MELANIE L. L E W S DEPARTMENT OF PLANT SCIENCES

Submitted in partial fuifilment of the requirements for the degree of

Master of Science

Faculty of Graduate Studies The University of Western Ontario

London, ON August 1998

0 Melanie L. Lewis t 998

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ABSTRACT

In many parts of the world atrazine is the most cornmonly used herbicide for control

of broad leaf weeds in a vanety of crops. Its intensive use and persistence in soil has led to

concerns regardinj gound and surface water contamination. Consequently there is

considerable interest in developing agricultural management practices that would minimize

the potential for ground and surface water pollution by atrazine and its metabolites. One

possibility is to use soil bactena that degrade atrazine to non-toxic metabolites. Atrazine-

degrading bacteria have recently been isolated From agncultural fields in Ontario and

Québec.

In the present study, rep-PCR using the consensus pnmers BOXAlR and

ERIC 1 WERIC2, plasmid profiles and biochemical reactions were used to characterize 1 2

gram-negative atrazine degraders isolated from a single site near Ottawa, Ontario. Results

indicated that there was very limited diversity among these isolates.

A unique atrazine-degrading gram-positive bactenum from Sr. Hyacinthe. Québec.

designated C 1 87, also was characterized. 16s rDNA sequencing indicated that C 1 87 was

a new species in the genus Nocardioides. Nocardioides sp. strain C 187 used atrazine as a

sole source ofnitrogen and as a carbon source. Although it could d e p d e atrazine it couid

not mineralize it to CO1 and ammonia. High pressure liquid chromatography revealed that

atrazine was degraded to the non-phytotoxic metabolite hydroxyatrazine under aerobic and

oxygen-free conditions. Hydroxyatrazine was also degraded under these conditions. This

suggested that the first two steps in the degradation pathway were catalyzed by hydrolases,

as has been described for Psezrdomonar sp. strain ADP (De Souza et al.. 1996 and Boundy-

... 111

Mills et al.. 1997). The hydrolases (atrazine chlorohydrolase and hydroxyatrazine

ethylaminohydrolase) synthesized by Psewdomonas sp. strain ADP are cncoded by the genes

atzA and utzB. Dot-blot hydridization with DIG-labelled probes prepared From at1.4 and ut=B

indicated no sequence similarity with purified jenomic DN.4 fiorn .Vocat-diozcles sp. strain

C 157. The gram-positive bactenum. Rhodococnrs corallinzrs YRRL B- l 5 U J R . degades

dethylaminoatrazine via the enzyme s-triazine hydrolase. which is rncoded by r ~ 4 (Shao et

al., 1995). Dot-blot hydndization usinp a DIG-labelled probe prepared from trz.4 indicated

no sequence homology with .Vocordiozdes sp. strain C 1%'. These results sugsest that the

hydrolases responsible for atrazine and hydroxyatrazine degradation by .Vucardiordes sp.

strain C 1 87 are not encoded by genes homologous to ar;-l. acB or rr-4 .

Cell free extracrs prepared bom strain C 187 cells gown with atrazine and without

a m i n e were analysed for their ability to degrade atrazine. For both treatrnents. no lag phase

was present, atrazine was degraded ro hydroxyatrazine at the same rate. and hydroxyanazine

was no t metabolized. These results suggest that anazine dec hloi-ination is constitutive.

The results of this study indicate that atrazine-degrading bactena isolared on a plot

scale are not diverse. This is the first report of a .Vocardioides species degrading atrazine.

Atrazine degradation by :Vocardzoides sp. C 1 87 is constitutive and the aromatic nucleus of

atrazine is not rnetabolized. While ,Vocardioides sp. strain CI87 degrades a m i n e

hydrolytically it does not possess sequences homologous to utz-f , ucB or r r -4 .

Keywords: atrazine, rnineralizaton, bacteria, degrading, hybridization, diversity.

.4CLiYOWLEDGEMENTS

A number of people both in the lab and outside of the lab whose contributions and

effons are highly appreciated were instrumental towards the completion of this project.

First and forernost. I would like to thank my supervisors Dr. Edward Topp and

Dr. Diane Cuppels. for their academic guidance throughout the past two years. 1 thank

my advisory cornmittee members, Dr. Andre Lachance and Dr. Susanne Kohalmi for

their advice and invaluable suggestions.

1 am extrernely gratefùl to Sandra LMillar, Henry Bork, Teresa Ainsworth, and Lou

Ann Verellen for their technical advice and assistance.

1 would like to thank M. De Souza, Dr. L. Wackett and Dr. M. Sadowsky at the

University of Minnesota, for providing me with Prer~domonar sp. strain ADP. piMD4

and pATZB-2. Thanks to Dr. D. Cuppels for providing me with bacterial strains that

were essential to many of my experiments.

Finally, I would like to thank Jeff Ivey for his endless help with the mathematical

and statistical analysis required for this thesis and for his emotional support as i

completed this project.

This project was financially supported by The University of Western Ontario and

Novartis Crop Protection.

TABLE OF CONTENTS

TITLE PAGE ......................................................................................................

CERTIFICATE OF EXAIMINATION .......................................................-.........

...................................................................................................... ABSTELKT..

ACKNOWL EDGEMENTS.. ...............................................................................

TABLE OF CONTENTS ..................,...........................................................

LIST OF FIGURES ............................................................................................

............................................................................................. LIST OF TABLES

............................................................................................ AB B REVIATIONS

Chapter 1 - INTRODUCTION ......................................................................-.

Chapter 2 - LITERATURE REVIEW ............................................................

........................................................................................ 2.1 Atrazine

2.2 Atrazine-degradation. ....................................................................

2.3 Pseudomonas sp. strain ADP.. ........................................................

2.4 Rhodococczis corallintrs NRRL B- 1 5 U R .....................................

Chapter 3 - MATERIALS AND METHODS ......................~...........-...-....-...-.

3.1 Bactenal strains and plasmids ..........................................-.~~--..--..-..

3.2 Media and growth conditions ..................................................--.....-

3.3 Characterization of bacteria ....................................................--......

3 -4 Analytical techniques.. .................................................-..........---......

vi

Page

1

* .

I l

. * .

111

v

vi

xiv

. . Atrazine minerahzatron ........................ .. ............................

High pressure liquid chromatogaphy (HPLC) analysis ......

Preparation of C 187 ce11 free extract (CFE) .......................

Detemination of rate of atrazine-degradation by cell free

.............................................................................. extract

Preparation of C 187 whole cells and determination

of the rate of atrazine-degradation .....................................

.......................................................... . 3 5 DNA manipulation techniques

................................................. 3 3 . 1 isoIation of plasrnid DN A.

3.5.2 Isolation of total genomic DNA .........................................

3 . 5 . 3 Preparation of DIG-labelled DNA ......................................

3.5.4 Recovery of DIG-labelled DNA

k a p e n t s from agarose .................... ............ ......................

- - 3 . 3 Dot blot preparation ..........................................................

33 .6 Hybridization and detection of DIG-labelled DNA

.............................................................................. probes

.......................................................................................... 3.6 tep-PCR

...................................................................... 3.7 16s rDNA sequencing

3.7.1 Preparation of PCR product for

....................................................... 16s rDNA sequencing

............................................. 3 J .2 16s rDNA sequence analysis

Chapter 4 - RESL'LTS .......................................................................................

4.1 Determination of diversity among atrazine-degrading

bacteria isolated from agicultural soils in Ottawa Ontario ................

4.1. I Assessrnent of biochemical diversity ...................................

. . 4.12 .Assessrnent of genetic diversity ..........................................

4.2 Characterization of C 187-an atrazine degrader from

St. Hyacinthe, Quibec. Canada .......................................................

4.3.1 Characterization of C 187 .............................................. ..-.

4.7.2 Determination of sequence sirnilarity with

Pseitdornonas sp. strain .4DP genes encoding atrazine

hydroiase and hydroxyatrazine ethylaminohydrolase and

with the Rhodococ~zî~ cora1liw.s 'IRRL B- l 5 W R

gene sncoding s-triazine chlorohydrolase ............ ............-.

4-23 'ulineralization of atrazine by C 1 57 ...... . . .. .. .... .-. - ... -. . - .-. . . -. . -

4.2.4 Degradation of atrazine to hydroxyatrazine by ce11

kee extract .... .... .. .. . ... .... . ... ... . ...... ..-. . . . . .. . . .. ... .. .-.... . .. .. . . . . . ...

4-23 DegradationofatrazinebywholrcellsofC1S7 ..................

Chapter 5 - DISCUSSION ........................................................... .....-... .-... .......-

5.1 Diversity and plasmid distribution in amine-degading

. . bactena isolated on a plot scale .........................................................

5.2 Characterization of a unique gram-positive bacteriun

from St. Hyacinthe, Québec .............................................................

. . . V l l l

. 5.2.1 IdentificationofC1S/ ..................... .. ............................. 7 5

5.2.2 Genetic and biochemical analysis of the initial steps

in the atrazine-degrading pathlva y. ..................................... 79

5 2.3 Regulation of atrazine degradation .............. ... .............. SZ

......................................................................................... REFERENCES

CLWCL!LLM VITI2 .............................................................................

LIST OF FIGURES

FIGURE DESCRIPTION PAGE

1. Chernical structure and properties of

- atrazlne.. ............................. ... .. 3

3 Structural and functionaI models of Photosystern iI 7 -, ..........

3. Proposed degadation pathways of atrazine

. . by sorl rnicroorganisms ..................................................... 13

4. Parhway for atrazine catabolisrn to cyanuric

acid in Psedomonas sp. strain A û P ............................... 15

5. Restriction rnap of Pset~rionionus sp. strain

ADP genomic DNA fragment cloned in

plasmid pMD 1. which contains the uc;l

and urrB senes .................... ... ....................................

Proposed pathway for the microbial degradation

of atrazine by Rhodococcrti cordlin~ls

NRRL B - l 5 W R ...........................................................

Dendrograms of the Ottawa amine-degrading

bacteria based on the biochemical responses to the

............................................. API 20E and ,srPI NFT test

Genomic fingerprints of anazine-degrading

bacteria isolated fiom Ottawa, Ontario, Canada

generated fiom ERTC-PCR. ............................................

X

Genomic fingerprints of atrazine-degading

bacteria isolated from Ottawa. Ontario. Canada

yrnerated Frorn BOX-PCR ............. ... ..........................

Plasmid profiles of selected atrazine-degading

bacteria isolated fiom Ottalva Ontario. Canada.. ............

'lucleoride sequence of the 16s rDNA jene

fiom Cl87 ......................................................................

Phylogene~ic tree showing the positions of C 1 S7.

rnembers of the genus iVocartiioides

and other related species .............. .... ..........................

Dot blot hybridization of purified DNA from C 187 with

DIG-labeiled nrr.4 and tznB probes ............... ..... ..........

Dot blot hybridization of purified DNA tiom C 157 with

.............................................. DIG-labelled tc.4 probe ....

Tram formation of atrazine to hydroxy atrazine

by CFE from C 187 when cells were g o w n

with atrazine and without atrazine, as demonstrated

by reverse phase HPLC analysis .......... ... .....................

Dissipation of anazine and formation of

hydroxyatrazine by CFE from C 187 .................................

Degradation of atrazine by Cl 87 whote cells as

demonstrated by HPLC analysis .......................................

Dissipation of atrazine. formation of hydroxyatrazine

and subsequent degradation of hydroxyatrazine by

C 187 whole cells ..............---...................-..-....--... . .-. . .. -. .-.

LIST OF T-ABLES

DESCRIPTION

.\nazine-degrading bactena used in this study ............................... 23

Plasmids and additionai bacteria used in this study ......................... 24

Primers used in this study 34 ............................................................

Biochemical responses of atrazine-degading bacteria isolated

tiom Ottawa. Ontario to .QI IOE biochemicai tesrs ...................... 43

Biochernicai responses of atrazine-degrading bacteria isolated

from Ottawa. Ontario to API >TT biochemical tests .............. .... ... W

ai

h p

* 4 i i S

bp

BCtP

CFE

cm

Cm

CFU

CC

Ci

Da

DIG

ABBREVTATIONS

- Percentage

- Degrees Centigrade

- Microgram (s)

- Microliter (s)

- Micrometer (s)

- Micromolar (s)

- Micromole (s)

- Absorbante

- Active Ingredient

- Ampicillin

Atrazine iMineral Salts

Basepair (s)

5-Bromo-4-Chloro-3- tndoly 1 Phosphate

Cell Free Extract

Centimeter (s)

Chlorarnphenicol

Colony Forming C'nit (s)

Cubic Centimeter (s)

Curie (s)

Dalton (s)

Digoxigenin

xiv

CSPD

JNTP

DN A

dLTP

DPM

ERIC

EDTA

EMBL

0 3

ha

HPLC

h

Km

kb

kg

LSC

L

LlMP

LB

- Disodium 3-(CMethoxyspiro { 1.2-Dioxetane-3.2'4 5-Chloro Tricyclo[3.3.1 .'.-IDecan! -4-yl) Phenyl Phosphate

- Deoxynucleoside Triphosphate

- Deosyribonucleic Acid

- Deosyuradine Triphosphate

- Disintegration Per Minute

- Enterobactenai Reperitive Intergenic Consensus

- Ethy lenediamine Tetraacetic Acid Disodium Salt

- European Molecular Biology Laboratory

- Hectare

- High Pressure Liquid Chromatogaphy

- Hour ( s )

- Kanamycin

- Kilobase (s)

- Kilogram (s)

- Liquid Scintilation Counter

- Liter ( s )

- Low Melting Point

- Luria-Bertani

- Milliarnpere (s)

- .Millicune (s)

min

$1

P P ~

PPm

Pen

pmol

nm

N

Nm

PV-

PCR

REP

- MilIigrarn (s)

- Milliliter (s)

- >Iillimeter (s)

- Millirnolar

- Minute (s)

- Mohr

- Mole(s)

- Nanometer (s)

- Nanomole (s)

- Nitroblue Tetrazolium

- Nutrient Broth-Yeast Extract

- Parts Per Billion

- Parts Pet MiIIion

- Penicillin

- Picornole (s)

- Nanometer (s)

- Normal

- Neomycin

- Pathovar

- Polymerase Chain Reaction

- Repetitive Extragenic Palindromic

- Repetitive Elernent Polymerase Chain Reaction

xvi

SSC

SDS

std, dev.

Sm

Su

Tc

Tra

Tn

TB E

Tris

TSA

G v

v

Vkm

v/v

w/v

- Resistant

- Revolutions Per Minute (s)

- Ribonucleic Acid

- Ribosomai Deoxyribonucieic Acid

- Ribosomal Ribonucleic Acid

- Sensitive

- Sodium Chloride Sodium Citrate

- Sodium Dodecyl Sulphate

- Standard Deviation

- Streptornycin

- Suppressor

- Tetracycline

- Transfer ( Conjugal)

Transposon

Tris Boric Acid EDTA Disodiurn

Tr ima Base (Tris [Hydro'rymethyl] Aminomethane

Tryptone Soy Agar

Ultraviolet

voit (s)

Volts Per Centimeter (s)

Vehme Per Volume

Weight Per Volume

xvii

1

Chapter 1. Introduction

Atrazine is the most widely used s-triazine herbicide for the control of annual broad leaf

weeds. About 36 x 106 kkg of atrazine are applied yearly in the Cnited States on crops such

as corn and sorghum (Mulbry, 1994). Atrazine was first introduced into Canada in 1960.

Current application rates are 1 to 1.70 kg a i h a depending on weed pressure. soil propenies,

climate conditions. and the crop planted (Ministry of Agiculture, Food and Rural

,4ffairs. 1996). The intensive use of atrazine in both the United States and Canada has led

to contamination of soil and gound water (Goodnch et al., 199 1 ; Belluck et al., 199 1 ; Frank

and Skons, 1985). Since atrazine may be a hazard to human health (Bouwer. 1989),

attention must be given to its fate in soil and to the chemical and biological processes

involved in its degradation.

In soil. biodegadation is the major pathway of atrazine dissipation (Erickson and

Lee, 1989). AIthough chemical degradation piays an important role. the present study

concentrates on microbial degradation. Initially atrazine was considered to be

nonmetabolized by most soil bacteria. It was no[ until the 1980s that microbial atrazine

degradation was proposed to occur by Y-dealkylation. resulting in the accumulation of

aminotriazine compounds (Behki and Khan, 1986; Giardina et al.. 1950, 1952;

Giardi et al, 1985). In the last 5 years pure cultures of bacteria have been described that c m

catabolize (mineralize) atrazine to CO, and amrnonia (Behki and Khan, 1994; Assaf and

Turco, 1994; Mandelbaum et al., 1995; Moscinski et al., 1996). Such bactena rnay be useful

for the decontamination of atrazine spill and waste sites. Atrazine degradation c m be

initiated by removal of the isopropyl and ethyl side chains. However recent studies

(De Souza et al.. 1995) indicate that degradation can also be initiated by hydrolytic

dechlorination. yielding the non-phytotoxic metabolite hydroxyatrazine. Funher degradation

ofthe ethylarnino and isopropylarnino side chains yields the metabolite cyanunc acid. which

can be used by many soit bacteria as a sole source of nitrogen (Cook. 1987; Cook et al.,

1985; Cook and Hütter, 1984; Eaton and Kams. 1991; Jutzi et al., 1952). Three genes

cncoding enzymes for atrazine catabolism to cyanuric acid have been cloned and sequenced

€rom the ritrazine-degrader Pseziï/olonronas sp. strain ADP (De Souza et al., 1996; Boundy-

Mills et al.. 1997: Sadowsky et al.. 19%). These genes. designated u c A BC. were shown to

be present in atrazine-catabolizing pure cultures isolated from sites throughout the world

(De Souza et al., 199s).

Atrazine-degrading bactena have been isolated from Canadian (Behki et al.. 1993;

Behki and Khan, 1957; Shao and Behki, 1996), American (Assaf and Turco, 1994;

Mandelbaum et al.. 1991) and French (Bouquard et al., 1997; Topp et al.. 1998) soils

previously exposed to atrazine. Recently our lab has isolated two groups of atrazine-

degrading bacteria (gnm-positive and gram-negative) from diverse soils in the agicultural

regions surrounding Ottawa, Ontario, St. Hyacinthe, Quebec and Winchester, Ontario (Topp

et al.. 1997). The two main objectives of this thesis are to deterrnine the divenity of the

gm-negative atrazine-degrading bacteria from Ottawa and to characterize the initial steps

involved in atrazine-degradation by a gm-positive aû-azine-degader from St. Hyacinthe.

Figure I . Chernical struct~ire of the a-icuitural herbicide atrazine i 2-cliloro-4-

ethy lamino-6-isopropylamino-s-tnazine) ( Spencer. 1 087).

Figure 2: Structural (-A) and hnctional (B) models of Photosytem 11. rr. a tyrosine

residur acting as an zlectron donor to p 6 W . the arrows show thr: direction

of sicctron tram fer: OEC. oxygen-svohng cornpiex: p68li. PSI1 reaction

cenrre: phaeo. phaeophytin a: Q., and Q,. quinones: PQ. mobile plastoquinonc

pool. Figure modi tied kom Cobb ( 1 YI?).

s

and mobility in soi1 (Topp et al.. 1W5L Atrazine is classifïed as a moderately persistent

herbicicic nith a hrilf-Iik rringing tiom 15 co 20 days in estuanne sediments to sc\.eral

months. up ro a year. in soi1 (Jones cr al.. 1983. .*y atrazine rcmaining in thc soi1 may be

triinsportrd into groundwater. surface waters. streams and tile drainage via surtace ninoff and

~roundwatcr intsrtlow. Jones et al. ( 1W3) reported that approsimately O. 1"% to 3% of the - atruinc placed on agicultural ficlds is iost to the aquatic environment. In many cases the

lzvrls o t'atrazint detectrd in gound watzr. surface w t s r s and jtreams esceeds the masimilm

contaminant Ievei sct by the Cnitcd Statcs Environmental Protection Agncy of 2 pans per

billion ( ppb) ( Ktllo. 1 'IS9). -4trazinc hÿs also been detrcred in the atrnospliere and min watcr

(Nations and Halberg, 1992). The solubiiity of atrazine in wiiter at 25 'C is 33 mgL. Once

airuine and othcr pesricidrs reacli pundwater and aquifers t k y c m persist t'or many yean.

For those who use goundwatsr as the sole source al'drinkiny watcr this can prcseni major

hralth prohlcms such as cancer. nen-ous systrm disorders. birth drfects and male strnlity

(Bouwer. 1089). In lin attrmpt to detoxify contaminated aquifers expensive physical and

chsmical treatment plants have bccn devrloped to rcmovc the contaminants at the whole

h o u x point of zntry (Goodrich ct al.. 199 1 j. Although trcatrncnt plants arc necessa-

(because ofprevious conramination). prevcntativc methods arc prcfcrrcd sjnce tliey are less

expznsivc and long tem. Consequently. there is considerable inrerest and need for the

dcvelopmsnt oT agicultural mamyement pncticas that minimize the potential for atrazine

to enter goundwater and surface water. One such application being studied is the addition

of organic amendmrnts. such as manure. to the soi1 in an attempt to alter the rate ot'atrazine

degndation. Topp et al. (1996) and Entry and Emmingharn ( 1995) both found that the

9

addition of Jairy manure to tields cropped to corn increassd the rats ofatrazins degradation.

[n order ro devclop iurther practicrs. characterizarion of atrazine degradation in the soi1 is

essential.

2.2 Atrazine-deyradation.

In the soi1 citrazine can be degradcd non-biologically and biologically. Non-

biological degrridation can occor \via photodccornposition. volatilization. soi1 ~dsorption. and

b y two soi 1-associarcd chernical rwctions: hydrosy lation and deal kylation ( Jordan ct al..

1 970). Although these processes are occumng. biological cisgadat ion. speci fically microbial

degradation. is the major pathway for atnzine dissipation in the soi1 iErickson and Lee.

19S9) . While çarlicr studies only reponed atrazine degradarion by rnixed rnicrobial

consoniü. more recent repons have indicated that several pure microbial srrains can degrade

atrazine and cise i t as a source of nitroyen and'or carbon ro suppon yrouth. Tliese strains

include Rhorioc*occirs strain B-30 ( Behki and Khan. 1994). Rliodococczts sp. strain NIS6iZ1

( Nagy et al.. 1995). Rlrodococcw sp. strain T E 1 ( Sliao and Behki. 1993). .-lci~zerob~rcrer

~dco~ierictts ( Mirgain et ai.. 1993 ), a new bacterial species closely related to .-lgrohacrericmr

i-citiiohmtcr ( Radosevich et ai.. 1 3 V o r i i Giardina ct ni., 1980. 1982:

Giardi et al.. 1985). and several strains of Pseltdonloms including Pseltdomorzus sp. strain

ADP (Mandelbaurn et al.. 19953. It is believed that atrazine c m be dejraded in the presencc

or absence of oxygen. depending on the organisrn in question. For instance. De Souza et al.

( 1996) showed that Psezrdomonus sp. stnin A D P could degrade atrazine under both aerobic

and oxygen-limiting conditions, while Behki et al. ( 1993) demonstrated that strains of

I O

R/~othcoc.cus could only degrade atrazins under aerobic conditions.

TIic tirst merabolic srep in the biodegradation ofatrazine has been sugsested to be

ï-dealkylation. wirh remocd of the cthyl sidr chain prcceding rernoval of the isopropyl sidc

chain i Radosevich st al.. 1995 ). Howvcr. Behki and a i a n i Ic)S61. reporteci that sorne

species of Pse~idoi~zomrs dralkylate the isoproppl yroup tirst. in eirhrr case the

microoganisms are using the olkyl side choins 3s a carbon source. Man)- microorganisms

do not fiirther metabolize rhe cierilkylated met3bolites. Sincc .V-dcalkylation is not sufficicnt

to desrroy the phytotoxic p r 0 p e ~ k of atrazine and the rnctaboiitcs may have unknow-n

effects on mimals and othcr organisms ( Kaufinan and Blake. 1970: Kaufman and Kramey,

1970) funher degradation is desirable. Additional depda t ion of arrazine metabolites by

Jeamination. cicchlorination and'.or ring cleavage i mincralization) lias been ubsewed in some

species i Boundy-Mills et ai.. 1997). Howcvcr. dechlorination to producs hydrosyanalojs

is suficient to dcsiroy phytotosicity ( Mandelbaurn ct al.. 19%). While the mechanism for

chcmicril dechlorination by hydrolysis is well undcrstood. the mechanism and rolc of

rnicrobial dechlorination (by hydrolysis or osygenic dcchlonnation) is less clear (Behki and

Khan. 1986). Cntil recently ir was believcd that soi1 bacteria ciid not have the snzyrnatic

systems to hydrate the aromatic carbon-halogen bond. Howevrr. recent studies of atrazine

dejradation by .-lgrohocreritrrn J I Jri (Moscinski et al.. 1996). Ralsroniü SA9 1-3

(Radosevich et al.. 1995), Hhodococc~rs sp. (Shao and Behki. 1995; Shao ct al.. 1995).

Rliizohirmi sp. (Bouquard ct al.. 1997). and Pselrdonionrir sp. s tn in ADP (De Souza et al..

1996; .Llandelbaum et al.. 1993. 1995) have indicrited that microbial dehalogenation is

occurring and in Fact may be the îïrst step in atrazine biodegradation by Psercdonioncls sp.

I I

strain ADP.

Cornpicte mineralization of otrazine occurs by clravage of the s-triazine ring. Dircct

attack on atrazine to sleaw the s-triazins ring has not been reported to date. How-ever.

studies by Cook et ai. ( 19S5). Ericltson and Lee i 19891. Radosevich et al. (1995) and

\Iandeibaurn et al. ( 19931 have shown that some organisrns. including soms ritrazine-

degraders are catabolizing the metabolite cljanuric acid to carbon dioxide and arnrnonia.

Althoush the exact pathwa'; in mazinc-dcgraders is unclear- i t has been hypotheslzed that

cyanuric acid is clcanxi cnzymatica11y to produce biurct and carbon Jioxide. Biuret is thcn

coniw-ted to urea. n-hich is dcgraded to the final products of mineralization. ornrnonia and

carbon dioxidc ( Figure 3 ).

2.3 Pseudomonas sp. strain ADP.

F'seucfonto~~cls sp. strain ADP is a gram-negati r-è ritruine degrader that was isolatsd

(rom a herbicide spi11 site in Minnesota. CS.;\. r Mandelbaum et al.. 1995). Pserdoi~roiras

sp. stmin ADP is capable of mineraiking the r-tnazine ring of atrvine via the intermediates

hydrosyatrazine and c~~anuric acid. in doing so i t uses ritrazine as its sole nitrosen source.

Most of our current undsrstanding of the genes and enzymes involved in atrazine

degradation cornes tiom studies of Pserdonzonu.~ sp. strain ADP. The enzymes for atrazine

catabolism to cyanuric acid (by Pserrdonio~ius sp. snain ADP) are encodcd by the genes utz.4.

rlcB and rrïzC. which are located on a self-transmissible plasmid (De Souza et al.. 1998).

The tirsr gene in the degradation pathway. c ic .4 . catalyzes atrazinc dechlorination to the non-

phytotoxic metaboiite hydroxyauazine (De Souza et al.. 1995) (Figure 4). The urz.4 gene is

Figure 3. Proposrd degadation pathways of atrazine by soi 1 microor~anisms exc luding

P.seiidomorr~rs sp. strain ADP. 3. dechlonnation: b. .V-dcalkylation: c .

deamination; and ci, ring c l s w a g .

Atrazine -

Deethylatrazine H ydroxyatrazine Deisopropylatrazine

Deeth yl hydroxyatrazine Deethyldeisopropylatrazine De i~~pr~p!~ lhydr~~yat raz i f le

Deethyldeiso propyl hyd roxyatrazine (Ammeline)

Cyanuric acid

Biuret

U rea

COz+ NH,

Figure 4. Pathway for atrazine catabol km to cyanunc acid in Psezctlon~o~im sp. strain

ADP (De Souza ct al.. 1998). L i r r l . ~ i r A 3 . and 'icC encods the enzymes

a i ru ine chlorohydroiase. Iiydrosymxzinc cthylarninohydrolase and Li

isopropylarnmelide isopropylaminohydrolase respectively.

16

located on a 2 l .j kilobase ( k b ) EcoR l fragment. This fragment. ~vhich was cloned into

pLAFR3 and designatcd pMD 1. cncoded atrazine degradation activity in E. coli DHju

( Figure 5 ) ( De Souza et al.. 1995 ). Atrazinc degradation was dcmonstratcd by a zone of

cleariny on agar rncdium containing crystallinc atrÿzinc. .A Scnc con ferring the atrazine-

clraring plienotype \vas s~ibcloned as a 1.9 kb .-fiuI fragment in pXCYC 1 8-1. which was

dcsignatrd pMD4 (Dc Souza et al.. 19951. The same 2 1.5 kb EcoRl hyment also contained

thc second gene in the degradative pathway. utzB t Figure 5 ) . <icB is located 8 kb

downstrcam o f ~ r 1 z - f and encodes the enzyme hydroxyatrazinc cthylarninohydrolase. which

rrmsfomis hydroxyatnzins to .V-isopropylammelide tBoundy-Mills et al.. 1997) (Figure 4).

trcC: thc third gene in the dcgradativc pathway. encodcs the enzyme Y-isopropylamrnelide

isopropylaminoliydrolase ( AtzC) which transfoms X-isopropylarnmclide to cyanuric acid

and isopropylaminr (Figure 4). rrcC has been cloned and cxpressed in E, coli DH5u

(Sadowky et al.. 1998).

Rrcent studics by De Souza et al. ( 1998) h a ~ e shown that ti\-e geographically-distinct

atrazine-deyrading bacteria (.4lculigmes strain SG 1. .Agi-oh~icrer-hnr strain J 1 Ja, Strain 3Si38,

R~rlstorritr strain M9 1-3 and Clasihcrcter-) contained genes homologous to nc.4. -B. -C. This

suggests that atrazine-catabolic genes are conserved in diverse bacterial genera thereby

making Psercdomoriirs sp. strain .4DP an ideal mode1 system for the study of atrazine-

A, Restriction map of Psezldornonas sp. strain ADP smomic D.N-4

hagnent clonrd in plasmid pMD 1. which contains the uc.4 and L I ~ B jenes.

B. Rsstnction rnap of subclons pATZB-2. -4 4.0 Kb Chi frrigmcnr w s

subcloned into p .KYC 184 to produce pATZB-2. This figure is kom the

journal article published by Boundy-Mills st al. i 190-) and used n i<h their

permission.

t .4 Rhodococcus coralliri ris X RRL B- 1 5444R.

R . cor-crliirtrrs N R R L B- 15444R is a gram-positik-e coryneform bacterium that \vas

obtiiincd tiom soi Is tliat were exposrd to higli Isvels of s-triazine herbicidzs for '0 years.

R. ru)-dliiriis NRRL B- 1 5 - W R is capable of uti lizing deisopropy laminoatrazinc and

destliylaminoatrazine but not atrazins (or simazine) as a solc nitrogcn source for growth

(Cook and Hütter. 1986). Deisopropylaminoatrazine. also called deethylsirnazine is a

product of aerobic atrazine and simazine degradation. whereas deeth y laminoatrazine is a

product of atrazine degrcidation only. R. coruilorrrs NRRL B- I j - I U R has the ability to

dechlorinate md deaminate deisopropylaminoatrazine (and deethylaminoatrazinc), both

aerohically and anasrobicnlly. to the metabolites .V-ethy 1-ammclinc and :V-cthy 1-ammelide

respectivrly (Figure 6 ) (Cook aid Hütter. 1986). The enzyme for deamination lias not been

isolated ro h i e . Cook and Hüttcr (19S6) purifid 2 hydrolases (180 000 Da. and 450 000

Da.) lrom R. coi-riiltirru NRRL B - I 5 4 U R which wcrc capable of dechlorination of

deisopropylaminoatrazine (and deethylaminoatrazine). However. Mulbry ( 1994) in an

attrmpt to reproducs the exprriments was only able to purify onc hydrolase composed of

four identical subunits of 54 000 Da. The enzyme. s-triazine hydrolase is encoded by the

sene r i z 4 (Shao et al., 1995).

Although atnzine is not a substratr for s-triazine hydrolase. gene probes for the gene

sequence would be useful for isolatinj reiated atrazine degradation genes from other

microbial isolates. In addition. manipulation of the genes responsible for degradation may

yield enzpat ic activities that do attack atrazine.

Figure 6. Proposed pathway for the microbial degradation of deisopropy latrazine b y

Rlto<lococctcs cor-dliir iis NRRL B- l54WR ( Mulbry. 1 994). Dec hionnation

of deisopropylatrazine occurs via rhc snzymc s-tria~ine hydrolase and is

encoded by try.4.

Deisopropylatrazine

Chapter 3. Materials and methods

3.1 Bacterial strains and plasmids

Thc bacterial strains and plasmids usrd in this study arc listed in Tablcs 1 a and 1 b.

Thc iitrazine-degadiny bacteria from Ottawa were isolated from soils described by Topp et

ai. ( 1996). C 1 S7 was isolatcd from soi1 ncar St. Hyacinthe that u s cropped with mixed

ccrcais and ne\-rr trcated with litruine. Major soi1 properties are as follows: texture: loarn.

3S0,1 sand. 30" (1 silt. 32"0 clay. I .-ln.n organic rnatter. and pH 6.

3.2 Media and orowth conditions

AI1 atrazine deyraden were g r o m at W C on atrazine mineral salts (.;LLLS) ( 1 -6 L

K2HPOJ. 0.4 SL KH,PO,. 0.2 g L MgS0,-7H@. 0.1 NaCl. 0.03 CnCI-ZHZO and 1 mL!L

trrice elements [IO mg. L ZnS0,-7H,O. 3 mgL MnC1,-4H,O. 30 m g L HjBOI. 20 m y L

CoC1,-6H,O. 1 rngL CuC1,-2H:O. 2 rn*L - 'iiC1,-1H,O. and 3 m-L N~LVOO,-LH~O

(modified from Lapage and Mitchell. 1 W O ) ] ) medium (modi fied from Mandelbaum et al..

1993) supplemcnted u n i t 1 1 citrazine (500 m j in 2 mL methanoliL). sodium citrate dihydrate

( 1 g L). Lynch vitarnins ( 1 mL, L ) (Lynch et al., 1980) and FeS0,-6H20 (5 mg/L). Lynch

vitamins and FeS0,-6HI0 were filter stenlized (0.2 um acrodisc. Gelman Sciences) and

added to cooled, autociaved media. The degradation of atrazine by bacteria was indicated

by a zone of clearing surrounding the colonies. P. syriiigue pt . [onzc~to "as gro\vn on Kings

B medium (King et al.. 1954) at ZYC. E. coli strains were grown on Luria-Bertani (LB)

agar (Sambrook et al., 1989) at 30' C. When requircd. chloramphenicol (30 @mL) and

kanamycin (50 pJrnL) were added to the medium. Clmibacter nriclzigarie,isis subsp.

Table 1 a. Atrazine-degrading bacteria used in this study.

S train number Geographical origin Source rind/or re ferencc

C 147. C 1%. C 1%. C 1 OO. Ottawa, Ontario C163. C17-L. C175. C176. C177. C l X C179. Cl80

Pserulonzoti~~s sp. strain .AD P

St. Hyacinthe. Quebec

Winchester. Ontario

Minnesota. US..\.

E. Topp: Topp ct al.. 1996

E. Topp

E. Topp

blandeibaum et al.. 1995

Table 1 b. Plasmids and additional bacteria used in this study.

Strain! plasmid Relevant characteristics Rcferencs and or source

Bacteria

DC3000 spontaneous Ri P derivatiw of NCPPB 1 106.68-kb

E. coli

host E. cok 5 3 . 146-kb, Tra' . h p r Km' Penr Su

D. Cuppels. 1'186

BRL

J . Dick

E. Topp

Shao et al.. L995

host E. coli CVASO3, Selvaraj and lyer. 30-kb, Cmr Kmr, TnS donor 1983

Table Ib continueid. Plasmids and additional bacteria used in this study.

S train/ p lasrnid Rele~mt characteristics Rzference andor source

Plasmids

pRK70 1 3 host E. coli HB I O 1. -CS-kb. Km' Nm' Tr3'. mobilizins factor. hcipcr plasrnid

host E. coli 55-3. 93-kb. Km' Ampr Smr Su Cm'

host E. coli D H k . vcctor pXCYC 1 84. MI-4

host E. coli DHSu. vcctor p X Y C 1 84. 'nrB

Boiindy-kIilIs ct al.. 1 9'17

36

/triclrig~irre~rsls JDS3- I \vas grown on t y t o n c soy agar (TSX) ( DIFCO. Detroit. MI) at room

tcmperature. For long terni storagc bacteria were suspended in 30°G glycerol or nutrient

brotli-yeast -tract <NB)-) (Vidin-er. 19671 brotli containing 15% ylycerol and stored at

-70 - C.

3.3 Characterization of bacteria

The Gram stain reaction \vas iised to discriminate between gram-positive and

gram-nryatiw atrazinc dryraden. .Al1 atrazine-degadins strains wsre grown on XVlS agar

supplsmentrd with atrazinc for 6 days at W C . The sram-positive control. Cim:ihacier

nrichigmeiuis subsp. niichtgmlemïis JDS3-1. uas grown on TSA for 72 h at room

temperature. The gram-ncgativc control. Pserrdotuoii<is siet-i~tgtre pv. rorrimo DC3000 was

crown on hB\i agar m room temperature for 72 h. The cells were Gram-stained according Cr

to tlie nianuf~cturer's instructions (Signa Cliem. Co.. St. Louis. Mo.). Cslls which retaincd

the p n m q dyr (crystal violet) u w e considered ,mm-positivc. Cclls which lost the pnmary

dye during the decoloration step were considered gram-negative.

The potassium hydroxide (KOH) test (Suslow et al.. 1952) was used to confirm the

Gram-reaction. .A drop (50 uL) ot'jO/o (wi'v) aqueous potassium hydroxide was placcd on

a clean siide. Bacterial cells were transferred to the KOH with a stcrilc wooden toothpick-

The cells were mixed thoroughly on the slide. After sevcral seconds. the toothpick was

alternately raised and lowered in order to detect a stnnging effect. The KOH test was

considered positive if the viscosity increased and stringing occurrcd within 15 seconds. A

positive rcaction is typical of gram-negative bacteria.

-4trazinc-degadins bacteria were testrd for cytochrome c ovidase acti\:ity according

to the procedure describsd by Smibert and Kneg ( 198 1 ). -411 strains. including the negativs

control. E. coli. azre y0a .n on NB)- ayar for 72 h nt 30 'C. Psrtr~lonioi~~~s~liro,.esceiis. the

positive control. \vas yrown on hB1- aga . îbr -IS h at 25 'C. Bacterial colonics were smeared

tvith a stenle loop ont0 \Chatman 3 C N filter paper rnoistençd rvith I o , ) ( w L-) trtrmeth-1-p-

phenylenzdiarnine dihydrochlonde (Sigma). Dcvsloprnsnt of a purple color wirhin 30

seconds was scored as a positivc reaction.

Substrate utilirarion w s cletetmineci for stnins C 147. C 155. C 156. C 160. C 163.

C 174. C 1 77. C 175. C 179 and C 1 SO usiny thc .et X E and .-\PI ';FT stnps ris recommmded

by rhc manufacturer ( .AP I Laboratory Products. Ltd.. St. Laurent. QuGbec. Canada). Samples

n-erc incubated at 30 'C and rcad at 24. 48 and 72 II.

3.4 .Inalvtical techniques

3.4.1 Atrazine rnineralization

Steri le A M S supplemsntcd with uni fomily nnj-labrllsd [lJC]atrazine

(50000 disintegrations pet minute I D P W ~ L ) (specific xtivity 4.5 mCi. mmol. radioactive

puri- 95%. S i l a ) \vas cidded to sterils microtitre wells i 200 u L well) (96 WeIl Cc11 Culture

Clustcr. Costar Corporation). tndividual \vrlls were inoculatcd wirh one colony of C 1 S7 or

Pserrrloniotlus sp . srrain ADP (positive conrrol). The remainin~ wells were supplied with

stcrile distilled water to maintain humidity levels. The microtitre plate was incubated at

3O'C. in a Zipiock bag for 48 h. .A Falcon tube (Fisher Scientific Ltd.) containing 1 mL of

1 'I XaOH was addrd to the Ziplock bas as a I4COZ trap. Samples (50 uL or 100 u L ) were

2s

takcn after 24 h and 48 h. The total radioactivity remaining in the samples \vas quanti fied

by Liquid Scintillation Counting ( LSC) (Beckman LS 580 I . Beckman Instruments Inc..

[mine. CA) iising I O rnL of CnivsrSol cocktail KN. Costa Mesa. CA) and corrected for

quenching tvith an cxtemal standard.

3.4.2 High oressure liauid ch romoto~raohv (HPLC) analvsis

Atrazine disappcarance and metabolite formation \vas determined using HPLC

analysis (Topp et al.. 1993). Microtitre plate wells u-ere fiilcd with 200 PL of .LMS

containing 92-76 UM (20 mgL) atrazine and the wells were inoculated with C 157. C 147. or

Psezrdonzo~rtzs sp. strain ADP. The microtitre plate was incubated at 3O'C overnight. .\fier

incubation. 100 u L samples were addcd to an equal volume of methanol and centrifused at

15 400 .u g for 4 min at room temperature to remove cslls and othcr insoluble matenals-

HPLC analysis of the supcmatant was perfomed with n U-atcrs 5 1 O Liquid Chromarogaph

Systsm equipped \vit11 a Waters 490 programmable-wavelength detector. Atrazine and its

metabolites were resol\-cd by using an analsicd C,, reverse phase CVhatnan HPLC column

(4.6 mm x 250 mm. 10 um diameter. irrejular packing). The opcntin_o conditions were as

follows: injection volume. 40 PL: drtector waveizngth. 210 nm: mobile phase. 7096

CH ,OH. 30% 5 mM 'iaH,PO, (pH 9.0) or 30% CH,OH. 5096 10 mk1 ammonium acetate:

flow rate. 1 mLimin. Identification of cornpounds \vas detemined by cornparin: retention

times with analytical standards. Concentrations (mg/L andor uM) were determined by

comparing peak areas with peak areas of known concentrations of analytical standards.

Resultant chrornatop.rns were generated using the sohvare prograns Lotus 1-14 97 (Lotus

' 9

Devsloprnental Corporation. Cambridge. MX) and Origin (Version 4.1 O. Microcal Sofnvare.

Inc.. 'lorthampton. M A ) .

3.4.3 Preoaration of C 187 cell free extract (CFm

Mid esponential-phase crlls grown in sither NBY broth containing 5 9 L jlucose and

1 S j 3 2 UM (40 mg.! L) atrazine or ' IBY broth containing 5 5 L glucose were han-csted by

centn fugation ai 12 300 s y for 10 min at 4-C and ivashcd tlvice in I O mL of 10 mM sodium

phosphate buffer (pH 7 . 2 ) (Topp ct al.. 1993. The cclls were resuspended in fresh bufler

to an A,,,,, of 4.5 (4.5 s iO" CFC1mL,. Cnide ceIl extracts Lvere prepared bp sonicating the

cells for ;i total of 1 O min ( 2 min. biirsts. 1 min. rest intenals) using ;i Braun-Sonic 2000

ultrasonic Iiomogenizçr (intermediate 13T titanium probe tip [3.:SW]. low powcr position.

approsimately 60 Watts. B. Braun Instruments. Burlinyame. C.1.j. The supernatant s a s

clanfied by centrifugation at 12 300 s y for 10 min nt 4 'C. The cmdc cxtract was kept on

ice and used immediately. To determine the minimum sonication time required to obtain

maximum protein. cclls t 4.5 s 10" CFC:m L ) w r e sonicatcd for O. 2. 4. 6 , S. 10. 12. 14 and

1 6 min. After rach time inrenal 100 uL samples were removed and cinalysed for protein

concentration. A 7 mL simple of cmde cxtract \vas removed and autoclaved (5 min. 121 ' C l

.Auroclaved CFE (200 u L ) and non-autoclaved CFE (100 u L ) \vas stored at -10'C for protein

mal ysis. The remairtins CFE \vas used io determine the rate of atrazine degradation (Section

3 -3.4).

The protein content was assayed using the Bio-Rad Protein Micro hssay (Bio-Rad).

Dilutions of protcin standard (bovine semm albumin) containing 3 to 18 p g m L were

30

prepared. CFE set aside for protein analysis was diluted 10-fold (SO u L sarnple into -20 u L

srenle sodium phosphate buffer [pH '2. 10 mM]). .-\ 0.8 mL aliquot of standard. diluted

sample. or sterïle sodium phosphate butEr (blank) (pH 7.1. I O mu) w;is miscd with 0.2 mL

of Jye rengcnt concentratc (Bio-Rad) m d lightly vortexed. .-\lier 10 min the A;.,I

detrrminsd for a c h sample using ;i Beckman DU 6-10 spcctrophotometsr (Beckman

Instnirnenrs Inc. ). -4 standard ciin-e plotting .AI,,, and standard protein concentration (3 pg-

I 5 ,LE) was prepared rach time the assa' \vas performed. The protein concentration of each

sample was cietermined from the standard curve.

3.4.4 Determination of rate of atrazine deuradation hy ceil free estract

Cd1 free extract was prepared as descnbed in section 3.4.3. Cell iiee extract and 10

mbl sodi~im phosphate buffcr (pH 7.1) containing 92.76 UM (20 m g L ) strazine were

prehearcd to 30-C. One rnL ol'CFE \vas mised with an equal volumc oisodium phosphate

butTcr ( 1 0 mhl. pH 7.2) in a 3 - m L sçnirn bottls. The reaction mixtures wsre incubated at

30 -C with shaking ( 1 50 rpm ). 1 00-uL samplcs a w c removed at 0. 1 0. 30. 30. -10. 50. GO.

90. 120. 151). 11 0, 271) and 1440 min intemals. and mixed with 100 u L of 95'6 methanof.

The samples were ccntnfuged at 15 400 s g for 4 min at room temperaturc. Xtrazine

dissipation and metabolite formation werc rcsolved using re-wrse phase HPLC analysis as

described in section 3.6.3. Caiculations of rates and rate constants were bascd on the

assumption that atrazine-degradation followed f i n t order kinetics. Exponential c w e fitting

was used to determine first order rate constants. The derivative of the decreasing exponential

at T=O was also used to demonstrate the rate of atrazine-degradation. Each rate was

3 1

normalized based on the mount of prorein in the reaction. Intercxpenmenral \-ariabilitp was

determinrd iisiny .WOV.\ (Statistica. Version 4.5. Tulsa. OK). Significant differences

benvecn treatments i \v ith atrazins \.ersus wi thout atrazine) w r e detennined using the paircd

t-test (Statistica. Version 4.5. Tulsa. OKA.

3.4.5 Preoaration of Cl87 whole cells and determination of the rate of atrazine-

de~radation-

Cclls growiny cxponentially in NBY broth containing 5 JL glucose and I S5 ULM (40 m2L)

atrazine were hm*ested by centrifugation at 12 300 x y for 20 min at 4-C and washed twice

in 1 O mL of 1 O mM sodium phosphate hutrer ( pH '3. Tlic crlls were resuspended in fresh

bufier to an .i ,,*,, o f 4.5 (4.5 Y 10" CFC mL). Three millilitcr samples of the cells were

preheated to 3O'C anci placed into 4 25-rnL serurn bottles. The scntrn bottles w x e scaled

wich geey butyl nibber stoppers md aluminurn cnmp tops. In order to crcatc m osyyen -kc

environment ovygen \vas evacuated under vacuum from nvo o f the sealcd s e m bottles and

backfiilcd to iitmosplieric pressure with nitrojen gas iising a r-acuum desiccaror. Three

rnilliliters of osyyen-tiee sodium phosphate buffer ( 1 O mM. pH 7.2. preheated to 3O'C)

containing 97.76 uM (20 myL) atrazine \vas added to al1 4 serurn boitles using an osygen-

Cree 10 cc alerile syringe. The reaction mixtures were incubated at 3O'C with shaking ( 1 50

rpm). Samples ( 100 yL) were removed at 0. 10. 20. 30.40. 50. 60.90. 110. 150. 11 0. 270

aiid 1440 min. mixed with 100 u L of 959th methanol and centrifuged at 14 000 rpm for 4 min

at room temperature. Atrazine dissipation and metabolire formation were resolved using

reverse phase HPLC analysis of the supernatant as descnbed in section 3 - 6 2 Data analysis

33

was performed usiny the proccddures descnbed in section 3.4.4 sxccpt that rate constants and

rates ivere nomalized based on the CFL mL rather than thc protein mnccntration.

3.5 DNA mani~ulation techniaues

3.5. f Isolation of ~îasrnid DNA

Cncut plasmid DNA was isolated from strains C 1-17. Cl 55. C 156. C 160. C 1 b3.

C 177. CI 7s. C 1 3 . and CI SO ticcording to the alkaline Iysis rnethod of Kado und Liu ( 198 1 ).

Atrazine degraders wrre grown on NBY agar for 73. h at 30 ' C. E. c d i strains wçre grorvn

on NBY agar supplernented with 50 p y m L of kanamycin for -48 h at 30 'C. One bactenal

colon- was picked Iiorn the plate. suspended (with gentle tlicking) in 150-uL lysing solution

i3'k SDS in 50 m'l Tris. pH 12.0) and then incubated for 60 min at 60-C. Tu the ceil lysatc

2 volumes of iinbuffercd phenol:chioroform ( 1 : 1 ) wxs added. Ccil &bris was removsù by

centrifugation at 15 400 .; 2 for 4 min at room temperature. Plasmid D'IX was visualized

by Iiorizontal gzl clectrophoresis in { J . j 0 i ultra pure agarose (Life Tschnoio~ies. Inc..

Gaithersburg. MD) at 32 V'cm for 19.5 h using O.5X TBE buffer ( IOX: 0.S9 M Tris. 0.89 .M

borïc acid. 0.01 .LI EDTA disodium. pH 8.4). Gels werc stained in dilute ethidium bromide

( 1 .U u g m L ) for 2U min. destained in distilled water for 20 min. and photogaphed under LV

light with a Polaroid camera (Kodak film. type F665. Rochester. YY).

3.5.2 Isolation of total genornic DNA

Punfied total genornic DNA was isolated from C 157. C 193. Psetulomonus sp. strain

ADP. and DC3000. C 157. C 192. and Pseirdomonus sp. strain ADP were Town on Abils

33

plus atrazins piates at 30 -C for 6 days. DG000 was yrown on Kings B medium at room

temperature Ior '2 h. Cells were waashsd from thcir plates wirh 2 mL of sterile distilled water

and placed in s t ede i 5 m L microfugs tubes. The suspensions were L-onsxed vigorously.

The cclls wrrc pelleted by centrifugation for I O min at U O O s g and the supernatants

discardrd. The pellets were then suspcndcd in ISO u L of lysozyme incubation but'fsr

(20 mgmL lysozyme. 30 mM Tris-HCl (pH S). 2 rn.M EDTA. 1 -39;) Triton) and incubated

60 min at 37'C. .A 35 ,uL aliquot of Proteinase K and 100 u L ofbuffer A L (Qiamp Tissue

Kit. Qiayen. Chatswonh. c.41 ivas added to çach and mised by uxtexing. Sarnples were

incubated at 70 -C for 30 min and 9 Y C for an additional 30 min. Total senomic DXA was

extractcd iising the Qiamp Tissuc Kit following the manufacturer's instructions (procceding

from step 1). Absorbancc values (.Alb, and .Azso) were obtained iisinj a B e c h a n D L 640

spectrophotometer (Becliman Insrniments Inc.). The A,,,, A,,, ratio for al1 samples was

behveen 1 .S and 1 -9. The final DNA concsntntion was calculated from the absorbante value

at 760 nm iising the rquation 1 A?,, = 50 pg, mL (Sambrook et al.. 1989).

3.5.3 Preparation of DIG-labeiled DNA

DIG-labelled probes were generated with the PCR DIG Probe Sythesis Kit

(Boehnnger Mannheim Biochemicals. Lavai. Québec). DIG-labelled probes mere generated

by incorponting DIG- 1 1-dUTP dunng a PCR according to manufacturer instructions

(Boehringer Mannheim Biochemicals). Custom internai primers (Table 7) were designed

specifically for ur1.4 and atzB with the software prograrns OLLGO (Ver 4.0. National

Biosciences, Inc., Plymouth, MN) and D N A i h Y (Version 2.5. Lynnon BioSott, Vaudreuil.

Table 3. Primers used in this studv

Primer Primer sequence* Region Rekrcncc and or amplitisd source

Dl

6SjrZ

530f

1 1 OOr

926f

l@?r

ERIC 1 R

ERIC2

BOXA 1 R

1 O7SF 1

1 S2iRZ

2358F1

2435R1

16s rDXA

16s rDN.4

16s rD3i.A

16s rDN.4

16s rDIi-4

16s rD'i.4

ERIC

ERIC

BOXA

tf l1.4

L I C Z . ~

a d 3

a c B

Lans. 1991

Martin et al.. 1992

This study

This study

This stud';

This study

35

Que.. Canada) and primcrs were synthesized by Li fc Technologies Inc. Xfter ampli fication.

ri 4-UL iiliq~iot of the 50-uL reaction mixture was analyzed by horizontal gel elec trophoresis

using 1. joo a;aros<r in 1 S TBE bufkr 3t 100 V For 45 min. The gel was stained brietly in

cthidium brornide ( 2 m g LI. destained and visualizcd under short ~vavclcngth UV light. The

vield of DIG-[abelled probe sas estimated by a colorirnetric assay with nitroblue tetrazolium

( NBT) ( Boehringer blannhcim Biochrmicals) and 5-bromo-4-c hloro-3-indol y 1 phosp hatc

(BCLP) (Boehringer Mannheim Biochemicals) as substrates for alkaline phosphatase

conjugated to anti-DIG mtibody. Scrial dilutions of the DIG-labelled probe were compared

n-ith serial dilutions of controi DiG-labclled DNA of known concentration. The DIG-

Iabellcd probe was stored at -10'C.

3.54 Recover>* of DIG-labelled DY.4 fragments from agarose

DNA bands werc cscised from agarose gels according to the msthods described by

Kutlu and Cuppcls ( 1997). DIG-labcllcd DNA wÿs electrophoresed in 1 % low-rnelting point

(LMP) a p o s e in IS TBE buffer at 60 Vkm for 70 min at 1'C. The gel was stained for 15

min in rthidium bromidc (2.0 m y L ) and visualized under short wavelength LW light. The

band of interest was escised. weighed and incubatrd with 0.04 volumes of 25X asarase

buffer (750 mM Bis-Tris. 250 mM EDTA) for 15 min at 65'C. The molten agarose was

cooled to 45 T and digested with 2.1 Units of DNAase-free agarase ( 1 Unit per 100 mg of

aiprose) at 45'C overnight. Remaining oligosacchandes were removed by addin;

0.1 volumcs of 3 M NaOAc (pH 5.5) and incubatins the solution for 15 min on ice. The

oiigosaccharaides were removed by centrifugation (1 5 100 x g for 10 min) at 1'C. DNA was

recovered from the supematant by cold ( -NT) ethanol precipitation.

3.5.5 Dot blot ~ r e ~ a r a t i o n

For preparation of dot blots (Cuppels ct al.. !990) whole ceIl suspensions were

prepared by suspending colonies that were gown on .\.LIS supplemcnted with atrazine. LB

plus kanamycin (50 ug,mL) or XBY agar in 500-uL of s t ede distilled water to an A,,,,,, of

2.0 (2.0 u 10" CFC mL). For soms dot blots. puri tied genornic DN.4 was prepared (Section

3.5.2) and used nrher than ivhols cells. Aliquots of whole cells O pi) or purified gcnornic

DNA (200 ng) were pipetted onto uncharsed 52 mm diameter nylon membranes for colony

and plaque hybridization (Boehnnjer blannheim Biochemicals). The cclls were lysed and

the DNA cienatured by placing the membranes on Uhatman 3mrn papzr saturatcd with

denaturation solution (0.5 N NaOH. 1.5 hf XaCl) for 15 min. The membranes wcre then

neutnlized by placing the membranes on Alter paper saturatsd wirh nrutralization solution

( 1 .O hl Tris[pH SI. 1 .j 41 NaCl) for 15 min. Aftcr being incubated for 5 min on filter paper

moistzned with 2X SSC the membranes wçre air dricd and placed in a L1; cross-linker

(LVP. Inc.. Lpland. CA). The membranes were storcd dry in a desiccator untii required for

hybridization.

3.5.6 Hvbridization and detection of DIG-labelled DiVA probes

Target DNA immobilized on nylon membranes was hybridized with a DIG-labelled

DNA probe (Cuppels et al.. 1990; Sambrook et al.. 1999). The dot blots were incubated for

2 h at J Y C in a prehybridization buffer containing 5X SSC. 50% deionized formamide,

37

O. 1 " A N-[auroyl-sarcosine. 0.07''L SDS. and 5% blocking reayent (Boehringer Mannheim

Biochemicals). Thcy were then tnnslerred to new bags containing 10 ng'rnL of heat-

denatureci probe in 3 mL of prchybridization buffer and incubatcd ovemight at 42 ' C . After

hybridization thc membranes were washcd twice. 10 min per wash. in 2 X wash solution

( 2 N SSC. 0. SDS) at room ternpsrature followed by two washes. 1 5 min per wash. in

prewarmed O.5X wasli solution (O.5X SSC. 0.1 ?& SDS) at 68 'C. The membranes wcre

incubated in Genius buffer 1 ( 100 rnM Tris. 150 m M NaCl. pH 7.5) for I min and then in

Genius bufkr Z ( 1 %[wh-] blocking rcascnt in Genius bu ffer 1 ) for 2 h at roorn temperature

wi th gentlc shaking. A Aer a 30 min incubation in alkaline phosphatase-conjugated anti-D IG

anribody diluted 1 : 10000 in Genius buffer 2. the blot was washed twice. 15 min per wash.

with Grnius buffer 1 and then equilibratcd in Genius buffer 3 for 2 min. The probe-target

DN .A hybrids Lverc detected usin$ the cherniluminescent substrate disodium

3-(4-rncthosyspiro ; 1 .I-dioxetane-3.2'-(Schloro) tricycl0[3.3.1.' 'ldecan: -4-yl) phenyl

phosphate (CSPD) (Boehringer ~Mannheim BiochcmicaIs). The membranes were moistened

with 1 mL of CSPD (diluted 1 500 in Genius buffer 3), scaled in hybndization bags and

rxposcd to X-ray tilm (Kodak X-Omat AR. 14" x 17". Richardson .Y-Ray, London, ON) for

45 to 120 min. To stnp membranes OF DIG-labelled probe they were washed nvice. 10 min

per wash, in 0.2 N NaOHiO. 1 % SDS with shaking at 37°C. The membranes were then rinsed

in 2X SSC and stored dry.

3.6 r e ~ - P c R

.\trazine-degadins bacteria isolated from Ottawa (Table 1 a) wcrc fingerprinted using

rep-PCR and the consensus primcrs BOXAI R and Enterobacterial Repetitive Intcrgenic

Consensus ( ERIC) 1 R and ERIC? (modified From Pooler et al.. 1996). Wlole cells of each

isolate were g o w n on TSA for 4s h. Single colonics were removed from the plates and

suspcnded in 1 mL of stede distilled water. The ceIl concentration rvas adjusied to an A,

of2 ( 2 s 10" CFU.mL) and used as remplate for PCR. The oligonuclcotide pnmers (Table 2)

were syntliesizrd by BRL based on published sequences ( Hulton et al.. 1992: Martin et al.,

1 ) . ERIC- PCR reactions wcrc carricd out in a 25 uL volume containing 9.6 uL sten le

viater. 2.5 ,pL 1OX buffcr (Promesa. -Madison. WI). 3 u L MgCl: (Promega), 2.5 pL sterile

gelatin ( l rny'rnL). 0.2 g L dNTP (25 mM), 1 pL each of ERICIR and ERIC2 prirners

(50 PM) and 0.7 UL of T C I ~ polynierase (Promepa). BOXA- PCR rcactions were cmied out

in a 25 u L volume containins 10.6 u L stenle water. 2.5 uL 1OX bufler (Promega), 3 pL

MgCI? (Promcga). 2.5 pL s tede gelatin ( 1 mymL) . 0.1 uL dNTP (3 miCl). 1 pL of the

BOXA 1 R primer (50 uM), and 0.2 pL of Tclq polymerase (Promcga). Each reaction tube

was covered with one drop of stcnlc minera1 oil. The amplifications were perfomed with

a H ybaid OmniGene Thermocycler (Interscience Inc., Markham. ON.. Canada). For both

the ERIC primers and BOXA primer (Table 1) the cycles used were as follows: 1 cycle at

94-C for lmin, 30 cycles at 9J3C for i min. at 52'C for 1 min. and ar 72'C for 1 min. ARer

amplification 4 pL of the BO1U and ERIC PCR products were separated by horizontal gel

electrophoresis in 1.5% agarose (Nusieve 3: 1 ) in 1 X TBE buffer (lOX: 0.89 M Tris. 0.89 M

boric Acid. 0.02 M EDTA disodium, pH 8.4) at 100 V For 45 min. A 1 pL aliquot of 1-kb

39

DKA Laddcr ( Li tè Technologies Inc.) was addcd to the first and last lane of each gel. Gels

\vere stained in dilute cthidiiim bromide ( 1 p ~ m L ) for 20 min. destained in distilled water

tUr 20 min and photographed under L1; liyht \vit11 a Polaroid camerci (Kodak film. type

F665).

3.7 16s rD.i.4 seauencing

3.7.1 Preoaration of PCR oroduct for 16s rDN4 seauencin~,

Strain Cl S7 was groum on . h i l S plus atrazine agar for 6 days. Cells were washed

from the plarcs with 2 mL of sterilc distilled tvater and placed in a stenle disposable test tube.

The cell concentration \vas adjusted to an A,,,,, of 2 ( 2 x 10'' CFUimL) and used as templatc

Ior PCR. PCR was carried out in a 75 u L volume containing 17 ,uL sterile distillcd water,

7 .5 u L 1 O S buffer (Promeja). 4.5 pL 41gClI (25 mM) (Promega), 0.6 uL 25 mM dNTP.

3-75 uL of rach opposing primer ( 1 O PM) and 0.1 uL of T.«q polymcrasc (Promega). Each

reaction mixture was coverrd with I drop of minera1 oil. PCR-fragments were amplificd

using a Hybaid OmniGene Thermocycler (Interscience Inc.. Markham. 03.. Canada) using

the following program: L cycle at 94'C for 3 min. 30 cycles at 94'C for 1 min

(denaturation), at 502C for 1 min (annealing) and at 72°C for 3 min (elongation); 1 cycle at

72-C for 5 min (final extension): and stabilization at 3O'C for 1 min. Published primcrs

(Table 2) specific fior the 16s rDNA molecule were purchased fiom Life Technoiogies inc.

The primers were paired together as follows: 926f-1492r; tD 1 -68jrI; and 530f-l l OOr. Three

PCR products of approximately 500 bp in length were obtained. The PCR products were

concentrated to a volume of 2 1 p L using Microcon- 100 microconcentrators (Amicon Inc.,

40

Bevsrlp. MA) 3s recommendrd by the manufacturer. Final DNA concentrations were

calculated liorn the absorbance values ar 160 nrn using the equation drscribed in section

3 - 5 2 The PCR products t 30 n j u L ) and corresponding primers ( 1.2s pmol.',uL) were sent

to The John P. Robans Research Instirute DNX Sequencing Facilit- (Cni\.rnic) of Western

Ontano. London. ON.. Canada) for sequencing (AB[ Prism Mode1 3 77 scquencer. Pcrkin

Elmer Applied Biosystems. Foster City. CA). Threr sequences wsre generatcd.

3.7.1 16s rDN.4 sequence analpis

For each strmd. the 3 srquencrs wrre mer@ into one 1462 bp sequrnce usinj the

so fhvarc program DX.AlM.4N (Version 2.7; Lynnon BioSoti. Vaudreuil. Qué.. Canada). The

seqlience was edited manuail- by cornparisons of the two strands to clirninate discrepancies.

Ambiyuous bases were resolvsd by esamination oithc t.lectrophoregrarns. Selected 200 bp

sequenccs from the 1500 bp sequence were cornpared nith 16s rDNA sequences h m

Gcnbank md E'VIBL sequencc databases. The 16s rDNA sequrnce of C 187 \vas then aligned

with 16s rDNA sequences of al1 species of the jenus .Vocctniioides and other related species.

using the algonthm Clustal W included in D'IAiLIh'l. The following nucleotide sequcnces

were obtained from the Genbank and EMBL databases iinder the following accession

niirnbers: AFOOjO 17 (,,Locardioicles fiiliws). AF005007 (rVoccinlioides 1~trelt.s). hFOO5006

( A'ocurdioides jer~ser~ii), S53 2 1 1 (iVoc~irtlioides dbtis), AF0050 1 5 (iVocurclioiciesjlav~is),

AF005007 (~Voccrrdioicies luteus). AF00500S (rVocnrriioicies plunrarum). AF0050 18

(LVoccrrclioities rlzermoliltci~ttis), AF005009 (iVocurdioides simplex). AF005070

(iVoccu-tlioitles sp. ATCC 2941 9). AF005021 (rVoccrrdioides sp. N S P 4 1 ). X92364

4 1

( (;eo<len~zorop/lil~~s sp. ). X93 1 S 5 (.4 crirtopiitles hrmilietrsis 1. SS3 806 ( C~'11dotnottc~s

[ru-hanr 1. DS-974 (.VociirdÏoirles sp. i . C9USS tStrepron1~res ilze~-rnoci~rho~~~~loi~o~-c~~zs DSXl

-14395 1. L-10627 ( Fr-cïiikici sp. 1. CO I 298 ( .Vuc~u.dioides sp. OS-4). XS067 1 ( Rlio~iococn~s

- o i ~ i i Sirnilanty \ducs and c\.olutionary distances wsre calculated from thc alignrnent

i DN.OI.AW .A phylogenetic tree \vas constructed by neighbour-joining with 1000 bootstrap

iterations.

Chapter 4. Results

4 1 Determination of diversity amonp atrazine-de radina bacteria isolated from

aericu ltu ral soils in Ottawa.

4.1.1 .\ssessrnen t of biochernical diversim.

Szvcral atrazine-de-adiny bacteria werc isolatrd tiom ihe Central Espenmental

Farm in Ottawa (Table 1 a. ). On .\.LIS pIus atrazine medium they produccd small whire

colonies ivith a distinct zone of cleanng around each colony. The Gram stain ÿnd KOH tests

revealed that al1 the isoiates were sram-nrgativc. ;\II of the isolates were cytocliromc c

ovidase positive. The cytochrome c ouidase-positive control strain P. jlziot-mcrris and thc

ouidase-nçgativc control strain E. roli gave the rxpecred rcactions. HPLC andysis. to

confirm that a11 the isolates degaded atnzine. showed rhar one of the isolatrd strnins. C 154.

\vas unablc to degrade atrazine (data not shown). Since C 174 was unable to degrade atrazinr

i t Lvas nor considered hrther. The .VI ZOE md .VI NFT biochemical strips were used to

comparc di fferenccs in biochemical reactions berwsen oach isolatc (Table 3a. Table 3b ). The

. V I X E systrm tests for fermentarive bacterio and rhe .&PI ';FT system tests for non-

fèrmentative bacteria. Dendrogarns slicited by cluster anaiysis OC the biochemical responses

to API X E and AP t 'iFT by atrazine-degading isolates formcd a dense clustcr in wliich

C 155 acted as tin outlier (Figure 7). Examination of the raw data indicatcd that C 155 differed

from the otherç in its ability to reduce nitrate cornplercly to nitroyen sas. Based on the API

ZOE strip al1 the isolates reacted negatively to tlic hllowing biochemical tests: transformation

of lysine into cadaverine. transfomarion of ornithine into putrecine. utilization of citrate as

Figure 7. A. Dendrognm of the Ottaua atrazine-degrading bacteria based on the

biochcrnicai responses to the .\PI 20E test. B. Dendro gram of the Ottawa

atrazinr-degrading bactcria based on the biochemical responses to rhs .\PI

'IFT test. In each case the dendrogam was simplified from that created by

equally weighted average linkagc cluster analysis to show the number of

biochemical responscs which ivcre di f i rent betwecn each isolatr.

Number of differences

Num ber of differences

1 7

the sole sourcc of carbon. the production of hydrogen sulfidc tiorn thiosulfatc. metabolisrn

of tnprophan. and the producrion of acetoin from sodium pyw-atc. .\II of the isolates were

riiso linable to ferment the crirbohydrates mannitol. inositol. sorbito 1. rhrirnnosc, mxose.

tneiibiose and amygdalin into an acid. Based on the APT NFT bioctiernical tests 211 rhe

isolatcs were unable to assirnilate D-gluconic acid. capric acid. adipic acid. L-arabinose and

L-rnalic acid. Both thc .VI ZOE and . V I NFT biochemical strips showsd a negati~qe reaction

for rhe followin~ biochemical tests: liqustàction of sslatin by protcolytic enzymes.

Iiydrolysis of ONPG to iiirrophcnol and yalacrose bu p-galxtosidasc. tomarion of indok

tiom the substnte rnptophan. transfomation of arginine into ornithine. ÿmmonia and carbon

Jioxide. and krmentation of $ucosc. .As w c l l both biochemical strips showed that al1 ofthe

isolates u-erc able to reduce nitrate to nirrite. Csin; the . l P I 30E biochcmical snip C 175 and

C 155 ivtire cinabie to produce ammonia î?om irea a. However. the .\PI SFT biocliemical s~r ip

show-sd that al1 of the isolates were capable of urea hydrolysis.

4.1.2 Xssessment of oenetic diversi-.

DNA tingerpnnts to determine the gcnctic di\-rrsitp of the atrÿzine-drgrading

bacteria [rom Ottawa wsre yenerated using rcp-PCR. Rep-PCR iising BOXA and ERIC

consensus primers. generated cornplex tingerprint patterns consistins of 20 or more PCR

products that ranged in size Iiom 0.2 kb to 1.6 kb ( Figure S and 9). Fingerprint profiles for

both primer sets were very sirnilar. with minor differences limited to the prescncc or absence

of one or two hi$ molecular weight bands (Figure S. lanes S. 9. 10. and 12). Thcsc results

suggest limited genetic divrrsity among the isolates. E. colt D H j a (Figure 8 and 9. lans 13)

Figure S. Rcp-PCR generated DNA tïngerprints of atrazine-degrading bactcna from

Ottawa. The Iinyerprint patterns were derived from whole cells using the

ERIC primer set t ERICIR and ERIC?: Table 2 ) . PCR products were

slectrophoresed in a 1 .jOG agarose gel and stained with srhidium bromidc.

Limes: 1 and 15: 1 kb DiI-4 Ladder: lane 2. C 147: lane 3. C 155: lime 4.

C 156: lane 5. C 160: lane 6. C 163: lane 7. C 175: lane S. C 176. lane 9. C 1 77:

Iane 10. C 178: lane 1 1. C 179: lane 12. C I SO: lane 13. E cofi DH5u: and lane

14. water control.

Fiyure 9. Rcp-PCR gensrated D-iA tingerpnnts of atrazine-degrading bactsria (rom

Ottawa. Tlis finyerpnnt patterns werc den\-sd frorn whole cclls using the

BOXA primer ( BOXA I R; Table 3. PCR products lwre stectrophorcsed

in a 1 . j o 6 agarose gel and stained with cthidium bromidc. Lanes: 1 and

15: 1 kb DNA Ladder: Ime 2. C 147; lane 3. C 155: lane 4. C 156: lanc 5.

C 160: lane O. C 163: lane '. C I 75: lane S. C 176. lane '1. C 1 - 7 lanc 10.

C 1 7s: lane 1 I . î 1°C): lane 12. C I SO: lane 13. E. c.oir DHSu; and lane 14.

water conrrol.

52

\\.as used as an outlicr and gave a distinct banding pattern cornparcd to the other isolates.

To tiirtlier compare the Ottawa isolates plasmid protiles were yenented u s i n j the

alkaline Iysis nicthod of isolation describecl by Kado and Liu ( 198 1 ). The plrisrnid protiles

arc shonm i n Figure 1 O. The size and nuniber of plasmids varicd for cach isolate. howevcr.

C 147. C 1 -T and C 1 7 s had siniilar profiles as did C 1 55 and C 156. .A sinjle 97-kb plasmid

\vas cornmon to al1 isolates. The s i x of the cornmon plasmid was dctcrmincd by creating

a standard cL1n.e iising plasmids ofknown molrcular weight from vanous bactcria (Figure

1 O. lanes 1 to 3 ).

4 . Characterization of C187-an atrazine-de~rader from St. Hvacinthe. Ouébec,

Canada.

4.2.1 Characterization of C 187.

On AMS supplemcntcd with atrazine C 1 S7 produces small (0.5 to I mm) white

colonies with a clear zonc surrounding rach colon-. The presrncc of thcse halos \vas

attributed to the metabolism of atrazine to soluble products. The Gram stain and KOH test

rcvealed Cl87 to be gram-positive nnging from rods to cocci in shape. The cytochrome c

oxidase test indicated that C 157 was oxidase positive. The 16s rDKA gene of C 187. which

\vas 1462 bp long (Figure I I ) . was sequcnced and compared with seqiiences published in

Genbank and EMBL. The 16s rDNA sequrnce of C l87 was aligncd with the 16s rDNX

sequences of al1 known iVocrirdioides species and other related species. Similarity values

and cvolutionary distances were deterrnined and a phylogenetic tree (Figure 12) was

constmcted by the ncighbour-joining mcthod. C 187 foms a distinct lineage within the

Figure 1 O. Plasmid profiles of selectrd anazinr-drgading bacrerin isolated fiom Ottawa.

Lncut plasmids isolated by the alkaline !-sis rnrthod of Kado and Liu ( 19s 1

were subjcctrd to horizontal agarose gel elrctrophoresis in 0.jU;, ayarosc ycls

at 3 2 V cm for 1.1.5 In. Size and mobilitp of fi1-e uncut reference plasmids

(pR4O.A [146 kb]. pR1 [93 kb]. DC3OOO [6S kb]. pRK7013 [AS kb] and pGS9

[30 kb]) are indicatrd on the leA ( [anes 1 to 5 ). Lane 6. C 147: lane -. C 155:

lane S. C 1 j6; Iane 9. C 160: lane 1 O. C 163: Ianr 1 1. C 1 ": lane 11. C 1-S:

lanc 1 3. C 1 79: and lane 1-4. C 1 W .

Fisure 1 1. Nucleotidr sequence of the 16s rDNA gene from C 187. The cornpiete

nucleotide sequence ( 1462 bp) of both strands was arnplified using published

primen (Stahl and ;\mman. 1991 ). The PCR product was sequenced at the

John P. Robarts Research Institute DPiX Ssquencing Facility (LWO.

London, ON).

CCTCTTTCAG AGW.GMGC.2

AGAGGTATGC SIWATGCGC.1 SGTTCTCTGG

ZGGAGGAAGG

CGTCTCAGST

CACGAAAGTT TGTSGAGGGA

1 4 4 1 GAAYTCGTAA

GGCGGCGTGC CTTCGYGGGT G,;iGC.WTCTG Û A 4 Y C TAV--S-- ~ r - - r - du L uGFAAG ATSAGCTTGT CC-UGTAGCCS TGAGACACGG MTATTGGAC CGSGAGGGAT CAGGGACGAA CCC-GZC.nJ.CT TÛCGAGCGTT -AGULGGTC'ïG CCCTGAGZCI AGGGGAGXiC GATATCAGGA G C A T T X C T G CASGATTAGA GCGCTAGGTG GCT-WCYCAT A b v ~ i M.?GAC GGCGGAGCAT TTACCTAGGT 2AGGTLCGT.A GTCAAGCTCGT G A G C C C S C C GGGACTCATA TGGGGATGAC CTTCACGCAT ATCGCAAGAT CGGATTGGGG GCTAGT19ATC CCCGGGCCTT GGCW-CACCC GCCGTCÛAAT CAAGGTMGCC

TTAACACATG ACACYAGCÛY SCCTTC-zlCAT SCSI\&TAC SA TTP ,;LCGGCGGT TGÛTUGGGTA GCCTGAGAGG CCCAGACTCC .UTGGGCGAA GACYGCCTTC GCGCAAGTGA A C G T G C C A K CTCCGGPATT TCACGTCGGG GCTTCCSATA GG.WTTCCTG GGAACACCGG ACGCTGAGGA TACCCTGGTA TC-GGACTCAT 7,AAGCGCCCC TCA.UGGAF.7 GCGGATTGAT TTGACATATA AYGOTCCTAT GTLGTGAGAf CTLGTCTTAT AGAGACTGCC G T C M G T C A T GCTACPATGG GGAGCGAATC TCTGCAACTC GCAGATC.4GC GTACACACCG GAAGCCGGTG GTGGGGCGAG GT

cr.AGTCG?.GC CGAACG(JYT5 ZS.GGAT-?J-C'T LC?.CTTC%L-G GTiAG GAA*r ..CSGCC,CACC STGACCEGCC TACSGGAGGC AGCCTGATCE GGGSTGTWJ. CGGTACCTSC AGCCGCGGTA ASTGGGZG'ï>. AGTGAPtMCT ICC-GGCAGHCT GTGTAGCGGT TGGCG=GGL G C G M G T G T STCCACACCG TCCACGAGTT GCCTGGGGAG 3GF.LGSGGGC TCGATGCFAC GGGFAATCTS ACAGGSGGTG STTGGGTT.AA GTTGCC.4GC.A GGGGTCAACT CATGCCCCTT CLSÛTACAPA CCXLV4.A-GCC GACCCCATGA AACGCTGCGG CCCGTCACGT GCCCA?-CCCT C G A T T G G G K

Figure 12. Phylogenetic tree showing the positions of C 157. members of the genus

:Vocardioides and other reiated species. The tree was generated using the

neighbour-joining method and was based on a cornpanson of the 16s rDNA

sequences. The numben at the nodes indicate the lsvels of bootstrap support

based on data for 1000 replicares. The bar scale indicatss 0.05 substitution

per nucleotide position.

9 Nocardioides sp. ATCC 3941 9

''1- Nocardioides jensenii

Nocardioides s p.

4 Nocardioides plantarum

Nocardioides sp. OS4

::cl- Noca rdioides simplex

r-'- Nocardioides sp. NSP 41

130 Nocardioides luteus

Nocardioides f h vus l m

Nocardioides albus

I Actinoplanes brasiliensis

1 q-Streptom yces thennoca box ydovorans r

32 I Noca rdioides themolila cin us r 1

Cellulornonas turba ta

7 os( Geodematophilus sp.

'1' Frankia sp.

Rhodoccocus rhodinii

senus .Vocrr~'ciioide.s and is most similar to .V. jerrsenii.

4.2.2 Determination of sequence similari with Psertdomonas sp. strain ADP ~ e n e s

encodin~ atrazine hvdrolase and hydrosvatrazine ethvlaminohvdroiase and with the

Rliodococcrrs coraffin ris 'VRRL B- 15444R oene encodin~ s-triazine ch lorohvdrolase.

Dot blot hybridization was used to determine if CI 87 had sequence sirnilarity with

the genes encoding atrazine cnlorohydrolase ( 4 ) and hydroxyatrazine

ethylarninohydrolasc (trtzl3) from the atrazine-degrader Pserrdornonus sp. strain ADP. PCR

using custom primers (Table 7 ) was used to ampli- and DIG-label sequences internai to the

NI-4 and utzB yenes fiom E. coli DHSu pMD4 m d E. coli DH5u pATZB-2 respectiveiy.

Amplification of ricB yielded a single product of approximately 3 0 bp in length (dara not

shown). .hplification of c r c 4 yielded 3 producrs of approximately 7500. 1700 and 900 bp

in length (dara not shown). The 900 bp product. which was the evpected product size based

on primer design. \vas purified from agarose sel and used the probe. The 900 bp DIG-

labelled product did not cross-hybridize with crcB. When the cntire PCR product mixture

(which included al1 three product sizes) tvas used as the ~rt--..l probe there was

cross-hybridization with r i t 3 . suggesting that the nvo additionai products ( 1700 bp and 2500

bp) were the result of non-specific primer binding during PCR. Both the DIG-labelled utzA

probe and the DIG-labelled mzB probe did not hybridize to purified genomic DNA from

Cl87 (Figure UA. l3B, lane 1 ) indicating that this strain had no sequence sirnilarity with

the two genes (crrz.4 and atzB) From Pseiidonionas sp. strain ADP.

Dot blot hybridization also was used to determine if CI 87 had sequence sirnilarity

F e 1 3 Dot biot hybridization of purified DNA From Cl87 with DIG-labeied ntz.4

(-4) and DIG-labeied atzB (B) probes from E. coli D H j a (pbfD4) and E. coli

D H j a (pATZB-2) respectively. 1. C 187: 2, Cl 93: 3. DC3000: 4. E. coli

DHja (piMD4); and 5. E. coli DHja pATZB-2.

62

with the gene rncoding s-mazine hydrolase from Rlrodococcrrs cot-dli)rzrs hiRRL B- I j J J J R .

A 600 bp BamHI fragment from the r r i A gene was DiG-labelled using rmdom pnming

(Zhu. unpublished). The rrs.4 probe did not hybridize to punfied genomic DNA from

C 187 (Figure 14. lane 1 ) indicating no sequence similarity with the rr-s.-l gene fiom

Rl~odococc~rs cordlinrrs 'IRRL B- 1 5 W R .

4.3.3 Mineralization of atrazine bv Cl 87.

To determine if C 157 was capable of metabolizing the aromatic nucleus of citrazine.

cells were incubatrd in .LMS medium containinj [C-ring "Cl atrazine. The remaining

radioactivity was quantified afier 14 and 4 3 h by LSC. The ndioüctivity decreased by 4%

afisr 48 h incubation for C 157. G% for the uninoculated control. and 94% for the positive

connol Pretîdotno>tus sp. strain ADP. Sratistical analysis using the t-test indicated that there

was no ~i~gnificant difference in the decline in radioactivity benvcen the uninoculated control

and C 187. There was a significant difference (p< 0.000 1. n= 4) in the mineralization

capabilities between C l S7 and Psezrdoino>rus sp. strain .-P. Thus C 1 57 is unable to

mineralize atrazine to CO1 and mmonia.

42.4 Degradation of atrazine to hvdroxvatrazine bv cell free estract.

In order to determine the regulation of amine-degading enzymes. CFE from C 157

gown in the presence of atrazine or in the absence of a m i n e was cvaluated for the ability

to degrade atrazine. CFE combined with PB (pH 7.1. 10 miM) containing atrazine was

analysed by HPLC at varying time intervals, and the resultant chromatograms were

Figure 14. Dot blot hybridization of purified DNA kom C 187 with DIG-labeled t r i -4

probe fiom Rhodacocczls cordlinr~ NRRL B- I j W R . 1. C 157; 2. DC3000;

and 3. R. cornllr'nrrs NRlU B- 1 5 W R .

65

comparecl with those of atrazine and hydrox yatrazine standards. Atrazine was no longer

detected afrer 24 11 of incubation. The disappearance of atrazine correlatcd with the

accumulation of a single metabolite with a rctcntion time of 4.6 min or 7.1 min depcnding

on the solvcnt system used (Figure 15). The rctention tirne of the metabolite corresponded

to thai of a hydroxyatrazine standard (4.4 min with 70041 methanol: 304% watrr. 7.15 min with

5O0h methanol: 50?" 1 O rnM ammonium acetate).

Figure i 6 shows the degradation of atrazine and the formation of hydrouyatrazine b y

CFE prepared tiom celis gown in NBY with (40 m g L) and without ütrazine. In both cases

(with and without atrazine) there was no lag precedins atrazine degradation. The rate

constant ( k ) of atrazine-degradation per mg protein for cells g o w n in atrazine was 5.5 u 1 0 '

min-' ( n = 4 standard deviatbn [std. dev.]= 2.4 x 10' min") and the rate (V,,- ,,,) was

30.6 nrnobmj protein - min ( n = 4 std. dev. = I 5.6 nmol/ms protein - min). For cslls grown

without atrazine k=5.7 'r min-' ( n = 4 std. dev.= 2.7 u 10.' min ' ) and V, , ,,,= 28.1

nrnoiim~ protein - min (n=4. std. dev.=14.9 nmol/mg protein - min). The variation

demonstrated by the larse standard deviations can be partially explained by intcrcxpcrimental

variability (.kVOV.4. p= 0.023). The assumption that degradation kinetics were tirst order

was substantiated by the linearity of the log rransformcd concentration versus time graph (6

> 0.93 for a11 graphs). The paired t-test showed no significant differencc bcnvcen the rate

of degradation for cells gown in atrazine versus those grown without atrazine. Thesc results

suggest that atrazine degradation by C 187 is expressed constitutively.

Figure 15. Transformation of a tmine to hydroxyatnzine by CFE from C 1 S: when cells

were g o u m with atrazine (B) and without asruine t C ) . as demonstrateci by

reverse phase HPLC. ( -4) Authentic standards of atrazine (46.38 uM) and

hydroxyatrazine (46.38 uM). Mobile phase. X O h CHIOH. ~ O ' ' O 5 mM

NaH2POI at a tlow rate of 1 mlmin .

d - 5 --

TU - - Hydroxyatrazine

J 1 6 3 'O

Retention Time (minutes)

Figure 16. Dissipation of atrazine (+) and formation of hydroxyatrazine ( W ) by CFE

from Cl57. ( A ) Activity of CFE when cells were groown in the presence of

atrazine. (B) Activity of CFE when cells were g o w n in the absence of

atrazine. Exponential cunre titting wri; used to determine first order rate

constants for atrazine degradation. Each rate constant was normalized based

on the arnount of protein per ml CFE. Error ban indicate the standard srror

among two replicates within one experiment.

Time (minutes)

4.2.5 Deyradation of atrazine bu whoie cells of Cl8%

Whols ccl ls gown in the presence of atrazinc w-erc ecaluared for rheir ribility to

degads atrazine under aerobic and oxyyen- ties conditions. .AM S containiny arrazinc \vas

inoculated with whole cclls and anaiysed by reverse phase HPLC at T=O and T=71 h.

Resultant chromatogams were cornparcd with thosr of atrazine and hydrosyatrazine

standards (Fiyurc 1 3 . Atrazine \vas no lonser dstected riftcr 23 h. .A metabolite ~v ich a

retention time of4.6 min was Jetscred rifter 24 hour. The retention time of the metabolite

corrcsponded ro that O t' a hyùrosyatrazinr standard 14.43 min ~ v i th 70"zh methanol: 30%

water 1.

To determine if the enzymes involved in atrazine-degradarion werc oxypen dependent

whole cclls of C 187 rnived with an q u a 1 volume of phosphate buffer conraining iitrazine

( 20 rns L) Lverc anaiysed for rheir abilit'. to ciegradc atritzins nhen incubated i\.ith 0s)-gcn

and without osygen. Figure 1 S shows the degradation of atrazine. accumulation of

Iiydrosyatrazinc and the subsequent degradation of hydroxyatrazine by tvhole cells for borh

incubations. The per cell rate (V, ,=,,,) of auazine degradation for cells incubated with oxygen

kvas 4.8 s 1W- nmol/CFC - h (n-6. srd. dcv.=j.l .; IO-'nmol.'CFC - hl (1.0 s I O - " 2 of

atrazine:CFC - h ) and the rate constant ( k ) per ce11 was 7 . 2 s 10"' h-i (n=6.

std. dev.=,.j x 1 9 " h-'1. In the absence of oxygen V,,=,, ,=l.4 s 10.' nmol!CFC - h (n=j .

std. dev.=l.O x 1WS nmol/CFC . 11) (3.0 x 10-" g oTatrazinuCFC . h ) and k=1.7 s 10'12 h- '

(n=5, std. dey. 4 . 7 s 1 O-" h+l). In al1 cases the 6 values for the 10% transfomed graphs were

yreater than 0.91. At 72 h hydroxyatrazine w u completely degraded under aerobic

conditions. Anaerobically, hydroxyatrazine was completely degadcd after 72 h (data not

Figure 17. Degradarion of atrazine by C 1 S7 whole c r l l s as drrnonstrated by HPLC

~inalysis. (-41 Authentic standards ot'arrazine 46-38 uM and hydroqatrazinc

40.3 S uM. ( B) C i 87 whole cells incubated with . A M containin3 atrazine and

sarnpled at T=O and T=14 hours. Mobile phase. 70% CH,OH. 30% 5 m.LI

NûHIP02 ai a tlow rate of 1 rnl'rnl.

Retention Tirne (minutes)

Figure I S. Dissipation of atrazine ( + 1. formation ofhydroxyatrazinc ( W ) and subsequent

degradation of hydroxyatrazine by Cl 87 whole cells. . Concentration

changes of anazine and hydroxyatrazine over 7 2 hrs whcn cells were

incubated aerobically . tB) Concentration changes of atrazine and

hydmxyatrazine over 73 hn when cells were incubated anarrobicallg. Inscn

gaphs show atrazine degradation and hydrouyatrazine formation in the iirst

hr for each incubation. Exponential curve fitting was used to detemine îïrst

ordcr rare constants for atrazins degradation. Error bars indjcate the standard

crror m o n g six replicates wirhin one sxperirnent.

Time (hrs)

75

iho~vn ). In both cases there ivas no accumulation of an additional rnctabolite. Statistical

anülysis using the t-test showcd a significant diffcrence between atrazine degradation

aerobically and anacrobically (p=0.000003. n=9. Cndcr maerobic conditions. atrazine was

degraded 3 tirnes s lowr . These results suggest that the enzymes reqiiired for atrazine and

hydroxyatrazine degradation are not dependent on mo lecular oxyjen.

Chapter S. Discussion

5.1 Diversitv and ~ ia smid distribution in rttrazine-de~radina bacteria isola ted on a l o t

scale.

Prokaryotic genomes contain srnall interspersed rcpeii tive sequences scparating

longer single-copy DNA scqurnces (Lupski and Weinstock. 1991). Thesr sequences are

non-encoding. between 1 5 and several hundrcd base pairs in length. and c m be ~ised as

oiigonucleoride binding sites for PCR rnediated genomic fingcrpnnting (Versalovic ct al..

9 1). -4lthough the sequcnces were idenrificd in a single species or yenus. several arc

conszrved and dispened in the genomes of rnmy diverse gram-negative and sram-posi tive

bacteria (\*crsaiovic et al.. 1994). Three such sequences are ERIC. repetitive extragenic

palindrornic (REP) and BOXA; REP and ERIC arc palindromic units. Genornic

fingrpnnting with BOXA. ERIC andior REP sequcnces is possible becausc individual

strains va- with respect to the number and distribution of these systcms in the cliromosome

( De Bruijn. 1992; Poolcr et a[., 1996; Judd et al.. 1993: Versalovic et al.. 199 1 ). In the

presenr study BOXA and ERIC consensus primers in combination with PCR were used to

determine the divenity of a group of gram-neyativc atrazine degraders isolated korn

a-gicultural soi1 near Ottawa. Xlthough there were occasional differences in the intensity and

prcsence of high molecular weight bands. the overall genomic fin~erprints of the soi1 isolates

suggests that the population of atnzine-degading bactcria is homogeneous. Some random

variation in the higher molecular weight region of the profiles is not uncxpected since

extension efficiency by Tkq polymerase decreases as the length of the target sequence

increases (Sambrook et al., 1989; Versalovic et al., 1994).

Bactena of diverse type and habitat are known to harbour plasmid DNA

(Stanisich. 1983. The çizc and number of plasmids a bacterium harbours can vary

tremendously ( Stanisich. 1985). .-\ varicty of plasmid isolation protocols haïe bccn

Jescribed (Kado and Liu. 198 1 ; Wheatcro fi and L~'i1liams. L 9s 1 ). d l of ichich exploit the

sniall çize (conipared to chromosornal DNA) and covalently clossd circle nature ofpiasmid

DNA i Sambrook et ai.. 1989). LVhile the plasmid profilcs of the airw.ine-degrading bacteria

were uniquc. one plasmid of iipprosirnatcly O X b \vas common to al1 isolatcs. Rccent

plasmid isolation studies b!. De Souza et al. ( 1998) on Pseridoi?rotit~s sp. strain .\DP

indicarcd that the genes for atrzine caiabolism to cyanunc acid: il[=-1. -B. and -C. are

locaiized on a 96-kb self-transmissible plvmid (pADP- I ). The rclationship benveen pADP-

1 and the 97-kb plasmid from the Ottawa isolates has yer ro b r detcrmincd. However.

knoiving rhat the Ottawa isolatcs haive sequencc similarity with ~ i r z . 4 anci -4tzB (data not

sliown) and tliat horizontal genc tnnsfcr (the transmission of DNA lragments between

different specics) (Hemck et al.. 1997) in narurally occumng microbial commitnities is nor

uncornmon (Fulthorpe and Wyndham. 1991: Hemck et al.. 1997: Lorcnz and

Wackemagcl. 1994). this 97-kb plasmid may in fact be the same one found in Psezr~lorm.mcrs

sp. strain ADP. As mentioncd above. the Ottawa isoiates yenerall y had di Kferent profiles.

Differcnccs between each isolate was not totally unespectcd since plsrnids Iiavs the ability

to be integnted into chromosornal DNA. c m be dispenscd by host cells under varying

growth conditions m d can underso horizontal tram fer (Madigan ct al.. 1997). Furthemore,

plasmid isolation techniques may result in plasmids of one isolate (and not another) being

nicked. resulting in different migration distances and hence the appearance of different

7s

plasmid profiles.

Substrate utilization and biochemicai reactions. using the .-\Pi systcms. were also

usrd to assess isolate Jiversity. Thsse spstcms which are standardizcd. miniatiinzed wrsions

of con\-entional biochemical procedures nllow for the quick identification and

characterization of E~treroh~icreriacme and other gam-negativç bacteria (bioblerieus Vitek

Inc.. 1095). .Amonj thc 1 1 strains. strain C 155 \vas the most uniquc. The major differencr

between Ci 55 and the othcrs was its ability to reduce nitrate ro nitrogen =as. Amongst the

remaining isolates thsrc wcre vcry f c ~ v differences in substra~s urilization and biochemical

- reactions as shown in Figure ,.

5.2 Characterization of a uniaue oram-oositive bacterium frorn St. Hyacinthe,

Québec-

5.1.1 Identification of Cl 87.

[dentification of C1S7 was bascd on 16s rDNA sequsncins. Traditionaliy the

classi fication a d identi ficntion of organisms has becn based on morphological.

developmental and nutntional characteristics ( Lane ct al.. I9S5: Moritz and Hillis. 1996).

However. these characteristics have the disadvantage in that they rire Iiighly intluenccd by

cnvironmental conditions and therefore directly affected bp cvolutionary convcrsence (when

organisms share a common phenotype because of common e v o l u r i o n ~ pressures) (Lane et

al.. 1985). For this rcason phylogenetic analysis based on macromolecules (,DNA. RNA.

protein and lipids) is more desirable. Of these macromolecules. ribosomai RNA (or DNA).

particularly the 10s rRNA coding region has been the rnost useful for establishing

79

relationships and identity between rnicroorganisms. since i t is universal. homologous. and

ücccssible (Hillis and Davis. 1986: Mindel1 and Honeycurt. 1990: Weller and Ward. 1989).

Cornpaison of the complete 16s rDKA sequencr tiom C 1 S7 ivith sequences published in

Gcnbank and EMBL indicatcd trhat C 1 S7 is a new species in the genus .Vocwdioides and is

most similar to .Y jemenii (AFQOjOO6). .LIernbers of the jenus .Loci~rtiioi&s are gram-

positive. pleomorphic. arrobic. mesophilic nocardiofom actinomycetss that r om well-

devcloped vegetative rnycçlia. Escept for the production of vegetativs myceliiim ( when

yrown on .\.LIS agar supplsmented with iitrazine). C1S7 hris a11 of ihese characteristics.

.Vocm-cltoitles sp. strain C l 87 may fom vegetative mycelium. However. the conditions

required for vrgeiative mycelium formation upere not defined in this study. .Vocrirciioides

species have been found to degrade a variety of organic pollutants including picnc acid

(2.4.6-trinitrophenol). pyridinc. dibenzothiophene and 2.4.5-tnchlorophenoxyacctic acid

iGolovleva et al.. lC19O: Rajan et al.. 1996: Yoon et al.. 1997: Sandhya et al.. 1995). While

other gram-positive bacteria including .locurciiu (Giardina et 31.. 1982) and Rlioducoccrrs

(Nagy et al.. 1995: Shao and Behki. 1996) san degrade ritruine (by .V-dealkylation), this is

the tint rcport of a .Voctrrdioirles species degrading atrazine.

5 2 . 2 Genetic and biochemical analvsis of the initial stem in the atrazine-degrading

pathwav.

Reverse phase HPLC analysis was used to follow a m i n e degradation by whole cells

oi'Xoccu-chdes sp. strain Cl 57 incubated aerobically and anacrobically. In reverse phase

HPLC. the stationary phase is non-polar and the mobile phase is polar. HPLC separates

50

molecules based on adsorption. ion-exchange and size esclusion. [ts superior resolution.

speed and hiyh sensitivip pcrmits the separarion. quantification and identification of srnall

qiiantities o t' metabolire. Howcvcr. HPLC doés not always provide conclusivc proof of

idcntity since thsrc is the possibility that some metabolites nmay have similar retznrion tirnes

whcn run in the sarne solvcnr system. Ldeally HPLC results would be coniïrmcd by linking

retention times with mass spectrometry (De Souza et ai.. S . Mass spectrometry

compares the complcte mass spectmm of a compound witli the mass spcctra of known

compounds (Eagle sr JI.. 1 0 9 1 I. Thc contirmation of identification (of metabolites) by mass

spectrometry was Sryond th<: scope of this study. Atrazine was dsgradsd by strnin C 187

under asrobic and anaerobic conditions. Howevçr. degradation was approsimately 3 times

hstcr aerobically. In both cascs whiic atrazinc was beziny drgraded hydrosyatrazine as

accurnulating. Once suazinc had dissipateLi .Vocu~-(fio~~irs sp. strain C 1 S- bcgan to degrade

hydrosyatrazins. Degradation of atrazinz and hydroxyritrazinc under osygrn-lirniting

conditions indicates that thc enzymes invoivcd are not mono or diosy,vsnases. which require

rnoircular oxygrn. but rathcr hydrolases. Most gram-positive bacteria that degradc ritrazine

are distinct in that degrridation is limited to .V-dealkylation and occurs under oxygen-rich

conditions by mcans o h PJ5,-like monooxygenase (Giardina ct al.. IW2; Nagy et al.. 1995;

Shao and Behki. 1996). However, RItodococcris cor-ullinrrs YRRL B-1SWR can

dechlorinate and deaminate dsisopropylarninoatrazine (but not atrazinc) anaerobically and

thcrefore presumably hydrolytically (Cook and Hütter. 1986). There has also been reports

o f gram-negative bacteria. including Psetcdomoiius sp. strain A D P (De Souza et al.. 1996).

degrading atrazine or its metabolites under oxygen-limirin; conditions

S I

(Radosetich ct ai.. 1995: Jessee et al.. 1983). Dc Souza et al. ( 1996) indicatcd that the

cnzymes Lvere hydro lases. The exacr rnechanism of the hydrolases synthesized b y strain

Cl 57 was not detsnnincd. Howsver. in the case of atrazine dechlorination. the enzyme

would catalyse the direct substitution ofchloride for hydroxide ( kom watcr). It is of interest

that the dec hlorination of pentachlorophenol to tcrrac hloro hydroquinone by

FZu-ohcicteri~rni sp. \vas originalIy thought to occur via a hydrolytic enzyme (Steisn and

Crawford. 1056) and was later shown to be catalysed by a monooxygrnasc (Sun and

Oner. 1991 ). . h y arnbiguity about atmzinc dechlorination by strain C 157 could be resolved

by enperimrnts using H,"O. howsver this mas not investigatrd in this study. If in fact the

cnzvrnc is a hydrolase tlien ['W]hydroxyatruine would bc yieided when whole ceils or CFE

of .Vocc~rdioides sp. strain C 157 are incubared with atrazine in H,"O. This result would ruls

out the possibility that the enzyme catal ysed cxchange aftcr an initial rnonooxygenasc

reaction that used traces of oxygen that remained in sealsd reaction vessels.

Nucltic acid hybridization offers a rapid approach to tht idtntificarion of

hornoloyous gene sequences (Cuppels et al.. 1990). Givcn that the appropriate DNA

scquence (probe) and hybridization conditions are chosen. thc sensitivity and specificity of

this mrthod is quite h i j h (Cuppels et al.. 1990). DG-labtllrd probes are preferred over

radio-labeilcd probes because thcy requirr shorter rxposure times and digosigenin poses no

health or environmental hazards. In addition. the short half-life of radioisotopes necessirates

kequent preparation of the probe. whereas DiG-labelled probes arc stable for up to one year

and can be reused several times (Boehnnger .LLannheirn Biochernicals. 19953. To determine

if .Vocnrd~ocrles sp. strain C 1 57 had seq uence similarity rvi th the Psetrdoinonas sp. strain

ADP ;enes sncoding atrazine chlorohydrolase

etliylarninohydrolase ( ~ l c B ) . dot blot hybridization

82

1 ) and Iiydrox yatrazine

usiny DIG-labeiled probes was

performed. ;\lthough .Vocui-dioides sp. s tnin CI S7 dechlorinates atrazine and degrades

hydroxyarrazine Iiydrolyticall y. its enzymes are not cncodcd by genes with Iiornology to K.-l

and KB. Thus. .Vbccir-diotdes sp. strriin C I S7 and Psetidolon~oizus sp. strain hDP may have

comp Iereiy different hydrolase genes.

To determine if .Vonri-diotdes sp. strain C 157 had scquence simiiarity with ri=.-l. the

gene cncoding s-{nazine Iiydrolase in Rliodococcz~s corolliittrs XRRL B - I 3 U R

( Shao et al.. 1095) dot blot hybridization us@ a DIG-labelled probe was performed. The

hydrolase responsibie for dechlorination of a m i n e is not cncoded by a gene with homolozy

to irz-1. susgesting that the Iipdrolase synthesized by strain Cl S Ï ( for dechlorination) is

cncoded by a diffcrcnt sene sequence.

5.2.3 Re~ulation of atrazine degradation.

Reverse phase HPLC was used to tollow atrazine degradation by strain C 1 S? CFE

in order to detennine enzyme regulation. CFE from .\ioccir-riioides sp. strain Cl S? cells

erown in NBY or NBY siipplernented with atrazine degraded atrazine at similar rates - (V,,.,,,=2S. 1 nmol/mghnin and V ,,.,$0.6 nrnolimgmin respectively ). In both cases

hydroxyatrazine was accurnulated: the concentration accumulated was equivalent to the

concentration of atrazine degradeci. Hydroxyatrazine was not degraded. Nearl y identical

rates dong with the absence of a lag phase indicates that the gene sncoding the hydrolase

requircd for dechlorination is continually transcribed. Thus. degradation of atrazine to

53

hydroxyatrazine by 3'occir-dioides sp. strain CI87 is constitutive. The accumulation of

hydroxyatrazine by CFE and not whole cells may suggest that the hydrolase required for

Iiydrosyatrazine degradation is an extracellular enzyme. however it is more likcly that the

protein responsible for hydroxyatrazine degradation was sensitive to the procedures

(sonication and centri fuytion) used to prepare the CFE.

Hydrosyatrazine is nonherbicidal and nontoxic (Gysin and Knuesli. I 9 G O ) and does

not leach t'rorn soi1 as reridily as atrazine (Erickson and Lee, 1989). Thus, hydrolytic

dechlorination is an idcal mechanisrn for degrading atrazine with the goal of bioremediation

of agricultural soils. In surnmary. this study presents the first report of a .Voc~it*dcoides

specirs capable of constitutive. hydrolytic degadation of atrazine and hydroxyatrazine. This

study Funher indicates that on a plot scale there is lirnited diversity among atrazine-degading

bactena.

84

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