Gene, 121 (1992) 149-153 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

149

0378-I 119/92/$05.00

06774

The aryldialkylphosphatase-encoding gene adpB from Nocardia B-l: cloning, sequencing and expression in Escherichia coli (Parathion

hydrolase;

biodegradation;

paraoxonase;

organophosphate;

pesticide;

sp.

strain

hydrolase)

Walter W. Mulbry Pesticide Degradation Laboratory. ARS, Received

by A.M. Chakrabarty:

USDA, Beltsville, MD 20705, USA

23 June

1992; Revised/Accepted:

22 July/27 July 1992; Received

at publishers:

3 August

1992

SUMMARY

Using degenerate oligodeoxyribonucleotides (oligos) derived from the N-terminal sequence of an aryldialkylphosphatase (ADPase) from Nocardia sp. strain B-l, an amplification reaction was used to isolate a DNA segment containing a 57-bp fragment from the adpB gene. Based on the nucleotide (nt) sequence of this fragment, a nondegenerate oligo was synthesized and used to screen a subgenomic library of strain B-l DNA for fragments containing adpB. A 3.55-kb PstI fragment containing adpB was cloned into Escherichia coli, and the nt sequence of a 1600-bp region containing adpB was determined. Under control of the lac promoter of pUC19, adpB expression in E. coli cultures was approx. 15fold higher than in strain B-l under the native adpB promoter. Comparison of adpB with the Flavobacterium ADPase-encoding gene, opd, revealed no significant homology at the nt or aa levels.

INTRODUCTION

Organophosphate (OP) compounds such as the insecticide ethyl parathion (O,O-diethyl-0-4-nitrophenyl phosphorothioate) have been used in agriculture for more than 30 years. Although these compounds generally have short half-lives in the environment, their extreme toxicity poses risks for ground and surface-water contamination. A group of microbial enzymes variously termed parathion hydrolases, phosphotriesterases, and paraoxonases (now termed

Correspondence to: Dr. W.W. Mulbry, Building 050, Rm 100, BARC-West, MD 20705, USA. Tel. (301)504-6417; Abbreviations: gene encoding ichia; EPN, base(s)

Pesticide Degradation Laboratory, 10300 Baltimore Ave., Beltsville, Fax(301)504-7976.

aa. amino acid(s); ADPase, aryldialkylphosphatase; adpB, ADPase; bp, base pair(s); DTT, dithiothreitol; E., Escher0-ethyl-0-4-nitrophenyl

phenylphosphonothioate;

or 1000 bp; K,,, Michaelis-Menten

constant;

oligo, oligodeoxyribonucleotide; OP, organophosphate; ing frame; PAGE, polyacrylamide-gel electrophoresis;

kb, kilo-

nt, nucleotide(s); ORF, open readPAM, point ac-

ceptable mutation; RBS, ribosome-binding site; SDS, sodium sulfate; TBE, 89 mM Tris_borate/2mM EDTA pH 8.3.

dodecyl

aryldialkylphosphatases (ADPases) (EC 3.1.8.1)) offer a biological means for detoxifying OP-containing solutions to prevent environmental contamination. ADPase activities have been reported from a variety of bacterial isolates (for review, see Mulbry and Kearney, 1991). Four unique microbial enzymes have been purified and characterized (Mulbry and Karns, 1989a; Serdar et al., 1989; Rothschild et al., 1990). However, only one class of ADPase gene (the opd gene of Flavobacterium sp. and Pseudomonas diminuta MG) has been isolated and characterized (Mulbry and Karns, 1989b; Serdar et al., 1989). Our laboratory has been involved in the characterization and manipulation of bacterial ADPase activities for use in detoxifying OP pesticide wastes. Although bacterial hosts containing the opd gene have proven effective in detoxifying solutions containing OP insecticide waste (Karns et al., 1987), other enzymes are needed for situations where the opd enzyme is inactive. The ADPase from Nocardiu strain B- 1 has been characterized previously (Mulbry and Karns, 1989a) and differs considerably from the opd-specified protein (Table I). As a first step toward manipulating this ADPase activity for the biodegradation of OPs, we sought

1.50 TABLE

I

Comparison

from Flawbacterium sp. and Nocardia strain B-l (adapted

of ADPases

Hydrolase

Mass (kDa)

Temp.

K,, (PM)~

optimum Native”

from Mulbry

( ” C) ’

Subunit b

Ethyl parathion

EPN

and Karns,

1989a)

Cellular

Relative hydrolysis

Substrate

location e

(“A of control)

turnover

with DTT’

(number/mm)”

I 10,000

Flavobacterium sp.

30.5

35

40

91

211

Membrane

< 1

Nocardia strain B-l

40

43

40

25

26

Cytoplasm

244

’ Determined h Determined

by size exclusion chromatography by O.l’, SDS-12Sb PAGE.

’ Using ethyl parathion d Determined let) fractions. r Calculated s Calculated

were subjected

After resuspension

and EPN concentrations to ultracentrifugation

of the membrane

as relative rates of ethyl parathion

in buffer containing

(Waters,

no additions

MA).

fraction,

hydrolysis

between

13 and 55 PM and 17 and 69 PM, respectively.

(105,000 x g, 2 h, 4°C) to separate both fractions

by extracts

were assayed

prepared

components

for hydrolase

in buffer containing

into soluble (supernatant)

and membrane

activity using ethyl parathion

1 mM DTT vs. the rate of hydrolysis

(pel-

as the substrate. by extracts

made

(control).

using purified hydrolases

(Mulbry

and Karns,

1989a) and ethyl parathion

to (i) identify, clone, and express in E. coli the strain B-l ADPase-encoding gene (which we term a&B); and (ii) determine the relationship of the adpB and opd genes.

EXPERIMENTAL

Milford,

as the substrate.

using ethyl parathion

e Cell-free extracts

using an SW300 column

26

as the substrate.

source and material from cattle dipping vats as the inoculum (Shelton and Somich, 1988). Initially characterized as a Gram- strain, strain B-l was a Gram + rod when grown on LB medium (Gibco, Madison, WI) at 30°C. Fatty acid analysis of the strain (Microbial ID, Newark, DE) indicates that strain B-l is a member of the genus Nocardia.

AND DISCUSSION

(b) N-terminal analysis of the strain B-l ADPase Previous purification of the strain B-l ADPase demonstrated that the enzyme is a 43-kDa monomer (Mulbry and

(a) Characterization of strain B-l Bacterial strain B-l was isolated from an enrichment culture with the OP insecticide coumaphos as a carbon

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 SerAlaGluPheValIleArgAsnAlaLysValPheAspG~yGluArgValTyrGluArg

U-R-9

25merllOZ4

degeneracy

CTR- 10

DNA AMPLIFICATION + 73 BASE PAIR PRODUCT

6 7 8 9 10 11 12 13 14 IleArgAsnAlaLysValPheAspGly CTR-11

/ ATCCGGAACGCGAAGGTCTTCGACGG 26-mer

26-mer11024 degeneracy

151

adpI3

c

I

Ser

I

I 0

I 400

Fig. 2. Restriction

maps of cloned fragments

from strain B-l were probed Subsequently, Membranes

I 1200

I 600

subgenomic (DuPont,

I 1600

libraries containing

Boston,

adpB from Nocardia strain B-l. Southern

containing

with the oligo CTRll

(Fig. 1) in order to choose

PstI fragments

the appropriate

of the N terminus

chloride is marked

in the hybridization

62018

PWM

70617

1 3550

bp

blots of restriction-endonuclease-digested enzyme for cloning

from strain B- 1 were screened by colony hybridization

MA), and tetramethylammonium

the ORF for the adpB gene. The position

1 3200

I 2600

I 2400

I 2000

pWM

and wash solutions

DNA

genomic DNA

fragments

adpt3.

containing

to CTRI 1 using Colony/Plaque (Ausubel

Screen

et al., 1989). The arrow shows

Ser

aa (Fig. 1).

which the 3.55-kb fragment is ligated to pUC19, demonstrated ADPase activity (Table II). A restriction map of the 3.55-kb insert in pWM1327 is shown in Fig. 2.

(c) Cloning of ad@ from strain B-l Attempts to clone adpB from strain B-l by screening E. coZi recombinants either on the basis of ADPase expression or by hybridization to degenerate oligo probes derived from the N-terminal sequence of the strain B-l enzyme were unsuccessful (data not shown). However, using degenerate oligos derived from the enzyme’s N-terminal sequence, a DNA amplification reaction was used to isolate a 73-bp DNA segment containing a 57-bp fragment from adpB (Fig. 1). From the nt sequence of this fragment, a nondegenerate oligo (CTRl 1) was synthesized and used to probe Southern blots of genomic DNA from strain B-l. A 3.55-kb PstI fragment that hybridized to CTRll was cloned from a subgenomic library of PstI fragments from strain B-l. E. coli cells containing the plasmid pWM1327, in

(d) Deletion analysis and expression of adpB in Escherichia coli A series of deletion mutants were generated from pWM1327 to define the approximate endpoints of adpB within the 3.55-kb PstI fragment (Fig. 2). In pWM62018, a deletion of approx. 570-bp downstream from adpB showed no significant effect on the ADPase activity present in cell-free extracts (Table II). Subsequent nt sequencing showed that the 570-bp deletion in pWM62018 extended 1 bp into the coding sequence of adpB (Fig. 3). Mutants of pWM620 18 were then generated in which regions upstream of adpB were deleted. One of the resulting mutants (pWM708 17) displayed a fourfold increase in ADPase activity and contained a deletion of approx. 1.7 kb of DNA upstream from adpB. In order to more precisely define the

Karns, 1989a). Using 30 pg of purified, desalted ADPase, N-terminal analysis yielded the sequence of the first twenty

Fig.

I. Strategy for generation

R, purine;

Y, pyrimidine;

N-terminal

sequence

of a gene probe for adpB from Nocardirr sp. strain B-l. Two highly degenerate

W, A or T) were synthesized

of the strain B-l ADPase.

for a DNA

The sequence

ADPase

with an EcoRI linker at its 5’ end. The sequence

sequence

with a PstI linker at its 5’ end. An amplification

DNA segment.

This DNA was isolated

ing to this unambiguous Freemont,

sequence

of oligo CTR9 corresponds

reaction coding

containing

primer concentrations

of

which would to residues

with PstI+EcoRI, for N-terminal

10 mM Tris (pH 8.3)/0.001%

1nM/25 units Taq polymerase/400

oligos, CTR9 and CTRlO (N, any nucleotide; amplify

of residues

6-14.

The nondegenerate

reactions

gelatin/50

encoding

were performed

N-terminal 73-bp

of a plasmid

oligo CTRl 1 was synthesized in an Eppendorf

the

of the strain B-I

14-19 of the protein’s

The nt sequencing

mM KCl/2.5 mM MgClJ0.2

ng of template

segment

sequence

DNA from strain B-l yielded the predicted

and ligated to the vector pUCI9. residues

a 73-bp DNA

l-6 of the N-terminal

to the complement

using CTR9, CTRlO and genomic

and used as a probe for adpB. DNA amplification

CA) using 25 pl reactions

and dGTP/oligo

sequence

reaction

of oligo CTRlO corresponds

from the gel, digested

taining this segment yielded the unambiguous

amplification

microcycler

con-

accord-

(Eppendorf,

mM each of dATP, dCTP, dTTP

DNA (both per ml). The temperature

profiles used were one

cycle at 94°C (5 min), 35 cycles of 52°C (90 s): 72°C (60 s): 95°C (45 s), and one cycle of 52°C (2 min): 72°C (5 min). Reaction products were separated by electrophoresis at 200 V using a 12f, polyacrylamide gel (30: 1) using TBE as the running buffer (Maniatis et al., 1982). After staining with ethidium bromide,

gel fragments

desalted

using centricon-

were excised and added concentrators

to tubes containing

(Amicon,

Danvers,

100 ~1 of TBE buffer. Following

MA), and the DNA was digested

overnight

incubation

at 4°C

these solutions

with 10 units each of EcoRI and PstI overnight

were

at 37°C.

After inactivating the restriction enzymes by heat treatment (20 min, 65”C), the solutions were desalted using centricon-10s. Approximately one tenth of the appropriate desalted, digested, amplified DNA product was ligated to 100 ng of pUC19 DNA which had been digested with EcoRI+PstI. Boiling plasmid minipreps (Maniatis et al., 1982) were used to isolate recombinant plasmids containing the amplified DNA product from transformed E. coli DH5r cells. The nt sequencing of double-stranded DNA templates was performed using a Sequenase kit (U.S. Biochemical Corp., Cleveland, OH) and plasmid DNAs

which were purified using Qiagcn

tip-20 columns

(Qiagen

Inc., Chatsworth,

CA).

1.52 TABLE

11

/~,\‘.nl.nl

“>lir:‘i,,

,’

of ADPase activities of extracts of Nocurdia strain B-l and E.ycherichiu coli cultures containing adpB plasmids” Comparison

i

“h’

;.i

L,. t,