ABSTRACT
Pentachlorophenol (PCP) is a highly toxic pesticide that was first introduced in the 1930s. The alphaproteobacterium Sphingobium chlorophenolicum, which was isolated from PCP-contaminated sediment, has assembled a metabolic pathway capable of completely degrading PCP. This pathway produces four toxic intermediates, including a chlorinated benzoquinone that is a potent alkylating agent and three chlorinated hydroquinones that react with O2 to produce reactive oxygen species (ROS). RNA-seq analysis revealed that PCP causes a global stress response that resembles responses to proton motive force uncoupling and membrane disruption, while surprisingly, little of the response resembles the responses expected to be produced by the PCP degradation intermediates. Tn-seq was used to identify genes important for fitness in the presence of PCP. By comparing the genes that are important for fitness in wild-type S. chlorophenolicum and a non-PCP-degrading mutant, we identified genes that are important only when the PCP degradation intermediates are produced. These include genes encoding two enzymes that are likely to be involved in protection against ROS. In addition to these enzymes, the endogenous levels of other enzymes that protect cells from oxidative stress appear to mitigate the toxic effects of the chlorinated benzoquinone and hydroquinone metabolites of PCP. The combination of RNA-seq and Tn-seq results identify important mechanisms for defense against the toxicity of PCP.
IMPORTANCE Phenolic compounds such as pentachlorophenol (PCP), triclosan, and 2,4-dichlorophenoxyacetic acid (2,4-D) represent a common class of anthropogenic biocides. Despite the novelty of these compounds, many can be degraded by microbes isolated from contaminated sites. However, degradation of this class of chemicals often generates toxic intermediates, which may contribute to their recalcitrance to biodegradation. We have addressed the stresses associated with degradation of PCP by Sphingobium chlorophenolicum by examining the transcriptional response after PCP exposure and identifying genes necessary for growth during both exposure to and degradation of PCP. This work identifies some of the mechanisms that protect cells from this toxic compound and facilitate its degradation. This information could be used to engineer strains capable of improved biodegradation of PCP or similar phenolic pollutants.
INTRODUCTION
Pentachlorophenol (PCP) is listed as a priority pollutant by the U.S. Environmental Protection Agency and is banned by the Stockholm Convention on Persistent Organic Pollutants. It is currently used in the United States as a wood preservative, and it is used more widely in China to kill snails that transmit schistosomiasis (1, 2). Concern about its use has arisen due to its inherent toxicity (3), recalcitrance (4, 5), and potential for long-range dispersal in the environment (6, 7). PCP is an endocrine disruptor (8) and a potential carcinogen (9). Alarmingly, it has recently been found in human fluid and tissue samples (10) as well as in the food chain (11).
Many microbes are capable of degrading PCP (12); the best characterized is Sphingobium chlorophenolicum, an alphaproteobacterium that mineralizes PCP (13–15) and has been used in studies aimed at improving PCP biodegradation (16–19). Degradation of PCP, which was introduced into the environment in the 1930s (20), is interesting from both an evolutionary and practical standpoint. Emergence of a new pathway for degradation of an anthropogenic compound requires recruitment of previously existing enzymes with at least a modest ability to convert the novel compound into a familiar metabolite that can be degraded by existing pathways. Not all microbes will have suitable promiscuous enzymes available to assemble a new pathway. The challenge of evolving a new pathway extends beyond recruitment of new enzymes, however. When a new pathway is introduced into a metabolic network, new metabolites may be toxic, either inherently or because they interfere with the functions of proteins that have not evolved to exclude previously unseen metabolites from sensitive sites (21).
Understanding the stresses caused by novel intermediates is also relevant for efforts to improve biodegradation, which often focus on improving flux through the pathway of interest by directed enzyme evolution (22, 23), tuning expression levels (24), and engineering posttranscriptional control (25). Understanding the stresses produced by the novel pathway and the defense mechanisms that contribute to survival may reveal additional strategies for improving biodegradation (26).
S. chlorophenolicum faces a number of stresses during PCP degradation. The toxicity of PCP itself has been attributed to disruption of the cell envelope (27–31) and dissipation of the proton motive force (PMF) (32–34). PCP is converted to tetrachlorobenzoquinone (TCBQ) by PcpB (Fig. 1) (15). TCBQ is highly toxic (LD50 of <1 µM in Escherichia coli [35]) because it reacts rapidly with nucleophiles, such as those found in glutathione, proteins, and DNA (36–38). PcpD reduces TCBQ to tetrachlorohydroquinone (TCHQ) while it is still within the active site of PcpB (39). TCHQ then undergoes reductive dehalogenation to 2,5,6-trichlorohydroquinone (TriCHQ) and then to 2,6-dichlorohydroquinone (DiCHQ); both reactions are catalyzed by PcpC (40, 41).
Degradation of PCP by S. chlorophenolicum and nonenzymatic oxidation of degradation intermediates. PcpB is PCP hydroxylase, PcpD is TCBQ reductase, and PcpC is TCHQ dehalogenase. Steps not shown include those catalyzed by PcpA (2,6-dichlorohydroquinone dioxygenase) and PcpE (maleylacetate reductase). PCP, pentachlorophenol; TCBQ, tetrachlorobenzoquinone; TCHQ, tetrachlorohydroquinone; TriCHQ, 2,5,6-trichlorohydroquinone; DCHQ, 2,6-dichlorohydroquinone; TriCBQ, 2,5,6-trichlorobenzoquinone; DCBQ, 2,6-dichlorobenzoquinone; GSH, glutathione.
Highly reactive benzoquinone and hydroquinone intermediates also occur during biodegradation of other widely used substituted chloro- and nitrophenols (42–45). 2,4,5-Trichlorophenol and 2,4-dichlorophenol are breakdown products of the pesticides 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. Nitrophenols are used in the production of pesticides such as parathion and nitrophen. Thus, problematic intermediates are a common problem during degradation of anthropogenic phenols.
The toxicity of PCP and its metabolites begs the question of how S. chlorophenolicum deals with the stresses associated with PCP degradation. The concentrations of chlorinated hydroquinone intermediates in cells during PCP degradation sum to approximately 61 µM (35). Exposure of HepG2 cells to 10 µM TCHQ produces significant amounts of reactive oxygen species (ROS) and decreases mitochondrial membrane potential (46). Exposure of primary splenocytes to 12.5 µM TCHQ produces ROS and substantially decreases viability (47). Treatment of hamster lung fibroblasts with 25 µM TCHQ for 1 h increases the level of 8-hydroxy-2-oxoguanosine, a marker of oxidative DNA damage, and induces single-strand breaks (48). Thus, the levels of the chlorinated hydroquinone metabolites during PCP degradation are in the range that causes toxic effects in mammalian cells. S. chlorophenolicum may be more or less capable of preventing ROS production and damage to macromolecules.
To explore the specific stresses perceived by S. chlorophenolicum during PCP degradation, we identified differentially expressed genes after exposure to PCP as well as a variety of other stressors using RNA-seq. The transcriptional response to PCP shared substantial overlap with responses to toluene and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which cause membrane disruption and dissipation of the PMF, respectively, but little overlap with responses to methylglyoxal and paraquat, which cause alkylation and ROS production, respectively. These results suggest that the majority of the transcriptional response during PCP degradation is caused by PCP itself and that S. chlorophenolicum largely avoids stresses caused by degradation intermediates, possibly because transcription of genes involved in defense against the downstream metabolites is already sufficient.
We identified genes that are important for fitness during PCP degradation using transposon sequencing (Tn-seq). Libraries in which every nonessential gene in the genome was disrupted by insertion of a transposon were grown for 20 to 25 generations in the presence of 200 µM PCP. We identified 76 genes whose disruption changes fitness in the presence of PCP but not in its absence. Notably, far fewer genes are important for fitness than change in expression in the presence of PCP, suggesting that much of the transcriptional response is wasteful. Genes encoding components of efflux pumps and membrane remodeling proteins are particularly important, suggesting that removal of PCP from the cell and membrane adaptation are important mechanisms of resistance to PCP toxicity. Interestingly, four genes are no longer important for fitness when PCP degradation is prevented by deletion of pcpB, which encodes the first enzyme in PCP degradation. Three of these genes may contribute to protection against the toxic degradation intermediates.
RESULTS
PCP degradation both detoxifies the medium and provides a novel carbon source.Although PCP degradation involves exposure to PCP and a number of toxic metabolites, it must benefit the organism. We examined the growth of wild-type, ΔpcpR, and ΔpcpB S. chlorophenolicum strains in the presence and absence of 200 µM PCP (Fig. 2A and B). (PcpR is the transcriptional activator for the PCP degradation genes.) We observed a 10% and 23% decrease in biomass yield of the ΔpcpR and ΔpcpB strains, respectively, compared to the biomass yield of the wild-type strain in the presence of PCP. Under these growth conditions, the wild-type strain depletes PCP from the medium, while the mutant strains endure constant PCP stress, but no stress from the metabolites. Not surprisingly, detoxification of the environment by PCP degradation is beneficial. However, this may not be the only benefit of degrading PCP. PCP could be used as a carbon source, although a relatively poor one because it is already highly oxidized.
Disruption of PCP degradation impacts growth in the presence of PCP. Representative growth curves for the wild-type, ΔpcpR, and ΔpcpB strains in SCD medium in the absence (A) or presence (B) of 200 µM PCP. (C) Final OD600 of three replicate cultures of wild-type, ΔpcpR, and ΔpcpB strains of S. chlorophenolicum grown in minimal salts medium (initial OD600 of 0.001) in the presence or absence of 200 µM PCP after 8 days incubation at 30˚C with shaking. *, P value of <0.001; n.s., not significant.
To test whether PCP can serve as a carbon source, starter cultures of wild-type, ΔpcpR, and ΔpcpB S. chlorophenolicum were inoculated into minimal salts medium in the presence or absence of 200 µM PCP. The wild-type strain grew 10-fold in the presence of 200 µM PCP, whereas neither mutant grew (Fig. 2C), demonstrating that S. chlorophenolicum can use PCP as a source of carbon and energy.
PCP exposure causes a massive transcriptional response.Given that PCP degradation is beneficial to S. chlorophenolicum despite the myriad challenges posed by PCP and its metabolites, we were intrigued by the question of how the bacterium responds to and manages