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Applied and Environmental Microbiology, May 2006, p. 3524-3530, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3524-3530.2006

A Single Monooxygenase, Ese, Is Involved in the Metabolism of the Organochlorides Endosulfan and Endosulfate in an Arthrobacter sp.

Kahli M. Weir,^1,2,3* Tara D. Sutherland,¹ Irene Horne,¹ Robyn J. Russell,¹ and John G. Oakeshott^1,3

CSIRO, Entomology, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia,¹ Charles Sturt University, PO Box 588, Wagga Wagga, New South Wales 2678, Australia,² Cooperative Research Centre for Sustainable Rice Production, Yanco, New South Wales 2703, Australia³

Received 23 November 2005/ Accepted 16 February 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this paper we describe isolation of a bacterium capable of degrading both isomers of the organochloride insecticide endosulfan and its toxic metabolite, endosulfate. The bacterium was isolated from a soil microbial population that was enriched with continuous pressure to use endosulfate as the sole source of sulfur. Analysis of the 16S rRNA sequence of the bacterium indicated that it was an Arthrobacter species. The organochloride-degrading activity was not observed in the presence of sodium sulfite as an alternative sulfur source, suggesting that the activity was part of the sulfur starvation response of the strain. A gene, ese, encoding an enzyme capable of degrading both isomers of endosulfan and endosulfate was isolated from this bacterium. The enzyme belongs to the two-component flavin-dependent monooxygenase family whose members require reduced flavin for activity. Nuclear magnetic resonance analyses identified the metabolite of endosulfan as endosulfan monoalcohol and the metabolite of endosulfate as endosulfan hemisulfate. The ese gene was located in a cluster of 10 open reading frames encoding proteins with low levels of sulfur-containing amino acids. These open reading frames were organized into two apparent divergently orientated operons and a gene encoding a putative LysR-type transcriptional regulator. The operon not containing ese did contain a homologue whose product exhibited 62% amino acid identity to the ese-encoded protein.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The organochloride (OC) insecticide endosulfan (Fig. 1) was first released for commercial use in 1954 (19). A relatively reactive sulfur moiety in this compound results in a half-life that is shorter than those of other OCs, and because of this endosulfan is the only OC insecticide registered for use in many countries. Compared to other available insecticides, such as the synthetic pyrethroids, endosulfan has low toxicity for many beneficial insects, mites, and spiders (12). It is therefore important in the management of pest species. However, endosulfan is extremely toxic to fish and aquatic invertebrates, and this has led to an interest in postapplication detoxification of this insecticide (27-30).

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FIG. 1. Compound structures and proposed pathway for endosulfan and endosulfate degradation by Arthrobacter sp. strain KW. Brackets indicate hypothetical compounds. The dashed line indicates a proposed pathway for which enzymes have not been identified yet. FMNH2, reduced flavin mononucleotide.

Commercial endosulfan is a mixture of two diastereoisomers (approximately 30% ß-endosulfan and 70% -endosulfan) which differ dramatically in their physicochemical and environmental properties (31). The environmental dispersion pathways include hydrolysis of the sulfur moiety to nontoxic endosulfan diol or oxidation to endosulfate. Endosulfate is more persistent than and as toxic as the parent isomers and thus poses a significant environmental problem (12).

Our laboratory is interested in isolating and characterizing bacteria and genes involved in detoxification of OC insecticides, particularly endosulfan and endosulfate, for the development of bioremediation technologies. We recently described Esd, an enzyme from a soil Mycobacterium species that degraded ß-endosulfan (27, 29, 30). Esd is a member of the two-component flavin-dependent monooxygenase (TC-FDM) family of enzymes that require reduced flavin supplied by an NAD(P)H-dependent flavin reductase. The Esd monooxygenase exhibited no activity with the -isomer of endosulfan or endosulfate (Fig. 1).

In this report we describe isolation and characterization of a gene, ese, from a soil bacterium that encodes an enzyme (Ese) capable of degrading both isomers of endosulfan, as well as endosulfate. Ese is a monooxygenase that requires reduced flavin mononucleotide as a cosubstrate for activity and can degrade endosulfate to endosulfan hemisulfate and the isomers of endosulfan to endosulfan monoalcohol (Fig. 1). The sequence of Ese is related to the sequence of Esd, albeit many closer, uncharacterized relatives were identified in other species. Ese is most similar to the product of a gene in a divergently orientated operon adjacent to the operon containing ese.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and growth conditions.
Bacterial strains and plasmids used in this study are described in Table 1. The sulfur-free medium (SFM) used for culture of endosulfan-degrading bacteria was the medium described by Sutherland et al. (27). The other media used in this study include Luria broth (LB) (25), low-salt LB (LB with 0.5 g · liter^–1 NaCl), and LB with 0.05% Tween 80. Ampicillin, hygromycin, and rifampin at a concentration of 100 µg · ml^–1 and kanamycin at a concentration of 25 µg · ml^–1 were included as required. OC compounds (99% pure; Sigma Diagnostics, St. Louis, MO) were prepared as 50 mM stock solutions in acetone and added to the culture medium at a concentration of 50 µM.

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TABLE 1. Nonstandard bacterial strains, DNA vectors, and constructs used in this study

Isolation and identification of soil bacteria.
A pure bacterium (strain KW) capable of degrading endosulfan and endosulfate was isolated from the mixed microbial culture of Sutherland et al. (28); the mixed culture was plated on low-salt LB agar, and then 35 colonies were screened for endosulfate-degrading ability in SFM with endosulfate, using thin-layer chromatography (TLC) as previously described (27). The 16S rRNA gene of strain KW was amplified by PCR from genomic DNA using the universal 27f and 1492r 16S rRNA gene primers of Lane (18) and the method of Bond et al. (3). Genomic DNA was extracted from strain KW using a method adapted from the method of Anderberg et al. (2), as described by Sutherland et al. (29).

Identification of metabolites.
The organic extractable metabolites resulting from OC degradation were characterized by TLC and gas chromatography (GC)-mass spectrometry (MS) as previously described (27). The endosulfan and polar endosulfate metabolites were examined by proton nuclear magnetic resonance (NMR) by Baseline Separation Technologies (Bulleen, Victoria, Australia), using a Bruker AC200 spectrometer. The endosulfate polar metabolite was produced in cultures of Mycobacterium smegmatis expressing ese in low-salt LB supplemented with endosulfate and then was purified using Oasis HLB reversed-phase chromatography cartridges (3 ml/60 mg; Waters, Rydalmere, NSW, Australia). After a cartridge was conditioned, 5 ml culture was added, the cartridge was washed, and the metabolite was eluted in 100% methanol. The endosulfan metabolite was extracted with ethyl acetate and purified after TLC separation. NMR spectra for the endosulfate metabolite were collected in methanol-d₄, and spectra for the endosulfan metabolite were collected in CDCl₃.

Cloning of the ese gene from genomic DNA.
A cosmid library (insert size, 30 to 40 kb) of strain KW was prepared after partial digestion of the genomic DNA with Sau3AI restriction endonuclease and ligation of the fragments into a BamHI-digested Mycobacterium-Escherichia coli shuttle vector (pYUB415). Ligated DNA was packaged into MaxPlax packaging extracts (Epicenter Biotechnologies, Madison, WI) and used to infect E. coli strain EPI305 cells, according to the manufacturer's instructions. Cosmid DNA was prepared using an Ultraclean mini plasmid prep kit (MoBio, Carlsbad, CA). The cosmid library was screened in M. smegmatis, as described by Sutherland et al. (30), for clones that conferred the ability to degrade -endosulfan. Concurrently, this library was also screened for clones that hybridized to the esd gene of Mycobacterium strain ESD (30). The esd probe was labeled using a Gigaprime oligonucleotide labeling kit (Gene Works, South Australia, Australia). Dot blot hybridization of cosmid DNA was performed overnight at 55°C, followed by low-stringency washes, as described by Sambrook et al. (25).

Construction and recomplementation of an insertional mutation in ese.
To validate the importance of ese in OC degradation, we disrupted the gene in strain KW. A 561-bp PstI and EcoRI internal fragment of ese was ligated into pJP5603 (23), an R6K-based suicide vector, to produce pJP5603-ese^int. This plasmid was mobilized from E. coli strain S17-1 pir (donor) into a rifampin-resistant isolate of strain KW (Arthrobacter sp. strain KW Rif^r; recipient) by plate conjugation, as described by Bonnett et al. (4). Exconjugants were selected on low-salt LB containing rifampin and kanamycin, confirmed by PCR analysis (data not shown), and shown by TLC to be unable to metabolize endosulfan or endosulfate in SFM. Recomplementation of ese in the disrupted Arthrobacter strain was performed after the full-length ese gene was cloned into the Mycobacterium expression vector pMV261 to produce pMV261-ese. The construct was transferred as a cointegrate on the replicative plasmid pR751::Tn813 by plate conjugation (4).

Expression of ese in a heterologous host.
The expression construct pMV261-ese was transformed into M. smegmatis electrocompetent cells as described by Sutherland et al. (30). Colonies were inoculated from plates into LB with 0.05% Tween 80 or SFM supplemented with either the - or ß-isomer of endosulfan, endosulfate, or other OC insecticides and grown at 28°C for 48 h. TLC analysis was used to monitor OC degradation.

Activity of Ese in cell extracts.
The ability of Ese to degrade endosulfan and endosulfate in cell extracts was measured after expression in E. coli. The ese open reading frame (ORF) was cloned into pET14b to produce pET14b-ese. Protein expression was performed at 25°C overnight without isopropyl-ß-D-thiogalactopyranoside (IPTG) induction, conditions that led to optimal expression of soluble protein. The cells were washed in 50 mM HEPES buffer (pH 6.9), resuspended in the same buffer, and lysed by sonication. OC degradation was measured in a reaction mixture as previously described (30). The mixture typically contained 20 µg protein · ml^–1 of cell extract and 500 µM OC substrate. Approximately 12 µg · ml^–1 of Mycobacterium flavin reductase (specific activity, 463 ± 40 µmol · min^–1 · µg^–1), expressed using the pET14b-MsFR construct as previously described (30), was included to provide reduced flavin. Quantitative data were obtained by GC analysis as described by Sutherland et al. (30), except that the herbicide thiobencarb was used as the internal standard.

Other assays.
To investigate the pathway of endosulfate metabolism in strain KW, we examined the ability of sulfatase to metabolize the polar endosulfate metabolite. Sulfatase assays with mixtures containing 50 µg of the polar endosulfate metabolite were performed with 100 µg sulfatase (type H-1; product no. S9626; Sigma Diagnostics, St. Louis, MO) in 3 ml of 50 mM HEPES buffer (pH 6.9) at 28°C. After overnight incubation, reaction samples were analyzed by TLC. No degradation of substrate in the absence of sulfatase was observed.

Endogenous sulfatase activity in cell extracts of M. smegmatis expressing ese grown with glutathione (200 µM) as the sole sulfur source was measured by resuspending recombinant cells in 50 mM HEPES buffer (pH 6.9), lysing the cells by sonication, and measuring the formation of 4-methylumbelliferone from 4-methylumbelliferyl sulfate with an excitation wavelength of 355 nm and an emission wavelength of 460 nm, using a PolarStar fluorometer with the FLUOimage software (BMG Lab Technologies, Offenburg, Germany) over a 30-min period.

Genomic organization and sequence comparisons.
The entire length of cosmid 3.1.6 was sequenced by the Australian Genome Research Facility (Brisbane, Queensland, Australia). Analyses of the nucleotide and translated amino acid sequences were carried out using the Fast Alignment Search Tool (FASTA) (22) and Basic Local Alignment Search Tool (TBLASTN and BLASTN) (1) programs (default settings).

A multiple-sequence alignment of protein sequences that exhibited similarity to Ese was performed using PILEUP (7) (default settings), and distance data sets were calculated by using the PROTdist and NEIGHBOR (24) algorithms. Phylogenetic analyses were performed with PHYLIP (10), and 100 bootstrap replications were carried out with SEQBOOT (9).

Nucleotide sequence accession numbers.
The nucleotide sequences reported in this paper have been deposited in the GenBank database under accession numbers DQ124296 (ese and adjacent ORFs described here) and AY913770 (16S rRNA).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and characterization of a soil bacterium that degrades endosulfan and endosulfate.
A bacterium (strain KW) capable of degrading -endosulfan, ß-endosulfan, and endosulfate was isolated from the mixed microbial culture of Sutherland et al. (28), which was grown under continuous pressure to use endosulfate as the sole source of sulfur. Growth was indicated by an increase in the optical density at 595 nm of more than 0.35 over 4 days. Growth was not observed in sulfur-free medium alone. Concomitant with growth in the endosulfan- and endosulfate-supplemented medium there was a reduction in the abundance of these compounds and previously described metabolites appeared (28) (Fig. 1). In the presence of 50 µM NaSO₃ and endosulfan or endosulfate, no metabolism of the OC compounds was observed. As OC degradation was restored after subsequent culturing in medium with endosulfan or endosulfate as the sole sulfur source, the absence of activity was not due to loss of the relevant genetic material.

Sequence analysis of the 16S rRNA gene of strain KW suggested that this strain fell in a radiation of Arthrobacter species most closely related to Arthrobacter pascens (98.4%), A. ramous (98.4%), A. crystallopoietes (96.7%), and A. histidiinolovorans (95.9%). The 1.6% difference in the 16S rRNA sequence from its nearest known relatives suggests that strain KW may be a separate species, and we have provisionally named this organism Arthrobacter sp. strain KW.

Identification of endosulfan and endosulfate metabolites observed in Arthrobacter sp. strain KW.
The metabolites found after degradation of endosulfan and endosulfate by Arthrobacter sp. strain KW were characterized by TLC and GC-MS analysis. One metabolite was observed after degradation of both - and ß-endosulfan. The mobility of this metabolite after TLC, the GC retention times, and the mass spectra were identical to those of the endosulfan metabolite produced by Mycobacterium sp. strain ESD and by the mixed soil culture from which strain KW was isolated (27, 28). As the metabolite accumulated in the bacterial growth medium, we were able to purify sufficient quantities for proton NMR analysis. This compound was identified as endosulfan monoalcohol (Fig. 1 and Table 2) rather than the putative endosulfan monoaldehyde structure previously proposed after GC-MS analysis (27).

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TABLE 2. NMR data and structures of endosulfan and endosulfate metabolites

Three metabolites of endosulfate were observed when Arthrobacter sp. strain KW was grown with endosulfate as the sole sulfur source. Their mobilities in two different TLC systems, their GC retention times, and their mass spectra suggest that these metabolites were endosulfan monoalcohol, endosulfan dimethylene, and endosulfan hydroxymethylene, the three metabolites found in the mixed soil microbial culture from which Arthrobacter sp. strain KW was isolated (28).

All of the endosulfan added to Arthrobacter sp. strain KW cultures at 28°C was metabolized within 4 days, and there was a concurrent increase in the cell density equivalent to that observed in the presence of equivalent molar amounts of NaSO₃. Endosulfan monoalcohol accumulated in the medium and did not appear to be metabolized further. Similarly, endosulfate was completely degraded; endosulfan monoalcohol and endosulfan dimethylene appeared concurrently in the culture, and endosulfan hydroxymethylene was formed later. Again, the endosulfan monoalcohol metabolite accumulated in the culture and apparently was not degraded further.

Isolation of a gene encoding an endosulfan- and endosulfate-degrading protein.
An Arthrobacter sp. strain KW genomic DNA cosmid library (384 clones) was screened by using pools of eight clones for the ability to confer -endosulfan-degrading activity on M. smegmatis. TLC analysis identified a single pool of cosmids (3.1.1 to 3.1.8) that resulted in the formation of a faint band with the same R_f as endosulfan monoalcohol. Parallel to this functional screening, the cosmid library was screened by low-stringency hybridization to esd from Mycobacterium sp. strain ESD. Since Mycobacterium sp. strain ESD (27) and Arthrobacter sp. strain KW produced apparently identical metabolites from endosulfan, the possibility that similar genetic systems might be responsible was considered. A single cosmid (3.1.6) that was obtained from the same pool of clones that had been isolated in the functional screening analysis was independently isolated from this screening.

Hybridization and sequence analysis of subclones of cosmid 3.1.6 resulted in identification of two ORFs which hybridized to esd. One of these ORFs was incomplete and truncated in the cosmid (orf1-trunc; 1.5-kb fragment). The other, ese, encoded a 476-amino-acid protein (Ese). Ese exhibited the highest level of identity to the protein encoded by orf1-trunc (62% identity over 375 residues). Significant levels of sequence identity were also observed for Esd (34.3%), the NtaA nitrilotriacetate monooxygenase (33.9%) of Chelatobacter heintzii (16), and the dibenzothiophene-degrading monooxygenase DszA (33.3%) from Rhodococcus strain IGTS8 (6). Thus, Ese exhibited sequence homology with other TC-FDMs that use reduced flavin as a cosubstrate for activity. The similarities of Ese to the proteins mentioned above extended along its full length, although Ese was between 20 and 60 amino acids longer than the other proteins and had no major insertional differences.

Analysis of OC-degrading activity of Ese.
A mutant with an ese insertional inactivation mutation did not degrade either endosulfan or endosulfate, indicating the importance of this gene and/or downstream genes in the metabolism of these OCs. Expression of ese behind a constitutive promoter (pMV261-ese) conferred the ability to degrade both isomers of endosulfan and endosulfate to M. smegmatis, thereby confirming the involvement of ese in endosulfan degradation. No degradation of dieldrin, chlordane, or heptachlor epoxide was detected.

Culturing recombinant M. smegmatis expressing ese in rich media led to the disappearance of endosulfate without concurrent formation of any metabolites visible by TLC. The rapid disappearance of endosulfate from the culture suggested that this compound was metabolized to a polar metabolite that was not extracted by ethyl acetate. We purified a polar metabolite of endosulfate by reverse-phase chromatography and identified a single isotope mass at 419.7 by liquid chromatography-MS analysis (average mass, 422.92). Subsequent NMR analysis identified this metabolite as endosulfan hemisulfate (Fig. 1 and Table 2).

Interestingly, when M. smegmatis expressing ese was grown with endosulfate as the sole sulfur source (i.e., under sulfur-limiting conditions), the metabolites endosulfan monoalcohol and endosulfan dimethylene accumulated in the culture medium. Under similar conditions, in Arthrobacter sp. strain KW cultures only endosulfan monoalcohol accumulated. In sulfur-rich conditions (SFM supplemented with inorganic sulfur) the recombinant M. smegmatis cultures did not produce detectable levels of endosulfate metabolites. The aryl sulfatase activity in lysed cells was high when the bacteria were cultured under sulfur-limited conditions, but it was below the level of detection of our assay under sulfur-rich conditions (data not shown). All this suggests that a sulfatase capable of desulfurizing endosulfan hemisulfate was induced under sulfur-limiting conditions. To confirm this, we incubated endosulfan hemisulfate with sulfatase from Helix pomatia, and we observed accumulation of endosulfan monoalcohol that mimicked the accumulation of this metabolite under sulfur-limiting conditions in recombinant M. smegmatis cultures.

Expression of Ese in E. coli yielded no endosulfan- or endosulfate-degrading activity in vivo. In the presence of flavin reductase, NADH, and flavin mononucleotide, Ese isolated from E. coli catalyzed the metabolism of both isomers of endosulfan, yielding endosulfan monoalcohol. The -isomer of endosulfan was metabolized more rapidly (specific activity, 0.13 ± 0.05 µmol · mg crude cell protein) than the ß-isomer (0.04 ± 0.02 µmol · mg crude cell protein). Endosulfate was metabolized at an intermediate rate (0.09 ± 0.02 µmol · mg crude cell protein).

ese gene is located in a cluster of ORFs encoding low-sulfur proteins.
Sulfur-regulated genes and genes encoding members of the TC-FDM family are generally found in operons (6, 15, 17, 32, 33). Sequencing around the ese gene in cosmid 3.1.6 confirmed that this gene is located in a cluster of 10 ORFs that encode low-sulfur proteins. This cluster was organized into two apparent operons that were 252 bp apart, were divergently oriented, and were flanked by a gene encoding a putative LysR-type transcriptional regulator (Fig. 2). One operon encoded a putative ABC transporter system comprising a substrate-binding protein, two permease proteins, and an ATP-binding protein and was followed by the incomplete orf1-trunc. The other operon contained ese, followed by four additional ORFs. The two genes directly upstream of ese encoded proteins with sequence similarity to putative oxidoreductases, and the other two ORFs had no known homologues in the GenBank database. Downstream (176 bp) from this operon was the divergently transcribed LysR-type transcriptional regulator gene.

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FIG. 2. Genetic organization and identification of ORFs in the vicinity of ese. The lengths and directions of arrows indicate the proportional lengths and directions of transcription of the corresponding genes. The putative identities of ORF products suggested from the amino acid sequence identitities to proteins in the GenBank database are as follows: 1, orf1-trunc, flavin-dependent monooxygenase; 2, ABC transporter (ATP binding protein); 3, ABC transporter (permease protein); 4, ABC transporter (permease protein); 5, ABC transporter (substrate binding protein); 6, Ese; 7, flavin mononucleotide-dependent oxidoreductase; 8, oxidoreductase; 9 and 10, no sequence homologues; and 11, LysR transcriptional regulator.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this paper we describe an enzyme capable of degrading both isomers of endosulfan and the endosulfan metabolite, endosulfate, which we isolated from an Arthrobacter strain with the same activities. The sequence and cofactor requirements of this enzyme indicate that it belongs to the ubiquitous TC-FDM family. The enzymes in this family comprise a single subunit that catalyzes oxidation of a flavin cosubstrate to provide reducing equivalents for the oxygenation of the substrate by molecular oxygen.

A search for proteins with similarity to Ese in the GenBank database (identity, >26.5%) revealed at least 50 homologues in gram-positive and -negative bacteria, as well as seven fungal species (data not shown). No homologues were identified in higher eukaryotes, plants, or archaea. The phylogenetic relatedness of the Ese homologues was often not consistent with the phylogenetic relationship of the host genomes, suggesting that there were a number of horizontal gene transfer processes among distantly related bacteria. However, all Ese homologues identified in fungi fell in the same radiation, which lacked any bacterial representation, suggesting that there was a single common ancestor and that there was minimal horizontal transfer between the bacteria and fungi. Esd, an enzyme that degrades ß-endosulfan (30), fell in a phylogenetic lineage distantly related to Ese, suggesting that its endosulfan-degrading activity may have evolved independently.

Ese acts on both isomers of endosulfan and endosulfate, and the oxidation state of the sulfur atom of the substrate appears to have little influence on rate of catalysis (endosulfate and -endosulfan are degraded at similar rates). The stereochemistry of the substrate has more influence, and the turnover rates of the ß-isomer are 1 order of magnitude less than those of the -isomer. We propose that Ese catalyzes the hydroxylation of one of the methylene groups adjacent to the sulfur esters (Fig. 1), similar to the hydroxylation adjacent to a nitrogen (16, 21, 34, 35), sulfur (8, 15, 20), or double-bonded carbon (11, 14, 36) described for other TC-FDMs. In the case of endosulfate, hydroxylation results in an unstable intermediate that spontaneously generates a dehydration reaction at one of the methylene groups, followed by bond cleavage to generate endosulfan hemisulfate. We did not detect endosulfan hemisulfite, the metabolite of endosulfan expected under this scenario, and we assumed that the sulfite moiety is rapidly released to form endosulfan monoalcohol. Subsequent enzymatic or spontaneous desulfurization results in the formation of either endosulfan dimethylene or endosulfan monoalcohol (Fig. 1). While endosulfan hydroxymethylene is found in Arthrobacter sp. strain KW cultures, it is not observed in ese-expressing M. smegmatis cultures or in Ese cell-free assays, suggesting that Ese is not directly involved in the production of this metabolite.

Under the control of its native promoter, the activity of the ese product was observed only under sulfur-limited conditions. Genes expressed in response to sulfur starvation are generally organized into operons to allow coordinate regulation under sulfur limitation conditions, and they generally encode proteins with low sulfur contents. Ese contains only five sulfur-containing amino acids (1.05%), and indeed, all five ORFs in the putative operon encode proteins with low sulfur contents (1.98% ± 0.69% [mean ± standard deviation]). Furthermore, this operon is located between another operon and a divergently oriented transcriptional regulator that also encode proteins with low sulfur contents (1.7% ± 0.67% [mean ± standard deviation]). In contrast, we calculated that the average sulfur content of the predicted proteins encoded in cosmid 3.1.6 not in the vicinity of ese is 3.1% (standard deviation, 1.8%), in close agreement with the 3.1% ± 1.3% that we found for the proteomes of 31 bacteria with sequenced genomes from various habitats.

Characteristic of TC-FDM enzymes is the use of reduced flavin as a cosubstrate. This cosubstrate is provided by a separate flavin reductase, such as that encoded by the gene located immediately downstream of ese (Fig. 2). The endosulfan-degrading activity of strain KW was removed by an ese insertional mutation, which also inactivated the downstream gene. We were unable to restore activity by reintroducing ese, suggesting that the downstream gene product is required to provide reduced flavin for Ese.

In contrast to most other operons encoding TC-FDMs, the ese gene was not genetically associated with genes encoding transporters. TC-FDMs are cytosolic enzymes that generally rely on ABC-type transporters to transport their substrates into the cytosol. When expressed behind a constitutive promoter in M. smegmatis, endosulfan degradation occurs in rich medium, suggesting that sulfur-regulated genes are not required to transport the compound into the cell to the cytosolic monooxygenase. This is perhaps not surprising given that the substrate is not charged at physiological pH. The dsz operon, which is responsible for desulfination of the uncharged compound dibenzothiophene, also does not encode transporters (13, 20).

Ese exhibited the highest level of sequence similarity to the truncated translation product of orf1-trunc (62% identity over 375 amino acids) in a comparison with other proteins in the GenBank database. A neighbor-joining tree showed that the orf1-trunc truncated product is the closest relative of Ese, suggesting that there was a relatively recent common ancestor. The full sequence of orf1-trunc and the substrate activities of its enzyme product are of particular interest in determining the molecular basis for the evolution of the endosulfan- and endosulfate-metabolizing ability of Ese.

 
We are grateful for the financial support of the Cooperative Research Centre for Sustainable Rice Production (project 1304), the Horticultural Research and Development Corporation (grant HG97340), and Orica Australia Pty. Ltd.

We thank Michael J. Lacey (CSIRO Entomology) for discussions on metabolites and pathways, Sue Dorrian for technical assistance, the Australian Genome Research Facility (University of Queensland, Brisbane, Australia) for sequencing cosmid 3.1.6, and Baseline Separation Technologies (Bullen, Victoria, Australia) for NMR analysis.

 
* Corresponding author. Mailing address: CSIRO Entomology, GPO Box 1700, Canberra, ACT 2601, Australia. Phone: 61 2 6246 4244. Fax: 61 2 6246 4173. E-mail: kahli.weir{at}csiro.au.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, May 2006, p. 3524-3530, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3524-3530.2006

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