Biosensors: Development and Environmental Testing

Investigators: Anne J. Anderson, Charles D. Miller and Joan E.

McLeanUtah State University

Logan, Utah 84322-5305

RD-83090701-0

Metals of interest

Copper: maximum drinking water level standard 1.3 mg/L

aquatic chronic exposure limit 30.3 µg/L

Human tolerance relatively highUsed for microbial pest controlSome lower organisms acute toxicity (phytoplankton, zooplankton,

amphibians)

Cadmium: maximum drinking water standard 0.005 mg/L

aquatic chronic exposure limit 2.7µg/L

Higher animal toxicity: accumulates in kidneys and liver, produces bone and blood problemsCarcinogenic (Group B1)Minor use as pesticide

Cell concentrations regulated by chelation, sorption, uptake, and efflux mechanisms

Cu is a trace element: required for certain enzymatic and carrier proteins

Cd no known cellular use—toxic- may disrupt Ca and Zn metabolism

Both cause oxidative stressBoth act on –SH proteins

MX

Biosensors

Final goals:

1) An array of promoter fusions that respond differentially and specificallywith light output upon exposure to toxic metals

2) A gene chip array to detect transcript abundance from cells responding to toxic metals. Pattern of gene activation would specify the metal.

P. putida KT2440

1.E+00

1.E+02

1.E+04

1.E+06

1.E+08

1.E+10

0.001 0.01 0.1 1 10 100

mg/L of Cu2+

# ce

lls p

er 1

ml

1 h 4 h

P. putida Corvallis

1.E-01

1.E+01

1.E+03

1.E+05

1.E+07

1.E+09

0.001 0.01 0.1 1 10 100

mg/L of Cu2+

# of

cel

ls p

er 1

ml

1 h 4 h

Cells of both pseudomonads are killed by concentrations of Cu

starting between 1 and 10 mg/L

Test organisms: P. putida strains

The pseudomonad cells show little change in culturability with Cd even at doses of 100 mg/L.

Both wild type isolates behave similarly showing resistance to Cd

P. putida Corvallis

1

10

100

1000

10000

100000

1000000

10000000

100000000

0.001 0.01 0.1 1 10 100

mg/L of Cd2+

# c

ells

pe

r 1

ml

1 h

4 h

P. putida KT2440

1

10

100

1000

10000

100000

1000000

10000000

100000000

1000000000

0.001 0.01 0.1 1 10 100

mg/L of Cd2+

# ce

lls p

er 1

ml

1 h

4 h

Generation of Luciferase Biosensor

Wild type

Light

Metal stimulus

promoter lux A/BORF

Protein product

Transcript Transcript

Normal cell Biosensor

P. putida Corvallis mutants-Cu [in .01M CaCl2]

0

5

10

15

20

25

30

0 0.01 0.1 1 10

mg/L of Cu2+

Lu

x

Cat A*10

Bfr

FeSOD

FeSOD mutant is most sensitive

Ca inhibits the Cd response

Cadmium detectionP. putida Corvallis mutants-Cd

[in .01M Ca(NO3)2]

0

2

4

6

8

10

12

14

0 1 10 50 100

mg/L Cd2+

Lu

x

Cat A*10

Bfr

FeSOD

P. putida Corvallis mutants-Cd [in .01M

KNO3 ]

0

2

4

6

8

10

12

14

0 1 10 50 100

mg/L of Cd2+

Lu

x Cat A*10

Bfr

FeSOD

Response of KT2440 mutants to Cu

0

0.5

1

1.5

2

2.5

3

3.5

68 18 45 47 41 4 43 66 70 69 7 31 46 21 3 30

Mutant strain

Rel

ati

ve

ch

ang

e in

lu

x (f

old

)

10 mg/L

1 mg/L

Response of KT2440 mutants to Cd

0

2

4

6

8

10

12

14

16

54 18 31 30 43 4 68 46 45 69 7 70 21 41 3 47 45

Mutant strain

Rel

ati

ve

ch

ang

e in

lu

x (f

old

)

100 mg/L

10 mg/L

1 mg/L

Control Treatment

(10 mg/l Cu++)

A B

pH 4pH 7

pH 4 pH 7Mr(kDa) Mr(kDa)

97.0

66.0

45.0

30.0

20.1

14.4

97.0

66.0

45.0

30.0

20.1

14.4

pI 4 pI 7 pI 4 pI7

Spot B: increased three fold

Corresponded to lipoamide dehydrogenase in TCA cycle

Cd responses Mr (kDa)

97.0

66.0

45.0

30.0

20.1

14.4

KT2440 0.01M Ca(NO3)2 100 ppm Cd

4-15%

pI 4 pI 7

Peptides of increasedIntensity.

Conclusions

RD-83090701-0

1) The two approaches for biosensor development are feasible

2) luxAB::Insertional mutants that detect Cu and Cd differentially have been identifiedGene loci await determination

3) Peptides that increase upon Cu exposure are detected and one has been correlated with a specific function