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Endogenous stress caused by faulty oxidation reactions fosters evolution of 2,4-dinitrotoluene-degrading bacteria.

Pérez-Pantoja D, Nikel PI, Chavarría M, de Lorenzo V - PLoS Genet. (2013)

Bottom Line: DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth.Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation.It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.

View Article: PubMed Central - PubMed

Affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología, CSIC, Campus de Cantoblanco, Madrid, Spain.

ABSTRACT
Environmental strain Burkholderia sp. DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth. To investigate this lack of physiological return of such an otherwise complete metabolic route, cells were exposed to DNT under various growth conditions and the endogenous formation of reactive oxygen species (ROS) monitored in single bacteria. These tests revealed the buildup of a strong oxidative stress in the population exposed to DNT. By either curing the DNT plasmid or by overproducing the second activity of the biodegradation route (DntB) we could trace a large share of ROS production to the first reaction of the route, which is executed by the multicomponent dioxygenase encoded by the dntA gene cluster. Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation. That both the old and the new substrate brought about a considerable cellular stress was indicative of a still-evolving DntA enzyme which is neither optimal any longer for naphthalene nor entirely advantageous yet for growth of the host strain on DNT. We could associate endogenous production of ROS with likely error-prone repair mechanisms of DNA damage, and the ensuing stress-induced mutagenesis in cells exposed to DNT. It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.

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Accumulation and removal of 4-methyl-5-nitrocathecol (4M5NC) in Burkholderia sp. DNT and derivatives exposed to DNT.(A) Cultures of the wild-type strain were exposed to DNT at a final concentration of 0.5 mM for 3 h, and flasks were photographed to evidence the production of 4M5NC as the medium acquires a yellowish color. Chemical analyses revealed that the yellow metabolite, which has an absorption peak at 420 nm, corresponds to 4M5NC [14]. (B) Time-course evolution of 4M5NC (the structure of which is shown in the inset) in cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain amended with DNT at a final concentration of 0.5 mM. Determinations were carried out as previously described by de las Heras et al. [14]. Error bars represent SD (n = 4).
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pgen-1003764-g006: Accumulation and removal of 4-methyl-5-nitrocathecol (4M5NC) in Burkholderia sp. DNT and derivatives exposed to DNT.(A) Cultures of the wild-type strain were exposed to DNT at a final concentration of 0.5 mM for 3 h, and flasks were photographed to evidence the production of 4M5NC as the medium acquires a yellowish color. Chemical analyses revealed that the yellow metabolite, which has an absorption peak at 420 nm, corresponds to 4M5NC [14]. (B) Time-course evolution of 4M5NC (the structure of which is shown in the inset) in cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain amended with DNT at a final concentration of 0.5 mM. Determinations were carried out as previously described by de las Heras et al. [14]. Error bars represent SD (n = 4).

Mentions: Since the most critical move in any biodegradative route of aromatics is the initial step that activates the ring for overcoming the resonance energy that stabilizes their structure [31], we concentrated on the leading reaction that converts DNT to 4M5NC (Fig. 1A). This compound, as is the case for other catechols, is highly toxic [32], [33]. Furthermore, it is known [11] that such yellow-colored intermediate transiently accumulates in the medium prior to be channeled towards the next step of the degradation route (Fig. 6A). The toxic effect of DNT on Burkholderia sp. DNT could thus originate in either faulty reactions of DntA on its substrate, in accumulation of 4M5NC or in both. Our strategy to distinguish these possibilities was to generate a strain that overproduced DntB (Fig. 1A). This enzyme is a monooxygenase that eliminates the remaining nitro substituent of 4M5NC to produce 2-hydroxy-5-methylquinone (2H5MQ; [34], [35]). Higher levels of DntB are thus predicted to drain 4M5NC faster towards 2H5MQ -thereby allowing us to separate the intrinsic physiological effect of DntA action from that of the toxic catechol that results from the reaction. To this end, we generated strain Burkholderia sp. DNT dntB↑, in which extra copies of the gene encoding the second oxygenase of the pathway were expressed from a constitutive promoter in a broad-host-range vector. Fig. 6B verifies such a prediction, as cultures of Burkholderia sp. DNT dntB↑ released a much lower amount (<20%) of 4M5NC to the medium. In the same setup, strain Burkholderia sp. DNT Δdnt did not accumulate any intermediate, while naphthalene would be biotransformed to 1,2-dihydroxy-1,2-dihydronaphthalene by any of the dntA+ strains. Once we had the three isogenic variants in hand, we set out to measure intracellular ROS production in wild-type, Δdnt and dntB↑ cells exposed to either DNT or naphthalene (Fig. 5A, B and C, respectively). These results provide an answer to the questions raised above. First, that DNT causes high ROS levels in both the wild-type and dntB↑ strains (Fig. 5A and C) clearly indicates that the bulk of oxidative stress can be traced to the very reaction of DntA with DNT, not to the product of the catalysis. Second, the effects of naphthalene were comparatively lower than those elicited by DNT, but they were still significant in respect to the untreated control conditions. ROS generated from the encounter of this substrate with the dnt-encoded enzymes must stem from uncoupled, faulty reactions with the leading DntA oxygenase, as there is no other enzyme that can recognize this aromatic compound as a potential but ultimately non-productive substrate. Since both the ancestral and the new substrate of the first dioxygenase release ROS when facing DntA, the cognate reaction has probably left behind a former biochemical optimum (i.e., when acting on naphthalene) but has not yet reached a new one with DNT.


Endogenous stress caused by faulty oxidation reactions fosters evolution of 2,4-dinitrotoluene-degrading bacteria.

Pérez-Pantoja D, Nikel PI, Chavarría M, de Lorenzo V - PLoS Genet. (2013)

Accumulation and removal of 4-methyl-5-nitrocathecol (4M5NC) in Burkholderia sp. DNT and derivatives exposed to DNT.(A) Cultures of the wild-type strain were exposed to DNT at a final concentration of 0.5 mM for 3 h, and flasks were photographed to evidence the production of 4M5NC as the medium acquires a yellowish color. Chemical analyses revealed that the yellow metabolite, which has an absorption peak at 420 nm, corresponds to 4M5NC [14]. (B) Time-course evolution of 4M5NC (the structure of which is shown in the inset) in cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain amended with DNT at a final concentration of 0.5 mM. Determinations were carried out as previously described by de las Heras et al. [14]. Error bars represent SD (n = 4).
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Related In: Results  -  Collection

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pgen-1003764-g006: Accumulation and removal of 4-methyl-5-nitrocathecol (4M5NC) in Burkholderia sp. DNT and derivatives exposed to DNT.(A) Cultures of the wild-type strain were exposed to DNT at a final concentration of 0.5 mM for 3 h, and flasks were photographed to evidence the production of 4M5NC as the medium acquires a yellowish color. Chemical analyses revealed that the yellow metabolite, which has an absorption peak at 420 nm, corresponds to 4M5NC [14]. (B) Time-course evolution of 4M5NC (the structure of which is shown in the inset) in cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain amended with DNT at a final concentration of 0.5 mM. Determinations were carried out as previously described by de las Heras et al. [14]. Error bars represent SD (n = 4).
Mentions: Since the most critical move in any biodegradative route of aromatics is the initial step that activates the ring for overcoming the resonance energy that stabilizes their structure [31], we concentrated on the leading reaction that converts DNT to 4M5NC (Fig. 1A). This compound, as is the case for other catechols, is highly toxic [32], [33]. Furthermore, it is known [11] that such yellow-colored intermediate transiently accumulates in the medium prior to be channeled towards the next step of the degradation route (Fig. 6A). The toxic effect of DNT on Burkholderia sp. DNT could thus originate in either faulty reactions of DntA on its substrate, in accumulation of 4M5NC or in both. Our strategy to distinguish these possibilities was to generate a strain that overproduced DntB (Fig. 1A). This enzyme is a monooxygenase that eliminates the remaining nitro substituent of 4M5NC to produce 2-hydroxy-5-methylquinone (2H5MQ; [34], [35]). Higher levels of DntB are thus predicted to drain 4M5NC faster towards 2H5MQ -thereby allowing us to separate the intrinsic physiological effect of DntA action from that of the toxic catechol that results from the reaction. To this end, we generated strain Burkholderia sp. DNT dntB↑, in which extra copies of the gene encoding the second oxygenase of the pathway were expressed from a constitutive promoter in a broad-host-range vector. Fig. 6B verifies such a prediction, as cultures of Burkholderia sp. DNT dntB↑ released a much lower amount (<20%) of 4M5NC to the medium. In the same setup, strain Burkholderia sp. DNT Δdnt did not accumulate any intermediate, while naphthalene would be biotransformed to 1,2-dihydroxy-1,2-dihydronaphthalene by any of the dntA+ strains. Once we had the three isogenic variants in hand, we set out to measure intracellular ROS production in wild-type, Δdnt and dntB↑ cells exposed to either DNT or naphthalene (Fig. 5A, B and C, respectively). These results provide an answer to the questions raised above. First, that DNT causes high ROS levels in both the wild-type and dntB↑ strains (Fig. 5A and C) clearly indicates that the bulk of oxidative stress can be traced to the very reaction of DntA with DNT, not to the product of the catalysis. Second, the effects of naphthalene were comparatively lower than those elicited by DNT, but they were still significant in respect to the untreated control conditions. ROS generated from the encounter of this substrate with the dnt-encoded enzymes must stem from uncoupled, faulty reactions with the leading DntA oxygenase, as there is no other enzyme that can recognize this aromatic compound as a potential but ultimately non-productive substrate. Since both the ancestral and the new substrate of the first dioxygenase release ROS when facing DntA, the cognate reaction has probably left behind a former biochemical optimum (i.e., when acting on naphthalene) but has not yet reached a new one with DNT.

Bottom Line: DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth.Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation.It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.

View Article: PubMed Central - PubMed

Affiliation: Systems and Synthetic Biology Program, Centro Nacional de Biotecnología, CSIC, Campus de Cantoblanco, Madrid, Spain.

ABSTRACT
Environmental strain Burkholderia sp. DNT mineralizes the xenobiotic compound 2,4-dinitrotoluene (DNT) owing to the catabolic dnt genes borne by plasmid DNT, but the process fails to promote significant growth. To investigate this lack of physiological return of such an otherwise complete metabolic route, cells were exposed to DNT under various growth conditions and the endogenous formation of reactive oxygen species (ROS) monitored in single bacteria. These tests revealed the buildup of a strong oxidative stress in the population exposed to DNT. By either curing the DNT plasmid or by overproducing the second activity of the biodegradation route (DntB) we could trace a large share of ROS production to the first reaction of the route, which is executed by the multicomponent dioxygenase encoded by the dntA gene cluster. Naphthalene, the ancestral substrate of the dioxygenase from which DntA has evolved, also caused significant ROS formation. That both the old and the new substrate brought about a considerable cellular stress was indicative of a still-evolving DntA enzyme which is neither optimal any longer for naphthalene nor entirely advantageous yet for growth of the host strain on DNT. We could associate endogenous production of ROS with likely error-prone repair mechanisms of DNA damage, and the ensuing stress-induced mutagenesis in cells exposed to DNT. It is thus plausible that the evolutionary roadmap for biodegradation of xenobiotic compounds like DNT was largely elicited by mutagenic oxidative stress caused by faulty reactions of precursor enzymes with novel but structurally related substrates-to-be.

Show MeSH
Related in: MedlinePlus