Sensing Superfund Chemicals with Recombinant Systems

Sapna K. Deo, S. Xu, D. Ghosh, X. Guan, A. Rothert, J. Feliciano, E. D’Angelo, Leonidas G. Bachas, and Sylvia Daunert Department of Chemistry and Department of Agricultural Sciences University of Kentucky Lexington, KY Molecular Recognition in Analytical Chemistry

• Proteins

• Cells

• High Throughput Screening • Whole Cell-Based Sensing Systems

Analyte No Analyte Signal

Reporter protein No Expression of Reporter Protein Arsenic Poisoning

• Applications • Agriculture • Treatment for diseases • Industrial uses

• Long exposure to low doses of arsenic • Skin hyperpigmentation and cancer • Other cancers • Inhibition of cellular enzymes

New Bangladesh Disaster: Wells that Pump Poison... New York Times November 10, 1998 Arsenic contamination in the USA

U. S. Geological Survey, Fact Sheet FS 063-00, May 2000 Arsenite Resistance in E. coli

O/P arsR arsD arsA arsB arsC Schematic Representation of the Antimonite/Arsenite Pump

- - AsO2 SbO2 Cytoplasm ADP ATP AsO - ATP 3- 2 AsO4 ADP ArsA ArsA ArsC

Periplasm Membrane ArsB

- - AsO2 SbO2 Fluorescent Reporter Proteins in Array Detection

Protein Excitation Emission

λ max λ max

GFP 395 (470) 509 EGFP 488 509 BFP 380 440 GFPuv 395 509 YFP 513 527 CFP 433 475 CobA 357 605 RFP 558 583 P H C 3 A Production of fluorescent A P

porphyrinoid compounds H3C N HN

oxidation NH N A A P A P H C 3 A A A P P P P NH HN H3C N HN UMT

NH HN SAM P A NH HN H3C A A A A A UMT P HN P P P P H3C N SAM urogen III Dihydrosirohydrochlorin (Precorrin-2) NH N A CH3 A-CH2COOH A P-CH2CH2COOH

SAM - S-adenosyl-L-methionine P P

UMT- methyltransferase III trimethylpyrrocorphin Construction of pSD601 plasmid

O/P arsR bla arsD PCR pRLUX arsR O/P luxAB arsR

+ pSD601 ampR cobA

cobA cobA

pISA417 PCR Time Study for Arsenite

10000

-5 1 x 10 M NaAsO2 8000

6000

4000

2000 Fluorescence (cps)

0

-2000 024681012 Time (h) ALA in Urogen Pathway

P A P A

COOH COOH A P A P COOH NH HN NH HN ALA PBG Urogen III HO O dehydratase deaminase synthase N NH HN NH HN H P A A A H N H N 2 2

ALA PBG A P P P HMB Urogen III ALA - δ- PBG - HMB- Urogen- Uroporphyrinogen Time Study of Arsenite with ALA

10000 NO ALA NO ALA 8000 1mM ALA

6000

4000

2000 Fluorescence (cps) 0

-2000 024681012 Time (h) Calibration Plot with ALA

45000

40000

35000

30000

25000

20000

15000

Fluorescence (cps) 10000

5000

0 -8 -7 -6 -5 -4 -3 log[sodium-m-arsenite] Selectivity Study

40

Induction time = 1 h 35 Analyte concentration = 5 x 10-6 M

(cps) 30

25

Intensity 20

15

10

Fluorescence 5

0 - - - 3- 2- - - Br Cl NO3 PO4 SO4 AsO2 SbO2 Field Challenges

• Conventional analytical techniques • Field-kits • Background signal • Viability of the cells • Freeze drying • Strips (β-galactosidase) Addressing Environmental Analysis with Self-Contained Kits

Lyophilized Reagents

“The bacteria are dim in tap water (top) but glow brightly when arsenic is present 0 0.1 0.2 0.5 1 (bottom).” Arsenite (µM)

Strip Sensors http://www.nature.com/nsu/030929/030929-7.html Centrifugal Microfluidic Platform for Micro-Total Analysis Systems

Low power and space requirements

Less reagent and sample consumption

Easy disposal

Short analysis time

Integrate washing, sample preparation, and calibration Prototype Compact Disc

7 6 5 4 3

1 2

1. Waste Reservoir 2. Optode 3-7. Solution Reservoirs Prototype CD for four simultaneous analyses Incorporation of Whole-cell Sensing System for Arsenite/Antimonite on the CD Platform

140

130

120

(Counts) 110

Fluorescence Intensity 100

0. y t i 0.7 s n 90 s l 6 l De

0.5 l Ce 0 a 15 c f 0.4 i 30 o 45 t 60 p Time (min) O Calibration Curves

Antimonite Arsenite 120 120 Detection Limit: 1 x 10-6 M Detection Limit: 1 x 10-6 M 100 100

80 80

60 60

40 40

20

Normalized Fluorescence 20 Normalized Fluorescence Normalized

0 0 -6.5 -5.5 -4.5 -3.5 -6.5 -5.5 -4.5 -3.5

log (Antimonite, M) log (Arsenite, M) Clc Operon and 3-chlorocatechol Pathway

clcR o/p clcA clcB clcD

O HOOC OH ClcA COOH ClcB ClcD COOH O COOH O COOH Cl Cl OH clcR clcA

clcB′ pSMM50R-B′

r amp lacZ Calibration Curves

5 (5 min induction) ) 5 3-clc

10 4 ×

3

2 4-clc 1 catechol

Light Intensity (counts Light Intensity 0 -10 -9 -8 -7 -6 -5 -4 -3 -2 log (concentration, M) Selectivity Study

1.5 1) 3-chlorocatechol; 2) 4-chlorocatechol; 3) 4-chlorobiphenyl ) 6 4-7) catechol, biphenyl, 2-chlorophenol, and 4-chlorophenol 1 10 ×

1.0

0.5 2

Light Intensity (counts Light Intensity 3 0 4-7 -10 -9 -8 -7 -5 -4 -3 -6 log (concentration, M) Challenges in Environmental Sample Analysis of Chlorocatechol

1. Extraction Method • Free Chlorocatechol Can be extracted by organic solvent O Fe • Bound Chlorocatechol O Cl OH Difficult to be extracted 2. Matrix Effect Selected Chemical Characteristics of Soils

Organi Electric cal Oxal atextractablee Soils car cbon onductivity pH P Al Fe -1 -1 (%) (µmhos•cm ) (mg kg ) Acid washed sand 0.02 12 6.1 8 32 54 Maury silt loam 3.3 53 5.0 704 1894 4126 Woolper 7.5 38 5.9 3203 3086 3203 Organic humus 15.3 1099 6.4 415 3575 1106

Optimized Protocol for Soil Analysis

Pellet (Intact, induced bacteria )

Supernatant 2 (Water + Bacteria) centrifuge Mixture 1 (10,000 ×g) (Soil + Water +Bacteria) Supernatant

Induction (2h), Precipitate (Discarded) centrifuge (400×g) (Soil) Results of Soils

HPLC Bacterial sensing system Sample Theoretical Experimental ± Theoretical Experimental ± matrix Value (mg•kg-1) SD (mg•kg-1) Value (mg•kg-1) SD (mg•kg-1) 0.5 0.49 ± 0.02 0.5 0.51 ± 0.02 Sand 2.0 2.03 ± 0.04 2.0 1.95 ± 0.08 10 9.75 ± 0.55 50 52.0 ± 2.6 Woolper 0.5 0.0 ± 0.0 0.5 0.52 ± 0.04 2.0 0.0 ± 0.0 2.0 1.90 ± 0.18 10 9.55 ± 0.75 50 54.0 ± 4.5 Maury 0.5 0.0 ± 0.0 0.5 0.43 ± 0.08 2.0 0.0 ± 0.0 2.0 2.24 ± 0.30 Organic 0.5 0.0 ± 0.0 0.5 0.43 ± 0.09 potting soil 2.0 0.0 ± 0.0 2.0 1.73 ± 0.28 Detection of PCBs Based on Dechlorination Followed by whole cell Sensing

Clx

Mg/K2Pd6 +H2O

Cly - +(X+Y)Cl+H2

PCB Dechlorination Reaction HPLC Chromatogram of Dechlorinated Product: Biphenyl Whole cell Sensing of Biphenyl Based on bph Operon from P. pseudoalcaligenes KF707

pE R1 A1A2(orf3)A3A4 B C

No biphenyl No Transcription

HOPD

O OH Transcription Biphenyl COOH pE Bphoperonof P. pseudoalcaligenes KF707

Biphenyl mol/L Increase in Light Intensity (%) lacZ pSD7001 1x 10-5 81.9 ± 9.5 ampR 1x 10-6 37 ± 6.2

1x 10-7 21 ± 4.8 Degradation Pathway of Hydroxylated-Biphenyl in the strain Pseudomonas azelaica HBP1

hbpR hbpC hbpA hbpD

OH OH COOH COOH + + NADH+H NAD O2 O H2O OH

OH OH COOH HbpC HbpD

O2 H2O HbpA Whole Cell-Based Sensing System for Hydroxylated PCBS

10000

7500

5000

2500 Relative Light Units

0 -9 -8 -7 -6 -5 -4 -3 -2 Log (2-hydroxy-3',4'-dichlorobiphenyl, M) Collaborators

Leonidas Bachas Marc Madou Barry Rosen Jan Roelof van der Meer

Acknowledgments

NIEHS-Superfund Basic Research Program the past and present members of the Daunert Group