A transposable class I composite transposon carrying mph (methyl parathion hydrolase) from Pseudomonas sp. strain WBC-3

R E S E A R C H L E T T E R

Atransposable class I composite transposon carryingmph (methylparathion hydrolase) fromPseudomonas sp. strainWBC-3Min Wei1,2, Jun-Jie Zhang1, Hong Liu1, Shu-Jun Wang1, He Fu1,2 & Ning-Yi Zhou1

1State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China; and 2Graduate School, Chinese Academy

of Sciences, Beijing, China

Correspondence: Ning-Yi Zhou, State Key

Laboratory of Virology, Wuhan Institute of

Virology, Chinese Academy of Sciences,

Wuhan 430071, China. Tel.: 186 27

87197655; fax: 186 27 87197655; e-mail:

[email protected]

Received 16 October 2008; accepted 5

December 2008.

First published online 7 January 2009.

DOI:10.1111/j.1574-6968.2008.01468.x

Editor: Hans-Peter Kohler

Keywords

IS6100 ; methyl parathion hydrolase;

Pseudomonas ; transposon.

Abstract

Pseudomonas sp. strain WBC-3 utilizes methyl parathion (O,O-dimethyl O-p-

nitrophenol phosphorothioate) or para-nitrophenol as the sole source of carbon,

nitrogen and energy. A gene encoding methyl parathion hydrolase (MPH) had

been characterized previously and found to be located on a typical class I

composite transposon that comprised IS6100 (Tnmph). In this study, the

transposability of this transposon was confirmed by transposition assays in two

distinct mating-out systems. Tnmph was demonstrated to transpose efficiently in a

random manner in Pseudomonas putida PaW340 by Southern blot and in Ralstonia

sp. U2 by sequence analysis of the Tnmph insertion sites, both exhibiting MPH

activity. The linkage of the mph-like gene with IS6100, together with the

transposability of Tnmph, as well as its capability to transpose in other phylogen-

etically divergent bacterial species, suggest that Tnmph may contribute to the wide

distribution of mph-like genes and the adaptation of bacteria to organopho-

sphorus compounds.

Introduction

Two types of organophosphorus degradation genes have

been identified in a number of bacterial strains isolated from

diverse geographical regions. The organophosphate degra-

dation gene (opd) was first identified in Flavobacterium sp.

ATCC27551 (Mulbry & Karns, 1989) and Pseudomonas

diminuta MG (Serdar et al., 1989), which were isolated from

the Philippines and Texas, respectively, in the late 1980s.

Recently, a methyl parathion (O,O-dimethyl O-p-nitrophe-

nol phosphorothioate) degradation gene (mph or mpd) was

reported in the Pseudomonas sp. strain WBC-3 (Liu et al.,

2005) and Plesiomonas sp. strain M6 (Zhongli et al., 2001)

isolated in China, but it showed only 12% identity to OPD

at the amino acid level (Liu et al., 2005). Subsequently, mph-

like genes have also been identified in seven other organo-

phosphorus pesticide-degrading strains of four different

genera at different locations in China (Zhang et al., 2006).

It has been demonstrated that bacterial genes for the

degradation of various xenobiotic compounds were often

located on transposable elements, resulting in the wide

distribution and rapid evolution of common catabolic path-

ways for microorganisms in the environment (Tan, 1999;

Top & Springael, 2003). opd was found to be flanked by one

copy of ISFlsp1 (a member of the IS21 family) and one copy

of a Tn3-like element encoding a transposase and a resolvase

in Flavobacterium sp., but no transposition events were

detected in a transposition study (Siddavattam et al., 2003).

However, because transposition is a complicated process,

this might be due to some unknown factors such as

nutritional or environmental conditions, or other genetic

factors present only in the natural host (Siddavattam et al.,

2003). opdA of Agrobacterium radiobacter P230 (Horne

et al., 2002), which shares 88% identity with opd at the

nucleotide sequence level, was located within an insertion

sequence derivative (TnopdA), whose transposase gene and

inverted repeat sequences are identical to those of IS6100

(Horne et al., 2003). It was demonstrated to be transposable

by an assay using an artificial transposable element with

disruption of the opdA gene (Horne et al., 2003), but no

attempt was made to investigate whether it jumped into the

recipients’ genome in a random manner.

Of all the strains that contain either type of organopho-

sphorus degradation genes, Pseudomonas sp. strain WBC-3

is the only one that is capable of utilizing methyl parathion

or para-nitrophenol as the sole source of carbon, nitrogen

and energy. The gene encoding methyl parathion hydrolase

(MPH, E.C.3.1.8.1), which catalyzes the hydrolyzation of

FEMS Microbiol Lett 292 (2009) 85–91 c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

methyl parathion to para-nitrophenol, was functionally

expressed (Liu et al., 2005), and the three-dimensional

structure of this enzyme was also resolved (Dong et al.,

2005). mph was localized to a typical class I composite

transposon (Tnmph), flanked by two isocopies of IS6100 at

each end in the same direction (Liu et al., 2005) (GenBank

accession no. AY251554), as shown in Fig. 1. In this study,

we report the transposability of this transposon and eluci-

date its potential role in horizontal gene transfer during the

evolution of microbial OPD.

Materials and methods

Bacterial strains, plasmids, and cultureconditions

The bacterial strains and plasmids used in this study are

listed in Table 1. Escherichia coli was routinely cultivated at

37 1C in Luria–Bertani (LB) media or on LB plates solidified

with 1.5% agar. Pseudomonas and Ralstonia strains were

cultured at 30 1C.

Streptomycin (1 mg mL�1 for selection of Pseudomonas

putida PaW340 or 100mg mL�1 for E. coli HB101), ampicillin

(100mg mL�1 for selection of Ralstonia sp. U2), tetracycline

(20mg mL�1), kanamycin (45mg mL�1), chloramphenicol

(34mg mL�1) or trimethoprim (50mg mL�1) was added to

the growth media, when necessary.

DNA manipulation and construction of plasmids

Established protocols were used for plasmid DNA prepara-

tion and manipulation (Sambrook et al., 1989). Genomic

DNA was isolated from strain PaW340 using the previously

described procedures (Ausubel, 2001). A 6.5 kb KpnI/Bam-

HI fragment carrying the complete Tnmph (Liu et al., 2005)

from strain WBC-3 was cloned into pEX18Tc to produce

pZWWM001. A kanamycin resistance gene (nptII), as a

selective marker, was amplified from plasposon pTnMod-

Okm (Dennis & Zylstra, 1998) and subsequently inserted

into the NotI site immediately downstream of mph in

pZWWM001 to produce pZWWM002, leaving the mph

gene intact. The physical map of pZWWM002 is illustrated

in Fig. 1. The KpnI/BamHI fragment of pZWWM002

carrying Tnmph with the nptII insertion was cloned into

pSTV29 to produce pZWWM003.

Transposition assay

The transposition of Tnmph in pZWWM002 was assayed by

biparental mating, as described previously (Williams et al.,

2002). Escherichia coli S17-1 (Simon et al., 1983) harboring

Fig. 1. Replicative transposition of Tnmph from

pZWWM002. Transposase-mediated fusion of

donor and target molecules generates a third

copy of IS6100 in the same orientation as the

original pair. The two possible results of trans-

position, depending on which IS6100 copy is

duplicated, are shown below the physical map

of pZWWM002. The solid and dotted lines in

pZWWM002 are, respectively, representative of

a 458-bp internal IS6100 fragment used as the

IS6100 probe (probe 1) and a 559-bp internal

fragment of sacB as the pEX18Tc probe (probe

2). In hybridization with the IS6100 probe, three

hybridized bands are expected: the 3.3 and

1.2-kb bands identical to those in the plasmid

pZWWM002 and a third one of variable size in

each independent transconjugant. In the case of

pEX18Tc probe, an 8.6-kb band was detected,

corresponding to that illustrated in Fig. 2b and d.

Primers P1, P2, P3 and P4 used for genome

walking are also indicated. mph, methyl para-

thion hydrolase gene; sacB, levansucrase gene;

tet, tetracycline resistant gene; nptII, kanamycin

resistant gene; P, PstI; K, KpnI; B, BamHI.

FEMS Microbiol Lett 292 (2009) 85–91c� 2009 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

86 M. Wei et al.

pZWWM002 was used as the donor strain and the plasmid-

free strain PaW340 (Jeenes & Williams, 1982), in which

pEX18Tc or its derivatives do not replicate, was used as the

recipient strain. The resulting transconjugants were screened

on LB plates containing 1 mg mL�1 streptomycin (selection

for strain PaW340) and 45 mg mL�1 kanamycin (selection for

Tnmph with nptII). The frequency of conjugation plus

transposition was calculated as the ratio of transconjugants

to recipients.

The transposition of a Tnmph derivative (kanamycin

resistant, Kmr) was also examined using a mating-out

experiment, as described previously (Tsuda & Iino, 1987;

Sota et al., 2002). For this purpose, E. coli DH5a harboring

pZWWM003 and plasmid R388 (trimethoprim resistant,

Tpr) (Ward & Grinsted, 1982), a conjugal plasmid free of

transposon, was used as the donor to mate with E. coli

HB101 (streptomycin resistant, Smr) on a membrane filter.

The transposition frequency was expressed as the number of

KmrSmr transconjugants per Tpr Smr transconjugants and

the plasmids in the transconjugants were further character-

ized by restriction digestion.

Conjugation assays were also performed using Ralstonia

sp. strain U2 (Zhou et al., 2001), as the recipient. Constructs

based on pEX18Tc were confirmed not to replicate in U2

before this conjugation. The resulting transconjugants were

screened on LB plates containing 100 mg mL�1 ampicillin

(selection for strain U2) and 45mg mL�1 kanamycin (selec-

tion for Tnmph with nptII).

Southern hybridization analysis

Genomic DNA from the PaW340 strain and transconjugants

was digested with PstI and subjected to 0.7% agarose gel

electrophoresis before the separated DNA fragments were

transferred from agarose gels to a positively charged nylon

membrane (Boehringer Mannheim, Germany). DNA probes

were prepared by PCR amplification with pZWWM002 as

the template, and hybridization was performed using the

DIG High Prime DNA Labeling and Detection Starter Kit I

(Roche Diagnostics, Germany) according to the manufac-

turer’s instructions.

Analysis of Tnmph insertion sites

Regions flanking Tnmph insertion sites were obtained by the

method of genome walking (Siebert et al., 1995). As

illustrated in Fig. 1, there are two theoretically possible

results of transposition, and so two groups of walking

primers are needed. To avoid unspecific amplification,

primers were not IS6100 specific, but were immediately

upstream or downstream of IS6100 (as indicated in Fig. 1),

depending on which direction of genome walking was

conducted, and the resulting fragment would contain both

IS6100 and its flanking section. DNA sequences were

determined by Shanghai Sangon Biological Engineering

Technology & Services Co. Ltd (Shanghai, China).

Results and discussion

Tnmph transposition onto the chromosome ofstrains PaW340 and U2 from a suicide plasmid

A transposition frequency of 10�5 was observed after bipar-

ental mating between the donor E. coli S17-1 [pZWWM002]

carrying Tnmph with a Kmr cassette inserted and the

recipient P. putida PaW340. It seemed far higher than that

of the recombination of the plasmid with the recipient’s

Table 1. Strains or plasmids used in this study

Strain or plasmid Genotype or phenotype Reference or source

Strains

E. coli S17-1 TprSmr recA thi pro hsdR M1 RP4::2-Tc::Mu::Km Tn7 lpir lysogen Simon et al. (1983)

E. coli HB101 hsdS20 recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 supE44 Ausubel et al. (1994)

E. coli DH5a F – recA1 gyrA96 thi-1 hsrdR17 supE44 relA1 deoRD(lacZYA-argF)U169 Woodcock et al. (1989)

Pseudomonas sp. WBC-3 p-Nitrophenol and methyl parathion utilizer, wild type Liu et al. (2005)

Pseudomonas putida PaW340 Mxy�Mtol� Strr Trp�MP� Williams & Murray (1974)

Ralstonia sp. strain U2 Naphthalene utilizer, AmprTcsKms Zhou et al. (2001)

Plasmids

pEX18Tc Tcr; oriT1 sacB1 gene replacement vector with MCS rom pUC18 Hoang et al. (1998)

R388 Tra1 Tpr Sur Ward & Grinsted (1982)

pSTV29 Cmr; cloning vector TaKaRa

pTnMod-Okm Kmr, source of neomycin phosphotransferase II gene (npt II) Dennis & Zylstra (1998)

pZWWM001 Tcr; pEX18Tc derivative carrying 6.5-kb KpnI and BamHI fragment

with the complete Tnmph

This study

pZWWM002 TcrKmr; pZWWM001 derivative carrying Kmr determinant at

unique NotI site immediately downstream of mph

This study

pZWWM003 CmrKmr; pSTV29 derivative carrying the KpnI/BamHI

fragment of pZWWM002

This study


87Methyl parathion catabolic transposon

genome, which was found to be lower than 10�8 in the

controls. This indicated that the kanamycin resistance of the

transconjugants resulted from transposition rather than

other recombination events.

Of the 500 Kmr transconjugants selected, all showed

resistance to tetracycline (Tcr), suggesting that pZWWM002

carrying Tnmph may have been entirely integrated into the

chromosome of the PaW340 strain (as illustrated in Fig. 1).

This was further confirmed by colony PCR to detect the

presence of mph, nptII (markers for Tnmph) and tet (tetra-

cycline-resistant gene, marker for pEX18Tc) genes in the

transconjugants obtained. Positive results were obtained in

each case among 20 transconjugants tested, indicating the

consistency between their genotypes and phenotypes. In

addition, all these transconjugants exhibited MPH activity,

as evidenced by the conversion of methyl parathion to

yellow-colored para-nitrophenol by these transconjugants.

The profiles of antibiotic resistance, diagnostic PCR analysis

and the presence of MPH activity indicated that cointegrate

molecules had formed between the donor plasmid

pZWWM002 and the target genomic DNA of the PaW340

strain.

Similar results have also been obtained for the U2 strain,

with a transposition frequency of 10�7. This is much higher

than that in the control, in which the frequency for the

recombination between the plasmid and the recipient’s

genome was found to be lower than 10�9.

Tnmph transposes in a random manner

To discriminate between SmrTcrKmr transconjugants either

as a result of transposition events or because of other

recombination events leading to plasmid integration, and

to whether the transposition occurred randomly, Southern

hybridization was performed on the PstI-digested genomic

DNA of transconjugants that were randomly selected from

the PCR diagnostically tested ones. This was conducted

twice in parallel, each with 10 transconjugants from an

independent conjugation experiment, using an internal

fragment of IS6100 or sacB as the probe (Fig. 2). Neither

probe hybridized to DNA from the control PaW340 strain

(lane 2 in Fig. 2a–d). In contrast, at least one extra fragment

with variable sizes was identified in 16 of the 20 transconju-

gants with the IS6100 probe (lanes 3–9 and 12 in Fig. 2a;

lanes 4–9, 11 and 12 in Fig. 2c), in addition to the two

original IS6100 fragments in pZWWM002 (lane 1 in Fig. 2a

and c). The sizes of the variable IS6100-hybridized frag-

ments differed between the transconjugants, indicating that

Fig. 2. Southern blot hybridization analysis of

the end products of Tnmph transposition in

Pseudomonas putida PaW340. Genomic DNA

(4 mg) was digested with PstI. After electrophor-

esis on a 0.7% agarose gel, the DNA was

transferred to a positively charged nylon mem-

brane and hybridized with DIG-labeled probes of

IS6100 (a and c) and sacB (b and d) (as described

in Fig. 1) at a hybridization temperature of 63

and 54 1C, respectively. Two independent South-

ern blots (a, b; c, d) were performed and in both

cases: lane 1, pZWWM002; lane 2, DNA from

strain PaW340; lanes 3–12, independent

SmrTcrKmr transconjugants.


88 M. Wei et al.

independent events had occurred at different genomic sites.

It also implied that there was no hot spot for the target site

of Tnmph, which was consistent with the characteristics of

IS6100-related transposable elements (Herron et al., 1999).

The hybridization with the sacB gene probe further con-

firmed the formation of a cointegrate of pZWWM002 in all

20 transconjugants, in 15 of which a single fragment

identical to the positive control in the PaW340 strain was

hybridized (lanes 3–12 in Fig. 2b; lanes 3, 4, 6, 10 and 11 in

Fig. 2d). For the other five samples, fragments shorter than

the control were observed (lanes 5, 7– 9 and 12 in Fig. 2d).

Interestingly, both phenomena were observed in one trans-

conjugant, with one band identical to the control and one

shorter than the control (lane 5 in Fig. 2d), indicating that

the recipients’ genome is in a dynamic state. This might be

due to some unknown genome rearrangements such as

deletion, recombination or reversion as a result of the activity

of IS6100. This unstable state of the recipients’ genome was

also found in our previous repetition of southern blot with

the transconjugants used in Fig. 2a and b (data not shown).

To detect the random transposition of Tnmph in Ralsto-

nia sp. U2 transconjugants (AmprTcrKmr), sequencing of

Tnmph insertion sites other than Southern blot was con-

ducted from seven transconjugants, using transposon-origi-

nated sequences as primers, as described in Materials and

methods. Sequence analysis could not only further confirm

the randomness of transposition but could also investigate

whether transposition occurred at a particular hotspot. The

result of insertion site sequencing revealed seven different

direct repeats flanking the insertion sites, as indicated in

Table 2.

These data indicate that the transconjugants were a result

of transposition, but not due to other recombination events

leading to plasmid integration. No transposition hotspot

could be detected, suggesting that this kind of transposition

occurred randomly. It is notable that in most transconju-

gants (six out of seven cases), an 8-bp direct repeat was

produced after transposition, which is consistent with the

characteristics of the IS6 family of insertion sequences, as

described in the ISfinder database (http://www-is.biotoul.fr)

(Siguier et al., 2006).

Observation of second transpositions

Second transpositions of IS6100 may have also occurred on

the chromosome, as suggested by the presence of more than

one extra copy of IS6100 detected in four transconjugants

(lanes 8, 9 and 12 in Fig. 2a; lane 4 in Fig. 2c). This is similar

to that seen in the study of the composite transposon DEH,

in which ISPpu12 transposed independently from DEH with

multiple insertions (Weightman et al., 2002; Williams et al.,

2002). Meanwhile, different hybridization patterns with

IS6100 for the same transconjugants were observed in

several cases during their propagation (data not shown),

implying its capability for second transposition. However,

only two copies of IS6100 were detected in four transconju-

gants (lanes 10 and 11 in Fig. 2a; lanes 3 and 10 in Fig. 2c).

This may be because either pZWWM002 had integrated into

the chromosome by abnormal recombination, or deletions

may have occurred after cointegrate formation, as proposed

in a study that used Tn610 (Martin et al., 1990).

Transposition of Tnmph from pSTV29 onto R388in a mating-out experiment

We also confirmed the Tnmph transposability using a well-

developed mating-out experiment (Tsuda & Iino, 1987; Sota

et al., 2002), although it does not identify whether random

insertions occurred during transposition. In this experi-

ment, plasmid pZWWM003, a pSTV29 derivative carrying

Tnmph inserted with the Kmr determinant, was constructed

and then used for the transposition assay. Conjugation

between the donor strain E. coli DH5a-containing plasmids

R388 and pZWWM003 and the recipient strain E. coli

HB101 yielded Kmr/Smr transconjugants at a transposition

frequency of 10�6 per R388 transfer. MPH activity was also

evident in all 10 randomly selected KmrSmr transconju-

gants. Subsequent restriction analyses revealed that each of

the transconjugants carried a plasmid of the same size,

which was presumably due to the formation of a cointegrate

between R388 and pZWWM003 in all cases.

Notably, Tnmph did not resolve from the target sites

during the transposition process after formation of the

cointegrate in either transposition assay. This is because the

Table 2. DNA sequences flanking the insertion sites after the random transposition of Tnmph in Ralstonia sp. U2 transconjugants

Transconjugant Sequence of insertion sites (50–30)

U2-01 GACCCGGCCGTGCTGGCGCAGGCCATCGACCGGGA-insertion site-GACCGGGATACCGGCGCGATCTTCCACCTGGCCGC

U2-02 GCGCGGCCATGGCGGTTTCGCCAGATTCGCTGGTC-insertion site-CGCTGGTCGATGCATGCTGGGCTTCGTCGATCATCA

U2-03 CGACAAGTACGCCCGGCTGCTCGCACGTTGCGAGG-insertion site-CGTTGCGAGGGCCTTCCGCCCATCCCGACCGCCGT

U2-04 AGGGTGTCGACGAGAACGCGCTGACCGTGCCGCAG-insertion site-TGCCGCAGCGCGCGCTGATCCGCAGCGCCCAGGCG

U2-05 GCCGGCACGCAACGACGACAGCCAGATCTATAACG-insertion site-CTATAACGAATATGTGTCGATGCAGTTGGCCCAGG

U2-06 GGACGTGCCGGTGGATGGCTTCTGGTCGGTCAGCG-insertion site-GGTCAGCGTCTACAACGCCAAGGGCTACTTCGAGA

U2-07 GTCAAGGAGTACGTGGTGTCGATCAAGGGCCCGTT-insertion site-GGCCCGTTGACCACGCCGGTTGGTGGCGGCATCCT

Underlined letters indicate the direct repeat produced after transposition.



IS6 family, to which IS6100 belongs, was distinguished by

the formation of a cointegrate molecule as the end point of

transposition, with the integration of the donor vector plus

one additional copy of the IS6100 sequence (Fig. 1) (Martin

et al., 1990; Guilhot et al., 1994; Smith & Dyson, 1995).

In contrast, in the two other functional class I transposons

identified, namely chlorobenzene dioxygenase transposon

(Tn5280), which comprised IS1066 (van der Meer et al.,

1991), and hydrolytic dehalogenases transposon DEH,

which comprised ISPpu12 (Weightman et al., 2002;

Williams et al., 2002), half the transconjugants acquired

the transposon without the integration of the full donor

plasmids.

Tnmph is structurally different from the opd or the opdA

transposable element, implying that diverse ways may have

been adopted in the evolution of these catabolic transposons

during the bacterial adaptation to organophosphorus com-

pounds. Interestingly, mph and opdA, two phylogenetically

divergent genes, are associated with similar transposable

elements, and in contrast, opd and opdA, two similar genes,

are associated with different transposons. This is yet another

example showing the divergent nature of recruitment

events, which bacteria have used to adapt themselves to

newly produced environmental contaminants.

In addition to the current case reported here, an mph-like

gene was also found in phylogenetically divergent bacterial

species and all were located in an identical Tnmph-like

organization (Zhongli et al., 2001; Liu et al., 2005; Zhang

et al., 2006). The linkage of mph to IS6100, their wide

dissemination in various phylogenetically divergent bacter-

ial species, as well as the transposability in a heterologous

host of Tnmph in this study, all suggest that Tnmph might

have played a role in horizontal gene transfer in bacterial

adaptation to organophosphorus compounds. If this is the

case, it is likely that strain WBC-3 has evolved from a para-

nitrophenol utilizer by acquiring the mph transposon

through a natural patchwork assembly, resulting in a com-

plete catabolic pathway for methyl parathion.

Acknowledgements

This work was supported by the National Natural Science

Foundation of China (grant no. 30570021), the National

High Technology Research and Development Program of

China (grant no. 2006AA10Z403) and the Knowledge In-

novation Program of the Chinese Academy of Sciences

(grant no. KSCX2-YW-G-009). We are also grateful to Dr

Masahiro Sota for supplying plasmids R388 and pSTV29.

Authors’contribution

M.W. and J.-J.Z. have contributed equally to this work.

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A transposable class I composite transposon carrying mph (methyl parathion hydrolase) from Pseudomonas sp. strain WBC-3