Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP 2,3,5,6-tetrachlorophenol 2,3,5-trichlorophenol 3,5-dichlorophenol 3-chlorophenol phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 mol (1 reactor volume)^–1 day^–1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.     

Purification and characterization of a tetrachloro-p-hydroquinone reductive dehalogenase from a Flavobacterium sp.

Tetrachloro-p-hydroquinone (TeCH) is the first intermediate in pentachlorophenol (PCP) degradation by Flavobacterium sp. strain ATCC 39723. We previously purified a PCP hydroxylase that oxidized PCP to TeCH. Subsequently, we identified the reductive dehalogenation of TeCH to 2,3,6-trichloro-p-hydroquinone and then to 2,6-dichloro-p-hydroquinone in a cell extract with the reduced form of glutathione as the reducing agent under anaerobic conditions. Here we report the purification of a TeCH reductive dehalogenase that reductively dehalogenated TeCH to trichlorohydroquinone and then to dichlorohydroquinone. The enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, and phenyl-agarose, anion-exchange, and gel filtration column chromatographies. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the protein has a molecular weight of about 30,000; nondenaturing polyacrylamide gel electrophoresis analysis suggests that the native enzyme exists as a dimer. The enzyme used glutathione but not NADPH, NADH, dithiothreitol, or ascorbic acid as the reducing agent. The optimal pH was close to neutral.     

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding–impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.     

A pulse--radiolysis approach to fast reductive cleavage of a disulfide bond to uncage enzyme activity

The essential thiol of the enzyme papain has been caged by linking to an aromatic thiol. The resulting caged protein is inactive but enzymatic activity is fully restored upon chemical cleavage of the protective disulfide bond. We have exploited the chemistry of this disulfide bond to uncage papain by pulse radiolysis. We have shown that up to 10% of the enzyme activity can be restored by reductive pulse radiolysis. This approach has been tested on a small-molecule model system, and experiments on this model compound show that pulse radiolysis of the mixed cysteine-aromatic disulfide results in selective reduction of the disulfide bond to generate a thiol in 10-20% yield, consistent with the radiolytically restored activity of the caged papain quantified by the biochemical assay.     

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 μm for bacitracin F to 1050 μm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.     

Dual substrate biodegradation of a nonionic surfactant and pentachlorophenol by Sphingomonas chlorophenolica RA2

The simultaneous biodegradation of the nonionic surfactant Tween 20 (Tw20) and pentachlorophenol (PCP) by Sphingomonas chlorophenolica sp. Strain RA2 (RA2) was measured. As a sole substrate, Tw20 biodegradation was best described by the Contois kinetic model. During concurrent biodegradation of Tw20 and PCP, the biodegradation rates of Tw20 were not significantly affected by 50 or 100 mg/L PCP, but were significantly inhibited by 500 mg/L PCP. Decreases in cell yield in the presence of PCP suggest that PCP was acting as an uncoupler. Cultures were pre-grown on PCP or Tw20 before degradation of PCP to evaluate enzyme induction effects, and long lags before PCP biodegradation after growth on Tw20 occurred. Although biokinetic models could accurately describe some of the data sets of RA2 growth and Tw20 and PCP degradation, finding a single set of kinetic parameters that predicted all dual substrate tests was not achieved. The complicating factors to modeling PCP and Tw20 interactions are described and may be more widely applicable to the biodegradation of toxic organic compounds in the presence of a biodegradable surfactant.     

Discrimination of multiple Dehalococcoides strains in a trichloroethene enrichment by quantification of their reductive dehalogenase genes

While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.     

Value of a hydrogen bond in triosephosphate isomerase loop motion

The motion of the active site loop (loop 6) in triosephosphate isomerase is investigated in solution by TROSY NMR spin-relaxation experiments. The data show clear evidence for motion with an exchange rate constant (kex) of 9000 s-1, consistent with opening and closing of this loop being partially rate-limiting to catalytic throughput. Similar rate constants are observed for residues in both the N- and C-terminal regions of loop 6, suggesting motional coupling of the loop hinges. Mutation of tyrosine 208 to a phenylalanine (Y208F) eliminates a hydrogen bond in the closed loop conformation. NMR experiments with this mutant enzyme indicate an increase in the population of the open conformer and concomitant increase in the opening rate constant and a decrease in the rate of loop closure. The destabilization of the closed conformer by approximately 3 kJ/mol is consistent with a similar decrease in affinity of Y208F for ligand. The site-specific nature of these experiments leads to additional insight into loop 6 motion and the role of a conserved residue in modulating this motion.     

Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds

The reductive biodegradation of a variety of haloaromatic substrates was monitored in samples from two sites within a shallow anoxic aquifer and was compared with freshwater sediment and sewage sludge. The metabolic capacity existing in methane-producing aquifer material was very similar to that in sediment in that three of four chlorobenzoates, five of seven chlorophenols, and one of two chlorophenoxyacetate herbicides were reductively dehalogenated in both types of incubations. The 2,4-dichlorophenoxyacetate was first converted to a dichlorophenol before dehalogenation occurred. Sewage sludge microorganisms dehalogenated four of seven chlorophenols tested and degraded both phenoxyacetate herbicides by first converting them to the corresponding chlorophenols, but the microorganisms did not transform the chlorobenzoates. In general, the same suite of initial metabolites were produced from a test substrate in all types of samples, as confirmed by cochromatography of the intermediates with authentic material. Aquifer microbiota from a sulfate-reducing site was unable to significantly degrade any of the haloaromatic substrates tested. Biological removal of the sulfate in samples from this site permitted dehalogenation of a model substrate, while stimulation of methanogenesis without removal of sulfate did not. These results demonstrate that dehalogenating microorganisms were present at this site but that their activity was at least partially inhibited by the high sulfate levels.     

Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed culture

A pentachlorophenol (PCP) degrading mixed culture contained three predominant strains identified as Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp. The relative abilities of the three strains to degrade PCP were tested individually and in combination. Rates of PCP degradation by individual isolates were lower than that observed for the three isolates combined. Of the individual strains, Flavobacterium gleum manifested highest PCP degradation ability. A biodegradation medium inoculated with a combination of the three isolates exhibited PCP degradation patterns similar to the original mixed culture. Varying low amounts of tetrachlorophenol were found in degradation medium inoculated with individual isolates, but this intermediate was absent from media inoculated with the mixed culture.     

Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds.

The reductive biodegradation of a variety of haloaromatic substrates was monitored in samples from two sites within a shallow anoxic aquifer and was compared with freshwater sediment and sewage sludge. The metabolic capacity existing in methane-producing aquifer material was very similar to that in sediment in that three of four chlorobenzoates, five of seven chlorophenols, and one of two chlorophenoxyacetate herbicides were reductively dehalogenated in both types of incubations. The 2,4-dichlorophenoxyacetate was first converted to a dichlorophenol before dehalogenation occurred. Sewage sludge microorganisms dehalogenated four of seven chlorophenols tested and degraded both phenoxyacetate herbicides by first converting them to the corresponding chlorophenols, but the microorganisms did not transform the chlorobenzoates. In general, the same suite of initial metabolites were produced from a test substrate in all types of samples, as confirmed by cochromatography of the intermediates with authentic material. Aquifer microbiota from a sulfate-reducing site was unable to significantly degrade any of the haloaromatic substrates tested. Biological removal of the sulfate in samples from this site permitted dehalogenation of a model substrate, while stimulation of methanogenesis without removal of sulfate did not. These results demonstrate that dehalogenating microorganisms were present at this site but that their activity was at least partially inhibited by the high sulfate levels.     

Purification, cloning, and sequencing of a 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1

A membrane-associated 3,5-dichlorophenol reductive dehalogenase was isolated from Desulfitobacterium frappieri PCP-1. The highest dehalogenase activity was observed with the biomass cultured at 22 degrees C, compared to 30 and 37 degrees C, where the cell suspensions were 2.2 and 9.6 times less active, respectively. The reductive dehalogenase was purified 12.7-fold to apparent homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular mass of 57 kDa. Its dechlorinating activity was not inhibited by sulfate and nitrate but was completely inhibited by 2.5 mM sulfite and 10 mM KCN. A mixture of iodopropane and titanium citrate caused a light-reversible inhibition of the dechlorinating activities, suggesting the involvement of a corrinoid cofactor. Several polychlorophenols were dechlorinated at the meta and para positions. The apparent K(m) for 3,5-dicholorophenol was 49.3 +/- 3.1 microM at a methyl viologen concentration of 2 mM. Six internal tryptic peptides were sequenced by mass spectrometry. One open reading frame (ORF) was found in the Desulfitobacterium hafniense genome containing these peptide sequences. This ORF corresponds to a gene coding for a CprA-type reductive dehalogenase. The corresponding ORF (named cprA5) in D. frappieri PCP-1 was cloned and sequenced. The cprA5 gene codes for a 548-amino-acid protein that contains a twin-arginine-type signal for secretion. The gene product has a cobalamin binding site motif and two iron-sulfur binding motifs and shows 66% identity (76 to 77% similarity) with some tetrachloroethene reductive dehalogenases. This is the first CprA-type reductive dehalogenase that can dechlorinate chlorophenols at the meta and para positions.     

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.     

Anaerobic biodegradation of pentachlorophenol in a liquor obtained after extraction of contaminated chips and wood powder

Recovery of 97.5% of the pentachlorophenol (PCP) in contaminated wood powder was obtained after extraction with 0.1% KOH solution at 60 degrees C for 75 min. Extraction with NaOH and Na2CO3 was less effective than KOH. The neutralized extract was treated using a methanogenic consortium in an upflow anaerobic fixed-film reactor. The reactor was operated at 29 degrees C for over 600 d. The best performance of the reactor was observed when the PCP liquor was supplemented with glucose and formate. Complete dechlorination of PCP and phenol removal was obtained for a PCP loading rate of 13.3-18.0 mg l(-1) of reactor volume d(-1) with recirculation of the effluent and a hydraulic retention time (HRT) of 0.5-0.6 d.     

Measurements of chromogranin B can serve as a complement to chromogranin A

OBJECTIVE: CgA has been shown to be an excellent marker for neuroendocrine tumours. However, there are two major drawbacks with CgA measurements; elevated levels are common in patients with decreased renal function and in patients on treatment with proton pump inhibitors. These problems are not seen with CgB measurements. We have recently presented the development of 13 region-specific radioimmunoassays for measurements of CgB. A region-specific assay was identified, which measured higher concentrations of CgB than the other assays and seemed to be very useful as a marker for neuroendocrine tumours. The aim of the present study was therefore to further explore the diagnostic potential of this assay in the clinical management of patients with neuroendocrine tumours. METHODS: Measurements of CgB with two methods were compared with CgA in plasma samples from patients investigated for neuroendocrine tumours (N=86), patients with decreased renal function (N=35) and patients on treatment with proton pump inhibitors (N=29). RESULTS: The diagnostic sensitivity for the new CgB assay was almost as good as that for CgA. Furthermore, with CgB measurements we could avoid the falsely elevated levels of CgA found in patients with decreased renal function and treatment with proton pump inhibitors. CONCLUSIONS: We conclude that the new CgB assay can serve as a complement to CgA measurements as an important tumour marker for neuroendocrine tumours.     

Isolation and characterization of Tn-Dha1, a transposon containing the tetrachloroethene reductive dehalogenase of Desulfitobacterium hafniense strain TCE1

A new 9.9 kb catabolic transposon, Tn-Dha1, containing the gene responsible for tetrachloroethene (PCE) reductive dechlorination activity, was isolated from Desulfitobacterium hafniense strain TCE1. Two fully identical copies of the insertion sequence ISDha1, a new member of the IS256 family, surround the gene cluster pceABCT, a truncated gene for another transposase and a short open reading frame with homology to a member of the twin-arginine transport system (tatA). Evidence was obtained by Southern blot for an alternative form of the transposon element as a circular molecule containing only one copy of ISDha1. This latter structure most probably represents a dead-end product of the transposition of Tn-Dha1. Strong indications for the transposition activity of ISDha1 were given by polymerase chain reaction (PCR) amplification and sequencing of the intervening sequence located between both inverted repeats (IR) of ISDha1 (IR junction). A stable genomic ISDha1 tandem was excluded by quantitative real-time PCR. Promoter mapping of the pceA gene, encoding the reductive dehalogenase, revealed the presence of a strong promoter partially encoded in the right inverted repeat of ISDha1. A sequence comparison with pce gene clusters from Desulfitobacterium sp. strains PCE-S and Y51 and from Dehalobacter restrictus, all of which show 100% identity for the pceAB genes, indicated that both Desulfitobacterium strains seem to possess the same transposon structure, whereas only the pceABCT gene cluster is conserved in D. restrictus.