Nucleotide Sequence and Genetic Structure of a Novel Carbaryl Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100

Abstract

Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine.
Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass (82 kDa), and
enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and 4-nitrophenyl acetate indicating
that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene (cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region containing cehA. No homologous sequences were found by a database homology search using the nucleotide and deduced amino acid sequences of
the cehA gene. Six open reading frames including the cehA gene were found in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion sequence-like sequence, suggesting that it makes part of a composite transposon.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2002, p. 1220–1227 Vol. 68, No. 3
0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.3.1220–1227.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Nucleotide Sequence and Genetic Structure of a Novel Carbaryl
Hydrolase Gene (cehA) from Rhizobium sp. Strain AC100
Masayuki Hashimoto,
1
Mitsuru Fukui,
2
Kouichi Hayano,
2
and Masahito Hayatsu
2
*
Bio-Oriented Technology Research Advancement Institution, Minatoku, Tokyo 105-0001,
1
and
Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529,
2
Japan
Received 27 August 2001/Accepted 6 December 2001
Rhizobium sp. strain AC100, which is capable of degrading carbaryl (1-naphthyl-N-methylcarbamate), was
isolated from soil treated with carbaryl. This bacterium hydrolyzed carbaryl to 1-naphthol and methylamine.
Carbaryl hydrolase from the strain was purified to homogeneity, and its N-terminal sequence, molecular mass
(82 kDa), and enzymatic properties were determined. The purified enzyme hydrolyzed 1-naphthyl acetate and
4-nitrophenyl acetate indicating that the enzyme is an esterase. We then cloned the carbaryl hydrolase gene
(cehA) from the plasmid DNA of the strain and determined the nucleotide sequence of the 10-kb region
containing cehA. No homologous sequences were found by a database homology search using the nucleotide and
deduced amino acid sequences of the cehA gene. Six open reading frames including the cehA gene were found
in the 10-kb region, and sequencing analysis shows that the cehA gene is flanked by two copies of insertion
sequence-like sequence, suggesting that it makes part of a composite transposon.
Carbamate insecticides such as carbaryl (1-naphthyl N-methyl-
carbamate) are broad-spectrum insecticides that comprise the
major proportion of agricultural pesticides used in today’s ag-
ricultural industry. These compounds are considered hazard-
ous because they potently inhibit acetylcholine esterase (9) and
the N-nitrosocarbamates formed are potent mutagens (8). On
the other hand, these pesticides generally do not persist in soil
for a long time, and the persistence of these compounds in
agricultural soil is due to repeated applications (10, 38). From
these perspectives, an understanding of the degradation mech-
anism is needed to control the persistence of these pesticides in
soil.
Soil microorganisms are thought to play a significant role in
the reduction of pesticides in soil, and many soil bacteria ca-
pable of degrading carbamate pesticides have been isolated
and characterized (4, 6, 16, 17, 21, 28). The biochemical char-
acteristics of carbamate pesticide hydrolases have also been
reported (5, 17, 18, 20, 25). However, little is known about the
genes for these enzymes. The mcd gene, which encodes a
carbofuran hydrolase, is located on a 100-kb plasmid called
pPDL11, and it has been cloned from Achromobacter sp. strain
WM111 (41). This gene was shown to be present in many
bacteria and to be encoded on a 100-, 105-, 115-, or 124-kb
plasmid found in diverse bacteria isolated from geographically
distant areas (6, 29). However, the structure of the carbamate
insecticide degradative gene has not as yet been reported,
although the nucleotide sequence of mcd gene is available
(accession no. AF160188).
The present study characterizes a carbaryl hydrolase purified
from Rhizobium sp. strain AC100. From the result of a plas-
mid-curing experiment, it was suggested that the carbamate
insecticide degradative gene is encoded on the plasmid. We
then cloned the degradative gene from the plasmid DNA and
determined the nucleotide sequence of the 10-kb region con-
taining the degradative gene. The sequence analysis suggested
that the genes in the sequenced region likely comprise a com-
posite transposon.
MATERIALS AND METHODS
Isolation and culture conditions. The minimal medium with carbaryl (MMC
medium) contained the following constituents (in grams per liter of 20 mM
potassium phosphate buffer [pH 7.0]): (NH
4
)
2
SO
4
, 1; NaCl, 0.2; MgSO
4
·7H
2
O,
0.1; CaCl
2
·H
2
O, 0.05; FeSO
4
, 0.02; ZnSO
4
, 0.002; yeast extract, 0.05; and
carbaryl, 0.1. Glucose (0.2%), Bacto Tryptone (0.4%), and yeast extract (0.2%)
supplemented with MMC medium instead of carbaryl were used as MMGTY
medium. Carbaryl-treated soil collected from agricultural fields with a history of
carbaryl application was inoculated into MMC medium, and then carbaryl-
degrading bacteria were enriched by repeating subcultivations. Appropriate di-
lutions of the enrichment culture were placed on MMC agar plates and incu-
bated at 30°C for a few days. We selected a colony that hydrolyzed carbaryl to
1-naphthol by spraying the colony with 0.5 mM carbaryl and 0.01% fast blue B
salt dissolved in sterilized distilled water (17). Colonies positive for carbaryl
hydrolysis quickly turned brown. Spontaneous rifampin-resistant (Rif
r
) and
streptomycin-resistant (Str
r
) mutants of strain AC100 were isolated by plating
MMGTY medium-grown cells on 1/2 nutrient agar supplemented with 25 ␮gof
rifampin or streptomycin per ml (16). Bacterial strains and plasmids used are
shown in Table 1.
Taxonomic identification. The strain was identified on the basis of classifica-
tion schemes published in Bergey’s Manual of Systematic Bacteriology (19). The
G⫹C content of the bacterial DNA was determined as described by Tamaoka
and Komagata (39). The quinone type was detected by high-performance liquid
chromatography (HPLC) using reference standards. The 16S rRNA gene was
amplified by PCR, and the nucleotide sequences of the PCR products purified
were determined as described (16). Computer analysis was performed using
DDBJ software. The sequence of the 16S rRNA of the strain was compared with
other published 16S rRNA sequences using the BLAST search option of the
DDBJ database to determine the closest phylogenetic neighbors.
Carbaryl hydrolase activity. Carbaryl hydrolase activity was assayed in a re-
action mixture containing 0.5 mM carbaryl, 50 mM sodium phosphate buffer (pH
7.0), and enzyme solution in a total volume of 1.0 ml. The reaction was started
by adding the enzyme and incubated at 30°C. The reaction was stopped by adding
100 ␮l of 2 mM HgCl
2
, and the amount of 1-naphthol formed was quantified as
described (17). One unit of enzyme activity was defined as the amount of enzyme
that produced 1 ␮mol of 1-naphthol per min at 30°C. In studies on the effects of
pH and temperature, carbaryl hydrolase activity was assayed with metolcarb
instead of carbaryl because carbaryl is unstable in alkaline solutions and at high
temperatures. To examine pH stability, the enzyme was incubated in buffers at
* Corresponding author: Mailing address: Faculty of Agriculture,
Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan. Phone and
fax: 81-54-238-4875. E-mail: ahmhaya@agr.shizuoka.ac.jp.
1220

various pHs for 14 h at 4°C, and the remaining activity was determined at 30°C.
For determination of the effects of metal ions and chemical reagents on the
enzyme activity, the purified enzyme was preincubated with 1.0 mM of various
compounds or metal ions in 1.0 ml of 50 mM sodium phosphate buffer (pH 7.0)
for 20 min at 30°C, and the remaining activity was determined at 30°C.
Enzyme purification. Shaking flasks containing 1/2 nutrient broth medium
were inoculated with a late-logarithmic-stage culture in the same medium and
incubated at 30°C on a reciprocal shaker. When the culture reached a late-
logarithmic stage, the cells were harvested by centrifugation at 10,000 ⫻gfor 20
min at 4°C. The cell pellets were washed with 50 mM sodium phosphate buffer
(pH 7.0). All operations were performed at 0 to 4°C, and sodium phosphate
buffer (pH 7.0) (P buffer) was used unless otherwise mentioned. Cells resus-
pended in the same buffer were disrupted by sonication, and the cell debris was
removed by centrifugation at 10,000 ⫻gfor 30 min. Protamine sulfate (2%) was
added to the supernatant to a final concentration of 0.2 mg per mg of protein.
After 15 min, the mixture was centrifuged at 10,000 ⫻gfor 30 min, and the
precipitate was discarded. Solid ammonium sulfate was added to the supernatant
to 25% saturation. After 30 min of stirring, the mixture was centrifuged at 10,000
⫻gfor 15 min, and the precipitate was discarded. The supernatant was brought
to 50% saturation with solid ammonium sulfate. After 30 min of stirring, the
precipitate was collected by centrifugation at 10,000 ⫻gfor 30 min. The pre-
cipitate was dissolved in 50 mM P buffer and dialyzed against 20 mM Tris-HCl
buffer (pH 8.0). The dialysate was applied to a column of Q Sepharose (pre-
packed HiTrap HP). The column was washed with 3 bed volumes of the same
buffer and eluted with a linear gradient of 200 ml of NaCl (0 to 300 mM) in the
same buffer. Active fractions were dialyzed against 20 mM P buffer (pH 7.0). The
dialysate was applied to a column of SP Sepharose (prepacked HiTrap HP). The
column was washed with 3 bed volumes of the same buffer and eluted with a
linear gradient of 200 ml of NaCl (0 to 500 mM) in the same buffer. The active
fractions were collected and concentrated to 6 ml using an ultrafiltration cell
(model 8050; Millipore, Bedford, Mass.) with a membrane molecular weight
cutoff of 10,000. The concentrated fractions were applied to a column of
Sephacryl S-200 HR (26/60) with 50 mM P buffer (pH 7.0). All column chroma-
tography was performed using ÄKTA explorer 10S (Pharmacia Biotech). The
protein was eluted with the same buffer, and the active fractions were dialyzed
against 50 mM sodium phosphate buffer (pH 7.0) containing 50% (vol/vol)
glycerol. The enzyme solution was stored at ⫺20°C.
Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed as described by Laemmli (22). The stacking and
resolving gels contained 4.5 and 10% acrylamide, respectively. Proteins in the gel
were stained with Coomassie brilliant blue R-250. Molecular mass standards for
SDS-PAGE were as follows: myosin (205 kDa), ␤-galactosidase (116 kDa),
phosphorylase b(97 kDa), fructose-6-phosphate kinase (84 kDa), bovine serum
albumin (66 kDa), glutamic dehydrogenase (55 kDa), egg albumin (45 kDa), and
glyceraldehyde-3-phosphate dehydrogenase (36 kDa).
Determination of substrate specificity and K
m
.The reaction mixture consisted
of 50 ␮mol of sodium phosphate buffer (pH 7.0), 0.5 ␮mol of substrate, and 0.01
U of the purified enzyme in a total volume of 1.0 ml. After an incubation at 30°C
for 10 min, the reaction was stopped with 1.0 ml of methanol. The enzyme
reaction was linear over time for at least 20 min. Substrate hydrolysis was
measured by HPLC using an octyldecyl silane C
18
column (ODS-UG-3 column;
4.6 by 35 mm; Nomura Kagaku Co., Aichi, Japan). The ability to hydrolyze
4-nitrophenyl acetate was measured by monitoring the A
400
. The K
m
values of
carbaryl hydrolase were determined by Lineweaver-Burk plots. The reaction
mixture consisted of 50 ␮mol of sodium phosphate buffer (pH 7.0), 10 to 200
nmol of substrate, and appropriate amounts of the purified enzyme in a total
volume of 1.0 ml.
Analytical methods. Protein concentrations were determined using the Bio-
Rad DC protein assay kit with bovine serum albumin as the reference standard.
Protein eluted during column chromatography was monitored by measuring the
A
280
. Pesticides and 1-naphthyl acetate were analyzed by HPLC on a C
18
column
with a mobile phase of acetonitrile-distilled water (50:50 [vol/vol]) at a flow rate
of 1.0 ml/min. The A
220
of the column effluent was monitored. The analytical-grade
pesticides carbaryl, xylylcarb (3,4-dimethylphenyl N-methylcarbamate), XMC (3,5-
dimethylphenyl N-methylcarbamate), metolcarb (3-methylphenyl N-methylcarba-
mate), propoxur (2-isopropoxyphenyl N-methylcarbamate), fenobucarb (2-s-butyl-
phenyl N-methylcarbamate), isoprocarb (2-isopropylphenyl N-methylcarbamate),
methylparathion (O,O-dimethyl-O-4-nitrophenylthiophosphate), chloropropham
(isopropyl-3-chlorocarbanilate), and carbofuran (2,3-dihydro-2,2-dimethyl-7-benzo-
furanyl methylcarbamate) and other chemicals were purchased from Wako Pure
Chemical Industries Ltd. (Osaka, Japan). The prepacked columns—HiTrap Q
Sepharose HP, HiTrap SP Sepharose HP, and HiPrep Sephacryl S-200 HR (26/
60)—were obtained from Pharmacia Fine Chemicals, Ltd.
Plasmid preparation and curing. Plasmids from strain AC100 and its deriva-
tive mutants were prepared by a modified alkaline-SDS extraction method (2,
15). For plasmid curing, single colonies of AC100 were inoculated into MMGTY
medium containing mitomycin C (4 ␮g/ml) and shaken at 30°C overnight. A
series of dilutions was made from the overnight culture and spread onto 1/2
nutrient agar plates. After several days of incubation at 30°C, mutants of AC100
unable to degrade carbaryl were selected by spraying the colony with 0.5 mM
carbaryl and 0.01% fast blue B as described above.
Cloning of carbaryl degradative gene(s). Plasmid DNA of strain AC100 was
digested with BamHI, SalI, or SmaI, and the fragments were inserted into a
broad-host-range plasmid pBI101 at the appropriate restriction enzyme sites,
respectively. Escherichia coli HB101 transformed with the recombinant plasmids
and strain AC1012 (Table 1) were used for triparental conjugation as recipient
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristic(s) Source or reference
Strains
E. coli
HB101 supE44 hsdS20 (r
B⫺
m
B⫺
)recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5
mtl-1
Takara, Osaka, Japan
JM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi ⌬(lac-proAB)
F⬘[traD36 proAB
⫹
lacI
q
lacZ⌬M15]Takara, Osaka, Japan
Rhizobium sp.
AC100 Wild type This study
AC101 Car
⫺
This study
AC1012 Car
⫺
Str
r
Rif
r
This study
Plasmids
pRK2013 Km
r
tra
⫹
; triparental mating helper, ColE1 replication 13
pBI101 Km
r
; broad-host-range cloning vector Clontech
pBluescript II SK(⫹) Amp
r
; sequencing vector Stratagene
pCEH-B 23-kb BamHI fragment from pAC200 cloned into pBI101 This study
pCEH-Sa 6.2-kb SalI fragment from pAC200 cloned into pBI101 This study
pCEH-Sm 7.2-kb SmaI fragment from pAC200 cloned into pBI101 This study
pCEH-EVB 3.2-kb EcoRV-BamHI fragment from pCEH-B subcloned into pBI101 This study
pCEH-EVSc 2.7-kb EcoRV-SacII fragment from pCEH-B subcloned into pBI101 This study
VOL. 68, 2002 GENETIC ANALYSIS OF A NOVEL CARBARYL HYDROLASE GENE 1221

and donor, respectively. Triparental matings were performed as described by
Ditta et al. (7). Donor, recipient, and helper (E. coli HB101 harboring pRK2013)
were cultured for 16 h and then washed, mixed, and incubated for 16 h on 1/2
nutrient agar plates. After appropriate dilution, cells collected from the plates
with sterile water were spread onto 1/2 nutrient agar plates containing rifampin
and kanamycin. Carbaryl-degrading transconjugants were selected as described
above.
DNA manipulation. Digestion with restriction enzymes and DNA ligation
proceeded basically as described by Sambrook et al. (33). DNA fragments were
routinely subcloned in the plasmid vector pBluescript II SK(⫹). E. coli HB101 or
JM109 was transformed either by the method of Hanahan (14) or by electropo-
ration using an Electro Cell Manipulator 600 M (BTX, San Diego, Calif.). Total
DNA from strain AC100 was prepared by the method of Marmur (23). Southern
blot hybridization onto nylon membrane (Hybond-N⫹; Amersham, Bucking-
hamshire, United Kingdom) was performed by the capillary blot method (G
capillary blotter; Taitec Co., Japan). Southern hybridization experiments were
performed using a digoxigenin labeling kit (Boehringer, Mannheim, Germany).
Overlapping restriction fragments of pCEH-B or pCEH-Sm were subcloned into
pBluescript II SK(⫹) for sequencing. Nucleotides were sequenced using an
automated laser fluorescence DNA sequencer (model 4000L; LI-COR). PCR
amplification proceeded using Ex Taq DNA polymerase (Takara Biomedicals,
Osaka, Japan) and the following primers: A, 5⬘-CGCGCGAATTCCAGCTTT
GGCCAGACCTATTTCG-3⬘;B,5⬘-ATGGACCTA-CTCAGTGTGATCCGC
C-3⬘;C,5⬘-CGCGGGAATTCATGGACCAACCATTCAAAC-CAGATCG-3⬘;
D, 5⬘-CCCCCGAATTCAAGGCGCAGAAAATAGAAAGCGACC-3⬘;E,5⬘-T
CAAGCGGGTGATTTGTCCTTTCTG-3⬘; and F, 5⬘-GCCGCGAATTCACGT
TAAGT-CGCTTTCGGCGATGATCC-3⬘. Plasmid DNA of strain AC100 and
AC101 was used as templates, and the primer sets are indicated in Fig. 6.
Nucleotide sequence accession numbers. The nucleotide and amino acid se-
quence data reported in this study have been submitted to the nucleotide se-
quence databases under accession numbers AB069723 (Tnceh) and AB069724
(16S ribosomal DNA [rDNA]).
RESULTS
Isolation and identification. The enrichment procedure gen-
erated a pure culture designated AC100 that hydrolyzed car-
baryl to 1-naphthol. The following taxonomic properties of
AC100 were determined: cell shape, motile straight rods or
pleomorphic rods with a single polar flagellum and dimensions
of 0.5 to 0.8 ␮m in length and 0.6 to 2.0 ␮m in width; gram stain
negative; oxidase and catalase positive; nitrate reduction pos-
itive; urease negative; G⫹C content, 56.5 mol% ⫾2.0 mol%;
major quinone type, ubiquinone Q10. About 1,482 bases of 16S
rRNA of AC100 were determined. A phylogenetic tree was
constructed from evolutionary distances by the neighbor-join-
ing method (data not shown). After phylogenetic analysis,
strain AC100 was placed in a cluster making up the genus
Rhizobium. The highest degree of similarity was 98%, which
was obtained with the 16S rRNA gene of Rhizobium legumino-
sarum (DDBJ accession no. D14513) and Rhizobium mongo-
lense (DDBJ accession no. U89820). Based on these observa-
tions, the isolate was designated Rhizobium sp. strain AC100.
Purification and properties of carbaryl hydrolase. The pu-
rification scheme of the carbaryl hydrolase of AC100 is shown
in Table 2. The enzyme was purified about 614-fold, with a
yield of 27% from the cell extract of AC100. The purified
enzyme preparation migrated as a single band on SDS-PAGE.
The molecular mass of the purified enzyme estimated by SDS-
PAGE was 82,000 Da. The estimated molecular mass of the
native protein was 160,000 Da according to gel filtration on
HiPrep Sephacryl S-200 HR (26/60), suggesting that the en-
zyme was a dimer composed of identical subunits. The optimal
pH for carbaryl hydrolase activity was around 9 at 30°C, al-
though the range was broad as it retained over 80% of its
maximal activity between pH values of 6 to 11. The hydrolase
was stable within a pH range of 10 to 11. The optimum tem-
perature for the enzymatic reaction was about 45°C under the
conditions described in Materials and Methods. The enzyme
was stable for 10 min at temperatures up to 30°C, and 50% of
the initial activity was retained at 50°C. The purified enzyme
was incubated with a 1.0 mM concentration of various com-
pounds or metal ions in 1.0 ml of 50 mM potassium phosphate
buffer (pH 7.0) for 20 min at 30°C. Thereafter, the remaining
activity was measured as described in Materials and Methods.
The enzyme activity was completely inhibited by Hg
2⫹
, whereas
iodoacetamide, diisopropylfluorophosphate, and paraoxon had
no effect on the enzyme activity. The enzyme was incubated
with various concentrations of EDTA (0.1 to 10 mM) for 30
min at 30°C. The enzyme activity was not affected by EDTA,
suggesting the absence of a metal ion requirement. The K
m
value obtained for carbaryl as substrate was 62 ␮M. The sub-
strate specificities of the purified carbaryl hydrolase are shown
in Table 3. The specificity of the hydrolyzing activity was broad
with substrates of N-methylcarbamate insecticide. The enzyme
was less reactive with propoxur, fenobucarb, and isoprocarb.
Hydrolase activity was undetectable with the substrates of car-
bofuran and chloropropham. The purified enzyme hydrolyzed
1-naphthyl acetate and 4-nitrophenyl acetate, indicating that
the carbaryl hydrolase is an esterase.
TABLE 2. Purification of the carbaryl hydrolase
Purification step Total activity
(U)
Total protein
(mg)
Sp act
(U/mg)
Purification
(fold) Yield (%)
Crude extract 69.3 1,520 0.05 1.0 100
Protamine treatment 69.4 953 0.07 1.4 100
Ammonium sulfate (25–50%) 72.1 262 0.27 5.6 104
Q Sepharose 114 12.3 9.30 185 165
SP Sepharose 78.8 3.9 20.2 404 114
Gel filtration 18.4 0.6 30.7 614 27
TABLE 3. Substrate specificity of carbaryl hydrolase
Substrate Relative activity
a
(%)
Carbaryl.....................................................................................100
XMC..........................................................................................115
Metolcarb.................................................................................. 54
Xylylcarb.................................................................................... 61
Propoxur.................................................................................... 9
Isoprocarb ................................................................................. 21
Fenobucarb ............................................................................... 11
Carbofuran................................................................................ND
Chloropropham ........................................................................ND
a
Rates of hydrolysis are expressed as percentages of rate observed with car-
baryl. ND, not detectable.
1222 HASHIMOTO ET AL. APPL.ENVIRON.MICROBIOL.

Plasmid curing. The involvement of naturally occurring
plasmids in the degradation of synthetic organic compounds
has been extensively documented (34). Catabolic plasmids are
thought to play an important role in the evolution of pesticide-
degrading ability in microorganisms (4, 34). We therefore exam-
ined the plasmid content of Rhizobium sp. strain AC100. The
AC100 harbored three large plasmids, designated pAC100,
pAC200 and pAC300 (Fig. 1, lane 1). To confirm that the
carbaryl-degrading activity is controlled by these plasmids, we
performed a curing experiment with mitomycin C. When
AC100 was treated with mitomycin C, 0.4% of the cells lost
carbaryl hydrolyzing activity. These mutants unable to hydro-
lyze carbaryl harbored a smaller plasmid of pAC200 which was
termed pAC200d. The mutant was designated AC101 (Fig. 1,
lane 2). These results suggested that the carbaryl hydrolase
gene is encoded on pAC200.
Cloning and sequencing of carbaryl hydrolase gene. To
clone the degradative gene, a library of genes in plasmid DNA
from strain AC100 was constructed in E. coli HB101 by use of
broad-host-range plasmid pBI101. The plasmid DNA library
was mobilized from E. coli into Rhizobium sp. strain AC1012
by triparental mating. The transconjugants that complemented
the carbaryl-degrading ability were selected by a fast blue so-
lution spraying technique. Three positive clones that contained
a 23-kb BamHI fragment, a 6.2-kb SalI fragment, and a 7.2-kb
SmaI fragment of plasmid DNA from strain AC100 were fur-
ther analyzed. The activity of the clone containing the SmaI
fragment was very low. To determine the location of the deg-
radative gene, 3.2-kb EcoRV-BamHI and 2.7-kb EcoRV-SacII
fragments from pCEH-Sa were subcloned into pBI101, and the
resulting plasmids were mobilized into AC1012. The activity of
each transconjugant is shown in Fig. 2b. The results showed
that the gene for degradation of carbaryl is located in the
3.2-kb EcoRV-BamHI fragment. On the other hand, a com-
parison of the restriction maps of the cloned fragments showed
that the 23-kb large BamHI fragment contained most of the
region of pAC200 and that the SmaI fragment included the
remainder. Thus, we constructed a physical map of pAC200
(Fig. 2a).
The nucleotide sequence of the 3.2-kb EcoRV-BamHI frag-
ment was determined on both strands. This region contained a
large open reading frame (ORF) of 2,382-bp that encoded 794
amino acids. TTG was the estimated initiation codon of the
ORF, and it was preceded by the putative ribosome-binding
site AGGAAGG. The N-terminal amino acid sequence of pu-
rified carbaryl esterase (STDAIEPQPYFA) determined by au-
tomated Edman degradation was identical to that deduced
from the nucleotide sequence starting from Ser-30, indicating
that this gene encodes the carbaryl hydrolase. The gene was
designated cehA. The amino acid sequence from the putative
start codon to the N-terminal of the mature protein showed the
characteristic features of a signal peptide. The calculated mo-
lecular weight of the mature protein deduced from the DNA
sequence was 84,100, which was consistent with that of the
purified protein estimated by SDS-PAGE. No significant ho-
mology sequences with cehA were found by a database homol-
ogy search.
Nucleotide sequence of the cehA surrounding region. Since a
part of another ORF located at upstream of cehA was highly
homologous to istB of IS1600 from Alcaligenes eutrophus NH9
(26), the nucleotide sequence of the cehA surrounding region
FIG. 1. Plasmid profiles of Rhizobium sp. strain AC100 and its
mutant. Lanes: 1, strain AC100; 2, strain AC101.
FIG. 2. (a) Restriction map of pAC200. Cloned regions are indicated as following: 23-kb BamHI fragment, solid line; 7.2-kb SmaI fragment,
stippled line; 6.2-kb SalI fragment, striped line. (b) Scheme of a DNA region containing the cehA gene and its carbaryl-degrading activities.
Abbreviations: B, BamHI; E, EcoRI; EV, EcoRV; Sa, SalI; Sc, SacII; Sm, SmaI.
VOL. 68, 2002 GENETIC ANALYSIS OF A NOVEL CARBARYL HYDROLASE GENE 1223

(10-kb EcoRI-NaeI fragment) was determined on both strands.
It was found that the DNA fragment had six ORFs containing
cehA (Fig. 3). The deduced amino acid sequence encoded by
two ORFs located upstream of cehA gene showed significant
homology to IstA and IstB of IS1600 from A. eutrophus NH9
and of IS1326 from Pseudomonas aeruginosa, which belong to
the IS21 family (1, 26). The nucleotide sequence of the region
containing IstA- and IstB-like sequences showed 67.7% ho-
mology with IS1600 and 60.5% with IS1326. In addition, a long
terminal inverted repeat (IR) was found at both ends of the
region (Fig. 4), indicating that the region had an insertion
sequence (IS) of the IS21 family. Thus, the region and the two
genes were designated ISRsp3,istA and istB, respectively.
ISRsp3 was 2,490 bp long and was delimited by 30 bp of perfect
IR, and parts of the terminal IR were directly repeated (arrows
in Fig. 4). The two adjacent ORFs, istA and istB, overlapped by
8 bp. The IstA of ISRsp3 carried a putative helix-turn-helix
motif in the N-terminal region and a motif related to the
widespread integrase DDE motif but lacked the conserved K
or R residue (35). IstB of ISRsp3 contained the ATP- or GTP-
FIG. 3. Physical map of the locations of ORFs found in the sequenced region of pAC200. Open arrows indicate ORFs.
FIG. 4. Characteristic nucleotide sequences at the ends of ISRsp3. IRs of ISRsp3 are shown on a black background. Arrows indicate direct
repeats. The double-underlined region is the putative ribosome-binding site of the cehA gene. Boxed CTCGGGG sequences are target site
duplicated sequences of Tnceh.
1224 HASHIMOTO ET AL. APPL.ENVIRON.MICROBIOL.

binding motif. ISRsp3 was adjacent to the cehA gene, and the
putative ribosome-binding site and right terminal IR over-
lapped by 1 bp. We therefore presumed that the promoter of
cehA is located in the ISRsp3 region and that the expression of
cehA is affected by the insertion of ISRsp3.
Further analysis revealed an identical sequence to ISRsp3
downstream of the cehA gene (Fig. 3). Although no target site
duplication was found at either end of the ISRsp3s, a 7-bp
direct repeat (CTCGGGG) was found at both ends of the
10-kb region containing ISRsp3s and cehA (Fig. 4). Therefore,
the genes in the 10-kb region had a transposon-like structure
that was designated Tnceh. The target site direct repeat at both
ends of Tnceh suggested that Tnceh is transposed into a pre-
cursor plasmid of pAC200.
An ORF of considerable length was found in a 3-kb region
between cehA and lower ISRsp3, and it was designated orfX.
The deduced amino acid sequence of orfX showed homology to
the putative salicin receptor SalC (27.2%) from Azospirillum
irakense (36), ORF1201 (25.5%) from Sphingomonas aromati-
civorans (32), and the putative TonB-dependent receptor SftP
(23.5%) from P. aeruginosa (37). The amino acid sequences
exhibited all the characteristics of the FepA/FhuA family outer
membrane receptor that are implicated in the transport of
several molecules, such as siderophores or vitamin B
12
. How-
ever OrfX seemed to be nonfunctional, since the result of the
alignment analysis of OrfX and its homologous proteins re-
vealed that OrfX (663 amino acid) was suddenly interrupted by
lower ISRsp3 and it was 50 amino acids shorter at the end of
the C-terminal region than the homologous proteins.
Southern hybridization and PCR analysis. The genetic struc-
ture of the Tnceh region was examined by Southern blot hybrid-
ization and PCR analysis. Total DNA from strain AC100 and
AC101 was used for Southern hybridization (Fig. 5). Labeled
internal fragments of the cehA gene and ISRsp3 were used as
probes to examine total DNA digested with EcoRI and/or
BamHI. The sizes of all hybridized fragments from AC100
were consistent with sizes estimated from the nucleotide se-
quence (Fig. 5, lanes 1 to 3). On the other hand, AC101 did not
hybridize with the cehA probe and signals from the hybridized
ISRsp3 probe indicated that the EcoRI-digested DNA frag-
ment was truncated and that the BamHI site in pAC200 had
disappeared (Fig. 5, lanes 4 to 6). We analyzed the PCR prod-
ucts amplified using some primer sets (Fig. 6). Although a
2.8-kb fragment was coamplified with the entire transposon
region (Fig. 6, lane 5), the lengths of all amplified products
from strain AC100 agreed with the sizes estimated from the
nucleotide sequence. Therefore, the genetic structure of Tnceh
was further supported by the PCR results. On the other hand,
PCR analyses of carbaryl-nondegrading mutant AC101 were
carried out to disclose changes in the mutant. A 2.8-kb frag-
ment was amplified from strain AC101 using primers that
anneal to the flanking regions of Tnceh (primers A and D).
The results of restriction fragment length polymorphism anal-
ysis of the fragment revealed that the fragment contained an
ISRsp3. Thus, the catabolic region was deleted due to homol-
ogous recombination between the two ISRsp3s in strain AC101
during the curing procedure. This finding was consistent with
those of Southern hybridization. Furthermore, restriction frag-
ment length polymorphism analysis showed that the 2.8-kb
fragment from AC100 that coamplified with the entire trans-
poson region was identical to the ISRsp3 containing the 2.8-kb
fragment amplified from AC101. This indicated that the dele-
tion event occurred spontaneously while AC100 was in culture.
DISCUSSION
We analyzed the properties of carbaryl hydrolase and its
gene from Rhizobium sp. strain AC100. Some reports have
revealed that genes encoding carbamate pesticide hydrolases
are located in plasmids (11, 16, 28, 29, 41). The present study
constructed the restriction map of a carbaryl degradative plas-
mid (pAC200). In addition, this is the first report to our knowl-
edge on the nucleotide sequence of a gene encoding an enzyme
that hydrolyzes N-methylcarbamate insecticides, although only
one sequence was submitted to DNA databases. The submitted
mcd gene from Achromobacter sp. strain WM111 encodes an
enzyme that hydrolyzes carbofuran, which is a kind of carba-
mate insecticide (database accession no. AF160188). No ho-
mologous sequences were found by database homology search
using nucleotide and deduced amino acid sequences of the
mcd gene. Those of the cehA gene also had no homologous
sequences, and no significant homology was apparent between
cehA and mcd in either nucleotide and amino acid sequences.
Therefore, the two carbamate hydrolyzing enzymes are not
evolutionarily related, so cehA seems to be a novel gene. On
the other hand, the enzymatic properties, except for substrate
specificities, of the carbaryl hydrolase from strain AC100 were
very similar to those of Blastobacter sp. strain M501 (17). Fur-
thermore, 12 amino acid residues of the N-terminal sequences
of both enzymes were identical (unpublished data).
The results of the ORF analysis of the cehA gene did not
reveal a start ATG codon, and two potential start codons, both
of which were TTG, were found in the frame of cehA gene. The
FIG. 5. Southern blot hybridization of total DNA of strain AC100
and AC101 digested with EcoRI and/or BamHI. Hybridization pro-
ceeded overnight at 50°C. Lanes: 1 to 3, total DNA of AC100 digested
with restriction enzymes (lane 1, EcoRI; lane 2, BamHI; lane 3, EcoRI
and BamHI); 4 to 6, total DNA of AC101 digested with restriction
enzymes (lane 4, EcoRI; lane 5, BamHI; lane 6, EcoRI and BamHI).
(a) Labeled HindIII-SalI fragment containing internal sequence of the
cehA gene was the probe. (b) Labeled PstI-SalI fragment containing
internal sequence of upper IS Rsp3 was the probe.
VOL. 68, 2002 GENETIC ANALYSIS OF A NOVEL CARBARYL HYDROLASE GENE 1225

distances between the TTG codons and the codon of the N-
terminal amino acid of the mature protein were 87 and 36 bp,
respectively. Since the putative amino acid sequence from the
upper TTG codon to the N-terminal region of the mature
protein displayed the common feature of signal peptide, the
upper was predicted as a start codon. A putative ribosome-
binding sequence, AGGAAGG, was then found at 7 nucleo-
tides before the start codon. The distance between the IR of
ISRsp3 and the initiation codon of the cehA gene was 13 bp,
and the putative ribosome-binding site and the IR overlapped
by 1 bp. These results suggested that the promoter of the cehA
gene is located in the ISRsp3 region and that the transcription
of cehA gene is activated by the insertion of IS. Since ISRsp3 is
highly homologous to IS1600 of the IS21 family, ISRsp3 was
also classified into the family. IS21 was found in the R68
plasmid of P. aeruginosa and transcriptional activations by IS21
and other ISs belonging to the IS21 family have been suggested
(24, 30, 31). The nucleotide sequence and/or multiple repeated
structure of terminal IRs of the IS21 family seem to be in-
volved in the transcriptional activation. Ogawa and Miyashita
suggested that IstAs of IS1600 and homologous ISs are clus-
tered on the phylogenetic tree of the IstAs of the IS21 family
(27). The cluster included IS1600 from A. eutrophus,orfSA
from Pseudomonas sp. strain P51, IS1326 from P. aeruginosa,
and nmoT from Chelatobacter heintzii (1, 42). C. heintzii and
Rhizobium sp. strain AC100 are allied species that belong to
the ␣-subdivision of the Proteobacteria, but ISRsp3 is most
homologous to IS1600 of A. eutrophus, which belongs to the
␤-subdivision. The clustered ISs were identified from antibiotic
resistant and xenobiotic degrading bacteria. However, no re-
lationships were shown among the degradative genes, the sub-
strate compounds and the bacterial species. This indicated that
the ISs are involved in acquiring the effective ability to degrade
various foreign organic hydrocarbons.
The carbofuran-degrading bacterium Sphingomonas sp.
strain CF06 contains five plasmids, at least some of which are
required for the metabolism of carbofuran (11). Observations of
rearrangements, deletions, and the loss of individual plasmids
resulting in the loss of carbofuran-degrading ability suggested
the involvement of mobile DNA elements such as transposons
or ISs. However, the relationship between the carbofuran deg-
radative gene and the mobile elements has not been clarified.
On the other hand, the 10-kb region in Rhizobium sp. strain
AC100 has likely been transposed since the 7 bp of duplicate
sequences of the target site were found at both ends of Tnceh.
Besides, the partial sequence of the other region of pAC200
was homologous to the traA gene, which was concerned with
conjugation (data not shown). Many xenobiotic degradative
genes are located in transposons and plasmids (40). These
genes may have repeatedly transferred and evolved to adapt to
the variety of substrates and to degrade them more effectively.
Associations between plasmids or transposable elements and
the diversity of carbamate-pesticide degradative genes have
been predicted or observed (11, 12, 29). This agrees with the
present study report that the carbaryl degradative gene cehA is
located in a transposon on a plasmid. Desaint et al. observed
the genetic diversity of 128 carbofuran-degrading bacteria iso-
lated from English and French soils, using amplified DNA
restriction analyses of the 16S rDNA and 16S-23S rDNA
spacer region (6). These were classified into six widely spread
clusters, of high- and low-G⫹C-content gram-positive bacte-
ria; Cytophaga; and ␣-, ␤- and ␥-Proteobacteria. Although 58 of
the 128 studied strains harbor the mcd gene, there was no clear
relationship between the presence of the gene and the phylo-
genetic position of the strain. On the other hand, Cha-
palamadugu and Chaudhry have noted that 15 bacteria iso-
lated from North America soils do not contain sequences
homologous to the mcd gene (3). Since the N-terminal se-
FIG. 6. PCR analyses of wild type and mutants. (a) Agarose gel electrophoresis of PCR products. Plasmid DNA from strain AC100 was
template for lanes 1 to 5 and from AC101 for lanes 6 and 7. Primer sets for each lane were as follows: lanes 1 and 6, primer sets B and E; lane
2, primer sets B and F; lane 3, primer sets C and F; lane 4, primer sets C and E; lanes 5 and 7, primer sets A and D. (b) Scheme of primer annealing
sites in sequenced region. Nucleotide sequences of primers are described in Materials and Methods. Arrows show primer annealing sites.
1226 HASHIMOTO ET AL. APPL.ENVIRON.MICROBIOL.

quences of carbaryl hydrolases from strain AC100 and M501,
which were isolated from different sites in Japan, were identi-
cal, diverse carbaryl-degrading bacteria in Japanese soil are
likely to contain the cehA homologous gene. The worldwide
genetic diversity of carbamate degradative genes should be
studied in more detail.
Environments contaminated with carbaryl are regarded as
hazardous because the pesticide is considered to be an endo-
crine-disrupting chemical. Thus, a technique for rapid decom-
position of the compound is required. The effectiveness of
bioremediation can be measured by factors such as the survival
of the introduced microorganism, the stability of the genes
encoding the appropriate catalytic functions, and the degree of
contaminant removal. The catabolic region of strain AC100
was spontaneously deleted without selective pressure. Further
study of the gene stability and survivability of Rhizobium sp.
strain AC100 in various environments is needed before the
bacteria can be applied to the process of bioremediation.
ACKNOWLEDGMENTS
This work was supported by a program for the promotion of basic
research activities for innovative biosciences of the Bio-Oriented Tech-
nology Research Advancement Institution (Minatoku, Japan).
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