Catabolic characteristics of biphenyl-utilizing isolates which cometabolize PCBs

111

115

We designed and successfully implemented the use of in situ-synthesized 45-mer oligonucleotide DNA microarrays (XeoChips) for genome-wide expression profiling of Burkholderia xenovorans LB400, which is among the best aerobic polychlorinated biphenyl degraders known so far. We conducted differential gene expression profiling during exponential growth on succinate, benzoate, and biphenyl as sole carbon sources and investigated the transcriptome of early-stationary-phase cells grown on biphenyl. Based on these experiments, we outlined metabolic pathways and summarized other cellular functions in the organism relevant for biphenyl and benzoate degradation. All genes previously identified as being directly involved in biphenyl degradation were up-regulated when cells were grown on biphenyl compared to expression in succinate-grown cells. For benzoate degradation, however, genes for an aerobic coenzyme A activation pathway were upregulated in biphenyl-grown cells, while the pathway for benzoate degradation via hydroxylation was upregulated in benzoate-grown cells. The early-stationary-phase biphenyl-grown cells showed similar expression of biphenyl pathway genes, but a surprising up-regulation of C 1 metabolic pathway genes was observed. The microarray results were validated by quantitative reverse transcription PCR with a subset of genes of interest. The XeoChips showed a chip-to-chip variation of 13.9%, compared to the 21.6% variation for spotted oligonucleotide microarrays, which is less variation than that typically reported for PCR product microarrays.

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confidence: 99%

Biphenyl and Benzoate Metabolism in a Genomic Context: Outlining Genome-Wide Metabolic Networks in Burkholderia xenovorans LB400

Denef

Park

et al. 2004

111

115

“…Under anoxic conditions, highly chlorinated congeners can often be reductively dechlorinated (8,25), resulting in congeners that can potentially be degraded by aerobic bacteria. The lightly chlorinated congeners (one to five chlorines) remaining after reductive dechlorination can be either utilized as an aerobic growth substrate (12, 16) or cometabolically degraded in the presence of biphenyl (9,14).With few exceptions (5, 10), anaerobic PCB dechlorination rarely occurs at the ortho positions and generally results in accumulation of ortho-or ortho-and para-substituted congeners (19,21,27,28). Interestingly, ortho substitution of a PCB also greatly reduces aerobic biodegradability (4, 13).…”

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confidence: 99%

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confidence: 99%

Microbial Growth on Dichlorobiphenyls Chlorinated on Both Rings as a Sole Carbon and Energy Source

Sang-Goo

Picardal

2001

We have isolated bacterial strains capable of aerobic growth on ortho-substituted dichlorobiphenyls as sole carbon and energy sources. During growth on 2,2-dichlorobiphenyl and 2,4-dichlorobiphenyl strain SK-4 produced stoichiometric amounts of 2-chlorobenzoate and 4-chlorobenzoate, respectively. Chlorobenzoates were not produced when strain SK-3 was grown on 2,4-dichlorobiphenyl.Although bioremediation is an effective cleanup strategy for many contaminants, efforts to engineer bioremediation systems for polychlorinated biphenyl (PCB)-contaminated soils or sludges have been largely ineffective. One of the more promising PCB bioremediation strategies is sequential anaerobicaerobic treatment (1). Under anoxic conditions, highly chlorinated congeners can often be reductively dechlorinated (8,25), resulting in congeners that can potentially be degraded by aerobic bacteria. The lightly chlorinated congeners (one to five chlorines) remaining after reductive dechlorination can be either utilized as an aerobic growth substrate (12, 16) or cometabolically degraded in the presence of biphenyl (9,14).With few exceptions (5, 10), anaerobic PCB dechlorination rarely occurs at the ortho positions and generally results in accumulation of ortho-or ortho-and para-substituted congeners (19,21,27,28). Interestingly, ortho substitution of a PCB also greatly reduces aerobic biodegradability (4, 13). As reported by Furukawa (11), PCB isomers with two ortho chlorines, such as 2,2Ј-dichlorobiphenyl (2,2Ј-CB) or 2,6-CB, are rarely susceptible to aerobic cometabolic degradation. As a consequence, limited aerobic degradation of ortho-substituted congeners is a weak link in the development of effective anaerobic-aerobic PCB bioremediation strategies. Possible accumulation of ortho-substituted congeners such as 2,2Ј-CB may also have ecological and human health implications (7,22).Although numerous reports have shown that monochlorobiphenyls can serve as growth substrates (2,3,6,17,18,23), reports of unambiguous growth by natural isolates on CBs has been demonstrated only for Burkholderia sp. strain LB400 (20). To the best of our knowledge, growth on a di-ortho-substituted CB has not been demonstrated. Here we report the isolation and characterization of unique bacteria capable of using as growth substrates ortho-substituted CB congeners with chlorine substituents on both rings, including an isolate able to utilize 2,2Ј-CB.The CB-degrading bacteria were isolated from PCB-contaminated, tertiary lagoon sludge as previously described (16). One isolate (SK-3) was selected based on its ability to grow on both 4-chlorobiphenyl and 4-chlorobenzoic acid (4-CBA) (16). Another isolate (SK-4) was ultimately enriched and selected based on an ability to grow on 2,2Ј-CB.Cell growth and biotransformation of CBs were studied in Balch tubes sealed with Teflon-coated stoppers as reported previously (16). Experiments were done in a mineral salts medium (16) which was amended with 5 or 10 l of a solution of a single PCB congener dissolved in heptamethylnonane (...

“…The high cost and public opposition to current physical remediation technologies have stimulated interest in the use of microorganisms for bioremediation of CB-contaminated sites (25,43,46). Since the demonstration that two strains of Achromobacter could degrade mono-and dichlorobiphenyls by Ahmed and Focht (1), numerous studies have shown that a broad diversity of bacteria have the ability to degrade many of the different CB congeners that contaminate the environment (3,16,24,39). While natural microbial degradation of CBs is constrained by a combination of physical, chemical, and biological factors (25), development of effective bioremediation strategies will be aided by an understanding of the biochemical mechanisms involved in the mineralization of CBs.…”

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confidence: 99%

Influence of Chlorine Substituents on Rates of Oxidation of Chlorinated Biphenyls by the Biphenyl Dioxygenase of Burkholderia sp. Strain LB400

Arnett

Parales

Haddock

2000

Biphenyl dioxygenase from Burkholderia (Pseudomonas) sp. strain LB400 catalyzes the first reaction of a pathway for the degradation of biphenyl and a broad range of chlorinated biphenyls (CBs). The effect of chlorine substituents on catalysis was determined by measuring the specific activity of the enzyme with biphenyl and 18 congeners. The catalytic oxygenase component was purified and incubated with individual CBs in the presence of electron transport proteins and cofactors that were required for enzyme activity. The rate of depletion of biphenyl from the assay mixture and the rate of formation of cis-biphenyl 2,3-dihydrodiol, the oxidation product, were almost equal, indicating that the assay accurately measured enzyme-specific activity. Four classes of CBs were defined based on their oxidation rates. Class I contained 3-CB and 2,5-CB, which gave rates that were approximately twice that of biphenyl. Class II contained 2,5,3,4-CB, 2,3,2,5-CB, 2,3,4,5-CB, 2,3,2,3-CB, 2,4,5,2,5-CB, 2,5,3-CB, 2,5,4-CB, 2-CB, and 3,4,5-CB, which gave rates that ranged from 97 to 35% of the biphenyl rate. Class III contained only 2,3,4,2,5-CB, which gave a rate that was 4% of the biphenyl rate. Class IV contained 2,4,4-CB, 2,4,2,4-CB, 3,4,5,2-CB, 3,4,5,3-CB, 3,5,3,5-CB, and 3,4,5,2,5-CB, which showed no detectable depletion. Rates were not significantly correlated with the aqueous solubilities of the CBs or the number of chlorine substituents on the rings. Oxidation products were detected for all class I, II, and III congeners and were identified as chlorinated cis-dihydrodiols for classes I and II. The specificity of biphenyl dioxygenase for the CBs examined in this study was determined by the relative positions of the chlorine substituents on the aromatic rings rather than the number of chlorine substituents on the rings.