Transcript Assessing the Effects of Naphthenic Acids Using a Microbial

Assessing the Effects of Naphthenic Acids Using a Microbial
Genome Wide Live Cell Reporter Array System
Xiaowei Zhang1,2*, Steve Wiseman2, Hongxia Yu1, Honglin Liu1, John P. Giesy1,2,3,4, Markus Hecker2,5
1 State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, China; 2Toxicology Centre, University of
Saskatchewan, Saskatoon, SK, Canada; 3 Dept. Biomedical Veterinary Bioscience, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; 4 Dept. Biology &
Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR China; 5 ENTRIX Inc. Saskatoon, SK, Canada
Methods
100mg NAs/L
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ssb
ydcS
hemC
bolA
sodA
brnQ
ybjP
yjbJ
dppA
ycaD
ybaY
inaA
ilvC
yfcD
marR
araF
chbA
uhpT
90
120
Time (min)
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inaA
ilvC
yfcD
marR
uhpT
chbA
insA_2
yajO
ycaD
yjeB
yfbM
xthA
ypeA
rbsD
ileX
rluE
ppiD
gadW
ydcM
yahD
yaaW
yfbE
ybiH
alaS
insA_7
crl
rph
ybhK
ydgH
modF
ribA
feaB
serC
ppiB
greA
ptsG
yfdU
rpiA
gnd
araF
yfeN
sodA
ybjP
yjbJ
hemC
pitA
gadX
somA
ybgI
atpI
yjbQ
ymcC
ybeB
brnQ
ligA
cvrA
yjfI
trxA
b2641
yjgA
rob
ftsK
aceB
ihfB
htrL
rpsM
msrB
pmrD
yhiD
rrnA
accB
trmU
yejA
ycfD
bolA
dppA
ydcS
ybfE
ssb
ybaY
Figure 3. Clustering of genes modulated by NAs. A). Clustering of the concentrationand time-dependent expression of the 27 genes altered at least 2-fold change over
background by NAs. B). Clustering of the time-dependent expression of the NAs
altered genes selected by1.5-fold change cut-off. Gene expression in cells exposed to
1000 mg NAs/L are displayed. Classification and visualization of the gene expression
were derived by use of ToxClust (Zhang et al., 2009).
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Discrete variable
Continuous variable
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Discrete variable
Continuous variable
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B: 2 fold change cut-off
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ymcC
ybeB
somA
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A: 1.5 fold change cut-off
1000 mg/L
100 mg/L
10 mg/L
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Fold Change
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1000mg NAs/L
NAs Conc
1000mg NAs/L
Response (%)
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crl
rpiA
alaS
insA_7
gnd
Figure 1. The microbial promoter collection includes more than 1900 promoters for
the E. coli K12 strain MG1655. Each of the reporter strains has a bright, fastfolding green fluorescent protein (GFP) fused to a full-length copy of an E. coli
promoter (Zaslaver et al., 2006).
10mg NAs/L
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100mg NAs/L
Figure 2 A Venn diagram displaying
the differentially expressed genes
selected by 1.5 or 2.0 fold change
cut-off at three different NAs
concentrations, 10, 100, and 1000
mg/L. NAs induced a concentrationdependent response in the number of
differentially expressed genes.
.
0.1
10mg NAs/L
Fold Change >1.5
0
Mixtures of NAs, which include cyclopentyl and cyclohexyl carboxylic acids, have
been identified as major toxic components in the effluents discharged by the oil
sands industry. The present study for the first time demonstrated an application of
a high throughput bacterial live cell array in a genome-scale investigation of the
toxic mechanisms of environmental chemicals, a commercial NAs technical
mixture extracted from crude oil (Sigma).
Fold Change >2.0
Response (%)
Introduction
Results
0.2
Real time gene profiling of time- and concentration-dependent effects of exposure
to a commercial naphthenic acid (NA) mixture in live cells for three hours was
conducted using a library of 1800 fluorescent transcriptional reporters for
Escherichia coli growing in 384-well plates. Response patterns obtained after
exposure to NAs suggested that the primary cellular responses were up-regulation
of the pentose phosphate pathway, processes involved in the molecular function of
NADP or NADPH binding, and down-regulation of the ATP-binding cassette (ABC)
transporter complex. Transcriptional networks that were significantly modulated
by NAs included those that were regulated by transcriptional factors such as CRP,
RecA, and GadE. The down-regulation of the SOS response pathway suggested
that DNA damage might not be the direct results of NAs within the first three hours
of exposure. However, CRP-dependent genes modulated by exposure to NAs
indicated that the cellular level of cyclic AMP was altered immediately upon
exposure of cells to NAs. Furthermore, the linear range of the concentrationresponse curve of the selected promoter reporters encompassed a range of
concentrations between 10 -1000 mg NAs /L, which covers concentrations
typically observed in the environment. Thus, this assay system may represent a
promising tool for the detection of environmental chemicals such as NAs.
0.1
Abstract
Figure 4 Active functional modules of a
transcriptional network of patters of gene
responses in E coli exposed to NAs. The
level of gene expression in cells exposed to
1000 mg NAs/L is indicated by the color
gradient. Brown: >2 fold up regulation;
gradient from Red to white: from 2 to1 fold
up regulation; gradient from White to blue:
from 1 to 2 fold down regulation; Gray: >2
fold down regulation. For the three TFs (crp,
lexA and gadE) that displayed no significant
change in response to NAs, their roles in
the network modules are highlighted by in
aquamarine.
10
100
1000
NAs concentration (mg/L)
10
100
NAs concentration (mg/L)
1000
Figure
5.
Concentration-dependent
transcriptional response to NAs. In the
discrete variable approach, the number or
the percentage of genes affected was
used to describe the degree of chemicalinduced effects.
In the continuous
variable approach, the actual expression
level of all the selected genes were
integrated to differentiate the degree of
effect induced by different concentrations
of chemical.
Discussion
1.Biological pathways involved in NAs effects.
1) up-regulation of the pentose phosphate pathway, 2) up-regulation of NADP or
NADPH binding pathway, 3) down-regulation of the ATP-binding cassette (ABC)
transporter complex.
2. Stress responsive pathway affected by NAs exposure.
1) redox-response, 2) SOS-response, 3) osmotic-response
3. Transcriptional networks involved in NAs-induced effects.
Tanscriptional factors: CRP-, RecA-, and GadE
4.Potential biosensors for environmental NAs detection.
Acknowledgement
Jiangsu EMT grant (1012) and Canada WED grant (#6578 and 6807)
Reference
Zhang et al., Environ. Sci. Technol. 2008, 42 (17), 6762-6769.
Zaslaver et al., Nat. Methods. 2006, 3 (8), 623-628.