Download enhancing organophosphorus pesticides detoxification using

ENHANCING ORGANOPHOSPHORUS PESTICIDES DETOXIFICATION USING
INTERGENERIC PROTOPLAST FUSION BETWEEN ESCHERICHIA COLI AND TWO
DIFFERENT BACTERIA GENERA
By
Ayman H. Mansee1*, Mohamed Yacout2 and Manal R. Montasser3
1
Pesticide Chemistry Department, Faculty of Agriculture Alexandria University
2
Department of Genetics, Faculty of Agriculture
3
Central Lab of Pesticide, Agricultural Research Center, Alexandria
*Author E-mail address: [email protected]
Fax:+203-5922780
ABSTRACT
Recently we isolated certain E. coli strains from specific contaminated sites of Marriute
Lake Alexandria Province, EGYPT that showed capabilities for organophosphate pesticides (OP)
degradation. The promising strains of those isolates had been chosen for protoplast fusion
(hybridization) with Bacillus thuringiensis D55 and Agrobacterium tumefaciens. The protoplast
fusion capabilities for OP biodegradation were assayed and compared with their parental bacteria.
These fusions showed a superlative increase in OP biodegradation. Interestingly, the performance
of the hybrids for OP biodegradation was enhanced with time and showed a tremendous
efficiency (100%) even at high level of substrate concentrations. The hybrids degraded up to
250µg/ml of Paraoxon completely 54 days after hybridization.
INTRODUCTION
Several organophosphate compounds (OP’s) are used to control a wide variety of insect
pests, weeds, and disease-transmitting vectors. Among known insecticides, OP has been
intensively used in Egypt over many years (El-Sebea et al., 1993). For example, parathion is one
of popular pesticides used for agriculture crop protection in US with more than 7 million pounds
consumed in 1995 (EPA, 1997) The widespread use of this group of pesticides in different
applications especially for agriculture purposes resulted in many undesirable effects, generating
of wastewater and causing a lot of environmental pollution problems (Gill and Ballesteros, 2000).
The need to develop safe, convenient, and economically feasible methods for removal and
detoxification of OP’s compounds from the environment has been urgent (Grimsley et al., 1997).
In this regard, biotechnology has a growing place (poison) in the remediation of hazardous waste
sites allover the world, especially in developing countries where population density is high with
limitation in land and fresh water resources (Omenn, 1992). Recently, using isolated
microorganisms from contaminated environmental phases for pesticides decontamination is
getting a great attention. Ramanathan and Lalithakumari (1999) mentioned that the continuous
increasing of selection pressure due to pollution factors developed in many environmental phases
such as water and soil, promptly induces the formation of modified bacteria strains; specifically
characterized by their capability for bioremediation such inducing chemicals. In addition, isolates
from specific contaminated areas of naturally occurring bacteria, showed variable capabilities for
metabolizing OP’s (Mansee et al., 2004a). This provides the possibility for a safe and in-situ
detoxification of these compounds (Mansee et al., 2004a and Mulbry et al., 1998).
In this view, a number of soil bacteria have been found to be capable of degrading
parathion. These bacteria normally produced parathion hydrolase, which is encoded by the opd
for organophosphate degradation gene. Microbial enzymes such as parathion hydrolase are
thought to play a significant role in the degradation of parathion and related organophosphate
insecticides in the soil (Siddaramappa, et al., 1973 ), this observation has stimulated studying of
the organisms that are capable of synthesizing such enzymes (Karns et al., 1987, Mulbry and
Karns, 1989, Mulbry, and Kearney, 1991, Mulbry, 1992). In each of these organisms the opd
gene was found to be located on a large plasmid (Harper et al. ,1988, Mulbry et al., 1986 ,
Somara, and Siddavattam, 1995).
The plasmids were all unrelated as judged by Southern
hybridization, but sequencing of the opd genes showed that the nucleotide sequences were 98100% identical (Mulbry and. Karns, 1989a, Serdar et al., 1989, Somara, 2002). Studies of two
of the plasmids, pPDL2 from Flavobacterium sp. strain ATCC 27551 and pCMS1 from P.
diminuta strain MG, by restriction analysis and hybridization experiments indicated that the opd
genes were located in a highly conserved region that was estimated to extend approximately 2.6
kb upstream and 1.7 kb downstream of opd (Mulbry et al., 1986, Mulbry et al., 1987 ). No
homology was evident between the two plasmids outside this region, and the possibility that the
opd gene was carried on a transposon was discussed (Mulbry et al., 1987) which confirmed later
by Siddavattam et al., (2003). The capabilities of different bacterial species for OP’s
biodegradation and the influence of several factors on such capabilities were studied by several
investigators; Bacillus sp. (Bhadbhade et al., 2002b); and E. coli (Kim et al., 2002; Mansee et al.,
2004a,b and McLoughlin et al., 2004).
Recently, our observation demonstrated the remarkable ability of local isolates of E. coli
to degrade OP insecticides. Such observation presents a potential possibility of OP insecticides
bioremediation, in the Egyptian environment. A technology based on the use of local bacteria and
developing the efficiency of isolated strains by adaptation as described in our reports could be
introduced an efficient, cheap and environmental friendly technique for pollutants
decontamination (Mansee et al., 2004a,b). On the other hand, protoplast fusion has been used as a
tool for gene transfer intergeneric bacteria (Gokhale et al., 1984, and Gokhale & Deobagkar,
1994) and to enhance genetically many bacterial characters (Shweil et al., 1998).
To enhance the capability of isolated bacteria for OP’s biodegradation, the present study
was undertaken to investigate the possibility of hybridization, through protoplast fusion, between
Agrobacterium and Bacillus species; and isolated E. coli strains that previously indicated their
capability for OP biodegradation. Moreover, the efficiency of resulted bacterial hybridizes for
biodegradation of OP were investigated.
MATERIALS AND METHODS
Chemicals:
Paraoxon 98% (diethyl- p-nitrophenyl phosphate) was purchased from Chem. Service, Inc, West
Chester, PA, USA.
Luria-Bertani (LB) media was composed of (l-1 distilled water): Bactotryptone 1g; Bactoyeast
extract 5g; sodium chloride 10g; potassium dibasic phosphate 1g; and potassium monobasic
phosphate 3g. Nutrient broth (NB), buffer phosphate (sodium monobasic phosphate, sodium
dibasic phosphate and sodium citrate) buffered to pH 8.0, TES buffer (0.01M tris-HCL, then
0.025M Na-EDTA, and 0.6M sucrose, pH 8.0) and the selective media SM (MgSO4, MnSO4,
CaCl2, CuSO4, FeSO4, (NH4)2SO4, K2HPO4, and glucose) were also used.
Bacterial Strains
Esherichia coli strain was collected from water samples that are located along Marriute Lake of
Alexandria according to waste drainage outlets which was identified based on cultural and
physiological characteristics according to Bergey’s manual of systematic bacteriology (Krieg and
Holt, 1984 and Mates and Schaffer, 1992) and assayed its OP’s degradation ability, in our
previous research (Mansee et al., 2004a). Agrobacterium tumefaciens was obtained from
Department of Plant pathology, Faculty of Agriculture, Alexandria University and have been
maintained according to Yacout (1992). Bacillus thuringiensis D55 was obtained from
Department of Microbiology, Faculty of Science, Alexandria University and was maintained on
L.B slape medium (Puntambekar and Ranjekar, 1989).
Protoplast Formation, Fusion and Regeneration:
The protoplast fusants were isolated by protoplast fusion between Bacillus thuringiensis
D55, Agrobacterium tumefaciens and E. coli according to Weiss (1976) with some modifications
(Shweil et al., 1998). Bacterial strains were grown for 16 hours in 10ml of L.B medium. The cells
were centrifuged, washed and resuspended in 10ml TES buffer, containing lysozyme (1mg/ml).
Then, the suspension incubated at 26oC for 3 hours with shaking and 0.05M MgCl2 was added.
Protoplast formation was confirmed by microscopic examination after staining.
Protoplasts were regenerated on LB agar plates containing 0.6 M sucrose and 0.8% agar.
Identical numbers of protoplast from each were gently mixed and fused in presence of 30%
polyethelene glycol (PEG) 6000 (wt/v) for 3 minutes and washed with TES buffer. The fused
protoplasts were mixed in selective medium (SM) for isolation of hybrids. The hybrids were
purified and used for tests.
Growing and Preparing Bacterial Cells For Biodegradation :
The method described by Kearney et al., (1986) and Mulbry et al., (1998) were used to
grow the hybrids and single bacteria isolates by using liquid media (N.B) supplemented with
(0.1g ml-1) ampicilin. The cells were harvested 48 h after growth by centrifugation at 4°C, 5000
rpm for 10 min, washed with 40ml of 150mM buffer citrate-phosphate pH 8.0 twice and recentrifuged at the same conditions. The resulted pellets were weighted and then suspended in
10ml of buffer citrate-phosphate.
Capability of hybrids Bacteria for OP Biodegradation:
Ten l of the harvested bacteria suspension were incubated with serial paraoxon
solutions, prepared in 50 mM buffer citrate-phosphate, pH 8.0 and incubated at 37 °C for 48 h.
Three replicates was carried out for each concentration, as well as three for control which
prepared by incubated bacteria cells in paraoxon free buffer solution. All of them were incubated
at 37°C for 48 h and then analyzed the product of degradation by measuring absorbance
spectrophotometrically at 410nm for p-nitrophenol.
RESULTS
Protoplast Formation and Fusion:
High ratios of bacterial cells (about 80-90%) were converted to protoplasts, under the
standard conditions used. Successful fusions produced three types of hybrids, i.e. E. coli with A.
tumefaciens (EA), E. coli with B. thuringiensisD55 (EB), and A. tumefaciens with B.
thuringiensisD55 (AB). The three hybrids were confirmed by light microscope examination. Fig.
(1) Shows the difference between bacterial cells size in the three parental bacteria (E. coli A.
tumefaciens and B. thuringiensisD55) and their protoplast fusants (AE, BE, and AB). Hybrids
cells were obviously bigger than the parental cells strains and they formed in single cells, while
parental cells were smaller and forming chains. Those results agreed with Gokhale et al., (1984)
and Shweil et al., (1998) results.
Figure 1: Bacterial and hybrids cells " E. coli with A. tumefaciens (EA), E. coli with B.
thuringiensisD55 (EB), and B. thuringiensisD55 with A. tumefaciens (AB).
Degradation of Paraoxon:
Degradation of Paraoxon to p-nitrophenol using the isolated E. coli, and its hybrids with
either Bacillus thuringiensis D55 or Agrobacterium tumefaciens was assayed and presented in
Figures 2. Figure2a showed paraoxon degradation due to the effect of microorganisms after seven
days of hybridization. This figure illustrated the biodegradation efficiency powerful of E. coli (E)
isolates is slightly higher than those of Bacillus thuringiensits D55 (B) or Agrobacterium
tumefaciens (A). Also, the hybridization between A and B introduce hybrid AB that did not
enhance the paraoxon degradation. This observation showed the essential role of E. coli isolate
for producing effective hybrids for OP degradation.
For the hybrids between E and either A or B, an increase in paraoxon degradation were
recorded for preparations after 7 days of protoplast fusions (figure 2A). The degradation
percentages of such hybrids were decreased from 100% at low concentrations of paraoxon (10
and 30µg/ml) to less than 30% at the highest concentration (250µg/ml). The relation between the
paraoxon degradation and its concentration found to be reversed for all treatments.
New preparations of same three hybrids were tested for their biodegradation efficiency
30 days after hybridization and the data presented in Figure 2B. The results showed an
enhancement of biodegradation capabilities of hybrids EA and EB especially for the higher
paraoxon concentrations level. This enhancement of the efficiency was more distinct with EB
hybrid than those of EA one. Another preparation was carried out after 54 days following the
same methodology and presented in Figure 2c. This figure showed a rising in the efficiency
enhancement either for EA or EB hybrids. However; increasing the paraoxon concentration
above 250 µ/ml, decreased the hybrids-paraoxon degradation efficiency.
From the previous data it could be concluded that protoplast fusion enhanced paraoxon
degradation for bacterial hybrids, used in this study, than their parents. That enhancement varied
according to the parental bacteria genius where it was about 10-15% in the case of AB and 100%
for AE and BE hybrids. Also, the presence of A and B increased E biodegradation capabilities
five folds for the paraoxon concentration 250 µ/ml, while at paraoxon concentration of 750 µ/ml,
the degradation efficiency found to be 25%.
DISCUSSION
Enzyme types and content is the one of the limiting factors for introducing
microorganisms that capable of pesticides bioremediation. Sogorb and Vilanova (2002) reviewed
the enzymes involved in the detoxification of organophosphorus insecticides through hydrolysis.
Several types of enzymes are involved in the detoxification of OP’s. The most important
enzymes are esterases of an unknown physiological role, classified by the International Union of
Biochemistry as PTE (E.C. 3.1.8) (Vilanova and Sogorb, 1999). Siddavattam et al., (2003) cited
that the efficiency of bacteria to degrade OP pesticides is generally due to their content of the
enzyme organophosphorus hydrolase (OPH) that is encoded by a plasmid gene (opd).
McLoughlin et al., (2004) studied opd and other genes that enabled E. coli to grow using
paraoxon as the sole phosphorus source. The variation between degradation activity hybrids
preparations after 7 days and after 30 or 45 days could be due to differences between their
bacterial membrane activity and cell wall development. This conclusion agrees with reports of
Brown (1980) and Dumas et al., (1989) that OPH is a zinc-containing homodimeric protein found
in the bacterial membrane. Also, morphological differences shown that the hybrids retaining
common genes from both parents (Chen et al., 1987 and Puntambekar and Ranjekar, 1989).
The recent results showed the role E. coli isolates for OP biodegradation either when used
individually or after hybridization with the two other used strains. This were supported by the
results of McLoughlin et al., (2004) and Kang et al., (2002) who enhanced the detoxification of
organophosphates using recombinant Escherichia coli with co-expression of organophosphorus
hydrolase and bacterial hemoglobin. Moreover, the OP degradation enhancement of the hybrids
of E. coli with the other two strains: Bacillus thuringiensis D55 and Agrobacterium tumefaciens,
could be due to the DNA recombinations as result of protoplast fusion as well as improving the
gene action of opd gene of E. coli in the hybrids.
We thank Ahmed Ibrahim and Amel M. Ibrahim for their help with some of the experiments.
Figure 2. Efficiency of Bacterial strains and its hybrids for OP Degradation
A
B
C
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