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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Aim: The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. Methods and Results: A endosulfan-degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast-growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic-degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur-separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3-(N-morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta-endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. Conclusions: This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur-separation reactions. The endosulfan-degrading reactions are a result of the sulfur-starvation response of this bacterium. Significance and Impact of the Study: This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

The chlorinated cyclodiene insecticide, endosulfan (Thiodan, bicyclo-[2·2.1]-2-heptene-5,6-bisoxymethylene sulfite), has been used extensively for over 30 years to control numerous insect pests in a variety of crops. The commercial product contains a mixture of two diastereoisomers: alpha-endosulfan and beta-endosulfan in a ratio of 7 : 3, respectively. In comparison to the more recalcitrant cyclodienes such as aldrin and dieldrin, endosulfan is relatively labile in the environment (Van Woerden 1963; Maier-Bode 1968) and is considerably less toxic to mammals (Goebel et al. 1982). However, it is highly toxic to fish (Goebel et al. 1982) and aquatic contamination due to run-off from arable soils is a considerable concern. Additionally, while endosulfan itself does not bioaccumulate (Goebel et al. 1982), the toxic metabolite endosulphate can be stored in animal fat (Beck et al. 1966; Maier-Bode 1968; Dorough et al. 1978). As a result, contamination of pastures due to incorrect application practices can lead to unacceptably high endosulphate residues in locally grown production animals. These concerns have led to growing interest in the bioremediation of the insecticide in the environment.

Bioremediation requires a source of catabolic enzymes capable of degrading endosulfan. It is well established that adaptation of indigenous soil microbial populations occurs as a result of contact with chemicals (Lewis et al. 1984) and increasingly, contaminated soil has been utilized as a source of xenobiotic-degrading organisms. We have previously described the enrichment of an endosulfan-degrading mixed culture from soil with a history of repeated endosulfan exposure (Sutherland et al. 2000). Degradation of the insecticide by this culture included both oxidative and sulfur-separation reactions. The oxidation reaction favoured the alpha isomer and produced endosulphate, which has equivalent mammalian toxicity to the parent compound. The sulfur-separation reaction resulted in the release of the sulfur moiety and formation of a novel metabolite with properties predicted of endosulfan monoaldehyde. Removal of the sulfur moiety of endosulfan dramatically decreases the toxicity of the insecticide and the metabolites are readily degraded by a wide range of microbes (Goebel et al. 1982). The endosulfan monoaldehyde metabolite was readily further metabolized by the culture (Sutherland et al. 2000). We report here on the isolation and characterization of a Mycobacterium species from the mixed culture described in Sutherland et al. (2000) that demonstrates both the oxidative and hydrolytic and sulfur-separation endosulfan-degrading activities. The ability of this strain to degrade endosulfan is regulated by levels of alternative sulfur sources in the growth medium.

Bacterial culture

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Unless otherwise stated Mycobacterium strain ESD and the mixed culture from which it was obtained were grown and maintained in ‘sulfur-free’ broth (Sutherland et al. 2000) supplemented with 50 µm endosulfan as the only source of sulfur. Growth was measured by monitoring optical density at 595 nm. Cultures were incubated at 28°C on a rotary shaker at either 120 rev min−1 or 200 rev min−1 and subcultured upon reaching stationary phase as indicated by optical density measurements.

Escherichia coli strain TG1 was obtained from New England Biolabs, Beverley, MA, USA, Arthrobacter oxydans strain DSM 4044 was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunshweig, Germany culture collection, and Pseudomonas aeruginosa strain RH was isolated from soil inoculum and identified by 16S rRNA gene sequencing. Mycobacterium smegmatis strain mc2 was kindly provided by Dr H. Billman-Jacobe. All bacterial stocks were maintained in 25% glycerolat −80°C. Sulfur-free broth or agar containing endosulfan (50 µm) or sodium sulfite (200 µm), and tryptic soy agar (Oxoid, Melbourne, Australia) were used to culture bacteria.

Nucleic acid methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Cells (100 ml, mid-log phase) were pelleted by centrifugation at 3000 g for 10 min and pellets washed with 10 ml of 50 mm Tris-HCl (pH 7·0), 50 mm EDTA, 75 mm NaCl (lysis buffer). Cells were then resuspended in 5 ml of lysis buffer with 10 mg/ml lysozyme and incubated at 37°C for 90 min Sodium dodecyl sulphate (SDS) (1% final concentration) was added and cells were incubated at room temperature with gentle rocking for 16 h. The lysed cells were extracted twice with an equal volume of phenol and chloroform, and then the nucleic acids were precipitated with ethanol. The nucleic acid pellet was resuspended in 10 mm Tris-1 mm EDTA (pH 8·0) buffer with 100 µg ml−1 RNase A. After 1 h incubation at 37°C the DNA was extracted with an equal volume of phenol/chloroform and then precipitated with ethanol. DNA pellets were resuspended in TE (10 mm Tris, 1 mm EDTA, pH 8·0) buffer and stored at −20°C.

Amplification of the 16S rRNA gene from genomic DNA was performed using universal 27f and 1492r 16S rRNA gene primers (Lane 1991), according to the method of Bond et al. (1995). The sequence of the amplicon was determined using the dye terminator automated sequencing procedure (Applied Biosystems Incorporated, Foster City, CA, USA) and four 16S rRNA gene-specific oligonucleotide primers (Lane 1991).

Characterization of strain ESD

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Detection of endosulfan and metabolites was performed by thin layer chromatography (TLC) on alumina oxide plates developed in either 1 : 4 acetone: petroleum ether (v/v), or 1 : 3 ethyl acetate: chloroform (v/v) as described by Sutherland et al. (2000). Although the TLC method used in this report is not quantitative, it was chosen over gas chromatography (GC) to characterize endosulfan metabolism by strain ESD as not all the metabolites detected by TLC could be measured using the GC (Sutherland et al. 2000). Endosulfan is hydrophobic, volatile and subject to chemical hydrolysis, hence we regard the detection of metabolites as an essential indicator of degradation.

Strain ESD 16S rRNA gene sequence and similar sequences in the GenBank database were aligned using the Pileup program of the Genetics Computer Group (Devereux et al. 1994) and a dendrogram showing the phylogenetic position of strain ESD was generated by the distance neighbour-joining method using paup (Swofford 1998). The Biolog bacterial identification procedure was performed according to the manufacturers directions (Oxoid, Melbourne, Australia). Acid-alcohol fastness was determined using the Ziehl-Neelsen stain and beta galactosidase activity was determined by the formation of the coloured product from X-gal (5-bromo, 4-chloro, 3-indolyl-beta-D-galactopyranoside) in the presence of IPTG (isopropyl-beta-thiogalactopyronoside).

Response of strain ESD to sulfur limitation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Single colonies of strain ESD were used to inoculate duplicate cultures of sulfur-free media containing 200 µm either magnesium sulphate, sodium sulfite, MOPS, DMSO, methionine, cysteine, glutathione, or sulfolane. Optical density (595 nm) was measured at approximately 4 h intervals through log-phase to determine doubling time. Doubling times in the presence of technical grade endosulfan or endosulfan isomers were determined in the same way as described above but in the presence of 50 µm insecticide. When cells reached stationary phase, 100 µl of culture was used to inoculate fresh sulfur-free medium containing 200 µm the same sulfur source plus 50 µm technical grade endosulfan. Cultures were grown to late-log phase (as determined by optical density) then assayed by TLC for the presence of endosulfan metabolites and loss of endosulfan in the growth medium.

The growth medium used in this study is buffered at pH 6·9 and includes detergent to solubilize the insecticide. Control experiments indicate that these measures minimize loss of the insecticide through chemical hydrolysis, adsorption to glass surfaces and volatilization (Sutherland et al. 2000). Endosulfan-degrading activity was defined into one of four categories according to the ratio of endosulfan and metabolites present in the culture of the stationary phase cells as determined by visual comparison of the intensity of spots on TLC plates. These categories were (i) no metabolites detected (endosulfan degrading activity −) (ii) intensity of metabolite spots detected less than intensity of endosulfan spot (endosulfan degrading activity +) (iii) intensity of metabolite spots detected approximately equivalent to intensity of endosulfan spot (endosulfan degrading activity + +), and (iv) no endosulfan detected (endosulfan degrading activity + + +).

Chemicals

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

The isomers of endosulfan were purchased from Chem. Services Inc. (West Chester, PA, USA). Technical grade endosulfan (99% pure mixture of approximately 70%alpha-endosulfan and 30%beta-endosulfan) for bacterial growth was a gift from Hoechst Schering AgrEvo Pty Ltd (Kwinana, WA, Australia). All other chemicals used were at least of reagent grade.

Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

A pure bacterial culture capable of degrading endosulfan was obtained from the mixed culture previously described in Sutherland et al. (2000), after six months successive subculture (approximately 30 rounds with 1% inoculum), followed by three rounds of subculturing using very dilute inocula (1% to 0·001% late-log phase culture). In each of the latter three rounds, the most dilute inoculum to subsequently exhibit growth also demonstrated endosulfan metabolism and was used as the starting culture for another round of dilution subculturing. Plating of the final culture onto sulfur-free agar media with endosulfan, or tryptic soy agar media, gave rise to slow growing translucent colonies that became easily visible after 3–4 d and reached 3 mm diameter after 6 d. Broth cultures of individual colonies degraded endosulfan to produce the endosulfan monoaldehyde, endosulfan hydroxyether, and endosulphate (Table 1), as described in Sutherland et al. (2000) for the parental mixed culture. This isolate was named strain ESD (Endosulfan Degrading).

Table 1.  Qualitative TLC analysis of endosulfan and metabolites present in cultures of Mycobacterium strain ESD grown in sulfur-free media containing technical grade endosulfan *
 Presence of endosulfan metabolite
Age of culturealpha-endosulfanbeta-endosulfanendosulphateputative monoaldehyde productendosulfan hydroxyetherendodiol
  • *

    Approximately 70%alpha-endosulfan/30%beta-endosulfan

  • Analysis of endosulfan metabolism was performed as described in the Materials and Methods. + + + + +, major product; + + + + , + + + , + +, intermediate product; +, minor product; −, no product detected.

At inoculation+ + + + ++ + +
Late-log (200 r.p.m)+ + + ++ ++ ++
Late-log (120 r.p.m)+ ++ + ++ + ++

Analysis of the 16S rRNA gene sequence of strain ESD revealed it to be within the genus Mycobacterium, most similar (98·3%) to Mycobacterium strain LB501T, which was described in a study of bacteria degrading polycyclic hydrocarbons (GenBank accession number AJ245702). A distance neighbour joining tree was constructed based on the comparison of related 16S rRNA gene sequences available on GenBank, which showed strain ESD clustered with the fast growing Mycobacteria (Fig. 1). This subgroup includes many other Mycobacterium species that have demonstrated xenobiotic-degrading activities (Fig. 1, Table 2).

image

Figure 1. Dendrogram showing the phylogenetic position of strain ESD based on 16S rRNA gene relatedness. Strains isolated or characterized because of xenobiotic-degrading activities are shown in bold. Sequences were aligned using the Pileup program of the Genetics Computer Group (Devereux et al. 1994) and the dendrogram was generated by the distance neighbour-joining method using paup (Swofford 1998). Numbers are percent bootstrap values after 1000 bootstrap replications. Only values above 50% are given. GenBank accession numbers are shown in brackets

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Table 2. Mycobacterium species isolated or characterized because of their xenobiotic-degrading activity that are related to strain ESD by 16S rRNA gene comparison
XenobioticStrain or speciesReference
  • *

    GenBank accession number.

Polycyclic aromatic hydrocarbonsLB501TAJ2450702*
RJG11·135, PYR-1Govindaswami et al. (1995)
BB1X81891*
C2-3AF165182*
M. hodleriKleespie et al. (1996)
3 and 4 ring aromatic  and aliphatic hydrocarbonsCH-1Churchill et al. (1999)
TolueneLAB2Juteau et al. (1999)
T103, T104Tay et al. (1998)
PentachlorophenolM. chlorophenolicumHaggeblom et al. (1994)
Fluoranthene/PyreneWF2, RF002U90877*, U90876*

Morphological properties

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Mycobacterium strain ESD is a Gram-positive rod that forms mostly rough, convoluted and some smoother, cream-coloured colonies after 3 d at 28°C on either tryptic soy agar, or sulfur-free medium with endosulfan. The ability to form more than one kind of colony is characteristic of many Mycobacterium strains (Wayne and Kubica 1986). Cells were nonmotile, did not form spores and tended to clump in broth media. Log-phase and stationary phase cells were not detectably acid-alcohol fast as determined using the Ziehl-Neelsen staining method.

Physiological and biochemical properties

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

No growth of strain ESD was observed in minimal medium without an added sulfur source but it did grow when a variety of different compounds were added as sole sources of sulfur (Table 3). Two exceptions for which no growth occurred were thioglycolate and sulfosalicylic acid, although control experiments demonstrated that these compounds were not toxic to strain ESD, when included in the medium in the presence of alternative sulfur sources. Sulfur sources were included in the minimal medium at a final concentration of 200 µm, except in the case of technical grade, alpha- or beta- endosulfan that were included only at 50 µm. The latter is still significantly higher than the solubility limits of these compounds in aqueous solutions (alpha-isomer, 0·32, beta-isomer, 0·33; Pesticide Manual 1994). The detergent Tween 80 was included in the endosulfan-containing medium at 0·05% (v/v; critical micelle concentration, 0·0016%), both to emulsify the insecticide (Sutherland et al. 2000) and to prevent it from partitioning to glass surfaces. For consistency, the detergent was included with all sulfur sources. Strain ESD, like many Mycobacteria species, can utilize Tween 80 as a carbon source (see Results below). Presumably as the bacteria metabolize the detergent they come into contact with endosulfan, facilitating metabolism of the insecticide.

Table 3.  Analysis of late-log phase Mycobacterium strain ESD cultures grown in sulfur-free medium in the presence of 50 µm endosulfan or with 50 µm endosulfan and 200 µm various other sulfur sources
  Doubling timeEndosulfan-degradingMetabolites detected
Sulfur source in mediaSulfur type(hours)activity*at (200rpm)at (120rpm)
  • *

    Analysis of endosulfan metabolism was performed as described in the Materials and methods. + + +, degradation of endosulfan to undetectable levels; + +, metabolites detected at levels approximately equal to levels of endosulfan; +, metabolites detected at levels less than levels of endosulfan; −, no metabolites detected.

  • EM, endosulfan monoaldehyde; EH, endosulfan hydroxyether, ESO4, endosulphate; the dominant products indicated in italicised boldface.

  • Approximately 70%alpha-endosulfan/30%beta-endosulfan.

  • §

    MOPS, 3-(N-morpholino)propane sulphonic acid.

  • DMSO, Dimethyl sulfoxide.

EM, EHE, ESO4    EM, EHE, ESO4
technical grade endosulfansulfite diester19·2+ + +EM, EHE, ESO4 
alpha-endosulfansulfite diester29·8+ + +EM, EHE, ESO4EM, EHE
beta-endosulfansulfite diester9·5+ + +EM, EHE, ESO4
MgSO4inorganic sulphate6·6
NaSO3inorganic sulfite11·6EM, EHE, ESO4
MOPS§sulphonic acid7·8+ +EM, EHE, ESO4EM, EHE, ESO4
DMSOsulfoxide8·0+ +EM, EHE, ESO4
methioninethiol ether8·4EM, EHE, ESO4
cysteinethiol8·3+ +EM, EHE, ESO4EM, EHE, ESO4
glutathionethiol8·7+ + +EM, EHE, ESO4ESO4
sulfolanesulphone7·8+ESO4 

When strain ESD was grown in sulfur-free media in the presence of 50 µm technical grade or beta-endosulfan, cell densities reached optical densities above 0·8 but did not increase further when additional sulphate was added to stationary phase cells, indicating that sulfur was not limiting growth. When grown in the presence of alpha- endosulfan at 200 r.p.m., cultures only grew to an optical density of 0·3 and then grew further (i.e. exceeded an optical density of 0·8) when 100 mm magnesium sulphate was added. Presumably the oxidation of alpha-endosulfan to form the quasi-terminal metabolite, endosulphate (Sutherland et al. 2000) acted as a sulfur sink and limited the amount of sulfur available for growth. At 120 rev min−1, cultures grown in the presence of alpha-endosulfan eventually exceeded an optical density of 0·8. Degradation in these cultures was less biased towards the oxidative pathway (Table 3).

Doubling times of Mycobacterium strain ESD were comparable when grown in minimal media with the various added sulfur sources except with alpha-endosulfan, where they were considerably longer (Table 3). Doubling times were faster in rich media (data not shown).

Growth of strain ESD in sulfur-free medium with endosulfan occurred at temperatures in the range of 22°C to 30°C, but growth was poor at 37°C, and no growth was detected at 16°C or 42°C. No growth occurred in the presence of 5% NaCl. Strain ESD was catalase positive, β-galactosidase negative and did not grow on MacConkey agar. Results from the Biolog Bacterial Identification System were inconclusive when performed as specified by the Biolog manual as all tests returned positive. According to the manufacturers directions this result is characteristic of many soil bacteria, which can store endogenous substrates leading to a high background level of respiration in the Biolog MicroPlates. The following substrates resulted in detectable respiration after strain ESD was incubated in sulfur-free broth containing 50 µm magnesium sulphate without glucose or Tween 80, for 4 h prior to incubation in the MicroPlates: Tween 80; Tween 40; l-arabinose; d-fructose; d-gluconic acid; α-D-glucose; maltose; d-mannitol; d-mannose; d-psicose; l-rhamnose; d-ribose; d-tagatose; turanose; xylitol; d-xylose; α- and β- hydroxybutyric acids; methyl pyruvate; methyl succinate; propionic acid; pyruvic acid; succinamic acid; alaninamide; l-alanyl-glycine, and glycerol.

Regulation of endosulfan-metabolizing activity

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

Mycobacterium strain ESD did not degrade endosulfan when sulphate, sulfite or methionine were included in the growth medium in addition to the insecticide (Table 3). Conversely, endosulfan metabolism was observed in medium when the insecticide was included in the presence of glutathione, 3-(N-mopholino) propane sulphonic acid (MOPS), dimethyl sulfoxide (DMSO), cysteine and sulfolane, albeit levels in these cultures were not as great as when either, or both, isomers of endosulfan were provided as the sole source of sulfur (Table 3). Decreasing the aeration of the cultures did not detectably alter rates of the disappearance of endosulfan from the medium but did significantly affect the ratio of endosulphate to the nonsulfur containing metabolites, endosulfan monoaldehyde and endosulfan hydroxyether such that levels of endosulphate were increased when the aeration of the culture was increased (Table 3).

When strain ESD was grown in the presence of both 20 µm magnesium sulphate and 50 µmbeta-endosulfan a biphasic growth pattern was observed (Fig. 2). An initial lag phase was followed by a period of exponential growth during which time thin layer chromatographic analysis indicated that no endosulfan metabolites accumulated in the culture medium (data not shown). This was followed by another lag phase then a second period of exponential growth at a reduced rate, during which time beta-endosulfan disappeared and the metabolites endosulfan hydroxyether and endosulfan monoaldehyde (described in Sutherland et al. 2000) appeared in the culture medium. Exponential growth rates were similar to those observed when magnesium sulphate or beta-endosulfan were provided as single sulfur sources (see doubling times, Table 3).

image

Figure 2. Growth of Mycobacterium strain ESD with limiting sulphate (20 µm) and beta-endosulfan (50 µm) as the only added sulfur sources in sulfur-free minimal medium. Straight lines (A, B) indicate approximate linear portions of exponential growth of the culture. The doubling time for growth of strain ESD in the presence of sulphate is 6·6 h and 9·5 h in the presence of beta-endosulfan. No growth was observed in the absence of added sulfur sources. Cultures were grown at 28°C and 200 r.p.m.

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Metabolism of endosulfan by other bacterial species

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

To our knowledge, endosulfan degradation has not been investigated previously in a sulfur-limited environment. To determine if the metabolism by strain ESD was part of a general response to sulfur limitation we tested the ability of Mycobacterium smegmatis strain mc2, Escherichia coli strain TG1, Pseudomonas aeruginosa strain RH, and Arthrobacter oxydans strain DSM 4044 to metabolize endosulfan under sulfur-starved conditions. Growth was observed by all four species in sulfur-free media with 50 µm added sulfite (the presumptive product of endosulfan hydrolysis) but not in media containing 50 µm endosulfan as the sole source of sulfur after 10 d (37°C, 200 r.p.m., data not shown). After more than 10 d, growth was observed at 37°C in all cultures. TLC analysis of sterile media containing endosulfan demonstrated an accumulation of endodiol after 10 d, indicating significant levels of chemical hydrolysis at this temperature. Presumably the sulfite released by this chemical degradation was being utilized for growth. After more than 10 d incubation low levels of endosulphate were detected in the M. smegmatis culture.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

To our knowledge this study is the first report of the enrichment and subsequent isolation of a pure bacterium able to metabolize the insecticide endosulfan. Using endosulfan as the only source of available sulfur we enriched soil inocula for microorganisms capable of releasing the sulfur from the insecticide, thereby providing a source of sulfur for growth. Initial attempts at isolation of the endosulfan-degrading bacterium from the enriched soil inoculum were hampered by the slow growth of the active organism with respect to the rest of the consortium of microorganisms, many of which formed obvious colonies on agar plates in 24 h. Repeated subculturing of the mixed culture led to an increase in numbers of the endosulfan-degrading strain, which eventually allowed the isolation of strain ESD.

Comparison of the 16S rRNA gene sequence of strain ESD with other 16S rRNA gene sequences in the GenBank database suggested strain ESD to be a member of the genus Mycobacterium, clustering with the rapid-growing Mycobacteria. Morphological and physical characteristics are consistent with this classification. For example, it is an aerobic, Gram-positive, nonspore forming, nonmotile rod. Cells did not appear to be acid-alcohol fast. Whilst Mycobacteria are generally acid-alcohol fast at some stages of growth, cells of rapid growers may be less than 10% acid fast and in some species this characteristic is partially or completely lost at some stages of growth (Wayne and Kubica 1986).

Bacteria of the Mycobacterium genus have extremely hydrophobic cell envelopes of exceptional thickness (Brennan and Nikaido 1995). The nature of this surface would favour contact with the hydrophobic endosulfan molecules, thereby increasing the availability of the insecticide for metabolism. Mycobacterium spp. are common in the environment (Barksdale and Kim 1977) and many have been reported to degrade a variety of environmentally hazardous chemicals, including volatile aliphatic, polyaromatic, and chlorinated hydrocarbons (Heitkamp and Cerniglia 1988; Kelly et al. 1991; Hartmans and deBont 1992; Haggeblom et al. 1994). Like endosulfan, these compounds are hydrophobic in nature and known to bind more strongly to soil with high organic matter content. The hydrophobic cell surfaces of Mycobacteria have been proposed to increase contact with the organic matter and therefore with the hydrophobic contaminant (Briglia et al. 1994). One study addressing this found adsorption capacity to be an inherent key parameter for degradation of pentachlorophenol by Mycobacterium chlorophenolicum (Brandt et al. 1999). In this regard, it is interesting that from a total of 20 soil samples that we have screened from different farms in the Narrabri district of NSW (Australia), we have only isolated two endosulfan-degrading bacteria. Analysis of the 16S rRNA gene of the second bacterium revealed it to be another Mycobacterium strain, with morphological and endosulfan-degrading characteristics indistinguishable from strain ESD. We have not characterized this second Mycobacterium sp. further.

Interestingly, both the sulfur-separation and the oxidative reactions of endosulfan by Mycobacterium strain ESD occur only in the absence of sulfite, sulphate or methionine. Similar regulation has been described in soil isolates with the ability to desulphonate a broad range of xenobiotic aromatic sulphonates (Zurrer et al. 1987; Dudley and Frost 1994; Kertesz et al. 1994a), and in Rhodococcus (Kayser et al. 1993) and Anthrobacter (Serbolisca et al. 1999) species with the ability to desulphonate dibenzothiophene. Synthesis of the responsible enzymes was strongly repressed in the presence of sulphate or cysteine (Kayser et al. 1993; Beil et al. 1996; Serbolisca et al. 1999).

It has been known for many years that bacteria can utilize numerous sources of sulfur (Roberts et al. 1955). These sulfur sources can be divided into two groups: compounds that lead to the expression of a set of proteins known as the ‘sulphate-starvation-induced stimulon’ (SSIS) phenotype, and those that suppress this phenotype (Kertesz et al. 1993; Quadroni et al. 1996; Van der Ploeg et al. 1996; Hummerjohann et al. 1998). Sulphate and other species-specific compounds are representative of the latter group, whilst many natural products and xenobiotics result in expression of the SSIS. The SSIS proteins are thought to play a role in scavenging alternative sulfur sources (Kertesz et al. 1994b) and in the natural environment may be critically important in the in situ degradation of sulfur-containing xenobiotics (Hummerjohann et al. 1998). The range of enzymes comprising the SSIS differs between species, although the regulatory mechanism controlling their expression appears similar across the small number of enteric and soil bacteria studied to date (Kertesz et al. 1993; Beil et al. 1996; Hummerjohann et al. 1998). The absence of endosulfan-degrading activity in the presence of sulphate and the biphasic utilization of sulphate then endosulfan as sulfur sources suggest that the endosulfan degradative activities observed in Mycobacterium strain ESD are part of the SSIS response in this strain.

In a previous study, Martens (1976) investigated the ability of 49 soil bacteria to degrade endosulfan in rich broth medium. Broth cultures of 15 of these species, including two Mycobacterium spp., showed some endosulfan degradation predominantly to endosulphate or endodiol after 10 d incubation. Endosulfan is sensitive to alkaline hydrolysis and as growth of the bacteria often raised the pH of the medium a substantial proportion of endodiol formation was attributed to chemical hydrolysis (Martens 1976). Aside from the alkalinity, it is difficult to compare the Martens (1976) study with the current one as the bacteria in the early study were grown in rich media containing ample sulphate. Under these conditions the endosulfan-degrading activities we observe in Mycobacterium strain ESD cultures would not be apparent. To our knowledge, endosulfan degradation by strain ESD is the first investigation of endosulfan metabolism in a sulfur-limited environment. As we do not observe endosulfan metabolism in M. smegmatis, E. coli, P. aeruginosa or A. oxydans under sulfur-limited conditions, we conclude that this is not a general SSIS activity.

Rates of degradation and low solubilities of hydrophobic xenobiotics often limit the growth rates of the bacteria utilizing the compounds (Thomas et al. 1986; Stucki and Alexander 1987; Volkering et al. 1992). This is not the case for technical grade or beta-endosulfan degradation by strain ESD. Estimations of rates of hydrolysis of the alpha-isomer are complicated by the competing oxidation reaction that serves as a sink for available sulfur. As doubling times were similar when technical grade or beta- endosulfan was the sole source of sulfur compared to growth in the presence of sulfite (the presumed product of endosulfan hydrolysis) we assume that endosulfan hydrolysis is not limiting growth. The slow growth rates of Mycobacteria are thought to be limited, in part, by the rate of their RNA polymerase (Harshey and Ramakrishnan 1977).

The requirement of the endosulfan-degrading activity of this strain for the absence of inorganic sulphate potentially restricts its usefulness in bioremediation of the insecticide. A similar problem has been addressed in studies of other sulfur containing xenobiotics. The operon encoding the metabolic pathway for dibenzothiophene desulfurization in Rhodococcus sp. IGTS8 is similarly sensitive to sulfur ion concentration (Piddington et al. 1995). Replacement of the native promotor with a constitutive one eliminated the dependence on very low sulfur concentrations for activity. With consideration of these results, we are attempting to clone the DNA encoding the enzyme(s) responsible for the initial step of endosulfan hydrolysis by Mycobacterium strain ESD.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References

We are grateful for the financial support of the Cotton Research and Development Corporation (CSE 77C), the Horticultural Research and Development Corporation (HG97340) and Orica Australia Pty Ltd. We thank Hoechst Schering AgrEvo Pty Ltd for providing technical grade endosulfan, Dr Billman-Jacobe from the University of Melbourne, Australia, for providing Mycobacterium smegmatis strain mc2, and Kahli Weir for 16S rRNA gene sequencing.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial culture
  6. Nucleic acid methods
  7. Characterization of strain ESD
  8. Response of strain ESD to sulfur limitation
  9. Chemicals
  10. GenBank accession number
  11. Results
  12. Isolation of a single bacterium that metabolizes endosulfan as a sole sulfur source
  13. Morphological properties
  14. Physiological and biochemical properties
  15. Regulation of endosulfan-metabolizing activity
  16. Metabolism of endosulfan by other bacterial species
  17. Discussion
  18. Acknowledgements
  19. References
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