the exponential phase. The only
exception was the chrA2 gene, from the megaplasmid,
which showed no significant difference in expression
between the growth phases (data not shown).
To investigate whether transcription of the chr genes is
affected by chromate, gene expression was also evaluated
after exposing B. xenovorans mid-exponential cultures
to subinhibitory chromate concentrations (the maximal
concentration of chromate that did not cause a significant
growth inhibition). As sulfate and chromate are known to
compete for the same cell transporter (Nies & Silver, 1989),
susceptibility tests were first conducted to determine the
chromate concentrations required for each sulfate level.
Subinhibitory concentrations were determined to be 2, 8,
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Burkholderia xenovorans CHR homologues
(b) 1.2
(a) 1.0
1.0
0.8
0.6
OD590
OD590
0.8
0.4
0.6
0.4
0.2
0.2
0.0
0.0
0
15
30
45
60
0
15
0.0
8
0
Chromate (mM)
2
(d) 1.0
0.8
0.8
0.6
0.6
OD590
(c) 1.0
OD590
Fig. 2. Chromate susceptibility of E. coli
transformants with B. xenovorans LB400 chr
chromosomal genes. (a–d) Cultures were
grown in M9 minimal medium with 2 mM
sulfate (a, b) or 0.2 mM sulfate (c, d) with the
indicated concentrations of K2CrO4 for 18 h at
37 6C, and OD590 was recorded (note that
different chromate concentrations were used
in high- and low-sulfate media due to competition for cell transport of these oxyanions).
(a, c) E. coli W3110 transformed with the
pACYC184 vector (#) or with recombinant
plasmids bearing LCHR homologues: ChrA1a
(&), ChrA1b (¤) and ChrA6 (.). (b, d) E. coli
W3110 transformed with the pACYC184
vector (#) or with recombinant plasmids
bearing SCHR homologues: Chr1NCa (&)
and Chr1NCb (¤). Data shown are means
from duplicates of three independent assays.
Bars, SE.
0.4
30
45
60
0.4
0.2
0.2
0.0
0
2
4
6
4
16 and 40 mM chromate for 50, 200, 800 and 2000 mM
sulfate, respectively (data not shown).
When the cultures were exposed to chromate, no
significant differences were observed in the expression of
chr genes from B. xenovorans chromosomes (Table 2);
relative expression values ranged from 0.2 (chrA1a) to
~120 (chr1NCb), which were similar to those obtained in
the assays from cultures without chromate treatment. In
contrast, expression of the chrA2 gene, from the megaplasmid, displayed a pronounced increase after chromate
exposure (Table 2); depending on the sulfate levels,
transcription of chrA2 by chromate treatment was 30- to
130-fold higher as compared with relative expression from
6
8
unexposed cultures. As in assays with exponential cultures,
no differences in expression were observed for chr genes
from B. xenovorans chromosomes in stationary-phase
cultures exposed to chromate; however, expression of the
chrA2 gene increased ~50-fold (data not shown).
chrA2 forms part of the chrBACF operon
As chrA2 forms part of the chrBACF gene cluster encoded
in the megaplasmid, this chr homologue was further
studied. As mentioned previously, sequence analysis
identified a putative promoter region at the 59 end of the
chrB gene (Fig. S1), but not at the 59 end of the chrA gene.
The presence of functional promoters was tested with the
Table 2. Relative expression of B. xenovorans LB400 chr genes
Cultures were grown in K1m medium with the indicated concentrations of sulfate and chromate to the mid-exponential phase, and total RNA was
isolated and processed as described in Methods. Values represent the mean of three independent determinations normalized with respect to the 16S
rRNA gene (61026)±SD, calculated as described in Methods.
Gene
chrA1a
chrA6
chr1NCa
chrA1b
chr1NCb
chrA2
50 mM sulfate
200 mM sulfate
800 mM sulfate
2000 mM sulfate
0 mM
chromate
2 mM
chromate
0 mM
chromate
8 mM
chromate
0 mM
chromate
16 mM
chromate
0 mM
chromate
40 mM
chromate
0.2±0.02
46.0±8.9
0.6±0.1
94.8±21
113.6±22
19.6±4.8
0.3±0.08
52.2±7.9
0.7±0.2
95.2±19.0
119.1±11.3
596.9±249.2
0.2±0.07
58.7±6.2
0.6±0.1
77.2±12
107.2±10.0
7.7±1.6
0.2±0.05
52.9±4.5
0.7±0.2
100.9±8.0
127.0±3.1
697.8±215.7
0.2±0.07
40.1±6.5
0.6±0.1
94.0±13
112.3±24
6.5±0.9
0.2±0.05
38.7±5.7
0.7±0.1
92.3±9.3
120.9±13.7
846±125.6
0.2±0.04
36.4±2.5
0.6±0.02
95.5±11
94.7±3.6
22.9±0.9
0.2±0.06
35.1±4.1
0.5±0.1
104.9±6.4
96.9±2.8
1709±112.8
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Y. M. Acosta-Navarrete and others
use of transcriptional fusions in the P. aeruginosa PAO1
strain, with constructions containing the 59 regions of the
chrB or the chrA gene and a promoterless lacZ reporter
gene from the pLP170 vector (Fig. 3a). High b-galactosidase activity (13 386±1830 MU) was obtained from
the chrB fusion, but no significant enzymic activity
(298±96 MU) was detected with the fusion containing
the 59 region of chrA. Similar results were obtained when
the transcriptional fusions were expressed in E. coli CC118
(data not shown). Their organization as a cluster and the
fact that chrA2 expression responds to chromate suggested
that chrBACF genes may constitute an operon. To analyse
this possibility, semiquantitative RT-PCR assays, designed
to identify co-transcription of adjacent chr genes (Fig. 3a),
were performed using total RNA from B. xenovorans LB400
cultures either untreated or exposed to chromate. As
shown in Fig. 3(b), amplification bands indicating
chromate-induced transcription were detected for all of
the intergenic regions; no corresponding signals were
observed with RNA from untreated cultures (data not
shown). These data indicate that the chrBACF gene cluster
constitutes a chromate-responsive operon.
with other bacterial genomes, which average 7.6 %
(±4.0 %); possession of multiple paralogues, as a result
of gene duplication, has been related to the high level of
metabolic versatility displayed by B. xenovorans LB400
(Chain et al., 2006). The largest number of redundant
genes in B. xenovorans pertains to transport proteins (230
paralogues), including 180 efflux systems, which comprise
21 heavy metal efflux pumps (Chain et al., 2006). Among
these transporters are the six homologues of the CHR
superfamily: four proteins from the monodomain LCHR
family and two protein pairs from the bidomain SCHR
family (Dı́az-Pérez et al., 2007).
In agreement with its possession of multiple CHR
homologues, B. xenovorans LB400 displayed chromateresistance behaviour when compared with E. coli and P.
aeruginosa standard strains; moreover, the resistance
phenotype was induced by prior chromate exposure,
suggesting a regulatory role of chromate in the expression
of chr genes.
Bacterial chr genes have been shown to behave distinctly
when transferred to E. coli. ChrA proteins from P.
aeruginosa (Cervantes et al., 1990) and Cupriavidus
metallidurans (Nies et al., 1990) did not confer chromate
resistance when expressed in E. coli; in contrast, CHR
homologues from Shewanella sp. ANA-3 (Aguilar-Barajas
et al., 2008) and Bacillus subtilis 168 (Dı́az-Magaña et al.,
2009) conferred chromate resistance on E. coli. To analyse
their relationship with chromate resistance, in this work
the six chr redundant genes were cloned individually from
B. xenovorans LB400 genomic DNA and transferred to E.
coli. Sulfate was tested as a candidate for regulation of chr
gene expression because it is considered an analogue of
chromate (Nies & Silver, 1989). The chrA2, chr1NCa and
DISCUSSION
Gene redundancy has been involved in providing genetic
robustness to living organisms (Gu et al., 2003). Higher
proportions of redundant genes are found commonly in
bacteria inhabiting perturbed environmental settings,
which correlate frequently with species possessing large
genomes (Jordan et al., 2001). This is the case for members
of the genus Burkholderia (Lessie et al., 1996). For example,
the B. xenovorans LB400 9.7 Mb genome displays 17.6 % of
redundant genes, a relatively large value when compared
(a)
700 bp
700 bp
chrB
374 bp
(b)
bp
400
chrC
chrA
334 bp
chrF
280 bp
M
chrBA
chrAC
300
chrCF
200
Fig. 3. The B. xenovorans LB400 chrBACF gene cluster constitutes a chromate-responsive operon. (a) Open arrows show chr
genes and direction of transcription. Lines above genes indicate the location and sizes of sequences utilized for construction of
transcriptional fusions. Small arrows indicate the location of primers utilized in the RT-PCR assays and the predicted sizes of the
amplified fragments. (b) RT-PCR assays carried out with complementary DNA from B. xenovorans LB400 grown in K1m
medium (50 mM sulfate) with 2 mM chromate using the primer pairs shown in (a) as described in Methods. The amplified
fragments (indicated on the right) were separated in a 1.5 % agarose gel. Molecular size markers (M) are shown on the left.
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Burkholderia xenovorans CHR homologues
chr1NCb homologues conferred chromate resistance on E.
coli under both high- and low-sulfate conditions; chrA1a
and chrA1b only afforded chromate resistance in highsulfate medium, whereas chrA6 did so only in low-sulfate
medium. These results demonstrated that all B. xenovorans
CHR homologues are able to confer chromate resistance on
E. coli and suggested that sulfate regulates the expression of
chr genes in the heterologous host. A regulatory role of
sulfate in the expression of chr genes from C. metallidurans
(Juhnke et al., 2002), Shewanella sp. (Aguilar-Barajas et al.,
2008) and Synechococcus elongatus (Aguilar-Barajas et al.,
2012) has been reported previously.
To test directly the effect of sulfate on the expression of chr
genes, RT-qPCR assays were performed with total RNA
from B. xenovorans LB400 grown in a minimal medium
with different sulfate concentrations. A wide spectrum of
expression values was found amongst chr genes, but
transcription levels were independent of the sulfate
concentration present in the growth medium. These data
contrast with those found in E. coli, where sulfate appeared
to be involved in the expression of chr genes. A correlation
between the results obtained from chromate susceptibility
tests with chr cloned genes in E. coli and expression data
from B. xenovorans was not found for all chr homologues.
The chr genes conferring the highest chromate resistance
on E. coli (chrA1a and chr1NCa) showed the lowest
expression values, whereas the gene with the highest
expression numbers (chr1NCb) only conferred moderate
chromate resistance. A correlation was found with the
chrA1b and chrA2 genes, with both conferring chromate
resistance and showing high expression values.
In general, chr homologues from chromosome 2 and the
megaplasmid showed the highest levels of expression,
whereas only the chrA6 homologue from chromosome 1
had a relatively high expression value. These four genes
were predicted to possess promoters regulated by s70
factors (the chrB promoter, in the case of the chrA2 gene).
However, the homologues with the lowest expression levels
(chrA1a and chr1NCa), both from chromosome 1, were
predicted to possess promoters regulated by s54 factors.
The fact that s54 controls the transcription of genes
devoted to a variety of functions (Reitzer & Schneider,
2001) suggests that these chr genes might express under
conditions distinct from those tested in this work. This
proposal is supported by the finding that chrA1a and
chr1NCa genes conferred clear chromate resistance phenotypes when assayed in E. coli.
In agreement with the promoter distribution of the chr
genes, the B. xenovorans LB400 genome encodes four rpoD
genes (encoding s70) and two rpoN genes (encoding s54) as
its main transcriptional factors (Chain et al., 2006).
Interestingly, chr genes encoding proteins from the same
subfamily (chrA1a/chrA1b and chr1NCa/chr1NCb), distributed in the two chromosomes, showed different predicted
promoter types and displayed contrasting expression
values, with homologues from chromosome 2 rendering
http://mic.sgmjournals.org
the highest transcription levels. These data correlate with
the proposal that dispensable genes in B. xenovorans are
more efficiently expressed from the ‘adaptive’ replicons,
chromosome 2 and the megaplasmid, as compared with
those encoded in the basic replicon, chromosome 1 (Chain
et al., 2006).
As sulfate appeared not to be involved in controlling
expression of chr genes in B. xenovorans exponential cultures,
a possible nutritional factor was then hypothesized as a
candidate for regulation. Thus, expression of chr homologues
was measured from stationary-phase cultures, a nutritional
stress condition known to affect bacterial gene expression
(Kobayashi et al., 2006; Sánchez-Perez et al., 2008). Under this
condition, a similar expression pattern as that obtained from
exponential cultures was found for all chr genes from B.
xenovorans LB400 chromosomes, except that expression levels
were two to seven times lower in the stationary phase.
Transcription of chr genes probably diminishes in favour of
secondary metabolism activities, or other stress protection
systems, mostly expressed in the stationary phase. In a
genome-wide analysis of expression profiling in B. xenovorans
LB400, the most differential expression of transport-related
genes (including inorganic ion transporters) was observed
after the transition to the stationary phase, with the
differences directed mostly towards a downregulation pattern
(Denef et al., 2004). The only B. xenovorans gene whose
expression was not affected significantly when changing the
culture’s growth phase was the chrA2 homologue, from the
megaplasmid; it appears that the regulatory circuit controlling
chrA2 expression (probably through the chrB gene product)
escapes from the apparent repression that occurs in the
stationary phase.
Testing of chromate as a predicted regulator of the
transcription of chr homologues showed that only the chrA2
gene responded to chromate exposure, increasing its
expression up to 130-fold depending on the sulfate levels of
the medium. Examples of bacteria possessing multiple
homologues with only one being induced by the related
heavy metal, whereas the remaining genes are silent or
constitutively expressed, have been reported (Nies et al., 2006;
Moraleda-Muñoz et al., 2010). Regulated expression could be
predicted for chrA2, as it is the only chr homologue whose
genome context involves genes associated with transcriptional
regulation, notably chrB. Accordingly, the chrB gene was
shown to contain a functional promoter and it was further
demonstrated that the chrBACF gene cluster constitutes a
chromate-responsive operon. Similar chrBACF clusters have
been identified in the pMOL28 plasmid of C. metallidurans
(Juhnke et al., 2002) and in transposons from Ochrobactrum
tritici (Branco et al., 2008), and from an ancient permafrost
Pseudomonas strain (Petrova et al., 2011). A relationship with
chromate resistance of the chrB, chrC and chrF genes was
reported previously for chr operons from C. metallidurans
(Juhnke et al., 2002) and O. tritici (Branco et al., 2008); the
sequences of the proteins encoded by chrBACF gene clusters
from B. xenovorans LB400 and C. metallidurans share 61, 66,
43 and 70 % amino acid identity, respectively.
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Y. M. Acosta-Navarrete and others
The high level of chromate resistance observed when chrA2
from B. xenovorans LB400 was expressed in E. coli, its
increased expression in both the exponential and the
stationary growth phases, and the fact that transcription of
the chrBACF operon responded to chromate exposure
suggest strongly that this chr determinant is mainly
responsible for the chromate resistance phenotype of this
strain. Supporting this hypothesis, B. xenovorans isolates
LMG-16224 (Chain et al., 2006) and CAC-124 (Martı́nezAguilar et al., 2008), which have been shown to lack the
megaplasmid and which did not show DNA amplification
bands in PCR assays with chrA2-specific primers, are more
sensitive to chromate than the LB400 strain (Y. L. LeónMárquez, unpublished results).
We are as yet unable to explain the presence of multiple chr
homologues in the B. xenovorans LB400 genome, because
this strain was isolated from a soil polluted with
polychlorinated biphenyls with no known chromate
contamination (Goris et al., 2004). It is possible, however,
that B. xenovorans LB400 had an evolutionary past that
included chromate exposure, which might have promoted
duplication of ancestral chr genes, or horizontal acquisition
of additional chr homologues, giving rise to multiple
paralogues able to express under different environmental
conditions. The latter event may be particularly true for the
chrBACF operon, which is located in a megaplasmid, this
replicon being absent in the majority of plant-associated B.
xenovorans strains (Chain et al., 2006).
A differential expression pattern of multiple B. xenovorans
LB400 homologues has been reported. B. xenovorans LB400
possesses three pathways for the catabolism of benzoate,
encoded by homologous genes distributed in two of its
replicons. Expression of these pathways displays a regulation pattern considered as a competitive advantage for
this organism (Denef et al., 2005).
In summary, multiple B. xenovorans chr genes are expressed
without involvement of sulfate levels in the culture
medium, but with chromate ions tightly regulating
expression of the chrA2 gene from the megaplasmid.
These varied expression patterns would be expected for
multiple adaptive genes functioning in an environmentally
versatile bacterium.
ACKNOWLEDGEMENTS
The present work was partially supported by grants from Consejo
Nacional de Ciencia y Tecnologı́a, México (Conacyt, no. 79190), and
Coordinación de Investigación Cientı́fica (Universidad Michoacana de
San Nicolás de Hidalgo; no. 2.6). Y. M. A.-N., Y. L. L.-M. and K. S.-H.
were recipients of fellowships from Conacyt.
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