Molecular Genetics of Pesticide Degradation by Soil Bacteria

Chapter 11

Molecular Genetics of Pesticide Degradation by Soil Bacteria Jeffrey S. Karns

Downloaded by UNIV OF AUCKLAND on May 3, 2015 | http://pubs.acs.org Publication Date: May 3, 1990 | doi: 10.1021/bk-1990-0426.ch011

Pesticide Degradation Laboratory, Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Although i t is generally accepted that the degradation of soil applied pesticides by microorganisms is responsible for many pesticide performance failures, little is known about the molecular mechanisms responsible for the evolution of new pesticide degradative capabilities. Mobile genetic elements such as plasmids and transposons have been shown to encode enzymes responsible for the degradation of several pesticides. The isolation of pesticide degrading microorganisms and the characterization of genes encoding pesticide degradation enzymes, combined with new techniques for isolating and examining nucleic acids from soil microorganisms, will yield unique insights into the molecular events that lead to the development of enhanced pesticide degradation phenomenon.

The

role

several The

of

soil

enhanced of

changes

(such

evolving In

microbial

or

as

the

case

source

of

utilized them

to

of

by

pesticides,

one

several

proliferate

to is

and

or

of

the

the

that

of

deal

the

other

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toxicity process

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a by

is

pesticide

of

the

rapidly

material

that

with

new

the or

microbial members

encodes conditions.

the

In into

it

is

hydrolysis, either

because

molecule

if

allows

community.

because

through

occurs

new which

community

of

relieved.

a

sulfur,

pesticide

pesticide that

result

c o m p o u n d may b e

a

of

(1-3).

environmental

phosphorous,

out-compete

selection

to

a pesticide)

introduced

members

degradation

documented

apparently

genetic

nitrogen,

transforming

reduction

a natural

introduction

the

well

respond

required to

microorganisms

them and by

oxidation, there

energy,

or

is

to

introduction of acquiring

enhanced

been

phenomenon

mechanisms

carbon,

Alternatively, toxic

the

the

has

populations

otherwise

biochemical

in

pesticides

degradation

ability

the

microorganisms

soil-applied

of

soil.

T h i s chapter not subject to U . S . copyright Published 1990 A m e r i c a n Chemical Society

In Enhanced Biodegradation of Pesticides in the Environment; Racke, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

case the

142

ENHANCED BIODEGRADATION OF PESTICIDES IN T H E ENVIRONMENT

There soil

are

several

microbial

rapidly

degrading

enzymes

that

also

catalyze

the

of

substrate.

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in

the

for

out

type

of

type

of

biodégradation the

problems. molecule

themselves

until

they

or

is

performance. which enhanced

able be

enough

conclusion

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seems

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ultimately

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degradability Another event for

must

take

of

place

at

molecule.

in

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gene

via

higher

higher Thus,

constantly

gene

so

the

organisms this might play

in

greater

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of

that

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be a

present role

capabilities

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in

within

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in the

the

may a l s o

degradation

soils.

it

the

is

form

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contribute Reanney

to

the

is

made of

from

mRNA

are

produced). in

a

population. pesticide

expressed to

rather would

be

at

or

high

than, a

levels or

in

population

a greater

cryptic,

normally not

pesticide

population

of

t h e DNA

potential

molecule are

expression.

on

result

is

silent,

soils.

particular

induced

result

that of

a

the

metabolite

amount

the

of

inducer

a b a c t e r i u m but evolution

cell

the

expression

gene

that

encodes

or,

as

for in

RNA (mRNA)

within

pesticide

a bacterial

DNA i n

bacteriophages in

the

possible

of

order

the

a natural

locations

each

in

involve

gene

by w h i c h p r o t e i n s

expression) substrate)

might

degrades

inducer

to

products.

community

pesticides

due

of

genetic

affinity

or

regulation

acting

lack

some

The i n c r e a s e d

activity

enzyme

the

degraded it

an

produced i n

that

natural

line,

as

in

one

failures

way

production of

affected

(pesticide

of

pesticide

manner

the

a

that

the

regulatory

process

enzyme

(constitutive

to

fate

on

the

degradation

higher

which messenger

enzyme

level

pesticide

addition

of

a mutation

degradation the

results

levels

at

a

a n enzyme

transcription.

translation

overall if

rate

via

of

for

encodes

affect

the

acts

that

that

has

numbers

incorporated

microbial it

the

pesticide

soils,

banned its

of

effects

describes

the

(either

their

of

Such an event

that

at

for

could

and/or

changes

enzyme

of

rate

members

rate

the

benefit

performance or

this

transform

of

increase

soil

enzyme

pathway

DNA t e m p l a t e

Along

the

metabolic

the

by

that

i n d u c t i o n causes

same

other

all

soil

substrate

which

The

the

will

that

form

hypothesis

occur.

Mutations

may a l s o

the

to

the

for

Indeed,

become

in

pesticide

responsible

observed

from

arise

being

natural

the

This

the

up

a

undoubtedly

noticeable

from

rate.

genes

expressed

degradation

(5). transposons, genesis

(6) d i s c u s s e d

be

to to

they

which the

that

due

molecules

slowly

accurately

suffer end

some

to

cause

is

it

that

several at

probably

organisms

phenomena

within

so

pesticide is

to

involves

degradation

pesticide

Usually,

will

either

a gene

with

obvious

scenario

enhanced

mutation

of

or

the

derive

scenario

degradation

because

of

may

natural

conditions.

pesticide

expected

pesticides rapid

if

to

threshold

this

ordinary

of

is

there

molecule

enzyme s

of

capable

w h i c h happen T

the

(4_)

cases

biodégradation

likelihood

concert

fast If

to

of

desirable

the

might

r e a c h some

degradation

under

However, are

in

they

rates

some

pesticide

molecule

elimination

pesticide

a

In

adaptation

which are

functions

co-metabolism

is

decreasing

population)

cellular

slow

pesticides

pollution by

normal

normal,

by w h i c h t h e

populations

may o c c u r .

pesticide

today's to

yield

transformation of

the

of

contributes

mechanisms

to

pesticide

most

environment,


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carry

resemblance part

theoretical

communities

the

of

and

enhanced

possible

role


of

11. KARNS such

Molecidar Genetics of Pesticide Degradation by Soil Bacteria

extra-chromosomal

organisms. described on

the

He a r g u e s in

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survival

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duplicate leaving to

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hope

introduction that

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the

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be

in

existing

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original

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to

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the

reader

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development

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this of

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cell

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selection,

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the

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in

genetics

enzymes

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occurrence

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the

the

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organism

the

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disappears.

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elements that

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to

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in

base

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the

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(Figure

1).

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3.7x10^

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is

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more


one

or

copies

143

144

ENHANCED BIODEGRADATION O F PESTICIDES IN T H E ENVIRONMENT

per

cell.

It

copy-number of

the

follows

plasmids

enzyme

encoded

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into

are

propagated DNA.

not

only

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at

its

bacterial

limited

by

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cases, Of

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laboratory

(Figure

Transformation i s from

the

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the

natural process

environment.

to

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to

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demonstrated been

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demonstrated

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the

the

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the

bacterium limits

DNA b e t w e e n labile

(9,10).

competence

on the

been

when

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bacteria

The s p e c i f i c i t y

the

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cycle

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(9^).

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through careful

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Haemophilus,

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when

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Mechanisms

3).

closely

bacteriophage

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discussion

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only are

to

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receptor

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11. KARNS

Molecular Genetics of Pesticide Degradation by Soil Bacteria


chromosome

Figure forms

Figure

DNA a r e

2.

encoding

degradation left

and

proteins

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normally

Generalized

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Types

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of

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3.

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the IS-R the

145

146


might

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genes

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role

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previously,

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play

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mobilized

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denotes contact.

and m o b i l i z a t i o n

plasmids. be

transduction

microorganisms.

ability

of

a

of

that

gene

as

gene

community.

Plasmids and Transposons i n Pesticide Degradation Although bacteria,

there

degradation. the from

most soil

bacteria

have

little

is

been

There are

significant are

reports

the

the

of

are

1)

the of

of

for

bacteria

harder

genetic

degradation

aspects

reasons

types

generally

subject

pesticide

genetic

p r o b a b l y many

are:

environments that

many

known a b o u t

to

of

this,

but

that

are

work w i t h

and m o l e c u l a r

by

this two

than

biology


of

isolated most

11. KARNS studies,

Molecular Genetics of Pesticide Degradation by Soil Bacteria and

inactivate

;

2)

transformation, selectable the

roles of

one

the

enhanced

enzymatic

plasmid

a

encoded

its

of

the

in

part

of

to

a

studied

of

2,4-D

evolve

isolates.

encoded

indicating these

a

role

two

in

types

(2,4,5-T) ability

plasmid the of

Although

plasmid

a specific

degradation shown

that

insertion involved cepacia a

role

this

of

not

have

the

by

the

been

evolution

a

periods.

single During

Pseudomonas PP3

original

be

the differed

community)

studies

revealed

strain

mobilized

have

PP3

from

from

of

various

organisms

that

these

independently some

share

of a

the

of

but

rather

genes

involved

common a n c e s t r y

degradative

have

capabilities

A 2,4,5-trichlorophenoxyacetic cepacia

a

rapid

was

rate

has

be

this

yet

to

organism,

shown

to any it

that

that

Since other

is

2,4,5-T

in

bear

have

these

strains

likely

for acid

the in

rich

involvement.

implicated

genes

(2_1).

in

lose

DNA s e q u e n c i n g

DNA s e q u e n c e s

close

to

upon s u b c u l t u r e plasmid

in

with

(19),

recombination events

the

have

organisms

diverse

a c i d d e g r a d a t i o n genes and

has

2,4-dichloro-

a c q u i r e d b y many

they

herbicides

demonstrated

hybridization studies

suggesting

of

the

of

a c q u i r e d by

DNA/DNA

ability

examined, of

the

could

indicative

found

(13).

bacterial

no

strain

d e g r a d a t i o n of

been

degradation been

sp.

two be

dalapon i n

herbicide.

phenoxyalkanoic acid

plasmids

repeated

located

2,4,5-T

that in

are

elements have

trait

2,4,5-T

there

in

sequences

in

evolution

a

to

degradation of

Numerous s t u d i e s

at

shown

prolonged

Subsequent

can

encoding

distinct

that

to

(15).

transfer

g r o w t h medium ( 2 0 ) ,

for

evolution

rapid

that

casualty proceed

been

the

a member o f

element

that

2,4,5-T

the

w h i c h had been

compounds.

degrade

have

degrading

dalapon.

d e g r a d i n g Pseudomonas

to

to

The genes

three

showed

has

shown

the

herbicide

dalapon but

of

chlorobenzoic

that

least

degrade

DNA s e q u e n c i n g

has

a of

several into

first

shown

chemostat

(1_6,18.)>

genes

spite

development

degradation of

Studies

degradative

of

the

the not

propionic acid which

(12).

the

(1_£,Γ7)·

homology

this

metabolism

played

(2,4-D)

the

been

capable

bacteria

plasmids

on

degrading Moraxella

at

PP1,

genes

degradation

common s e t

plasmid

(strain

has yield

a

to

metabolism of

acid

done in

is

In

insights

incorporated perhaps

witnessed

some d e t a i l .

substantial

2,4-D

in

dehalogenate

other

involvement

bacterial

they

transposable

in

phenoxyacetic

a

studies

dehalogenases

completely

culture.

bacterial

comparing

to

studied

P P 3 w h i c h was

PP3 i n t o

not

difficult.

may p l a y

soil

source

concert

of

dehalogenase

The

that

(14)

in

progenitor

were

did

the

chloroacetate

study

pure

ability

the

shown

a

capable

strain

that

the

in

from

phenomenon.

community c o n t a i n e d

was

herbicide

been

transposons

community m a i n t a i n e d

course

strain

very

genetic

dehalogenation

which acted

organism

its

been

c a r b o n and energy

original

from

studies

chloroaliphatic acid

microbial

in

and/or

and co-workers

members

t r a n s f o r m and

degradation phenotype

d e g r a d a t i o n phenomenon)

as

used

putida

have

degradation

through

the

pesticide

degradation of

be

The

bacteria

(2,2'-dichloropropionic acid,

distinct


there

plasmids

enhanced

Slater

the

the

d e r i v i n g any known b e n e f i t

making g e n e t i c

Bacterial dalapon

without

d e g r a d i n g b a c t e r i a w h i c h may p r o v i d e

that

the

many c a s e s

thus,

difficulties

pesticide

in

pesticides

that

degradative

the studies

have

resemblance

been

shown

to

to be

r e p e a t e d DNA of

Pseudomonas

they

have

capability

organism.

American Chemical Society Library 1155 15th St., H.W. In Enhanced Biodegradation of Pesticides in the Environment; Racke, K., et al.; Washington, D.C. Society: 20038 Washington, DC, 1990. ACS Symposium Series; American Chemical

played of

147

148

E N H A N C E D BIODEGRADATION OF PESTICIDES IN T H E ENVIRONMENT

Plasmids of

the

have

organophosphate been

shown

pesticide in

to

were

(25).

be

present

in

at

note

cloning

that

function the

in

(22,2^3,24).

in

the

addition,

insert the

the

causes

the

range

role

that

protein of of

to

be

these

While

of

the

genetics

the

above of

mentioned

implicate

plasmids

degradative

community,

there

are

pneumoniae

been to

to

a volume

that, to

reports

from

enzymes

responsible

several

other

cloned

gene

encoding

sp.

of

several

the

carbofuran hydrolase

(32).

large

best

of

(26,28). such

the

a

important

in

the

of

that

plasmids

This

enzyme gene

we w e r e

plasmid

the

as

Of

in

special

encode

enzyme

ability

are

the

the we

in

to

have

an

catalyze

(33).

probe

show t h a t

this

A

carbamothioate

a specific to

of

Klebsiella

phenomenon

insecticides

able

present

enables

I n my l a b o r a t o r y , has

of

microbial

studied.

that

the

and s p r e a d soil

(29).

degradation

cases

strongly

encoded

well

bromoxnil

of

the

plasmid

less

a nitrilase

on

studied and

evolution

of

are

N_-methylcarbamate

experiments,

the

the

a carbofuran hydrolase

hydrolysis

on

cases

(30,30.

Achromobacter

encoded

of

to

but

across

played

bacteria, the

degradation

and b u t y l a t e

cloned

in

date,

laboratories the

are in

among members

enhanced

EPTC

hybridization

(28).

membrane

lividans

have

which

functions

the

function

to does

does

diminuta strains

suggestive

herbicide

herbicides the

(26),

lividans

in

strongly

examples

to

the

for

protein

these

promoter

but

opd gene

to

encode

the on

from

opd gene

Streptomyces

degradation

several

degrade

interest

the

opd

different

interesting

organisms,

recombination

abilities

shown

is

elements

and r e c o m b i n a t i o n

degradation

has

and

very

Streptomyces by

in

gene.

pesticide

pesticide pesticide

is

it

has

that

and

USA, i d e n t i c a l

Escherichia coli

Pseudomonas by

demonstrated

opd genes

the

(for

hydrolase,

Philippines, the

the

hydrolase

excreted

opd

or

organism

regulatory

plasmid mobility

in

that

encoded

parathion

was

degradation

The opd

organophosphate

otherwise

gram n e g a t i v e

peptide

distinct

(2£»27.)»

shown

the

parathion

the

were that

putida

are

microorganisms

dissemination

plasmid

have

F l a v o b a c t e r i u m and

ability

wide

revealed

gram p o s i t i v e leader

processed

native

The

a

that

in

(22^23^.

It

in

MG, i s o l a t e d

Pseudomonas hosts

involved

three

isolated

identical

studies

original

function

has

absolutely

be

encoding

least

plasmids

DNA s e q u e n c i n g are

like

strain

on

to

parathion

gene,

bacteria

diminuta carried

organisms not


shown

degradation)

degrading

Pseudomonas

the

been

insecticide

F l a v o b a c t e r i u m ATCC27551,

genes

In

also

organophosphate

in

the

bacterium

By

the using

DNA/DNA

gene

is

(32).

Future of Genetic Research on Enhanced Degradation of Pesticides Much r e s e a r c h

remains

understanding

of

development in

the

to

bacterial

will

be

their

thorough understanding phenomenon. the

various

are

actually

of

to

that

actual

might

it

of

one

isolated

agents

require

basic no of

the

very

a role to

c a n be

play

enhanced

contribute the

there

that

seems

play

T o my k n o w l e d g e , causative

the

involvement

microorganisms

demonstration

before

factors

While

found

populations

pesticides,

done

and p r o p a g a t i o n

agricultural soils.

transposons

be

genetic

in

has

enhanced

the

likely

plasmids

the

rapid be

that

evolution

degradation

ecology

demonstrated of

degradation. of

of

without

of

the

that

any

and

of

determined

degraders

development

true

in

phenomenon

microbial as

any

role

degradation

the

cannot

a

any

of

pesticide Such a

an a g r i c u l t u r a l


a

11. KARNS version

of

isolated to

an

Molecular Genetics of Pesticide Degradation by Soil Bacteria Koch's

from

inactive

While

to

practices, Many

soil,

conduct soil

of

degradation Monoclonal pesticide

techniques

on

the

track

denatured,

tagged

(radiolabeled

reporter

of

of

colonies

to

or

This

have

been

enzymes

pesticide

genes

in

would

allow

as

other

polymerase

chain

the

amount

of

4).

This

that

sequences

of

complements

to

the

remelted

over

act

is

original

sample

increase

the

as help

only

PCR has

in

of one of

the

a

in

only After

20

Thus,

detection study been

of

to

bulk

the

shown

to

such that

genes of

a

probes

to

spread

The

greatly

amplify (Figure

and

to

known

The synthesis

of

new

specific duplicated. and

cycles was

this of use

there

present

would

in

technology interest in

The

the

molecular microbial be

from

genes

DNA s a m p l e

DNA i s

using

for

from

similar

were

synthesis

DNA s e q u e n c e

acids

community.

vitro

that of

from

with

specific it

primers are

round

copy.

if

interest.

the

the

interest another

already

samples

for

Since

c o u l d be

DNA i s o l a t e d

detect

homologous

of

detect

heterologous

DNA p o l y m e r a s e

gene

many t i m e s .

copies

sensitivity

certainly

environmental

primers 4).

allow

repeated

stable

a

small

to

degradation of

to

used

in

various

nucleic

highly

c a n be

By using

Using cloned the

even

microbial

and

electrophoresis

in

These gene

the

DNA c u t

possible

specifically

DNA o f

to

1 million

soils.

as

soil

the

bulk

extracting

be

powerful

possible

support

36),

involved

the

conditions.

of

highly

purified

DNA s e q u e n c e

within

DNA ( F i g u r e

be

In

pesticide

of

is

of is

study

supports

(38>1£>^£)·

a given

heat

a

solid

for

(PCR, 4 0

are

in

use it

DNA m o l e c u l e .

it

by a g a r o s e

would

the

reaction

oligonucleotides

procedure

within

procedure uses

nucleotide

or

many s o i l s .

a particular

oligonucleotides

strands

from

to

it

probe

the

recently

likely

the

solid

blots,

Techniques

t r a c k i n g of

organisms

to

highly

a very

advantage of

interest

microbes,

probes

organisms the

of

a

to

encoding

for

By

field

through attachment

a specific

(colony

developed

takes DNA t o

and s e p a r a t e d

37).

genes

takes

through the

under model

strands

stranded

plate

enzymes

which encode

two

T h e DNA f i x e d

blots,

the

cell.

used

the

specific

microorganisms.

c o u l d be

on

study

technique

antibodies

of

levels.

present

Using such probes

specific

field

enhanced

to

highly

detected

(35).

technique

tagged

the

used

bacterial

these

c a n be

organisms

the

a p o p u l a t i o n of

(Southern

of

(34,15).

antibodies

technique

DNA s e q u e n c e s

DNA.

restriction

will

that

DNA a s

on an agar

or

of

account. the

antigens

c o u l d be

particular

detecting

single

molecules)

levels

mixture

be

soils

soil

molecule

This

nature

fixing

is

surface

DNA/DNA h y b r i d i z a t i o n o f f e r s

enzymes.

with

the

of of

be

tillage

into

into

technique

complementary

DNA

insight

these

degrading

to

taking

associated

soil.

might

o r g a n i s m a l and m o l e c u l a r

and enumerate

means

active

it

etc.

of

specific

given

scale,

and spread

The

soils

newly

is

introduced

the

isolate

this

organism

for

techniques

simulations

the

perform,

rainfall,

gain

from

a

obtained

microscopy

to

greenhouse

is

to

in

to of

a fluorescent probe

fluorescent

soil

cells

surface

to

classically

both

ability

from

crop,

causative

phenomenon when

greenhouse

specific

those

putative the

simple

of

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attaching

low

type

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the

growth

them on a

antibodies of

possible

not

used

of

a

produce

be

distribution

specific

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can also

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where

to

and r e - i s o l a t e d

types,

the

biotechnology


shown

such experiments

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and

postulates,

soil,

the

in

the to soils

ecology

study

(42).


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149


150


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Diagramatic (PCR) used

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synthesis

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s t a b l e DNA the

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repeated


(D).

11. KARNS In that

Molecular Genetics ofPesticide Degradation by Soil Bacteria

order

degrade

pesticide to

be

this

one

reach

degradation

compared

in

classes

of

that

do

not

have

demonstrated

Once

the

genes

degree

determined,

of

interest genes

require

a

would of

would take

yield place

be

In

is

interest

some

with

given

must

be

known.

cooperation

Thus,

such

into

detects

the

gene genes

P C R DNA s e q u e n c e s experiments

and m o l e c u l a r

the

but We

(43).

degradation

between p e s t i c i d e

ecologists,

insights

use

in

among

sources

adequately to

functions,

diversity

have

is

several

experiments.

bacterial

order

would

there

may b e

identical of

organisms encoding

genes

there

pesticide

that

In

many genes

cloned

cases

a degree a

the

hybridization

DNA p r o b e s

microbial

These

from d i f f e r e n t in

and

how m u c h d i v e r s i t y

enzymes

developed.

incredible in

there

of

sophistication

cloned.

DNA/DNA

diversity

set

of

isolated,

determine

in

genes

unprecedented

microbiologists,

to

encode

that

of

be

function.

react

hydrolase

level

must

enzymes

that

cross

is

with

this

order

biochemical

parathion


to

a pesticide

biological

chemists,

would soil

biologists, processes

but

that

soils.

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8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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151

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