Indian Journal of Experimental Biology Vol. 40, July 2002, pp. 774-779

Localisation of identical organophosphorus pesticide degrading (opd) genes on genetically dissimilar indigenous plasmids of soil bacteria: peR amplification,

cloning and sequencing of opd gene from Flavobacterium balustinum

Sita Somara l, Bramanandam Manavathi l

, Christoph C Tebbe2 & Dayananda Siddavatam '* IDept. of Biochemistry, S. K. University , Anantapur 515 003, India

2Institute for Soil Biology, FAL, Bundessalle 50,38 146 Braunschweig, Germany

Received 19 November 2001; revised 28 February 2002

Plasmid borne organophosphorus pesticide degrading (opd) gene of Flavobacterium balustinum has been amplified us­ing polymerase chain reaction (PCR) and the resulting PCR product (1.25 Kb) was cloned in pUCI8. Further, a detailed re­striction map was determined to PCR product and subcloned as overlapping restriction fragments. The nucleotide sequence was determined for all subclones to obtain complete sequence of PCR amplified fragment. The sequence showed 98% simi· larity to opd genes cloned from other soil bacteria isolated from diversified geographical regions . The protein sequence pre­dicted from the nucleotide sequence was almost identical to parathion hydrolase, a triesterase involved in hydrolys is of tri­ester bond found in variety of op-pesticides. The signal sequence of parathion hydrolase contained recently discovered twin arginine transport (tat) motif. It appears that tat motif play a critical role in membrane targeting of parathion hydrolase.

Large amount of organophosphates have been used i agriculture as insecticides to control insect pests. Or­ganophosphorus pesticides (OP) by virtue of their anti-acetylcholinesterase (AchE) activity block nerve transmission I and thus pose threat to non-target or­ganisms including human populations. Persistent use of these pesticides in agriculture has led to accumula­tion of these chemicals as residues in almost all com­ponents of ecosystem. However, certain soil micro­flora acquired genetic capability to degrade op­pesticides2

• Pure cultures of these bacterial strains such as Pseudomonas diminuta and Flavobacterium sp. have also been used in clean up operation of op-pesticide polluted environment including industrial waste3

. In view of growing relevance and importance of bio-remedial measures in treating toxic wastes of op-pesticides, attempts have been made to identify op-pesticide degrading (opd) genes. In Pseudomonas diminuta and Flavobacterium sp. opd gene encoding parathion hydrolase was found on large indigeno s plasmids4

.5

. Subsequently, this plasmid borne opd gene has been cloned and successfully expressed in heterologous hosts6

.

We have isolated a Flavobacterium balustinum strain that could use methyl parathion as sole carbon source. A large indigenous plasmid, pBC9 found to be

*Correspondent author:- Fax: 08554-55244; e-mail: swethasl@hd2.dot.net.in

involved in degradation of op-pesticides, has been identified in this soil isolate7

. In the present study, we have reported PCR amplification, cloning and se­quencing of opd gene from F. balustinum.

Materials and Methods Bacterial strains, media and chemicals-The bac­

terial cultures such as E. coli HB 10 1 and F. balusti­num used in this study were grown in LB medium. When necessary ampicillin was supplemented to the

medium (250 J..I.g/ml). Restriction and other enzymes used in this study were purchased from Boehringer Mannheim and used following the manufacture's pro­tocols.

Polymerase chain reaction (PCR) - Based on the reported sequence information, highly conserved re­gions of opd gene were identified to design primers . The oligos were purchased from TIE , Berlin, Ger­

many. PCR reaction (50 J..I.I) consisted of 100 ng of plasmid pBC9, 5 J..I.I of lOX PCR buffer, S p moles of

each forward primer and reverse primer, 2 J..I.I of 4 mM

MgCh, 1.5 J..I.I of 200 J..I.M d NTP mix and 0.5 U of Taq polymerase. A similar reaction mixture without tem­plate DNA was taken as negative control. The reac­

tion mixture was over layered with 50 J..I. l of mineral oil and subjected to 35 temperature cycles using Hy­baid Thermal Cycler. Each cycle consisted of denatu­ration at 94°C for 90 sec, primer annealation 53°C for

i

SOMARA et al.: LOCALISATION OF IDENTICAL ORGANOPHOSPHORUS PESTICIDE 775

90 sec and extention at 72°C for 1 min. After comple­

tion, 8 fJ.1 of reaction mixture was analyzed on agarose gel to detect the amplified DNA fragment.

Cloning of PCR product-The vector pUC18 and PCR product were independently digested with Pst! and BamH!. The digested DNA were independently extracted with phenol: chloroform and ethanol pre­cipitated. The ligation of linearized vector and PCR product was used to perform following the standard procedures8

. The ligation mixture was used to trans­form E. coli HB 101 and the colonies containing the recombinant plasmid were selected as described else­where8

. The recombinant pl asmid pSS 15 was purified and a digested with EcoR! and Hindll! to assess the presence of insert. The fragment having the right sized insert was then selected for determining the de­tailed restriction map.

Sequencing of PCR product-After establishing the detailed restriction map, the PCR fragment was digested with various restriction enzymes to obtain overlapping fragments. These fragments were sub­cloned in pUC 18 and the sequence was determined following the standard procedures9

.

Results and Discussion Almost identical opd genes have been identified in

Pseudomollas diminuta and Falvobacterium sp. on otherwise dissimilar plasmids. Expecting homology in op-pesticide degrading gene of Flavobacterium bal­ustinum, appropriate primers were designed to am­plify it using PCR. A 21 mer oligo (5' ACC CCG GCA TTG ACA TCT GAC 3') corresponding to 5' end of the gene and a 21 mer reverse pri mer wi th a sequence of 5' CTG GCT GGA AGG A TC CAG A TG 3' corresponding to 3' end of the gene were de­signed. Using these two primers and plasmid pBC9 as template, PCR was performed as described earlier. Simultaneously PCR was performed using plasmid pCMS 110 or pEA3 11 as templates to serve as positive and negative controls respectively. A 1. 2 kb fragment was found amplified only in PCR mixture containing pCMS I and pBC9 as templates. Restriction pattern of PCR product showed 100% identity to restriction maps of opd genes of Pseudomonas diminuta and Flavobacterium Sp.4.12. Pst! and BamHI sites present at the flanking ends of PCR product, facilitated easy cloning of fragment into pUCI8. Resulting recombi­nant plasmid pSS 15 was digested and each fragment was then cloned in pUC 18 to generate clones with overlapping fragments.

Nucleotide sequence of PCR product (EMBL data bank accession No. AJ426431) revealed that an Open Reading Frame (ORF) started with A TG at nucleotide position 62 (Fig. 1 ) and ended with TGA at nucleotide position 1157. ORF (1095 bp) encode a protein of 365 amino acids with a predicted molecular mass of 39 KDa. The putative ribosomal binding site was found 7 bp upstream to translational start codon ATG. Up­stream to ribosomal binding site from nucleotide posi­tion 14 to 29, a sequence motif that showed some similarity to consensus RpoN (0' 54) dependent pro­moter was noticed. However, after careful considera­tion of thi s sequence motif, a gap of 11 bases was noticed between conserved dinucleotides GG and GC (Fig. 1). The RpoN dependent promoters are well­characterized in Klebsiella pneumoniae and other di­azotrophic bacteria l3. In all RpoN dependent promot­ers there is a gap of 10 bp between conserved dinu­c1eotides and hence these nucleotides are found on the same face of DNA helix. Alterations made either to enhance or to reduce the gap resulted in loss of pro­moter activi ty l4. When such is the necessity for RpoN dependent promoters attributing the promoter status to a sequence containing 11 bp gap between conserved dinucleotides is unreasonable. Unfortunately the se­quence information at the 5' end of ORF is not enough to find a more reasonable promoter motif. However, Karns and co-workers 15 have sequenced opd gene from Flavobacterium sp. They have found existence of a classical 070 dependent promoter (81 bp) upstream of translational start site. As 5' end of the sequence of PCR product is almost identical to the sequence reported by Karns and co-workers. It is quite logical to predict similar promoter motif in opd gene of F. balustinum.

Sequence of PCR product showed strong homology to six other genes available in the data bank (Fig. 2). Out of these six genes, four are from prokaryotic origin. The opd gene cloned from Flavobacterium sp. and Pseudomonas diminuta showed highest homol­ogy. These results confirmed that PCR product was opd gene of Flavobacterium balustinum that encoded a membrane bound parathion hydrolase. Interestingly, in the signal sequence of parathion hydrolase a sequence motif with high proportion of positively charged amino acids was identified. The positively charged region with a sequence of TRRVVLK resem­bled the consensus double arginine motif identified in variety of prokaryotic proteins that contain large co­factors 16. The consensus sequence of double arginine

776 INDIAN J EXP SIOL, JUL Y 2002

CTGCAGCCTGACTCGGCACCAGTCGCTGCAAGCAGAGTCGTAAGCAATCGCAAGGGGGCA 60 .... ---

M Q T R R V V L K S A A A A G T L L G GCATGCAAACGAGAAGGGTTGTGCTCAAGTCTGCGGCCGCCGCAGGAACTCTGCTCGGCC 120

R LAG CAS V A G S I G T G 0 R I N T GCCTGGCGGGGTGCGCGAGCGTGGCTGGATCGATCGGCACAGGCGATCGGATCAATACCG 18C

V R G PIT N SEA G F T L THE H I C TGCGCGGTCCTATCACAAACTCTGAAGCGGGTTTCACACTGACTCACGAGCACATCTGCG 240

G T SAG F L RAW Q E F F G S R K A L GCACGTCGGCAGGATTCTTGCGTGCTTGGCAGGAGTTCTTCGGTAGCCGCAAAGCTCTAG 300

A E K A V R G L R R A R A A G V R T I V CGGAAAAGGCTGTGAGAGGATTGCGCCGCGCCAGAGCGGCTGGCGTGCGAACGATTGTCG 360

o V S T F DI G R 0 V S L L A E V S M M ATGTGTCGACTTTCGATATCGGTCGCGACGTCAGTTTATTGGCCGAAGTTTCGATGATGG 420

V 0 V S L L A E T G L W FOP P LSI G TCGATGTCAGTTTATTGGCCGAGACCGGCTTGTGGTTCGACCCGCCACTTTCGATCGGAT 480

L R S VEE L T Q F F L REI Q Y G I E TGAGGAGTGTAGAGGAACTCACACAGTTCTTCCTGCGTGAGATTCAATATGGCATCGAAG

o T G I RAG I I K V A T T G KAT P F ACACCGGAATTAGGGCGGGCATTATCAAGGTCGCGACCACAGGCAAGGCGACCCCCTTTC

Q E L V L K A A A R A S L A T G V P V T AGGAGTTAGTGTTAAAGGCGGCCGCCCGGGCCAGCTTGGCCACCGGTGTTCCGGTAACCA

T H T A A S Q R 0 G E Q Q A A I F ESE CTCACACGGCAGCAAGTCAGCGCGATGGTGAGCAGCAGGCCGCCATTTTTGAGTCCGAAG

G L S P S R V C I G H SOD TOO L S Y GCTTGAGCCCCTCACGGGTTTGTATTGGTCACAGCGATGATACTGACGATTTGAGCTATC

L TAL A A R G Y L I G L 0 HIP H S A TCACCGCCCTCGCTGCGCGCGGATACCTCATCGGTCTAGACCACATCCCGCACAGTGCGA

I G LED N A S A SAL L G IRS W Q T TTGGTCTAGAAGATAATGCGAGTGCATCAGCCCTCCTGGGCATCCGTTCGTGGCAAACAC

R ALL I K A LID Q GYM K Q I L V S GGGCTCTCTTGATCAAGGCGCTCATCGACCAAGGCTACATGAAACAAATCCTCGTTTCGA

NOW L F G F S S Y V T N I M 0 V M 0 R ATGACTGGCTGTTCGGTTTTTCGAGCTATGTCACCAACATCATGGACGTGATGGATCGCG

V N P D G M A F I P L R V I P F L R E K TGAACCCCGACGGGATGGCCTTCATTCCACTGAGAGTGATCCCATTCCTACGAGAGAAGG

G V P Q E T LAG I T V T N PAR F L S GCGTCCCACAGGAAACGCTGGCAGGCATCACTGTGACTAACCCGGCGCGGTTCTTGTCAC

P T L R A S * CGACCTTGCGGGCGTCATGACGCCATCTGGATCC 1174

540

600

660

720

7 80

840

900

9 60

102 0

108 0

1140

Fig. I-Nucleotide sequence of peR product ( 1.25 kb) and its deduced amino acid sequence. The ribosomal binding si te is underlined. The nifpromoter like motif is shown with bullets.

SOMARA et at.: LOCALISATION OF IDENTICAL ORGANOPHOSPHORUS PESTICIDE

FLB FL PD

MM RR FLB FL PD MT EC

MM RR FLB FL PD MT EC

MM RR FLB FL PD MT EC

MM RR FLB FL PO MT EC

MM RR FLB FL PO MT EC

MM RR FLB FL PD MT EC

MM RR FLB FL PD

Alignment of Opd homologous sequences

MQTRRVVLKSAAAAGTLLGRLAGCASVAG MQTRRVVLKSAAAAGTLLGGLAGCASVAG MQTRRVVLKSAAA RTLLGGLAGCAT---

MSSLSGKVQTVLG -LVEPSQLGRTLTBEHLTMTFDSFYCPPPPCHEVTS KE PIMMKN 56 MSSLSGKVQTVLG-PVEPSQLGRTLTBEHLTMAFDSFYCPPPPCQEAASREPIMMKN 56

- --SIGTGDR INTVRG -PITNSEAGFTLTBEBICGTSAGF- --- --: - --- ----LRAWQ 44 ---SIGTGDRINTVRG-PITISEAGFTLTHEHICGSSAGF----- ---- ------LRAWP 44 --WLDRSAQAMRSlRARPITISEAGFTLTHEDIS--AA- - -- - --- ------- - --RQDS 38

MPELNTARG-PIDTADLGVTLMHEHVFIMTTEI- --------- --- -- AQNyp 37 MSFDPTGYTLAHEBLHIDLSG ---- ----- ------------ 2 1

* ** ** .:

LFWIQKNPYSHRENLQLNQEVGAIREELLYFKAKGGGALVENTTTGLS-RDVH-TLKWLA 114 LFWIQKNPYSHQENLQLNQEVEAVREELLYFKAKGGGAVVENTTTGLS-RDVR - TLKWLA 114 EFFGSRKALAEK----------AVRGLRR-ARAAGVRTIVDVSTFDIG- RDVS - LLAEVS 91 EFFGSRKALAEK--- --- ----AVRGLRR-ARAAGVRTIVDVSTFDIG-RDVS-LLAEVS 91 CVLGQSSSVAQSS - --------SAKGCERIARQSGWR-ANDCRCVDFRYRSRRQFIGRGF 88 EAWGDEDKRVAG-- ----- - --AIARLGE- LKARGVDTIVDLTVIGLG -RYIP - RIARVA 84 -FKNNVDCRLDQY-- ------AFICQEMNDLMTRGVRNVIEMTNRYMG-RNAQ-FMLDVM 7 0

EQTGVHIIAGAGFYV-- DAT-- ---HSAATRAMSVEQLTDVLINEILHGADG--TSIKCG 165 EQTGVHIIAGAGFYV-- DAT-----HFAATRAMSVEQLTDVLISEILHGADG--TSIKCG 165 MMVDVSLLAETGLWF--DPP-----LSIGLR - -SVEELTQFFLREIQYGIED--TGI RAG 140 RAADVHIVAATGLWF - -DPP-----LSMRLR--SVEELTQFFLREIQYGIED--TGIRAG 140 AGCRRSYLAATGLWF--DPP-----LSMRLR - -YVEELT-LVLPAVRFNMASKYTGIRAG 138 AATELNIVVATGLYTYNDVPFYFHYLGPGAQLDGPEIMTDMFVRDIEHGIAD--TGlKAG 142 RETGINVVACTGYYQ--DAFFP---EHVATR--SVQELAQEMVDEIEQGIDG -- TELKAG 121

:. :" : * ::. *

VIGEIGCSWP-LTDSERKILEATAHAQAQLGCPVIIHPGRNPGAPFQIIRILQEAGADIS 224 VIGEIGCSWP-LTDSERKVLQATAHAQAQLGCPVIIHPGRNPGAPFQIIRVLQEAGADIS 224 II-KVATTGK-ATPFQELVLKAAARASLATGVPVTTHTAASQRDGEQQAAIFESEGLSPS 198 II-KVATTGK-ATPFQELVLKAAARASLATGVPVTTHTAASQRDGEQQAAIFESEGLSPS 198 II-KVATTGK - ATPFQELVLKAAARAS LATGVPVTTHTAASQRDGERGRPPFLSPKLEPS 196 IL - KCATDEPGLTPGVERVLRAVAQAHKRTGAPISTHTHAGLRRGLDQQRIFAEEGVDLS 201 IIAEIGTSEGKITPLEEKVFlAAALAHNQTGRPISTHTSFST-MGLEQLALLQAHGVDLS 180

* ." • * *; * .

KTVMSHLDRTIFDKKELLEFAQLGCYLEYDLFGTELLNYQL----SPDIDMPDONKRIRR 280 KTVMSHLDRSIFDKKELLEFAQLGCYLEYDLFGTELLNYQL----SPOTOLPDDNKGLGG 280 RVCIGHSO-OTOOLSYLTALAARGYLIGLOHIPHSAIGLEONASASALLGIRSWQTRALL 257 RVCIGHSD-DTDDLSYLTALAARGYLIGLDHIPHSAIGLEDNASASALLGIRSWQTRALL 257 RVCIGHSD-DTDDLSYLTALL - RGYLIGLDHIPHSAIGLEDNASASPLLGIRSWQTRALL 2 54 RVVIGHCG-OSTDVGYLEELlAAGSYLGMDRFGVDVI--------SP------FQDRVNI 246 RVTVGHCD-LKDNLDNILKMIDLGAYVQFDTIGKNSY- -- -----YP - - ---- OEKRIAM 225 :. : .. . : VHFLVDEGYEORILMAHDIHT-- - ------KHRLMKYGGHG -YSH ILTNI VPKMLLRGLT 330 VRFLVNEGYEORILMAHDIHT---------KHRLMKYGVHG-YSHILTNVVPKMLLRGLT 330 lKALIDQGYMKQILVSNDWLFGFSSYVTNIMDVMORVNPDG- MAFIPLRVIPF LREKGVP 316 IKALIDQGYMKQILVSNDWLFGFSSYVTNIMDVMDRVNPDG-MAFIPLRVIPFLREKGVP 316 lKALIOQGYMKQILVSNDWLFGFSSYVTNIMDVMDRVNPOG-MAFIH--- -- - -- - -- - - 300 VARMCERGHADKMVLSHDACCYFOALP---EELVPVAMPNWHYLHIHNDVIPALKQHGVT 303 LHALRDRGLLNRVMLSMDITR---------RSHLKANGGYG -YOYLLTTFIPQLRQSGFS 275

. . * .::::: .. it

ERVLOKILIENPKQWLTFK---- 349 ERVLOKILRENPKQWLTFK--- - 349 QETLAGITVTNPARFLSPTLRAS 339 QETLAGITVTNPARFLSPTLRAS 339

777

Fig. 2-Comparison of amino acid sequence deduced from nucleotide sequence of PCR amplified fragment with sequence of parathion hydrolase of Flavobacterium sp. (FL) and Pseudomonas diminuta (PO). The Yhf V protein of E. coli (Accession No. EMBL Z92669), the proteins encoded by cON A designated as rpr- I (Davies et al. 1997) and mpr 56-1 (Houx et al. 1996) also show considerable homology with the deduced amino acid scquencc. The conserved histidine residues are shown with bold letters.

778 INDIAN J EXP BIOL, JULY 2002

moti f can be defined as (Srr) RRXFLK, where the argi nines are completely invari ant and the frequency of the occurrence of each of the other amino acids exceeds 50%16. Periplasmic proteins containing twin­argi nine motif are translocated across membrane th rough a sec- independent pathway known as twin­arginine transport pathway l7 . Parathion hydrolase is a membrane bound protein. The twin arginine motif fo und in the signal sequence of parathion hydrolase is identical to tat motif found in variety of membrane bound or peri pl asmic proteins. Therefore, it is logical to predi ct its ro le in membrane targeting of parathion hydrolase in soil bacteri a. However, further studi e are required to establi sh its role in membrane target­ing of parathi on hydrolase.

Interes tingly, sequence of PCR product showed strong homology to yhjV gene of E. coli (data bank accessions AE 0004 13 and AE 0004 14) and to an un­characteri zed ORF of Mycobacterium tuberculosis (Data bank accession number EMBL Z92669). A cON A clone des ignated as rpr-l of rat lS and mpr 56-1 of mouse l9 also showed considerable homology wit the sequence of PCR product. Extent of homology was much lower when compared to homology noticed among opd sequences. However, the deduced amino acid sequences fro m these genes showed more than 50% homology to amino ac id sequence of parathi on hydrolase. Especially the region containing hi stidine residues that bind to zinc ions are hi ghl y conserved (Fig. 2). Such a strong conservation in catalyti c do­main of these proteins showed the functional similari ­ti es among these enzymes.

Ex istence of identical cataboli c genes such as opd within structurally unrelated plasmids from soil bacte­ria arc reported in literature. Several detailed investi­gati ons have been undertaken to understand the struc­tural organi sation and mode of transfer of these iden­tical cataboli c genes20

.2 1

. These studies revealed exis­tence of cataboli c genes in the fo rm of complex trans­poson. Jacob and hi s co-workers22 are the first to re­port about the transpos ition of toluene catabolic genes and subsequent studies have shown number of fun -. I b I' 202 1 tiona cata 0 IC transposons . .

The op-pesticide degrading soil bacteria have been isolated from di versified geographical regions. [n three cases the op-pesticide degrading (opel) plasmids have been characterized 12.7. The opd plasmids have shown considerable di versity with respect to the size and genetic informati on. However, a DNA region of 7 kb among these opd plasmids is hi ghl y conserved l2

•

The 1.25 kb opd gene is part of thi s 7 kb conserved region. Though the sequence of opd genes is known, DNA fl anking to opd gene is yet to be sequenced. Sequence of entire conserved region as essential to gain insights about organi zati on and transfer of opd gene among soil bacteri a.

Acknowledgement The work is supported by DST, New Delhi. SS and

MB thank CSIR, New Delhi for awardi ng JRF.

References Corbett J R, Wright K & Baillie A K, The biochemical mode oj actioll oj pesticides, (Academic Press, London, New York) 1984,107.

2 Alexander M, Biodegrada tion of chcmicals of environmental concern , Sciell ce , 2 1 I ( 198 1) 131.

3 Munnccke 0 M, Enzy mat ic hydro lysis of organophosphorus insecticides, a-possible pes ticide disposal method, Appl Ellvi­ron Microbiol , 32( I ) (1976) 7.

4 Mc Dani el C S, Haper L L & Wild J R, Cloni ng and se­quencing of plasmid borne gene encoding a phosphotri­esterase, J Bacteriol , 170(5) (1988) 2306.

5 Scrdar G M, Gibson 0 T, Munnecke 0 M & La ncaster J H, Pl asmid in volve ment in parathion hydrolysis by Psec/olllolloS d illl il/uta, Appl Ellviroll Microbiol, 44( I ) (1982) 246.

6 Rowlands S S, Zu lty J J, Sathyamonhy M, Poge ll B M & Speedie M K, The effec t of signal sequences on the cffi­ciency of sec retion of heterologous phosphotriestcrase by Streptomyces lividalls, Appl Microbiol Biorechnol, 38 (1992) 94.

7 Somara S & Siddavatam 0 , Pl asmi d med iated orga nophos­phorus pes ti cide degradat ion in FlavobacteriulII balustilllllll. Biochelll Mol Bioi 1111, 32 ( 1995) 672.

8 Sam brook , Fritsch E F & Mani at is T, Molecular clollillg: A laboratOlY lIIallllal , (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y) 1989, 14. 1.

9 Sanger F, Delermina tion of nuc leotide sequence in dcoxy ri­bonucleic acid, Sciellce, 2 14 ( 198 1) 1205.

10 Serdar G M. Murdock 0 C & Rohde M F, Parath ion hy­drolase gene fro m Pseudomollas dilllilluta MG-Subc loning, complete nucleotide sequcnce and express ion of mature por­tion of the enzyme in E. coli , BiofTecllllOl, 7 (1989) 115 1.

II Singh M, Kleberger A & Klinmuller W, Location of nitrogen fi xa ti on (niJ) genes on indigenous plasm ids of Ellferobacter agglulll eralls, Mol Cell Cenet, 190 ( 1983) 373.

12 Mulbry W W, Kearney P C, Nelson 0 J & Karn s J S, Phys i­cal compari son of parathion hydro lase plasm ids from Psell­dOlllollas dilll ill llla and Flavobacterium sp. Plaslllid. 18 (1987) 173.

13 Merrick M & Edwards R A, Nitrogen control in bacteria, Mi­crobiol Rev, 59 (1995) 604.

14 Cannon W, Claverie M F, Austin S & Buck M, Idelll ification of a DNA-contacting surface in the transcription factor 054, Mol Microbiol, II (1994) 227.

15 Mulbry W W & Karns J S, Parathion hydrolase specified by the Flavobacterium balustinwl1 opd genc: Relationship be­tween the gene and protein , J Bacteriol , 171 (1 2) (1989) 6740.

SOMARA et al.: LOCALISATION OF IDENTICAL ORGANOPHOSPHORUS PESTICIDE 779

16 Berks B C, A common export pathway for proteins binding complex redox co-factors?, Mol Microbiol, 22 (1996) 393.

17 Weiner J H, Bilous P T, Shaw G M, Lubittz S P, Frost, Tho­mas G H, Cole J A & Jurner R J, A novel and ubiquitous sys­tem for membrane targeting and secretion of co-factor con­taining proteins, Cell, 93 (1998) 93.

18 Davies J A, Buchan V L, Krylova 0 & Ninkina N N, Mo­lecular cloning and expression pattern of rpr-I, a resinifera­toxin-binding, phosphotriesterase-related protein, expressed in rat kidney tubules, FEBS Lett. 410 (1997) 378.

19 Houx, Maser R L, Magerheimer B S & Calver J P, A mouse-

kidney and liver-expressed eDNA having homology with a prokaryotic parathion hydrolase (phosphotriesterase)­encoding gene: Abnormal expression in injured and polycys­tic kidneys, Gene, 168 (1996) 157.

20 Wyndham R C, Cashore A E, Nakatsu C H & Peel M C, Catabolic transposons, Biodegradation,S (1994) 323.

21 Tan H T, Bacterial catabolic transposons, Appl Microbiol Biotechnol, 51 (1999) I.

22 Hedges R W & Jacob A E, Transposition of ampicillin resis­tance from RP4 to other replicons, Mol Gen Genet, 132 (1974) 31.