Microbiology
- J Bacteriol 1998 Mar;180(6):1354-9
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Cloning and sequencing of
a 2,5-dichlorohydroquinone reductive dehalogenase gene whose product is involved
in degradation of gamma-hexachlorocyclohexane by Sphingomonas paucimobilis.
Miyauchi K, Suh SK, Nagata Y, Takagi M
Department of Biotechnology, The University of Tokyo, Japan. aa67118@hongo.ecc.u-tokyo.ac.jp
Sphingomonas (formerly Pseudomonas) paucimobilis UT26 utilizes gamma-hexachlorocyclohexane
(gamma-HCH), a halogenated organic insecticide, as a sole carbon and energy
source. In a previous study, we showed that gamma-HCH is degraded to
2,5-dichlorohydroquinone (2,5-DCHQ) (Y. Nagata, R. Ohtomo, K. Miyauchi, M.
Fukuda, K. Yano, and M. Takagi, J. Bacteriol. 176:3117-3125, 1994). In the
present study, we cloned and characterized a gene, designated linD, directly
involved in the degradation of 2,5-DCHQ. The linD gene encodes a peptide of 343
amino acids and has a low level of similarity to proteins which belong to the
glutathione S-transferase family. When LinD was overproduced in Escherichia coli,
a 40-kDa protein was found after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Northern blot analysis revealed that expression of the linD
gene was induced by 2,5-DCHQ in S. paucimobilis UT26. Thin-layer chromatography
and gas chromatography-mass spectrometry analyses with the LinD-overexpressing
E. coli cells revealed that LinD converts 2,5-DCHQ rapidly to chlorohydroquinone
(CHQ) and also converts CHQ slowly to hydroquinone. LinD activity in crude cell
extracts was increased 3.7-fold by the addition of glutathione. All three of the
Tn5-induced mutants of UT26, which lack 2,5-DCHQ dehalogenase activity, had
rearrangements or a deletion in the linD region. These results indicate that
LinD is a glutathione-dependent reductive dehalogenase involved in the
degradation of gamma-HCH by S. paucimobilis UT26.
PMID: 9515900, UI: 98175654
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- Biochem Biophys Res Commun 1996 Apr 25;221(3):755-61
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Novel polypeptides induced by the insecticide
lindane (gamma-hexachlorocyclohexane) are required for its biodegradation by a
Sphingomonas paucimobilis strain.
Adhya TK, Apte SK, Raghu K, Sethunathan N, Murthy NB
Division of Soil Science and Microbiology, Central Rice Research Institute,
Cuttack, India.
When exposed to the potent insecticide gamma-hexachlorocyclohexane or lindane, a
Sphingomonas paucimobilis strain rapidly synthesized 7 novel polypeptides and
concomitantly gained the ability to degrade lindane. Synthesis of these proteins
was switched-off subsequent to the disappearance of lindane from the medium.
Treatments which induced the synthesis of identical proteins also conferred on
cells the ability to degrade lindane. In contrast, cells blocked in protein
synthesis could not be induced to degrade lindane. The close correspondence
observed between expression of lindane-induced proteins and gamma-hexachlorocyclohexane
catabolism strongly implicates these novel proteins in lindane biodegradation.
PMID: 8630034, UI: 96205349
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Microbiol Res 1995 May;150(2):187-94
- Insecticides: their effect on
microorganisms and persistence in rice soil.
Das AC, Chakravarty A, Sukul P, Mukherjee D
Department of Agricultural Chemistry and Soil Science, Bidhan Chandra Krishi
Viswavidyalaya, Mohanpur, India.
A field experiment was conducted to investigate the effect of four insecticides,
HCH, phorate, carbofuran and fenvalerate, at recommended doses on the
preponderance of bacteria, actinomycetes and fungi. We also measured the
persistence of the insecticides in the rhizosphere soil of rice. HCH and
fenvalerate stimulated the proliferation of all of the microorganisms
significantly. Phorate increased the population of bacteria and actinomycetes.
Carbofuran accentuated the preponderance of actinomycetes in soil. Insecticides,
in general, did not have marked influence on the proliferation of Bacillus,
Streptomyces, Aspergillus and Fusarium in soil. However, we observed a
stimulation of growth of Staphylococcus, Proteus and Sarcina with HCH,
Pseudomonas, Corynebacterium, Erysipelothrix and Rhizopus with phorate, Serratia,
Corynebacterium, Klebsiella, Escherichia, Rhizopus and Humicola with carbofuran,
and Staphylococcus, Sarcina, Klebsiella and Nocardia with fenvalerate. On the
other hand, there was an inhibition in growth of Pseudomonas, Micrococcus,
Nocardia and Penicillium with HCH, of Pseudomonas, Micrococcus and Penicillium
with carbofuran, and of Pseudomonas, Micrococcus and Micromonospora with
fenvalerate. Different types of insecticides exhibited differential patterns of
dissipation in soil. HCH had the highest persistence followed by phorate,
carbofuran and fenvalerate, respectively.
PMID: 7541287, UI: 95323371
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- J Bacteriol 1994 Jun;176(11):3117-25
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- Cloning and sequencing of a
2,5-dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase gene involved in the
degradation of gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.
Nagata Y, Ohtomo R, Miyauchi K, Fukuda M, Yano K, Takagi M
Department of Agricultural Chemistry, University of Tokyo, Japan.
In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is
converted to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL), which is then
metabolized to 2,5-dichlorohydroquinone. Here, we isolated from the genomic
library of UT26 two genes which expressed 2,5-DDOL dehydrogenase activity when
they were transformed into P. putida and Escherichia coli. Both gene products
had an apparent molecular size of 28 kDa by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The first gene, named linC, located separately from the two
genes (linA and linB) which we had already cloned as genes involved in the
gamma-HCH degradation. The other, named linX, located about 1 kb upstream of the
linA gene encoding gamma-HCH dehydrochlorinase. A gamma-HCH degradation-negative
mutant, named UT72, which lacked the whole linC gene but had the intact linX
gene was isolated. The linC gene given in a plasmid could complement UT72. These
results strongly suggest that the linC gene but not the linX gene is essential
for the assimilation of gamma-HCH in UT26. Deduced amino acid sequences of LinC
and LinX show homology to those of members of the short-chain alcohol
dehydrogenase family.
PMID: 7515041, UI: 94252977
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- J Bacteriol 1993 Oct;175(20):6403-10
Cloning and sequencing of a dehalogenase
gene encoding an enzyme with hydrolase activity involved in the degradation of
gamma-hexachlorocyclohexane in Pseudomonas paucimobilis.
Nagata Y, Nariya T, Ohtomo R, Fukuda M, Yano K, Takagi M
Department of Agricultural Chemistry, University of Tokyo, Japan.
In Pseudomonas paucimobilis UT26, gamma-hexachlorocyclohexane (gamma-HCH) is
converted by two steps of dehydrochlorination to a chemically unstable
intermediate, 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), which is then
metabolized to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) by two steps
of hydrolytic dehalogenation via the chemically unstable intermediate
2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL). To clone a gene encoding
the enzyme responsible for the conversion of the chemically unstable
intermediates 1,4-TCDN and 2,4,5-DNOL, a genomic library of P. paucimobilis UT26
was constructed in Pseudomonas putida PpY101LA into which the linA gene had been
introduced by Tn5. An 8-kb BglII fragment from one of the cosmid clones, which
could convert gamma-HCH to 2,5-DDOL, was subcloned, and subsequent deletion
analyses revealed that a ca. 1.1-kb region was responsible for the activity.
Nucleotide sequence analysis revealed an open reading frame (designated the linB
gene) of 885 bp within the region. The deduced amino acid sequence of LinB
showed significant similarity to hydrolytic dehalogenase, DhlA (D. B. Janssen,
F. Pries, J. van der Ploeg, B. Kazemier, P. Terpstra, and B. Witholt, J.
Bacteriol. 171:6791-6799, 1989). The protein product of the linB gene was 32 kDa
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Not only
1-chlorobutane but also 1-chlorodecane (C10) and 2-chlorobutane, which are poor
substrates for other dehalogenases, were good substrates for LinB, suggesting
that LinB may be a member of haloalkane dehalogenases with broad-range
specificity for substrates.
PMID: 7691794, UI: 94012509-
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- Biosci Biotechnol Biochem 1993 May;57(5):703-9
Isolation and characterization of
Tn5-induced mutants of Pseudomonas paucimobilis UT26 defective in gamma-hexachlorocyclohexane
dehydrochlorinase (LinA).
Nagata Y, Imai R, Sakai A, Fukuda M, Yano K, Takagi M
Department of Agricultural Chemistry, University of Tokyo, Japan.
Pseudomonas paucimobilis UT26 grows on gamma-hexachlorocyclohexane (gamma-HCH)
as a sole source of carbon and energy. Tn5 mutation was introduced into UT26,
and two kinds of mutants defective in gamma-HCH degradation were phenotypically
isolated; one (UT64) completely lacked the activity to degrade gamma-HCH, while
the other (UT61) retained a very low level of activity. Tagging and sequencing
analysis showed that both mutants had a Tn5 insertion at the same site of the
linA (gamma-HCH dehydrochlorinase encoding) gene. However, UT61 had an
additional rearrangement, which could be the cause of its retaining a low level
of activity. An in vitro complementation test with a crude extract from UT64
plus partially purified LinA protein showed that LinA was essential not only for
the first-step reaction (gamma-HCH to gamma-pentachloro-cyclohexene; gamma-PCCH),
but also for the second-step reaction (gamma-PCCH to compound B) of gamma-HCH
degradation in UT26.
PMID: 7686793, UI: 93313284-
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- Schriftenr Ver Wasser Boden Lufthyg 1992;89:255-68
[Use of bacterial toximeters with
separate cell cultures for continuous water monitoring].
[Article in German]
Fritz-Langen H, Blessing B, Krebs F
Two commercially available bacterial toxicity monitors are compared. As test
organisms pure cultures of Pseudomonas putida are used in both systems. The
bacteria are grown continuously in turbidostatic ("Toxalarm", LAR,
Berlin, Germany) or chemostatic ("Stiptox-norm", Siepmann und
Teutscher, Gross-Umstadt, Germany) regulated cultures in a selective culture
medium in nonsterile devices. Toxic substances can be detected by continuously
working bacterial respiration tests. Oxygen consumption is the measuring
parameter. The bacterial test suspension is mixed continuously in a fixed
proportion with air-saturated test water in a measuring cell. The separate
culturing of the bacteria and the carrying out of the tests ensures that neither
the bacterial culture is endangered by toxic substances nor the sensitivity can
be changed by poison adaptation. Results of "Toxalarm"-tests with
several chemicals (e.g. Atrazine, Lindane, 2-Nitro-phenol,
Sodiumpentachlorophenolate) are presented. The registration of an alarm event
(River Rhine) by "Stiptox-norm" is shown.
PMID: 1284920, UI: 93355202-
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- J Bacteriol 1991 Nov;173(21):6811-9
Molecular cloning of a Pseudomonas
paucimobilis gene encoding a 17-kilodalton polypeptide that eliminates HCl
molecules from gamma-hexachlorocyclohexane.
Imai R, Nagata Y, Fukuda M, Takagi M, Yano K
Department of Agricultural Chemistry, University of Tokyo, Japan.
Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane
(gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in
Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After
2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH
degradation were detected. A 5-kb fragment from one of the cosmid clones was
subcloned into pUC118, and subsequent deletion and gas chromatography-mass
spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for
the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene.
Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp
within the fragment. The nucleotide sequence of the linA gene and the deduced
amino acid sequence showed no similarity to any known sequences. The product of
the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide
gel electrophoresis.
PMID: 1718942, UI: 92041564
Norio Kurihara* (Radioisotope Research Center, Kyoto University, Kyoto
606-8501, Japan)
J. Mass Spectrom. Soc. Jpn., 46(3), 157-172 (in
Japanese), 1998
Deuterium substitution of certain hydrogen atoms in a molecule may change its
metabolism rate in various biological systems. Some of the deuterium labeled
pesticides were metabolized in some in vitro systems, such as rat liver
microsomes and purified P450 reconstituted systems, in a slower rate than the
unlabeled counterpart. This sometimes caused a substantial bioactivity -
insecticidal activity, etc.- enhancement. Metabolic reactions of lindane,
DDT analogs, diuron analogs and methoxychlor are discussed. Measurement of
isotope effect values would provide us various information on the biological and
biochemical reactions. There are examples of metabolic reaction mechanism
studies of oxidative O-demethylations of methoxy-compounds, oxidative N-demethylations
of N-methylamine derivatives and benzene ring hydroxylations using
specifically deuteriated aromatic compounds. These studies revealed some details
of relevant metabolic reaction pathways, the information that cannot be obtained
by other methods. Analysis of very large isotope effect values observed in some
metabolic reactions of xenobiotics, such as indene derivatives, revealed a
branched metabolic pathway. Also, a tunnel effect in metabolic reactions was
proposed for some xenobiotic metabolism that exhibited a very large observed
isotope effect value, for example metabolic oxygenation of linoleic acid and
methane. Mass spectrometric analysis is an essential means in all these studies.
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