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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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Superoxide production in Burkholderia sp. DNT upon exposure to DNT and naphthalene.Cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain were exposed to either DNT or naphthalene (Naph) at a final concentration of 0.5 mM for 3 h. Superoxide levels were determined in vitro by treatment of the cells with NBT, and the results were normalized to control conditions (in which cells were added with DMSO, the DNT and naphthalene solvent carrier). Different lowercase letters identify significant differences within treatments at P<0.05 (ANOVA). Error bars represent SD (n = 4).
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pgen-1003764-g007: Superoxide production in Burkholderia sp. DNT upon exposure to DNT and naphthalene.Cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain were exposed to either DNT or naphthalene (Naph) at a final concentration of 0.5 mM for 3 h. Superoxide levels were determined in vitro by treatment of the cells with NBT, and the results were normalized to control conditions (in which cells were added with DMSO, the DNT and naphthalene solvent carrier). Different lowercase letters identify significant differences within treatments at P<0.05 (ANOVA). Error bars represent SD (n = 4).

Mentions: In a further effort to identify the specific types of ROS that result from the above mentioned processes, we tested cells treated with the various substrates with nitro blue tetrazolium (NBT), a reagent that is specific for superoxide production. Consistently with the flow cytometry data above, the highest indications of superoxide presence were found in wild-type and dntB↑ cells exposed to DNT, while a lower level was detected in the Δdnt strain and in bacteria exposed to naphthalene (Fig. 7). Since superoxide originated in defective redox reactions promotes hydroxyl-radical formation and consequent DNA damage [36], [37] we wondered whether ROS stemming from the faulty oxygenations discussed above could eventually translate into an insult to the genome of Burkholderia sp. DNT. One direct way of quantifying such damage is the measurement of the 8-hydroxy-2′-deoxyguanosine (8-oxoG) content of genomic DNA as a coarse descriptor of HO• attack to purines [37], [38]. On this background we resorted to an immunoassay for quantifying 8-oxoG levels in cells exposed to DNT, using H2O2 as a positive control of oxidative stress. As shown in Fig. 8A, DNT indeed caused a 1.4-fold increase in the share of damaged purines in the Burkholderia sp. DNT genome (as opposed to a 2-fold increase elicited by H2O2). As such a chemical damage to DNA bases triggers the SOS response and eventually increases the mutation rate of the bacteria that undergo the insult [36], we next wondered whether the ultimate consequence of the uncoupling of the ring-hydroxylating reaction performed by DntA with their current and ancestral substrates was to increase genetic diversity by enhancing mutation rates. Since virtually nothing is known about the SOS response in Burkholderia sp. DNT, we directly measured such mutation rates as a descriptor of emerging genetic novelty. For this, we employed the standard test of appearance of rifampicin-resistant (RifR) clones under the various conditions assayed. As shown in Fig. 8B, both naphthalene and DNT triggered a considerable increase in the appearance of RifR colonies which was not noticeable in the strain deleted of the dnt genes The mutagenic effect of naphthalene in this context was slightly more pronounced than what could be expected from the sheer data on ROS production shown above. We speculate that ROS could react chemically with this bicyclic aromatic compound and generate additional DNA-intercalating agents (e.g. naphtoquinones and naphtodiols [39]) with a separate mutagenic action on DNA. In any case, it is worth noticing that maximum novelty (as measured with this procedure) is accompanied by an acute lethality (Fig. 2), so that the survivors to DNT exposure are more capable to explore the possible solution space to the next adaptive challenge (metabolic or otherwise) than those which had not been diversified because of the phenomena described here.


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)

Superoxide production in Burkholderia sp. DNT upon exposure to DNT and naphthalene.Cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain were exposed to either DNT or naphthalene (Naph) at a final concentration of 0.5 mM for 3 h. Superoxide levels were determined in vitro by treatment of the cells with NBT, and the results were normalized to control conditions (in which cells were added with DMSO, the DNT and naphthalene solvent carrier). Different lowercase letters identify significant differences within treatments at P<0.05 (ANOVA). Error bars represent SD (n = 4).
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Related In: Results  -  Collection

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pgen-1003764-g007: Superoxide production in Burkholderia sp. DNT upon exposure to DNT and naphthalene.Cultures of the wild-type strain, the Δdnt strain and the dntB↑ strain were exposed to either DNT or naphthalene (Naph) at a final concentration of 0.5 mM for 3 h. Superoxide levels were determined in vitro by treatment of the cells with NBT, and the results were normalized to control conditions (in which cells were added with DMSO, the DNT and naphthalene solvent carrier). Different lowercase letters identify significant differences within treatments at P<0.05 (ANOVA). Error bars represent SD (n = 4).
Mentions: In a further effort to identify the specific types of ROS that result from the above mentioned processes, we tested cells treated with the various substrates with nitro blue tetrazolium (NBT), a reagent that is specific for superoxide production. Consistently with the flow cytometry data above, the highest indications of superoxide presence were found in wild-type and dntB↑ cells exposed to DNT, while a lower level was detected in the Δdnt strain and in bacteria exposed to naphthalene (Fig. 7). Since superoxide originated in defective redox reactions promotes hydroxyl-radical formation and consequent DNA damage [36], [37] we wondered whether ROS stemming from the faulty oxygenations discussed above could eventually translate into an insult to the genome of Burkholderia sp. DNT. One direct way of quantifying such damage is the measurement of the 8-hydroxy-2′-deoxyguanosine (8-oxoG) content of genomic DNA as a coarse descriptor of HO• attack to purines [37], [38]. On this background we resorted to an immunoassay for quantifying 8-oxoG levels in cells exposed to DNT, using H2O2 as a positive control of oxidative stress. As shown in Fig. 8A, DNT indeed caused a 1.4-fold increase in the share of damaged purines in the Burkholderia sp. DNT genome (as opposed to a 2-fold increase elicited by H2O2). As such a chemical damage to DNA bases triggers the SOS response and eventually increases the mutation rate of the bacteria that undergo the insult [36], we next wondered whether the ultimate consequence of the uncoupling of the ring-hydroxylating reaction performed by DntA with their current and ancestral substrates was to increase genetic diversity by enhancing mutation rates. Since virtually nothing is known about the SOS response in Burkholderia sp. DNT, we directly measured such mutation rates as a descriptor of emerging genetic novelty. For this, we employed the standard test of appearance of rifampicin-resistant (RifR) clones under the various conditions assayed. As shown in Fig. 8B, both naphthalene and DNT triggered a considerable increase in the appearance of RifR colonies which was not noticeable in the strain deleted of the dnt genes The mutagenic effect of naphthalene in this context was slightly more pronounced than what could be expected from the sheer data on ROS production shown above. We speculate that ROS could react chemically with this bicyclic aromatic compound and generate additional DNA-intercalating agents (e.g. naphtoquinones and naphtodiols [39]) with a separate mutagenic action on DNA. In any case, it is worth noticing that maximum novelty (as measured with this procedure) is accompanied by an acute lethality (Fig. 2), so that the survivors to DNT exposure are more capable to explore the possible solution space to the next adaptive challenge (metabolic or otherwise) than those which had not been diversified because of the phenomena described here.

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