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Detection of dichloromethane with a bioluminescent (lux) bacterial bioreporter

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Abstract

The focus of this research effort was to develop an autonomous, inducible, lux-based bioluminescent bioreporter for the real-time detection of dichloromethane. Dichloromethane (DCM), also known as methylene chloride, is a volatile organic compound and one of the most commonly used halogenated solvents in the U.S., with applications ranging from grease and paint stripping to aerosol propellants and pharmaceutical tablet coatings. Predictably, it is released into the environment where it contaminates air and water resources. Due to its classification as a probable human carcinogen, hepatic toxin, and central nervous system effector, DCM must be carefully monitored and controlled. Methods for DCM detection usually rely on analytical techniques such as solid-phase microextraction (SPME) and capillary gas chromatography or photoacoustic environmental monitors, all of which require trained personnel and/or expensive equipment. To complement conventional monitoring practices, we have created a bioreporter for the self-directed detection of DCM by taking advantage of the evolutionary adaptation of bacteria to recognize and metabolize chemical agents. This bioreporter, Methylobacterium extorquens DCMlux , was engineered to contain a bioluminescent luxCDABE gene cassette derived from Photorhabdus luminescens fused downstream to the dcm dehalogenase operon, which causes the organism to generate visible light when exposed to DCM. We have demonstrated detection limits down to 1.0 ppm under vapor phase exposures and 0.1 ppm under liquid phase exposures with response times of 2.3 and 1.3 h, respectively, and with specificity towards DCM under relevant industrial environmental monitoring conditions.
1 23
Journal of Industrial Microbiology &
Biotechnology
Official Journal of the Society
for Industrial Microbiology and
Biotechnology
ISSN 1367-5435
Volume 39
Number 1
J Ind Microbiol Biotechnol (2012)
39:45-53
DOI 10.1007/s10295-011-0997-5
Detection of dichloromethane with a
bioluminescent (lux) bacterial bioreporter
Nicholas Lopes, Shawn A.Hawkins,
Patricia Jegier, Fu-Min Menn, Gary
S.Sayler & Steven Ripp
1 23
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ORIGINAL PAPER
Detection of dichloromethane with a bioluminescent
(lux) bacterial bioreporter
Nicholas Lopes Shawn A. Hawkins
Patricia Jegier Fu-Min Menn Gary S. Sayler
Steven Ripp
Received: 4 March 2011 / Accepted: 6 June 2011 / Published online: 19 June 2011
Society for Industrial Microbiology 2011
Abstract The focus of this research effort was to develop
an autonomous, inducible, lux-based bioluminescent bior-
eporter for the real-time detection of dichloromethane.
Dichloromethane (DCM), also known as methylene chlo-
ride, is a volatile organic compound and one of the most
commonly used halogenated solvents in the U.S., with
applications ranging from grease and paint stripping to
aerosol propellants and pharmaceutical tablet coatings.
Predictably, it is released into the environment where it
contaminates air and water resources. Due to its classifi-
cation as a probable human carcinogen, hepatic toxin, and
central nervous system effector, DCM must be carefully
monitored and controlled. Methods for DCM detection
usually rely on analytical techniques such as solid-phase
microextraction (SPME) and capillary gas chromatography
or photoacoustic environmental monitors, all of which
require trained personnel and/or expensive equipment. To
complement conventional monitoring practices, we have
created a bioreporter for the self-directed detection of
DCM by taking advantage of the evolutionary adaptation
of bacteria to recognize and metabolize chemical agents.
This bioreporter, Methylobacterium extorquens DCM
lux
,
was engineered to contain a bioluminescent luxCDABE
gene cassette derived from Photorhabdus luminescens
fused downstream to the dcm dehalogenase operon, which
causes the organism to generate visible light when exposed
to DCM. We have demonstrated detection limits down to
1.0 ppm under vapor phase exposures and 0.1 ppm under
liquid phase exposures with response times of 2.3 and
1.3 h, respectively, and with specificity towards DCM
under relevant industrial environmental monitoring
conditions.
Keywords Bioluminescence Bioreporter
Dichloromethane Lux Methylene chloride
Introduction
Dichloromethane (DCM, or methylene chloride, CAS No.
75-09-2) is a colorless organic solvent widely used in paint
removers and various chemical processing applications. Its
industrial prevalence, low boiling point (volatility), and high
water solubility make it a frequently encountered environ-
mental contaminant. Worldwide production approaches
approximately 520,000 metric tons. It is estimated that 86%
of discharged DCM is released into the atmosphere where it
has a half-life of 79–110 days, while the remaining 14%
accumulates in water, soil, and groundwater where, it can
remain for over a millennium [1,30].
A variety of adverse health effects occur during DCM
exposure. Fatal intoxication due to DCM inhalation as well
as numerous cases of non-fatal poisonings by both inha-
lation and oral ingestion have occurred [4,10,15,17,27].
Lightheadedness, nausea, and fatigue have been reported in
the workplace as acute side-effects of short-term
N. Lopes S. A. Hawkins P. Jegier F.-M. Menn
G. S. Sayler S. Ripp (&)
The Center for Environmental Biotechnology,
University of Tennessee, 676 Dabney Hall, Knoxville,
TN 37996, USA
e-mail: saripp@utk.edu
S. A. Hawkins
The Department of Biosystems Engineering and Soil Sciences,
University of Tennessee, 676 Dabney Hall,
Knoxville, TN 37996, USA
F.-M. Menn G. S. Sayler
The Joint Institute for Biological Sciences,
Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
123
J Ind Microbiol Biotechnol (2012) 39:45–53
DOI 10.1007/s10295-011-0997-5
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exposures. Extensive studies conducted in mouse, rat, and
human models have provided sufficient evidence for the
U.S. Environmental Protection Agency (EPA) to classify
DCM as a ‘‘probable human carcinogen’’. More recent re-
evaluations have reduced the estimated risks but the
mechanisms of DCM toxicity continue to remain unclear
[8,9,28]. Once absorbed through the lungs, skin, or gas-
trointestinal tract, DCM is metabolized by two separate
pathways, both yielding noxious by-products including
carbon monoxide, carbon dioxide, hydrogen chloride, and
formaldehyde [3,9]. Among the many DCM metabolites,
carbon monoxide and formaldehyde are of particular note
because acutely, carbon monoxide is a known central
nervous system depressant and is toxic to both the liver and
cardiovascular system. Chronically, formaldehyde is a
potent carcinogen. While most long-term health concerns
involve the genotoxic effect of DCM metabolites, immedi-
ate and lethal DCM poisoning has been confirmed in several
individuals after high-level occupational exposures [10,15,
18]. Occupational safety standards are generally designed to
minimize personal exposure through adequate ventilation.
However, situations where breathing air is recycled or
unventilated present cause for further precaution. For
example, DCM is considered a high-priority airborne
contaminant by NASA due to its known accumulation in
Shuttle and International Space Station atmospheres, where
Spacecraft Maximum Allowable Concentrations (SMACs)
range from 100 ppm for 1 h exposures to 1 ppm over
1,000 day exposures [20]. More relevant to us here on Earth,
the Occupational Safety and Health Administration (OSHA)
has set permissible air concentration limits at 25 ppm as an
8-h time-weighted average (TWA) and 125 ppm as a short-
term exposure limit (STEL). The odor threshold of DCM is
approximately 250 ppm. U.S. EPA safe drinking water
standards are set at 5 ppm.
The detection and monitoring of workplace and envi-
ronmental contaminants can be achieved via whole-cell
bacterial bioreporters that sense and respond to targeted
metabolites. Bioreporter bioassays for numerous contami-
nants have been demonstrated in water, sediment, soil, air,
and wastewater environments using genetically modified
bacteria typically incorporated with colorimetric, fluores-
cent, or bioluminescent signaling elements [24]. For the
bioreporter described herein, bioluminescence derived
from the bacterial lux series of genes (luxCDABE) was
applied to provide real-time, reagentless signaling and
assessment of target analyte bioavailability. As a first line
of detection for environmental contaminants, biolumines-
cent bioreporters offer a rapid, inexpensive, simple, and
sensitive method of monitoring, unlike traditional time-
consuming analytical techniques that require specially
trained personnel and high operational cost. We developed
alux-based bioluminescent bioreporter specific to DCM by
employing the evolutionary adaptation of the methylo-
trophic bacterium Methylobacterium extorquens (formerly
M. dichloromethanicum) DM4 to scavenge for and
metabolize DCM as a sole carbon source. The genes for
DCM metabolism by strain DM4 are located within the
dcm operon, which includes the dcmA and dcmR genes
(GenBank accession #M32346) [12]. The dcmA gene
encodes for a DCM dehalogenase while the dcmR gene
encodes for a repressor that negatively controls the dcm
operon. In the presence of DCM at low ppb concentrations,
the repressor is inactivated and transcription ensues from
the dcmA promoter, generating a 50–80-fold increase in
dichloromethane dehalogenase [13]. Genetically engineer-
ing M. extorquens DM4 with the inducible bioluminescent
reporter plasmid pCM66–dcmA/RluxCDABE, containing
a transcriptional fusion between the luxCDABE gene cas-
sette from Photorhabdus luminescens and the dcmA/R gene
of the dcm degradation operon, permitted quantifiable
determination of DCM concentrations based on biolumi-
nescence response profiles after DCM exposure.
Materials and methods
Bioreporter construction
M. extorquens strain DM4 (DSM No. 6343), a pink pig-
mented facultative methylotrophic bacterium, was used as
the host strain for development of the DM4
lux
bioreporter.
Genetic construction involved fusion of the dcmR/Agenetic
region (GenBank accession #M32346) of strain DM4 to the
P. luminescens luxCDABE gene cassette (GenBank acces-
sion #M90093) downstream to which was ligated the rrnB
T
1
T
2
transcriptional terminator derived from the cloning
vector pKK223-3 (GenBank accession #M77749) (Fig. 1).
The 1,450-bp dcmR/Aregion was PCR amplified from
M. extorquens DM4 genomic DNA using the forward primer
5’-TCTAGACCTCCAAGGCTTGAAC-3’ containing a
unique XbaI site (underlined) and the reverse primer
5’-GAGCTCCACGTTATCCTCCCTT-3’ containing a
unique SacI site (underlined) and placed within a pCR4-
TOPO vector (Invitrogen, Carlsbad, CA, USA). The
luxCDABE gene cassette was PCR amplified from P. lumin-
escens genomic DNA using the forward primer 5’-ATT
AAATGGATGGCAAATAT-3’ and the reverse primer 5’-
GTCGACAGGATATCAACTATCAAAC-3’ containing a
unique SalI site (underlined) and placed within a pCR-
XL-TOPO vector (Invitrogen). The rrnB transcriptional
terminator was PCR amplified from pKK223-3 using the
forward primer 5’-GTCGACAAGAGTTTGTAGAAAC
GC-3’ and the reverse primer 5’-GTCGACCTGTTT
TGGCGGATG-3’ each containing a SalI site (underlined)
and placed within a pCR4-TOPO vector. The luxCDABE
46 J Ind Microbiol Biotechnol (2012) 39:45–53
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gene cassette was then ligated to the rrnB transcriptional
terminator by cleaving the rrnB pCR4-TOPO vector with
SalI to remove the rrnB sequence and ligating it into the
pCR-XL-TOPO-luxCDABE vector linearized via cleavage
with SalI with proper orientation confirmed by restriction
mapping. This produced a pCR-XL-TOPO-luxCDABE-rrnB
vector. DNA isolations were performed with Wizard Mini-
preps and Midipreps (Promega, Madison, WI, USA) and
purified when necessary with the Geneclean Spin Kit (MP
Biomedicals, Irvine, California, USA). PCR reactions were
carried out in an MJ Research DNA Engine tetrad (Waltham,
MA, USA) using Ready-To-Go PCR beads (Amersham
Biosciences, Piscataway, NJ, USA). DNA was sequenced at
all steps with the ABI Big Dye Terminator Cycle Sequencing
reaction kit on an ABI 3100 DNA Genetic Analyzer (Applied
Biosystems, Foster City, CA, USA).
To construct the bioreporter, each of the TOPO isolated
gene sequences above were inserted into the multicloning
site of the broad host range cloning vector pCM66 [19].
The dcmR/Agenetic region was excised from its pCR4-
TOPO vector with an XbaI/SacI double digest and direc-
tionally inserted into pCM66 similarly cleaved with XbaI
and SacI. The luxCDABE-rrnB sequence was removed
from its pCR-XL-TOPO vector via digestion with EcoRI
and then ligated into the dcmR/A-pCM66 vector linearized
with EcoRI. Proper orientation was confirmed by restric-
tion digest mapping and sequencing. This finalized
pCM66–dcmA/RluxCDABE vector was electroporated
into M. extorquens DM4 (2.5 kV, 400 X,25lF) with
transformants plated on nutrient agar plates containing
20 lg kanamycin/ml. Exposing the plates to DCM vapor at
approximately 50 ppm allowed bioluminescent colonies to
be selected for using a Caliper Life Sciences IVIS imaging
camera (Hopkinton, MA, USA). The brightest of these
colonies, designated DCM
lux
, was propagated for further
analysis.
Growth conditions
The DCM
lux
bioreporter, taken from -80C frozen stock
cultures, was maintained on nutrient agar plates containing
kanamycin at 20 lg/ml to select for the lux reporter plas-
mid. The strain, being a slow grower, required incubation
at 30C for 4 days. Plates were refreshed from -80C stock
on a weekly basis. Liquid cultures were started from plates
via inoculation into nutrient broth followed by incubation
for up to 2 days at 30C with shaking at 200 rpm to
achieve an optical density at 600 nm (OD
600
) of 0.15.
Cultures were centrifuged at 10,000 rpm for 10 min and
washed once in an equal volume of sterile phosphate buf-
fered saline (PBS; in g/l, NaCl, 8; KCl, 0.2; Na
2
HPO
4
,
1.15; KH
2
PO
4
, 0.2). Cell pellets were then resuspended in
1/10th strength (0.19) sterile minimal salts media (MSM;
19stock solution in g/l, KH
2
PO
4
, 0.68; K
2
HPO
4
, 1.73;
MgSO
4
7H
2
O, 0.1; NH
4
NO
3
, 1.0; pH 7.0) to an OD
600
of
0.25 for application in vapor and liquid-phase biolumi-
nescent assays.
DCM vapor phase bioluminescent assays
For vapor phase testing, DCM
lux
cultures were immobilized
in 0.7% agarose in 0.1 9MSM. Approximate 10 ml cul-
tures of strain DCM
lux
were prepared in MSM at an OD
600
of 0.25 as explained above. Liquefied 40C agarose (2 ml)
was added to 4 ml of this culture which was immediately
and gently vortexed. From this mixture, 4 ml was pipetted
into an 8-cm-long 91.5 cm
2
20-ml total volume quartz
flowcell. Once solidified (*10 min), this formed an even,
immobilized layer of DCM
lux
culture with an upper surface
area that could be exposed to DCM gas as it passed through
the flowcell. DCM gas was purchased from Airgas Spe-
cialty Gases, Port Allen, LA, USA at 1,000 ppm. DCM
concentrations were regulated using a mass flow controller/
flow tubes (Aalborg, Orangeburg, NY, USA) and a diluting
supply of Grade D breathing air (Airgas) to provide biore-
porter exposures at 100, 50, 10, 1, and 0.1 ppm. The DCM/
breathing air mixture was continuously metered through
sterile, chemically inert 3.1-mm-diameter Masterflex PTFE
tubing (Cole Parmer Instrument Co. Chicago, IL, USA)
connected to a 900-cm
3
SPME glass sampling chamber,
Fig. 1 The pCM66 vector [19] was used as the backbone for
construction of the dichloromethane bioreporter DCM
lux
. The dcmR/
dcmA regulatory promoter region from M. extorquens DM4 was
ligated upstream of the P. luminescens luxCDABE cassette (see text
for specific details. Kn kanamycin resistance)
J Ind Microbiol Biotechnol (2012) 39:45–53 47
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then through the quartz flowcell positioned within the IVIS
Lumina imaging chamber (Caliper Life Sciences), and
finally to a waste outlet (Fig. 2). Bioluminescent imaging
was monitored online in the IVIS Lumina with readings
taken in photon counts per second (CPS) every 10 min over
4-h exposure durations using the integrated Living Image
software package (Caliper Life Sciences) [5]. Control
flowcells containing non-induced DCM
lux
cultures exposed
to breathing air without DCM additions were run simulta-
neously alongside test cultures to establish background
bioluminescence. All DCM exposure experiments were
performed in triplicate.
Paint stripper as a test medium
Bacterial cultures were grown and then immobilized in the
quartz flowcell in the same manner as for the DCM vapor
phase bioluminescent assays and exposed to dilute off-gas
vapors from Crown brand Handi-Strip Semi-Paste Stripper
(Packaging Service Company, Pearland, TX, USA). One
milliliter of paint stripper was placed in an empty 3-l glass
flask sealed with a Teflon-lined screw-cap lid. Metered
breathing air was pumped continuously through an inlet
located at the bottom of the flask to produce a DCM-con-
taminated air supply that was directed into the 900-cm
3
SPME sampling chamber, then to the flowcell in the IVIS
imaging chamber, and finally, out to waste. Paint stripper
exposures were performed over 4-h periods with IVIS
images obtained every 10 min. Control flowcells contain-
ing non-induced DCM
lux
cultures exposed to metered
breathing air without paint stripper additions were run
simultaneously alongside test cultures to establish back-
ground bioluminescence. All paint stripper exposure
experiments were performed in triplicate.
DCM liquid-phase bioluminescent assays
A 13,000 ppm (0.15 M) stock solution of DCM was pre-
pared by adding 490 ll of concentrated HPLC-grade DCM
(Fisher Scientific, Fair Lawn, NJ, USA) to 49.5 ml sterile
HPLC-grade H
2
O. Cultures (100 ml) of strain DCM
lux
were prepared in MSM at an OD
600
of 0.25 and DCM from
the stock solution was added to produce initial concentra-
tions of 100, 10, 1, 0.1, and 0.01 ppm. Cultures were
immediately placed on a magnetic stirrer and continuously
mixed at room temperature (*20C) while being circularly
pumped at a flow rate of 340 ml/min through a sealed
system consisting of the quartz flowcell positioned inside
the IVIS Lumina imaging chamber. Experimental expo-
sures were performed over 4 h with readings taken every
10 min. All associated tubing was sterile, chemically inert
3.1-mm diameter Masterflex PTFE. Control flowcells
containing non-induced DCM
lux
cultures (0.1 9MSM
without DCM) were run simultaneously alongside test
cultures to calibrate for background bioluminescence. All
DCM exposure experiments were performed in triplicate.
Analytical measurements of DCM concentrations
All gas-phase DCM samples were analyzed using a Hewlett
Packard gas chromatograph (Model 6890) equipped with a
mass spectrometer detector (MSD, Model 5973N) with an
inert source. A DB-5MS column with 10-m DuraGuard
(30 m 90.25 mm i.d., J&W Scientific, Folsom, CA, USA)
was used for sample separation with helium gas as the
carrier gas at a constant flow rate (1.0 ml/min) maintained
by an electronic pressure control module. Manual injection
was used for sample analysis. The oven temperature was
held at 45C for 10 min, and then increased to a final
temperature of 300C with a 10-min hold. The injection
temperature was set at 280C and the MS source tempera-
ture was 250C. The MS was operated in the selected ion
mode (SIM) and DCM (m/z 84) was monitored. Gas-phase
DCM samples were collected with a SPME fiber assembly
(75 lm carboxen/polydimethylsiloxane; Supelco, Bella-
fonte, PA, USA). Fibers were inserted for 15 min into the
900-cm
3
SPME gas-tight sampling chamber (Fig. 2) during
the vapor phase bioluminescence assays (n=3). For the
liquid-phase bioluminescence assays, the SPME fibers were
inserted for 15 min into the culture flask headspace (n=3).
DCM measurements were taken hourly with the SPME
Vent to
hood
GC/MS gas
analyzer
Innova
photoacoustic
gas monitor
Flowcell
SPME
sampling
chamber
IVIS imaging chamber
CCD camera
Immobilized
bioreporter
O2
DCM
Flowmeter
Fig. 2 Flow-through system for exposing the M. extorquens DCM
lux
bioreporter to vapor phase DCM
48 J Ind Microbiol Biotechnol (2012) 39:45–53
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fibers and immediately analyzed by GC/MS [23]. SPME
fibers were cleaned daily by insertion in the 280CGC
injection port for 5 min.
Liquid calibration standards were prepared at six dif-
ferent initial DCM concentrations ranging from 0.0013 to
130 ppm in 4.0 ml glass vials containing 2 ml of solution
and sealed with a Teflon-lined septum and screw-cap lid.
These standards were stirred for 30 min prior to SPME
fiber exposure to assure equilibrium distribution of the
DCM between the gas and liquid phases within the vials
[16]. The equilibrium headspace DCM concentrations were
calculated using Henry’s law constant for DCM
(0.00196 atm-m
3
/mol at 1 atm and 20C) and plotted
against the GC peak area measurements to establish a
calibration curve [34]. This allowed direct measurement of
the DCM exposure concentrations during the gas-phase
bioluminescent assays. During the liquid-phase biolumi-
nescence assays, the exposure concentrations were inferred
using the equilibrium headspace DCM concentration and
the Henry’s law constant.
A factory-calibrated Innova Model 1412 photoacoustic
gas monitor (LumaSense Technologies, Ballerup, Den-
mark) equipped with optical filter UA0980 was used to
confirm the gas-phase DCM measurements made with
SPME. Automatic sampling occurred every 2 min with a
sample integration time of 20 s. This provided a manu-
facturer’s specified lower detection limit of 0.05 ppm at
20C and one atmosphere pressure. DCM concentrations
measured with the Innova were maintained within 10% of
the SPME vapor-phase measurements (data not shown).
Induction of M. extorquens DCM
lux
by related aliphatic
chlorinated industrial solvents
To characterize the specificity of the DCM
lux
bioreporter, it
was exposed to several related aliphatic chlorinated
hydrocarbons commonly used as industrial solvents (tri-
chloroethylene, tetrachloroethylene (otherwise known as
perchloroethylene or PERC), 1,1,1-trichloroethane, and
chloroform). Since testing each of these compounds at
several different concentrations was too time-consuming
using the flowcell system, they were instead tested for
false-positive bioreporter induction in a higher throughput
96-well microtiter plate format in a Biotek Synergy2
microplate reader (Biotek, Winooski, VT, USA). The
DCM
lux
bioreporter was grown and resuspended in MSM
to an OD
600
of 0.25 as for the liquid-phase experiments
above. The first column of wells in a black 96-well
microtiter plate (Dynex Technologies, Chantilly, VA,
USA) contained 200 ll of bioreporter culture at a final
chemical exposure concentration of 8,500 ppm. Dilutions
of 1:10 were then performed throughout successive wells in
200-ll volumes to achieve a lower concentration limit of
approximately 0.85 ppm. Plates were prepared on three
different days using three separately grown cultures (n=3
among experiments) with each plate containing each
compound in triplicate wells (n=3 within experiments).
Each plate contained triplicate wells of strain DCM
lux
exposed to DCM at 10 ppm (positive control) and triplicate
wells of strain DCM
lux
not exposed to a chemical to
establish background bioluminescence (negative control).
Plates were sealed with Breath-Easy membranes (Diversi-
fied Biotech, Boston, MA, USA) and monitored for bio-
luminescence in the Biotek Synergy2 at room temperature
every 10 min for 24 h.
Results
Bioluminescent response kinetics of the DCM
lux
bioreporter to vapor-phase DCM and paint-stripper
volatiles
The vapor-phase bioluminescent response profile of the
DCM
lux
bioreporter was established in a flowcell format at
DCM concentrations of 100, 50, 10, 1, and 0.1 ppm (actual
DCM concentrations as measured by SPME (n=3) were
101.0 ±0.03, 50.8 ±0.02, 10.5 ±0.05, 0.97 ±0.03, and
0.14 ±0.02 ppm). Significant bioluminescent responses
(three standard deviations above control) were observed at
all concentrations except 0.1 ppm and ranged from 1 h at
100 ppm to 2.3 h at 1.0 ppm (Fig. 3). Using the upper
response time of 2.3 h, a linear bioluminescent response
curve to increasing concentrations of DCM was generated
(R
2
=0.99) (Fig. 3, inset). The paint-stripper vapor-phase
DCM concentration was estimated to be 34.5 ppm using
this bioluminescent response curve. SPME measurement of
paint stripper volatiles at the 12.3-h time point was
39 ±3.55 ppm. The earliest significant response during
the paint stripper exposure occurred at 1.2 h.
Bioluminescent response kinetics of the DCM
lux
bioreporter to liquid-phase DCM
A recirculating pump-driven flow-through system was used
to establish the response profile for M. extorquens DCM
lux
upon exposure to liquid-phase DCM at concentrations of
100, 10, 1, 0.1, and 0.01 ppm (DCM concentrations
inferred using SPME (n=3) were 100 ±5.71, 50 ±2.45,
10 ±0.70, 1 ±0.001, 0.1 ±0.30, and 0.01 ±0.07 ppm).
Significant bioluminescent responses (three standard devi-
ations above control) were observed at all concentrations
except 0.01 ppm (Fig. 4). Response times ranged from
0.5 h at 100 ppm to 1.3 h at 0.1 ppm. The bioluminescent
response was linear (R
2
=0.99) at the upper response time
of 1.3 h (Fig. 4, inset).
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Induction of the DCM
lux
bioreporter by related
chlorinated solvents
M. extorquens DCM
lux
was exposed in a 96-well micro-
titer plate to trichloroethylene, tetrachloroethylene, 1,1,1-
trichloroethane, and chloroform to assess the specificity of
the bioreporter bioluminescent response to DCM (n=3
within and between experiments). Exposure concentra-
tions ranged from 8,500 to 0.85 ppm. No significant
bioluminescent response (three standard deviations above
control) was detected in any of the samples at any of the
concentrations tested over the 24-h exposure period
(Fig. 5).
Discussion
The bacterial bioluminescent bioreporter M. extorquens
DCM
lux
was constructed by fusing the 1,450-bp dcmR/
Aregulatory region from M. extorquens DM4 upstream of
a promoterless P. luminescens luxCDABE gene cassette.
The bioreporter proved capable of autonomously generat-
ing bioluminescence in response to vapor and liquid-phase
DCM at lower tested detection limits of 1.0 and 0.1 ppm,
respectively. These detection limits are well below the
threshold levels for potential health risks established by
OSHA for air contaminants (TWA of 25 ppm and STEL of
125 ppm) and EPA for safe drinking water (5 ppm).
Response times at these lower detection limits ranged from
2.3 h under vapor-phase exposures to 1.3 h under liquid-
phase exposures. Elevated DCM exposures (100 ppm)
initiated bioluminescence within 1 h or less under both
conditions. The parental M. extorquens DM4 strain can
grow at an upper DCM concentration of approximately
850 ppm [14]. Thus, the bioreporter has sufficient robust-
ness to tolerate much higher concentrations of DCM before
succumbing to a toxic, disabling response. Testing at such
high concentrations was not performed, however, since the
purpose of the bioreporter was to signal low concentration,
early warning contaminant exposures expected in work-
place environments.
Volatiles emanating from enclosed commercially
available paint stripper could be detected within 2.3 h at a
concentration of approximately 35 ppm. This is well below
the predicted DCM concentrations for consumer paint
stripper applications that range from 170 to 453 ppm in
residential, low-ventilation rooms [31]. Occupational
exposures, for example, during aircraft paint stripping
operations, produce DCM at concentrations estimated to
range from 20 to 525 ppm [29,33]. Thus, the DCM
lux
bioreporter could therefore effectively pre-alert consumer
and industrial workers to harmful DCM exposures.
Hours
01234
Normalized bioluminescent response (CPS)
0
2x106
4x106
6x106
8x106
10x106
12x106
100 ppm
50 ppm
10 ppm
1 ppm
0 ppm
Paint stripper
Log DCM concentration (ppm)
0.0 0.5 1.0 1.5 2.0
Log bioluminescence (CPS)
4.0
4.5
5.0
5.5
6.0
6.5
7.0
99.0
226.5680.0
2
=
+=
R
xy
Fig. 3 Bioluminescent response profile in counts per second (CPS) of
the M. extorquens DCM
lux
bioreporter exposed to vapor-phase DCM
at concentrations ranging from 1 to 100 ppm and to paint stripper.
The normalized bioluminescent response was calculated by subtract-
ing induced culture bioluminescence from background (un-exposed)
culture bioluminescence for all concentrations. Inset Linearity of the
bioluminescent response to vapor-phase DCM. Points were taken
from the bioluminescent profile at the upper response time of 2.3 h
and plotted over concentrations ranging from 1 to 100 ppm. The white
circle represents the 2.3-h exposure response of the DCM
lux
bioreporter to paint stripper volatiles
50 J Ind Microbiol Biotechnol (2012) 39:45–53
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No significant bioluminescence induction was observed
from the DCM
lux
bioreporter upon exposure to other com-
mon industrial solvents (trichloroethylene, tetrachloroeth-
ylene, 1,1,1-trichloroethane, and chloroform), thus
confirming its specificity for DCM and validating minimal
false-positive signaling under the tested environmental
conditions. Hyphomicrobium sp. DM2 (ATCC #43129),
which also harbors a dcmA DCM dehalogenase gene [21],
has been applied as a reporter in its native state in a multi-
transducer flow-calorimeter/chloride-sensitive electrode
biosensor format, with a detection limit of 10 ppb in water
[13]. It demonstrated similar specificity but was predictively
cross-sensitive to dihalomethanes besides DCM, such as
bromochloromethane and dibromomethane. However, the
minimal to nonexistent industrial and commercial applica-
tion of these other dichloromethanes makes the DCM
lux
bioreporter DCM specific within its intended monitoring
environments.
In its current state, the DCM
lux
bioreporter could be
applied as a sensor within bioreactor treatment schemes
designed for biological removal of DCM from air- and
liquid-flow streams. Biotrickling filters, bioscrubbers, and
similar bioreactor fabrications containing biodegradative
microorganisms such as M. extorquens DM4 and Hypho-
microbium sp. DM2 have been shown to effectively reduce
DCM concentrations in polluted air streams and industrial
and municipal wastewater effluents [2,11,22]. The inte-
gration of DCM
lux
bioreporters into bioreactors would
serve not only to degrade DCM but to additionally report
on its bioavailability, as has been previously demonstrated
with, for example, the bioreporter Pseudomonas putida
TVA8 and its targeted degradation and sensing of toluene
Hours
01234
Normalized bioluminescent response (CPS)
0
2x107
4x107
6x107
8x107
100 ppm
10 ppm
1 ppm
0.1 ppm
0 ppm
Log DCM concentration (ppm)
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0
Log bioluminescence (CPS)
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
99.0
843.6271.0
2
=
+=
R
xy
Fig. 4 Bioluminescent response profile in counts per second (CPS) of
the M. extorquens DCM
lux
bioreporter exposed to liquid-phase DCM
at concentrations ranging from 0.1 to 100 ppm. The normalized
bioluminescent response was calculated by subtracting induced
culture bioluminescence from background (un-exposed) culture
bioluminescence for all concentrations. Inset Linearity of the
bioluminescent response to liquid-phase DCM. Points were taken
from the bioluminescent profile at the upper response time of 1.3 h
and plotted over concentrations ranging from 0.1 to 100 ppm
Chlorinated Organic Solvent Concentration (ppm)
8500 850 85 8.5 0.85
Fold increase above background control
0
100
200
300
400
500
600
700
800
Trichloroethylene
Tetrachloroethylene
1,1,1-Trichloroethane
Chloroform
DCM
Negative Control
Fig. 5 Specificity of the peak bioluminescent response as fold
increase above background control bioluminescence for the M.
extorquens DCM
lux
bioreporter exposed to potentially interfering
liquid phase chlorinated organic solvents (trichloroethylene, tetra-
chloroethylene, 1,1,1-trichloroethane, and chloroform) in a 96-well
microtiter plate at final concentrations ranging from 8,500 to 0.85 ppm
J Ind Microbiol Biotechnol (2012) 39:45–53 51
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in a packed bed reactor [26]. The measurement of resulting
bioluminescent signals within the bioreactor is then typi-
cally achieved via the connection of fiber optic cables that
terminate to a photomultiplier tube (PMT) or direct con-
nection of the PMT or other light gathering device to the
bioreactor itself [7]. Smaller-scale chip-based biosensors
can also be functionally applied for target chemical sensing
using bioreporter interfaced microelectronic circuitry.
Integrated circuit microluminometers and avalanche pho-
todiodes have both been mated with living bioluminescent
bioreporters to produce self-contained biosensors on a
platform of only a few square millimeters [6,25,32]. The
potential exists to similarly assimilate DCM
lux
bioreporters
into analogous on-chip detection schemes. The ability to
effectively encapsulate these bioreporters into agar-based
matrices and exploiting their slow growth rate to maximize
longer-term viability attests to such potential suitability.
Acknowledgments Research support was provided by the NASA
Advanced Environmental Monitoring and Control Program under
cooperative agreement number NNJ04HF02A and the National Sci-
ence Foundation Division of Biological Infrastructure (DBI) under
award number DBI-0963854.
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Thesis
Le dichlorométhane (DCM ; CH2Cl2) est un polluant chloré toxique émis dans l’environnement principalement par les activités industrielles. Ce polluant peut être dégradé par des bactéries méthylotrophes qui utilisent des composés en C1 réduits comme seule source de carbone et d’énergie. La protéobactérie Methylorubrum extorquens DM4 porte quatre gènes dcm au sein du transposon catabolique dcm très conservé chez les bactéries dégradant le DCM. Le gène dcmA code la DCM déshalogénase de la famille des glutathion-S transférases essentielle à la croissance avec le DCM. Son activité est modulée par DcmR, un facteur de transcription qui régule l’expression de dcmA ainsi que son propre gène, orienté de manière divergente par rapport à dcmA. DcmR porte un domaine hélice-tour-hélice de fixation à l'ADN et un second domaine appelé methanogen / methylotroph, DcmR sensory (MEDS) potentiellement impliqué dans la fixation d’un composé hydrocarboné ligand. Les objectifs de ma thèse étaient de répondre à plusieurs questions : i) quel est le niveau d’expression des transcrits dcm et des protéines correspondantes par rapport à d’autres gènes et protéines dont l’abondance est modifiée en réponse à la croissance sur le DCM ? ii) Comment le facteur de transcription DcmR intervient-il dans la régulation des gènes dcm ? iii) Quelle est la variabilité du gène dcmR et de son environnement génétique in situ ? Des méthodes globales « -omiques » de transcriptomique et de protéomique ont permis d’inventorier les ARN et les protéines dont l’abondance varie chez la souche sauvage DM4 cultivée soit avec le DCM ou le méthanol, le substrat de référence de la méthylotrophie. Les gènes dcm sont parmi les plus exprimés en présence de DCM, ce qui confirme leur régulation en présence de DCM. Deux approches complémentaires ciblant la détermination des sites d’initiation de la transcription (TSS-Seq) et de la traduction (N-terminome) ont permis la recherche de motifs de régulation dans les régions 5’UTR (5’-untranslated terminal region) et les promoteurs de gènes régulés en réponse à la croissance avec le DCM. Le rôle régulateur de dcmR a été étudié en comparant les phénotypes de croissance, l’activité promotrice par fusion transcriptionnelle, la quantification des ARN et des protéines des gènes dcm chez la souche sauvage par rapport à des mutants du gène dcmR seul ou combiné à d’autres gènes dcm mutés. Ces travaux ont permis de confirmer qu’en absence de DCM, DcmR inhibe la transcription de son propre gène ainsi que celle de dcmA. Outre DcmR, la répression nécessite aussi l’expression d’au moins un des autres gènes dcm et ceci par un mécanisme indépendant des boîtes de 12 pb conservées dans les promoteurs des gènes dcmR et dcmA. Lors de la croissance en condition DCM, l’absence du gène dcmR confère une vitesse de croissance ralentie, qui ne résulte pas d’une différence de production des ARN et des protéines codées par le transposon dcm. Pour que l’expression du gène dcmA soit activée, l’ensemble de la région intergénique entre dcmR et dcmA doit être présente, ce qui suggère la présence de sites de régulation pour la fixation d’un facteur de transcription indépendant de DcmR. L’ensemble de ces résultats a permis de proposer un nouveau modèle de régulation des gènes dcm. Alors que le gène dcmR a été détecté en quantité similaire à celle du gène dcmA par qPCR dans des échantillons de sites contaminés par le DCM, une analyse bioinformatique à partir des données de séquences indique que des gènes dcmR-like sont trouvés dans d’autres contextes génétiques que celui du transposon catabolique dcm. Ainsi, DcmR pourrait exercer un rôle de régulateur dans d’autres contextes ouvrant de nouvelles pistes pour l’identification des ligands du domaine MEDS.
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