The phosphotriesterase gene opdA in Agrobacterium radiobacter P230 is transposable

FEMS Microbiology Letters 222 (2003) 1^8

www.fems-microbiology.org

The phosphotriesterase gene opdA in Agrobacterium radiobacter P230 is transposable Irene Horne

a;


b

, Xinghui Qiu b , Robyn J. Russell a , John G. Oakeshott

a

a CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, PR China

Received 15 January 2003 ; received in revised form 30 January 2003 ; accepted 3 February 2003 First published online 16 April 2003

Abstract We report a transposase gene (tnpA) upstream of the opdA phosphotriesterase gene of Agrobacterium radiobacter P230, as well as inverted repeats indicative of insertion sequences, flanking the two genes. Both the tnpA gene and the inverted repeats resemble the Tn610 transposon from Mycobacterium fortuitum. Two additional putative open reading frames separate opdA and tnpA with inferred translation products with similarity to two proteins encoded on the Geobacillus stearothermophilus IS5376 transposon. To test the proposition that these genes were contained on a transposon, an artificial composite transposon was constructed. This artificial transposon was then delivered into Escherichia coli DH10L cells. Transposition was demonstrated by the presence of opdA on the E. coli chromosome and confirmation of insertion by inverse polymerase chain reaction. The data presented suggest a possible role of transposition in the distribution of the opd/opdA genes across a wide range of soil bacteria. 4 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : OpdA; Phosphotriesterase; Transposition

1. Introduction Searches for bacterial enzymes capable of degrading organophosphate (OP) insecticides have now identi¢ed the same gene/enzyme system from various organisms and diverse geographic locations [1^4]. The gene has been called opd for organophosphorus degradation gene. Identical opd sequences have been identi¢ed in both Flavobacterium sp. ATCC 27551 and Brevundimonas diminuta MG [3,4]. These identical genes were located on di¡erent plasmids in the two organisms: on a 43-kb plasmid in Flavobacterium sp. ATCC 27551 [5] and on a 66-kb plasmid in B. diminuta MG [6]. A 5.1-kb region of these plasmids surrounding the opd gene appeared to be homologous, as determined by Southern hybridisation [7]. A gene homologous to opd was also detected by highstringency hybridisation on the chromosome of a Pseudomonas isolate using Southern hybridisation [1] and a further sequence similar to opd was recently identi¢ed in an

* Corresponding author. Tel. : +61 (2) 6246 4110; Fax : +61 (2) 6246 4173. E-mail address : [email protected] (I. Horne).

Agrobacterium isolate. This gene, called opdA, was approximately 88% identical at the nucleotide level to opd [2]. Both opd and opdA encode OP hydrolytic enzymes but the Pseudomonas strain and the Agrobacterium strain lack the plasmids found in both Flavobacterium sp. ATCC27551 and B. diminuta MG. The presence of opd-like genes has also been demonstrated in several bacterial genome sequencing projects [7,8], although these sequences are much more distant (30^40% identity at the amino acid level) from those found in Flavobacterium sp. ATCC27551, B. diminuta MG and Agrobacterium radiobacter P230. Of the OPD homologues identi¢ed in these genome sequencing projects, only the Escherichia coli OPD-like protein, called PHP (phosphotriesterase homology protein) has been puri¢ed and characterised. No enzymatic activity could be attributed to this protein [9]. Therefore the native function of these OPD homologues is not known. Transposons are one means by which genes can move between otherwise distantly related bacteria [10]. They have been involved in the mobilisation of both antibiotic and heavy-metal resistance through microbial communities in hospitals and heavily polluted environments, respectively [11,12]. Here we examine the possibility of

0378-1097 / 03 / $22.00 4 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1097(03)00211-8

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2. Materials and methods

GCC; nt 4325^4342). The cycling was as follows : an initial denaturation of 94‡C for 3 min, after which 1 Wl of PfuTurbo (Stratagene) was added and the reaction allowed to cycle 30 times (94‡C/1 min, 48‡C/1 min 35 s, 72‡C/5 min) and a ¢nal extension of 72‡C/10 min.

2.1. Bacterial strains and plasmids

2.4. Construction of OPD-homologue expressing plasmids

The E. coli strains used in this study were both recA3 strains: JM109 Vpir (endA1, recA1, gyrA96, hsdR17 þ (r3 K , mK ), supE44, v(lac-proAB), [FP, traD36, proAB, lacIq , lacZvM15] lysogenised with Vpir; [13]), DH10L (F3 mcrAv(mrr-hsdRMS-mcrBC)P80dlacZvM15vlacX74 endA1 recA1 deoR v(ara, leu)7697 araD139 galU galK nupG rpsL V3 ; Gibco BRL), and BL21(DE3) (F3 dcm, 3 ompT, hsdS(r3 B , mB ) gal V (DE3); Stratagene). Mycobacterium smegmatis mc2 (a gift from H. Billman-Jacobe, University of Melbourne) was grown as previously described [14]. Plasmids used in this study are listed in Table 1. E. coli was grown at 37‡C on Luria^Bertani (LB) medium [15]. When used in the medium, ampicillin, kanamycin and spectinomycin were at concentrations of 100, 25 and 25 Wg ml31 , respectively.

The Drosophila OPD homologue was ampli¢ed using the primers dprnde5 (5P-GATCGTCATATGTCAACCGTACAGACCGTT, with NdeI site underlined) and dprbam3 (5P-GTCTAAGGATCCCATGATAAGAGATGCTAC, with BamHI site underlined) and the plasmid pBSdpr as the template. The PCR product was digested with NdeI^BamHI and cloned into similarly digested pET14b. The M. smegmatis OPD homologue was PCRampli¢ed from genomic DNA using the primers myopd5 (5P-CATATGGTGCCAGAACTAAATACC, with NdeI site underlined) and myopd3 (5P-GGATCCTCACTGATAGCCGCCCTG, with BamHI site underlined) and the PCR product was digested with NdeI^BamHI and ligated with similarly digested pET14b, to create pETmyopd. The expression of both was according to the method supplied by Novagen.

transposition as a means by which the opdA gene can move around microbial populations.

2.2. DNA manipulation 2.5. Phylogenetic analysis Isolation of chromosomal DNA was performed according to Gardiner et al. [16]. Standard DNA manipulations were carried out according to Sambrook et al. [15]. All cloning into pJP5603 derivatives was carried out in the E. coli strain JM109 Vpir. Polymerase chain reaction (PCR) detection of the opdA gene on chromosomal extracts was performed using the upstream and downstream primers, 5P-GATCGTGGATCCCCAATCGGTACAGGCGATCTG and 5P-GATCGTAAGCTTTTCATCGTTCGGTATCTTGACGGGGAAT, respectively, which amplify from nt 3703 to 4747. Prior to PCR, DNA was denatured at 94‡C for 3 min, and then 0.5 units of Taq DNA polymerase (Gibco BRL) was added. There were then 30 cycles of 94‡C/35 s, 48‡C/1 min 10 s and 72‡C/2 min, followed by a ¢nal extension of 72‡C/5 min. The GenBank accession number for TnopdA is AY043245. 2.3. Inverse PCR E. coli chromosomal DNA was completely digested with BamHI in a volume of 60 Wl. This was then diluted with TE bu¡er to 300 Wl and ligated according to the manufacturer’s instructions (Fermentas). The ligated DNA was then precipitated with sodium acetate/ethanol at 320‡C for 2 h after which it was pelleted by centrifugation at 13 000Ug for 20 min at 4‡C, air-dried and resuspended in 10 Wl TE bu¡er. An aliquot of this (0.1 Wl) was used in the inverse PCR reaction, which was performed with the primers tnpa2 (5P-CGAAGTCATGCGAGCCCT ; nt 229^212) and opdA3seq (5P-TGAGCTACCTAACCG-

OPD and its homologues from Mycobacterium tuberculosis H37Rv [8] and E. coli [7] were used in a TBLASTN and BLASTP analysis against all genomes in the NCBI database. Homologues of approximately the same length showing conservation of the histidines involved in metal chelation in OPD [17] were then assembled into a dataset for phylogenetic analyses. These sequences were aligned using the PileUp programme from the Genetics Computer Group (GCG; [18]), with default settings (gap weight 3.0 and gap length weight 0.1). Their similarities were calculated using the FASTA algorithm [19]. A distance neighbour joining tree was then created using the Jukes^Cantor distance correction method and Growtree (GCG). GenBank accession numbers for the genes used in this analysis are as follows : Bacillus halodurans, NC001496; Deinococcus radiodurans, NC001263; E. coli, NC000913 ; Klebsiella pneumoniae, NC003486 ; Listeria monocytogenes, NC003210; Mesorhizobium loti, NC002678 ; Mycobacterium leprae, NC002677; M. smegmatis, NC002974; Mycobacterium bovis, NC002945; M. tuberculosis, NC000962; Geobacillus stearothermophilus, NC002926; Salmonella typhi, NC003198; Mycoplasma pulmonis, NC002771; OPD, M20392; Drosophila, AE002593; rpr-1 (rat homologue), NM022224; mpr-1 (mouse homologue), NM008961; Agrobacterium tumefaciens C58, NC003063 ; Burkoholderia fungorum, NC003371 ; Rhodobacter sphaeroides, NC002718; B. diminuta IAM1269T 16S rRNA gene, AB021415; Flavobacterium sp. ST-92 16S rRNA gene, AB075232. Bootstrap resampling of the trees was per-

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formed for all analyses to provide con¢dence estimates for the inferred topologies. A total of 1000 replicates were used.

B

1

2

P

P

3

3

4

5

H

7

6 N

H

8 B

2.6. Analysis of transposition tnpA

orfA

orfB

opdA

Fig. 1. Physical map of the 8-kb BamHI fragment from A. radiobacter P230 containing opdA. The top line represents the length of DNA in kb. The open arrows correspond to the transposase (tnpA) and opdA genes. The solid arrows represent the left (LIR) and right (RIR) inverted repeats. Restriction sites are: P, PstI; B, BamHI ; H, HindIII; N, NotI. A spectinomycin-resistant cassette was placed in a NotI site in the 5P region of the opdA gene.

As the opdA-containing DNA fragments (8-kb BamHI and 4-kb HindIII fragments) had no selectable markers, the spectinomycin resistance gene from pUI1188Sp was inserted into the opdA gene at a unique NotI site. The opdA-containing fragments with the inserted spectinomycin resistance cassette were then cloned into the plasmid pJP5603. This plasmid contains an R6KQ origin of replication requiring the Vpir gene. All the pJP5603 derivatives were transformed into E. coli DH10L via electroporation [15] to allow for maximum transformation e⁄ciency. The transformation mix was diluted and plated onto LB plates containing no antibiotics, LB plates containing spectinomycin, and LB plates containing kanamycin. The number of colonies on each plate was quantitated after overnight incubation.

ti¢ed between opdA and tnpA using the GenScan programme on Bionavigator [20]. The ¢rst (orfA, nt 1116^ 2495) would encode a product of 460 amino acids with a molecular mass of 50.1 kDa. This protein has approximately 60% sequence similarity to a putative transposase for insertion sequence IS5376 in G. stearothermophilus ([21] ; Fig. 2). However, it does not possess a helix^turn^ helix motif at the N-terminus noted in the transposase of IS5376 that would be involved in DNA binding. So we ¢nd it unlikely that this is a functional transposase. The second putative orf (orfB, nt 2618^3475) would encode a 32.1-kDa protein of 285 amino acids. This protein has sequence similarity (77%) to a hypothetical protein in the Novosphingobium aromaticivorans genome (GenBank accession number ZP_00094124) as well as a putative ATP-binding protein insertion sequence element in Streptomyces coelicolor and putative ATP-binding protein (59%) associated with insertion sequence IS5376 of G. stearothermophilus ([21,22] ; Fig. 2). One element of an inverted repeat was also identi¢ed immediately downstream of the tnpA gene on the 8-kb BamHI fragment (nt 13^25, Fig. 1). This sequence is identical to the left inverted repeat of Tn610 from M. fortuitum [23]. A right inverted repeat unit identical to that of Tn610 (5PTTGCAACAGAGCC; nt 4817^4829) was found downstream of the opdA gene (nt 3619^4773, Fig. 1). All these aspects of the architecture from nts 13^4829 suggest that the transposase gene (tnpA) and the opdA gene are con-

2.7. Biochemical techniques Phosphotriesterase activity assays were performed as previously described [2].

3. Results 3.1. Sequences upstream of opdA resemble transposon-related genes Fig. 1 shows the location of open reading frames (ORFs) identi¢ed by sequencing the 8-kb BamHI fragment from A. radiobacter P230 that contains opdA from A. radiobacter P230 [2]. The ¢rst ORF, which we call tnpA, runs from nt 831 to 67 (5P to 3P) and is identical in sequence to the transposase of Tn610 from Mycobacterium fortuitum (accession number X53635). Sequence similarity with the IS6100 transposase is reduced immediately after the stop codon of the gene. Two putative orfs were idenTable 1 Plasmids used in the study Plasmid pJP5603 pBluescript KSþ (pBS) pB1 pJPB1 pJPB1Sp pUI1188Sp pBSdpr pET14b pETdpr pETmyopd

Relevant characteristics

Reference R

R6K-based suicide vector, Km Cloning vector, ApR 8-kb BamHI fragment containing opdA in pBS 8-kb BamHI fragment ontaining opdA in pJP5603 pJPB1 with 6Sp in NotI site in opdA 6Sp in pBS dpr-1 in pBS his-tag expression vector dpr-1 in pET14b M. smegmatis opdA homologue in pET14b

[19] Stratagene [2] This study This study [36] Jamie Davies Novagen This study This study

Ap, ampicillin ; Km, kanamycin ; Sp, spectinomycin; 6Sp, spectinomycin-resistant cassette £anked by transcription terminators.

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A. orfA

3

G. s

62

orfA

63

G. s

121

orfA

123

G. s

179

orfA

182

G. s

238

FLRERVTAFPDLTASRLTREIREMGYVGAYTAVKRYLAAIRPDHDPKPYEVRFETKAGVQ +L++R+ + +L EIR+ GY G T +K Y+ R + K Y VR+ET G Q YLQKRMLEDGVFNSEKLFFEIRQQGYTGGKTILKDYMKPFR-ETAKKKYTVRYETLPGEQ GQVDFARFVVEFTDEPGVQRIVWLFSLVLGYSRFLFLFSLVLGYSRFLFARYVLHQDLQT QVD+ + V E E G + + LF LGYSR +LF LGYSR +A + QD + MQVDW-KEVGEVVIE-GKKVKLSLFVATLGYSRMKYLFVATLGYSRMKYAVFTTSQDQEH LLRCHMQAFEALGGVPIEILYDRMKTAVTGEDDQGHIVYNRSLLALAQHYRFSPGV-PPY L+ C +Q+F+ GGVP ++L+D MKT G +QG + +N+ A +Y F P V PY LMECLIQSFKYFGGVPKKVLFDNMKTVTDGR-EQGVVKWNQRFSEFASYYGFIPKVCRPY SDQTKARLRRSFRYIREDFFLGRSLHNLD QTK ++ R+ +YI + F++G + +++ RAQTKGKVERAIQYIMDHFYVGTAFESIE

B. orfB

20

G. s

12

orfB

80

G. s

72

orfB

140

G. s

132

LKMPRALEILDATLRRIEQGQIDGIEALDDLLGEELSLRENRRVKAALRMARLPVVKTLA 79 L +P E A I E L LL E+ ++ R ++ +++++LP KT+ LHLPVMAERWSAMAEYASTHNISYSEFLFRLLEAEIVEKQARSIQTLIKLSKLPYRKTID 71 GYDFSFQPSLDKNRILALAGLDFIERAEVVHLLGPPGTGKSHIATALAVEAVRAGKSVYF 139 +DF+ QPS+D+ RI L L FI+R E + LGPPG GK+H+A ++ +EA+ G YF TFDFTAQPSVDERRIRELLTLSFIDRKENILFLGPPGIGKTHLAISIGMEAIARGYKTYF 131 IPLADLIAQLAKAEREGTLREKIRFLXRASLLVVDEI 176 I DL+ QL +A++EG L +K+R + ++L++DE+ ITAHDLVNQLRRADQEGKLEKKLRVFVKPTVLIIDEM 168

Fig. 2. A: Sequence alignments orfA with a putative transposase from IS5376 from G. stearothermophilus. B: orfB with a putative ATP-binding protein associated with insertion sequence IS5376 of G. stearothermophilus.

tained on a transposon-like fragment, which was designated TnopdA. 3.2. Transposition of the opdA gene To determine if the DNA fragment could indeed transpose, the 8-kb BamHI fragment was cloned into the vector, pJP5603, to create pJPB1. The vector pJP5603 requires a trans supply of the Vpir gene product for maintenance [13] and is therefore a suitable vector for insertional inactivation in E. coli strains that generally lack this gene. To facilitate the screening of possible transposition events (generally 1035 ^1037 ), the spectinomycin resistance cassette from pUI1188Sp was cloned into the unique NotI site (nt 3650) of the opdA insert in pJPB1 to create pJPB1Sp. E. coli DH10L is a recA3 strain and is therefore unable to perform homologous recombination events. Furthermore, pJP5603 derivatives cannot be maintained in this strain as it does not contain the Vpir gene. The plasmid, pJPB1Sp, was therefore transferred into E. coli DH10L by transformation. The transformation mix was then diluted and plated onto LB agar plates containing various antibiotics: with control plates lacking any antibiotics (to select for all E. coli DH10L cells), plates with kanamycin (to

select for transformants containing the vector pJPB1Sp), and plates with spectinomycin (to select for transformants containing, speci¢cally, the spectinomycin resistance cassette and therefore presumptively the opdA gene). The number of colonies on each plate was then quantitated. More spectinomycin-resistant transformants were obtained than kanamycin-resistant transformants (9.6U109 of 4.3U1011 total number of transformants per ml compared with 0 for KmR ). Also the frequency of spectinomycin-resistant transformants was about 1% of all transformants, assuming a 100% transformation e⁄ciency. Since no spectinomycin- or kanamycin-resistant transformants were detected in the E. coli control, it is assumed that the spectinomycin-resistant colonies were not due to spontaneous antibiotic resistance. Therefore we conclude that the opdA gene of A. radiobacter P230 is contained on a transposable element. 3.3. Con¢rmation of insertion of TnopdA on the chromosome of E. coli In order to con¢rm that the opdA gene had been transferred onto the chromosome of E. coli DH10L from the construct pJPB1Sp, eight spectinomycin-resistant, kanamycin-sensitive transformants were selected randomly

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mouse rat Drosophila A. tumefaciens R. sphaeroides K. pneumoniae Me. loti S. typhi OpdA OPD M. bovis M. tuberculosis M. avium M. smegmatis G. staerothermpophilus D. radiodurans B. halodurans E. coli L. monocytogenes My. plasmpulmonis Bu. fungorum

15 15 11 8 8 8 8 8 43 44 15 11 23 8 8 25 8 1 8 6 8

5

55| |57 VEPSQLGRTLTHEHLTMTFDSFY VEPSQLGRTLTHEHLTMAFDSFY ITPNLLGRTLTHEHVALDFEHFY IATDQLGVTLMHEHILNDCRCWW IPSSALGHTLMHEHLQNDCRCWW LPINEMGVTLMHEHILLDASGKW VAIADMGVTLMHEHILLDGSTSW VAHTDMGLTLPHEHLFNDLSSVV IPVSEAGFTLTHEHICGSSAGFL ITISEAGFTLTHEHICGSSAGFL IDTADLGVTLMHEHVFIMTTEIA IDTADLGVTLMHEHVFIMTTEIA IDTAALGVTLMHEHVFIMTTEIA IDTADLGVTLMHEHVFIMTTEVM VPVEQLGKTLIHEHFLFGYPGFQ VAAAQLGATLPHEHVIFGYPGYA IKPEEFGVCACHEHLHIDLSRI. MSFDPTGYTLAHEHLHIDLSGF. IAPEQLGFTYSHEHI.VCVPAYW IDPKQLGVVDCHDHL...IKNYG ITPDQAGSTLTHEHIRYAYPGCE . .* . *.*.

Fig. 3. Alignment of the N-terminal region that was used to identify OPD homologues. Two of the histidines (H55 and H57, according to OPD nomenclature) that are involved in metal binding [17] are indicated.

and examined in more detail. No pJP5603-derivative plasmids could be extracted from these transformants, suggesting that spectinomycin resistance was conferred from the chromosome. PCR was used to examine the presence of opdA in extracted chromosomal DNA from these transformants (data not shown). All of the transformants examined possessed the opdA gene. Therefore both the spectinomycin resistance cassette and the opdA gene were transferred to the chromosome of the transformants, and spectinomycin resistance correlates with the presence of the opdA gene. Tn610 belongs to the Tn6 family of transposons that form stable cointegrates by virtue of the inability of their transposases to resolve the transposon. Perhaps the role of either orfA or orfB is to do this as the E. coli DH10L cannot do this. The insertion of TnopdA into the E. coli chromosome was con¢rmed by inverse PCR to be in the glutathione oxidoreductase gene in one transconjugant and at the junction of the phnJ/phnK genes involved in phosphonate usage in another transconjugant (GenBank accession number NC000913). 3.4. The phylogenetic relationships of OPD/OpdA do not match those of their hosts A comprehensive dataset of Opd(A)-like sequences was compiled using OpdA and several known OPD homologues [8] in TBLASTN and BLASTP analyses against all genomes (both ¢nished and un¢nished) in the NCBI database. Further analyses were then restricted to a subset of 21 proteins. Homologues shared conservation of all histidines involved in zinc chelation in OPD [17], conservation of size (approximately 340 amino acids) and the presence of an ^HEH^ motif in the N-terminal region of

the protein (Fig. 3). These proteins had at least 50% similarity with OpdA over the entire protein. Notably, one of these was a chromosomal copy identi¢ed in the genome sequencing project for A. tumefaciens C58 (29.0% identity with OpdA). A phylogenetic tree of these amino acid sequences was then constructed by the neighbour-joining method with bootstrap resampling (see Section 2). The same phylogenetic methods were then used to construct a tree of 16S rRNA genes of the organisms in which the Opd(A) homologues were identi¢ed, or in two cases, their nearest relatives (Fig. 4). In the latter cases, the 16S rRNA gene sequence was not available for Flavobacterium sp. ATCC 27551 or B. diminuta MG so we used the 16S rRNA gene of Flavobacterium sp. ST-92 [24] and B. diminuta MG IAM12691T [25] instead. In most respects the OPD tree is consistent with the 16S organismal tree, although there is less resolution (lower bootstrap scores) in the OPD tree. The eukaryotic mouse, rat and Drosophila homologues form one clear clade. Among the bacterial homologues the mycobacterial proteins form one clear assemblage as do the K-proteobacteria A. tumefaciens and R. sphaeroides. Importantly, however, OpdA sits with OPD as a clearly distinct clade. OpdA does not cluster with the A. tumefaciens homologue, nor does OPD, which was identi¢ed in both B. diminuta and Flavobacterium, cluster with either of those organisms. This strongly suggests that OPD and OpdA have been acquired by their current hosts via a lateral gene transfer mechanism. If anything, OpdA and OPD appear to cluster with the mycobacterial homologues, which might suggest they have a mycobacterial origin. Perhaps this is consistent with the ¢nding that Tn610 was identi¢ed in a mycobacterial strain [23].

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Fig. 4. The phylogenetic tree of (a) the 16S rRNA genes (excluding eukaryotic species) and (b) their corresponding OPD homologues from various bacterial genomes.

Of the homologues identi¢ed, only the E. coli protein has been reported in the literature to be examined for phosphotriesterase activity and did not possess any [9]. We have also tested the Drosophila homologue and it lacks activity against coumaphos/coroxon/parathion/paraoxon (data not shown). We have tested the mycobacterial homologue (from M. smegmatis) and it has slight hydrolytic activity against the OP, coumaphos but no detectable activity against parathion.

4. Discussion This study demonstrates that the opdA gene from A. radiobacter P230 is transposable. Several lines of evidence suggest that OPD may lie in the same transposable element. Firstly, identical opd genes were found on plasmids from Flavobacterium sp. ATCC 27551 and B. diminuta MG, even though the two organisms are well-separated phylogenetically. Secondly, restriction map comparisons of the opd-containing plasmids from Flavobacterium sp. ATCC 27551 and B. diminuta MG indicate a region of homology of approximately 5.1 kb in the two plasmids (2.6 kb upstream and 1.7 kb downstream of opd; [5]). TnopdA also covers almost 5 kb. We therefore suggest that one interspeci¢c transposition event occurred prior to

the sequence divergence between opd and opdA, with another, more recent horizontal transfer to account for the ¢nding of identical opd sequences in B. diminuta and Flavobacterium. In the last 50 years, there has been a selective pressure on heavy-metal- and antibiotic-resistant genes to move via transposition through microbial communities, as these compounds are highly toxic to bacteria [11,12]. However, OPs are not toxic to bacteria, so why would opd be transferred so readily in the environment ? It is suspected that the spread of the TOL genes, which encode enzymes involved in the catabolism of toluene and related phenolics and are also contained on a transposable element [26], is because these pollutants provide a rich carbon source [27,28]. Perhaps the nutritional value of OPs has driven the spread of the opd/opdA genes. OpdA was isolated by virtue of the ability of its host, A. radiobacter P230, to grow with OPs as a phosphorus source [2]. Phosphate in soil can be limiting to growth as it is organically bound [29]. OPs can also serve as carbon sources in opd-containing bacteria; Flavobacterium sp. ATCC 27551 was isolated by virtue of its ability to utilise diazinon as a carbon source [30] and a Pseudomonas strain containing opd was able to use methyl parathion as a carbon source [1]. The widespread distribution of Opd(A)-like sequences across prokaryotes and eukaryotes clearly suggests an an-

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cient origin for this multigene family, probably pre-dating the divergence of Archaea, Prokaryota and Eukaryota. However, our phylogenetic analysis clearly points to some lateral gene transfers during the evolution of these homologues. OPD/OpdA do not cluster according to the phylogeny of their ‘host’ organisms but instead emerge as most closely related to homologues from Gram-positive bacteria, in particular Mycobacteria. All this is consistent with the proposition that a mobile sequence that confers OP hydrolytic activity has been e¡ectively selected for in many microbial communities in agricultural soils over the last 50 years. One apparent anomaly in this respect is the 10% amino acid sequence divergence between OPD and OpdA. At least in the absence of diversifying selection such a level of divergence would normally require much longer periods of time to accumulate. We note, however, that OPD and OpdA di¡er signi¢cantly in their activities against the various classes of OP insecticides [2], so it may be that their divergence has been driven by rapid, recent diversifying selection due to di¡erent OP insecticides. Interestingly, many more distantly related members of the much larger amidohydrolase multigene family containing OPD/OpdA are involved in the catabolism of nitrogencontaining compounds, for example urease and hydantoinase [31]. Indeed a nitrogen-regulated promoter was identi¢ed upstream of opd [32] and appeared to be retained in opdA (our unpublished data). We have tested OpdA for urease and hydantoinase activities using the assays of Kaltwasser and Schlegel [33] and Abendroth et al. [34]. In no case were we able to recover detectable activity, although we clearly cannot preclude the possibility of other nitrogen-related hydrolytic activities. Many insertion sequences have been identi¢ed during bacterial genome sequencing projects. For example, phage, transposon and plasmid insertions represent 2% of the 4.6-Mb E. coli chromosome [7]. Clearly, these mobile elements can be responsible for moving DNA fragments around [35]. The data herein suggest another case in which there has been rapid adaptive transfer across phylogenetically disparate bacterial species.

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12] [13]

[14]

[15]

Acknowledgements [16]

We wish to thank Michelle Williams for technical assistance and Associate Professor John Pemberton (University of Queensland) for providing pJP5603. This research was supported by Orica Australia Ltd and Horticulture Australia Ltd.

[17]

[18]

References [1] Chaudhry, G.R., Ali, A.N. and Wheeler, W.B. (1988) Isolation of a methyl parathion degrading Pseudomonas sp. that possesses DNA

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