Changes of the Proteome and Acetylome during Transition into the Stationary Phase in the Organohalide-Respiring Dehalococcoides mccartyi Strain CBDB1.

The strictly anaerobic bactGIerium Dehalococcoides mccartyi obligatorily depends on organohalide respiration for energy conservation and growth. The bacterium also plays an important role in bioremediation. Since there is no guarantee of a continuous supply of halogenated substrates in its natural environment, the question arises of how D. mccartyi maintains the synthesis and activity of dehalogenating enzymes under these conditions. Acetylation is a means by which energy-restricted microorganisms can modulate and maintain protein levels and their functionality. Here, we analyzed the proteome and Nε-lysine acetylome of D. mccartyi strain CBDB1 during growth with 1,2,3-trichlorobenzene as an electron acceptor. The high abundance of the membrane-localized organohalide respiration complex, consisting of the reductive dehalogenases CbrA and CbdbA80, the uptake hydrogenase HupLS, and the organohalide respiration-associated molybdoenzyme OmeA, was shown throughout growth. In addition, the number of acetylated proteins increased from 5% to 11% during the transition from the exponential to the stationary phase. Acetylation of the key proteins of central acetate metabolism and of CbrA, CbdbA80, and TatA, a component of the twin-arginine translocation machinery, suggests that acetylation might contribute to maintenance of the organohalide-respiring capacity of the bacterium during the stationary phase, thus providing a means of ensuring membrane protein integrity and a proton gradient.

Anaerobic degradation of 2,4,5-trichlorophenoxyacetic acid by enrichment cultures from freshwater sediments.

The anaerobic biodegradation of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was investigated using enrichment cultures from freshwater sediments at two different sites in the region of Halle, central Germany. 2,4,5-T and different organic acids or hydrogen were added as possible electron acceptor and electron donors, respectively. The primary enrichment cultures from Saale river sediment completely degraded 2,4,5-T to 3-chlorophenol (3-CP) (major product) and 3,4-dichlorophenol (3,4-DCP) during a 28-day incubation period. Subcultures showed ether cleavage of 2,4,5-T to 2,4,5-trichlorophenol and its stoichiometric dechlorination to 3-CP only in the presence of butyrate. In contrast, the primary enrichment culture from sediment of Posthorn pond dechlorinated 2,4,5-T to 2,5-dichlorophenoxyacetic acid (2,5-D), which, in the presence of butyrate, was degraded further to products such as 3,4-DCP, 2,5-DCP, and 3CP, indicating ether cleaving activities and subsequent dechlorination steps. Experiments with pure cultures of Dehalococcoides mccartyi and Desulfitobacterium hafniense demonstrated their specific dechlorination steps within the overall 2,4,5-T degradation pathways. The results indicate that the route and efficiency of anaerobic 2,4,5-T degradation in the environment depend heavily on the microorganisms present and the availability of slowly fermentable organic compounds.

Desulfitobacterium contributes to the microbial transformation of 2,4,5-T by methanogenic enrichment cultures from a Vietnamese active landfill.

The herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5-T via ether cleavage to 2,4,5-trichlorophenol (2,4,5-TCP), which was further dechlorinated to 3,4-dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5-T and subsequent dechlorination of 2,4,5-TCP. 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes, Bacteroidetes, Spirochaetes, Chloroflexi and Euryarchaeota. Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5-T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5-TCP. Despite an ether-cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2-bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms.

Anaerobic microbial dehalogenation and its key players in the contaminated Bitterfeld-Wolfen megasite.

The megasite Bitterfeld-Wolfen is highly contaminated as a result of accidents and because of dumping of wastes from local chemical industries in the last century. A variety of contaminants including chlorinated ethenes and benzenes, hexachlorohexanes and chlorinated dioxins can still be found in the groundwater and (river) sediments. Investigations of the in situ microbial transformation of organohalides have been performed only over the last two decades at this megasite. In this review, we summarise the research on the activity of anaerobic dehalogenating bacteria at the field site in Bitterfeld-Wolfen, focusing on chlorinated ethenes, monochlorobenzene and chlorinated dioxins. Various methods and concepts were applied including ex situ cultivation and isolation, and in situ analysis of hydrochemical parameters, compound-specific stable isotope analysis of contaminants, 13C-tracer studies and molecular markers. Overall, biotransformation of organohalides is ongoing at the field site and Dehalococcoides mccartyi species play an important role in the detoxification process in the Bitterfeld-Wolfen region.

Comparative genomics of the genus Desulfitobacterium.

The Desulfitobacterium genus comprises anaerobic Gram-positive bacteria, of which the majority are facultative organohalide respirers. We here present the genomes of eight strains of Desulfitobacterium spp., including five strains of Desulfitobacterium hafniense, one strain each from D. dichloroeliminans and D. metallireducens, and one strain that had not been assigned to any species prior to this study. The newly sequenced genomes were compared with four previously published desulfitobacterial genomes. The average genome sizes are 5.5, 4.3 and 3.4 Mbp for D. hafniense, D. dehalogenans and D. dichloroeliminans/metallireducens, respectively. The genomes encode up to seven reductive dehalogenases, the genomes of both D. hafniense DP7 and D. metallireducens 853-15AT did not encode any reductive dehalogenase. The latter result was a surprise as D. metallireducens 853-15AT has been reported to carry out organohalide respiration. Unlike reported for the pceABCT gene cluster, the other reductive dehalogenase gene clusters do not show any signs of being genetically mobile. All analyzed desulfitobacterial genomes encode a complete cobalamin synthesis pathway. A menaquinone synthesis pathway was found in all strains except D. dichloroeliminans DCA1T. The detailed analysis of the genome sequence of 12 desulfitobacteria from four different species confirmed that this genus has an extremely large metabolic repertoire.

A H2 -oxidizing, 1,2,3-trichlorobenzene-reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1.

Dehalococcoides mccartyi is a small, slow-growing bacterium of the phylum Chloroflexi that conserves energy using aliphatic and aromatic organohalides as electron acceptors, and hydrogen as sole electron donor. A recent study identified a protein complex in the membrane of strain CBDB1 comprising a Hup hydrogenase, a complex iron-sulphur molybdoprotein and a reductive dehalogenase (RdhA) that catalyses reduction of 1,2,3,4-tetrachlorobenzene. Using a combination of size-exclusion chromatography, in-gel hydrogenase activity-staining, immunological analysis and mass spectrometry, we identified here a large molecular mass protein complex solubilized from the cytoplasmic membrane of D. mccartyi strain CBDB1 that catalysed H2 -dependent reduction of 1,2,3-trichlorobenzene (1,2,3-TCB) to 1,3-DCB. In-gel zymographic staining revealed H2 :benzyl viologen oxidoreductase activity associated with the complex and immunological analysis identified co-elution of CdbdA195, the predicted catalytic subunit of the iron-sulphur molybdoenzyme, the chlorobenzene-specific RdhA, CbrA, and traces of HupL, the catalytic subunit of the Hup hydrogenase. Quantitative reverse transcriptase PCR analyses indicated that the expression of the hupL and cbdbA195 genes was induced by 1,2,3-TCB but not by hydrogen. Together, these data identify and describe a protein-based electron-transfer complex catalysing H2 oxidation coupled to chlorobenzene reduction.

The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1.

Reductive dehalogenases are essential enzymes in organohalide respiration and consist of a catalytic subunit A and a membrane protein B, encoded by rdhAB genes. Thirty-two rdhAB genes exist in the genome of Dehalococcoides mccartyi strain CBDB1. To gain a first insight into the regulation of rdh operons, the control of gene expression of two rdhAB genes (cbdbA1453/cbdbA1452 and cbdbA1455/cbdbA1454) by the MarR-type regulator Rdh2R (cbdbA1456) encoded directly upstream was studied using heterologous expression and in vitro studies. Promoter-lacZ reporter fusions were generated and integrated into the genome of the Escherichia coli host. The lacZ reporter activities of both rdhA promoters decreased upon transformation of the cells with a plasmid carrying the rdh2R gene, suggesting that Rdh2R acts as repressor, whereas the lacZ reporter activity of the rdh2R promoter was not affected. The transcriptional start sites of both rdhA genes in strain CBDB1 and/or the heterologous host mapped to a conserved direct repeat with 11- to 13-bp half-sites. DNase I footprinting revealed binding of Rdh2R to a ∼30-bp sequence covering the complete direct repeat in both promoters, including the transcriptional start sites. Equilibrium sedimentation ultracentrifugation revealed that Rdh2R binds as tetramer to the direct-repeat motif of the rdhA (cbdbA1455) promoter. Using electrophoretic mobility shift assays, a similar binding affinity was found for both rdhA promoters. In the presence of only one half-site of the direct repeat, the interaction was strongly reduced, suggesting a positive cooperativity of binding, for which unusual short palindromes within the direct-repeat half-sites might play an important role. IMPORTANCE: Dehalococcoides mccartyi strains are obligate anaerobes that grow by organohalide respiration. They have an important bioremediation potential because they are capable of reducing a multitude of halogenated compounds to less toxic products. We are now beginning to understand how these organisms make use of this large catabolic potential, whereby D. mccartyi expresses dehalogenases in a compound-specific fashion. MarR-type regulators are often encoded in the vicinity of reductive dehalogenase genes. In this study, we made use of heterologous expression and in vitro studies to demonstrate that the MarR-type transcription factor Rdh2R acts as a negative regulator. We identify its binding site on the DNA, which suggests a mechanism by which it controls the expression of two adjacent reductive dehalogenase operons.

Dehalococcoides mccartyi strain DCMB5 Respires a broad spectrum of chlorinated aromatic compounds.

Polyhalogenated aromatic compounds are harmful environmental contaminants and tend to persist in anoxic soils and sediments. Dehalococcoides mccartyi strain DCMB5, a strain originating from dioxin-polluted river sediment, was examined for its capacity to dehalogenate diverse chloroaromatic compounds. Strain DCMB5 used hexachlorobenzenes, pentachlorobenzenes, all three tetrachlorobenzenes, and 1,2,3-trichlorobenzene as well as 1,2,3,4-tetra- and 1,2,4-trichlorodibenzo-p-dioxin as electron acceptors for organohalide respiration. In addition, 1,2,3-trichlorodibenzo-p-dioxin and 1,3-, 1,2-, and 1,4-dichlorodibenzo-p-dioxin were dechlorinated, the latter to the nonchlorinated congener with a remarkably short lag phase of 1 to 4 days following transfer. Strain DCMB5 also dechlorinated pentachlorophenol and almost all tetra- and trichlorophenols. Tetrachloroethene was dechlorinated to trichloroethene and served as an electron acceptor for growth. To relate selected dechlorination activities to the expression of specific reductive dehalogenase genes, the proteomes of 1,2,3-trichlorobenzene-, pentachlorobenzene-, and tetrachloroethene-dechlorinating cultures were analyzed. Dcmb_86, an ortholog of the chlorobenzene reductive dehalogenase CbrA, was the most abundant reductive dehalogenase during growth with each electron acceptor, suggesting its pivotal role in organohalide respiration of strain DCMB5. Dcmb_1041 was specifically induced, however, by both chlorobenzenes, whereas 3 putative reductive dehalogenases, Dcmb_1434, Dcmb_1339, and Dcmb_1383, were detected only in tetrachloroethene-grown cells. The proteomes also harbored a type IV pilus protein and the components for its assembly, disassembly, and secretion. In addition, transmission electron microscopy of DCMB5 revealed an irregular mode of cell division as well as the presence of pili, indicating that pilus formation is a feature of D. mccartyi during organohalide respiration.

Bacillus pervagus sp. nov. and Bacillus andreesenii sp. nov., isolated from a composting reactor.

Two strains, 8-4-E12(T) and 8-4-E13(T), were isolated from a biowaste composting reactor. Based on 16S rRNA gene sequences, both strains belong to the genus Bacillus. Strain 8-4-E12(T) was most closely related to the type strains of Bacillus shackletonii, B. acidicola, B. sporothermodurans and B. oleronius (96.4, 96.3, 96.0 and 95.6 % 16S rRNA gene similarity, respectively), whereas strain 8-4-E13(T) was most closely related to the type strain of Bacillus humi (96.5 % sequence similarity). Strains 8-4-E12(T) and 8-4-E13(T) shared 94 % 16S rRNA gene sequence similarity. The fatty acid profile of strain 8-4-E12(T) was dominated by saturated iso- and anteiso-branched fatty acids (iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0), and also contained considerable amounts of C16 : 0. The fatty acid profile of strain 8-4-E13(T) showed a predominance of iso-C15 : 0 (65 %), with smaller amounts of other saturated branched-chain fatty acids along with an unsaturated alcohol. Both strains contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as major polar lipids. Additionally, strain 8-4-E12(T) contained an unknown lipid and strain 8-4-E13(T) two unknown (amino-)phospholipids. The diagnostic diamino acid found in the cell-wall peptidoglycan of 8-4-E12(T) and 8-4-E13(T) was meso-diaminopimelic acid. The predominant menaquinone was MK-7. The results of physiological and biochemical tests also allowed phenotypic differentiation of the two strains from each other and from related Bacillus species. On the basis of their phylogenetic, phenotypic and chemotaxonomic properties, strains 8-4-E12(T) and 8-4-E13(T) represent novel species of the genus Bacillus, for which the names Bacillus pervagus sp. nov. (type strain 8-4-E12(T) = DSM 23947(T) = LMG 27601(T)) and Bacillus andreesenii sp. nov. (type strain 8-4-E13(T) = DSM 23948(T) = LMG 27602(T)) are proposed.

Genome sequences of two dehalogenation specialists ­ Dehalococcoides mccartyi strains BTF08 and DCMB5 enriched from the highly polluted Bitterfeld region.

The genomes of two novel Dehalococcoides mccartyi strains, DCMB5 and BTF08, enriched from the heavily organohalide-contaminated megasite around Bitterfeld (Germany), were fully sequenced and annotated. Although overal lsimilar, the genome sequences of the two strains reveal remarkable differences in their genetic content, reflecting a specific adaptation to the contaminants at the field sites from which they were enriched. The genome of strain BTF08 encodes for 20 reductive dehalogenases, and is the first example of a genome containing all three enzymes that are necessary to couple the complete reductive dechlorination of PCE to ethene to growth. The genes encoding trichloroethene and vinyl chloride reductive dehalogenases, tceA and vcrA, are located within mobile genetic elements, suggesting their recent horizontal acquisition.The genome of strain DCMB5 contains 23 reductive dehalogenase genes,including cbrA, which encodes a chlorobenzene reductive dehalogenase, and a gene cluster encoding arsenic resistance proteins, both corresponding to typical pollutants at its isolation site.

Regulation of reductive dehalogenase gene transcription in Dehalococcoides mccartyi.

The remarkable capacity of the genus Dehalococcoides to dechlorinate a multitude of different chlorinated organic compounds reflects the number and diversity of genes in the genomes of Dehalococcoides species encoding reductive dehalogenase homologues (rdh). Most of these genes are located in the vicinity of genes encoding multiple antibiotic resistance regulator (MarR)-type or two-component system regulators. Here, the transcriptional response of rdhA genes (coding for the catalytic subunit) to 2,3- and 1,3-dichlorodibenzo-p-dioxin (DCDD) was studied in Dehalococcoides mccartyi strain CBDB1. Almost all rdhA genes were transcribed in the presence of 2,3-DCDD, albeit at different levels as shown for the transcripts of cbrA, cbdbA1453, cbdbA1624 and cbdbA1588. By contrast, 1,3-DCDD did not induce rdhA transcription. The putative MarR CbdbA1625 was heterologously produced and its ability to bind in vitro to the overlapping promoter regions of the genes cbdbA1624 and cbdbA1625 was demonstrated. To analyse regulation in vivo, single-copy transcriptional promoter-lacZ fusions of different rdhA genes and of cbdbA1625 were constructed and introduced into the heterologous host Escherichia coli, and expression levels of the fusions were measured. The cbdbA1625 gene was cloned into a vector allowing a regulation of expression by arabinose and it was transformed into the strains containing the rdh-promoter-lacZ fusion derivatives. CbdbA1625 was shown to downregulate transcription from its own promoter resulting in a 40-50% reduction in the ß-galactosidase activity, giving the first hint that it acts as a repressor.

Prevailing negative soil biota effect and no evidence for local adaptation in a widespread Eurasian grass.

BACKGROUND: Soil biota effects are increasingly accepted as an important driver of the abundance and distribution of plants. While biogeographical studies on alien invasive plant species have indicated coevolution with soil biota in their native distribution range, it is unknown whether adaptation to soil biota varies among populations within the native distribution range. The question of local adaptation between plants and their soil biota has important implications for conservation of biodiversity and may justify the use of seed material from local provenances in restoration campaigns. METHODOLOGY/PRINCIPAL FINDINGS: We studied soil biota effects in ten populations of the steppe grass Stipa capillata from two distinct regions, Europe and Asia. We tested for local adaptation at two different scales, both within (ca. 10-80 km) and between (ca. 3300 km) regions, using a reciprocal inoculation experiment in the greenhouse for nine months. Generally, negative soil biota effects were consistent. However, we did not find evidence for local adaptation: both within and between regions, growth of plants in their 'home soil' was not significantly larger relative to that in soil from other, more distant, populations. CONCLUSIONS/SIGNIFICANCE: Our study suggests that negative soil biota effects can prevail in different parts of a plant species' range. Absence of local adaptation points to the possibility of similar rhizosphere biota composition across populations and regions, sufficient gene flow to prevent coevolution, selection in favor of plasticity, or functional redundancy among different soil biota. From the point of view of plant--soil biota interactions, our findings indicate that the current practice of using seeds exclusively from local provenances in ecosystem restoration campaigns may not be justified.

Lactate in cystic fibrosis sputum.

BACKGROUND: Antibiotic therapy is thought to improve lung function in patients with cystic fibrosis (CF) by decreasing neutrophil-derived inflammation. We investigated the origin and clinical significance of lactate in the chronically inflamed CF lung. METHODS: Lactate was measured in sputa of 18 exacerbated and 25 stable CF patients via spectrophotometry and gaschromatography. Lung function was assessed via spirometry. Seven patients with chronic obstructive pulmonary disease (COPD) and three patients with acute lung inflammation served as control groups. Neutrophil and bacterial lactate production was assessed under aerobic and anaerobic conditions. RESULTS: In sputum specimens of patients with respiratory exacerbations lactate concentrations decreased significantly (p<0.005) from 3.4±2.3mmol/L to 1.4±1.4mmol/L after 2-3 weeks of intravenous antibiotics. Successful treatment was reflected in 16 patients (88.9%) by FVC increase associated with lactate decrease (p<0.05). In every single sputum lactate was detectable (3.0±3.1mmol/L, range 0.2-14.1mmol/L). Lactate was lower (1.6±0.8mmol/L) in sputa from seven COPD patients, and it was below the detection limit in three patients with acute lung inflammation. Neutrophil lactate production accumulated up to 10.5mmol/L after 4 days, whereas bacterial lactate production did not appear to contribute substantially to sputum lactate concentrations. CONCLUSIONS: Successful antibiotic therapy is reflected by a decrease in lactate concentrations. Neutrophils are the most likely source for lactate in sputum of CF patients. Therefore lactate may be used to monitor responses to antibiotic therapy as an adjunct to lung function measurements.

Anaerobic reductive dehalogenation of polychlorinated dioxins.

Polychlorinated dibenzo-p-dioxins and -furans (PCDD/Fs) are among the most harmful environmental contaminants. Their widespread distribution due to unintentional or unknown release coincides with environmental persistence, acute and chronic toxicity to living organisms, and long-term effects due to the compounds' tendency for bioaccumulation and biomagnification. While microbial aerobic degradation of PCDD/Fs is mainly reported for the turnover of low chlorinated congeners, this review focuses on anaerobic reductive dehalogenation, which may constitute a potential remediation strategy for polychlorinated compounds in soils and sediments. Microorganisms in sediments and in microcosms or enrichment cultures have been shown to be involved in the reductive dechlorination of dioxins. Bacteria related to the genus Dehalococcoides are capable of the reductive transformation of dioxins leading to lower chlorinated dioxins including di- and monochlorinated congeners. Thus, reductive dehalogenation might be one of the very few mechanisms able to mediate the turnover of polychlorinated dioxins by reducing their toxicity and paving the way for a subsequent breakdown of the carbon skeleton.

Transcription analysis of genes encoding homologues of reductive dehalogenases in "Dehalococcoides" sp. strain CBDB1 by using terminal restriction fragment length polymorphism and quantitative PCR.

The transcription of reductive dehalogenase homologous (rdh) genes of "Dehalococcoides" sp. strain CBDB1 was investigated during the growth and reductive dechlorination of 1,2,3- and 1,2,4-trichlorobenzene (TCB). A method was developed to monitor the expression of all 32 rdhA genes present in the genome based on reverse transcription-PCR amplification with 13 degenerate primer pairs and terminal restriction fragment length polymorphism (t-RFLP) analysis. With this approach, the upregulation of the transcription of 29 rdhA genes was indicated in response to 1,2,3- and 1,2,4-TCB added after a substrate depletion period of 72 h. The transcription of the remaining three rdhA genes additionally was detected using specific primers. While most rdhA genes were upregulated similarly in cultures after induction with 1,2,3-TCB or 1,2,4-TCB, three rdhA genes responded differentially to 1,2,3- and 1,2,4-TCB, as revealed by the comparison of t-RFLP profiles. The enhanced transcription of cbdbA1453 and cbdbA187 was observed in the presence of 1,2,3-TCB, while the transcription of cbdbA1624 was strongly induced by 1,2,4-TCB. Comparison of t-RFLP profiles obtained from cDNA and genomic DNA indicated a particularly high induction of the transcription of cbrA (=cbdbA84) by both TCBs. As indicated by reverse transcription-quantitative PCR, the transcription of these plus two other rdhA genes (cbdbA1588 and cbdbA1618) increased within 48 to 72 h by one or two orders of magnitude. Subsequently, transcript levels slowly decreased and approached initial transcript levels several days after complete dehalogenation. Finally, cbrA was transcribed to a level of 22 transcripts per cbrA gene, suggesting that cbrA mRNA could be an appropriate biomarker for the investigation of the natural dechlorination potential at chlorobenzene-contaminated sites.

Enrichment of a dioxin-dehalogenating Dehalococcoides species in two-liquid phase cultures.

Enrichment cultures capable of reductively dechlorinating 1,2,4-trichlorodibenzo-p-dioxin (1,2,4-TrCDD) were shown to dechlorinate 1,2,3-trichlorobenzene (1,2,3-TrCB) to 1,3-dichlorobenzene. To test if this activity can be used to enrich for dioxin-dechlorinating bacteria, a two-liquid phase cultivation with 200 mM 1,2,3-TrCB dissolved in hexadecane was established. During the dechlorination of 1,2,3-TrCB, the number of 1,2,4-TrCDD-dechlorinating bacteria increased by four orders of magnitude, eventually accounting for 11% of the total cell number. Characterization of the bacterial communities of the initial dioxin-dechlorinating culture and of the trichlorobenzene enrichments by restriction fragment length polymorphism (RFLP) analysis of cloned 16S rRNA genes revealed a proportional increase of nine different sequence types, one representing a Dehalococcoides strain. Inhibition of methanogens further enhanced the rate of chlorobenzene dehalogenation and also resulted in a rapid dechlorination of 1,2,3,4-tetrachlorodibenzo-p-dioxin that was applied via a hexadecane phase. The further enrichment was monitored by terminal RFLP, quantitative real-time PCR and microscopy, and aimed at the reduction of the accompanying non-dehalogenating populations by using different combinations of electron donors and the application of antibiotics. Hydrogen as the sole electron donor proved to be less efficient due to the co-enrichment of acetogens. The novel Dehalococcoides strain DCMB5 was enriched up to 50% by the cultivation with organic acids, hydrogen and vancomycin, and was finally purified by conventional isolation techniques.

Microbial dehalogenation of trichlorinated dibenzo-p-dioxins by a Dehalococcoides-containing mixed culture is coupled to carbon isotope fractionation.

An anaerobic enrichment culture reductively dehalogenated 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) almost exclusively at peripheral positions forming the main products 1,3-dichloro-(DiCDD) and 2-monochlorodibenzo-p-dioxin (MCDD) from 1,2,4-TrCDD and 2,3-DiCDD from 1,2,3-TrCDD. Dehalococcoides was monitored in the mixed culture by quantitative real-time PCR. A yield of 2.5 x 10(8) to 2.75 x 10(8) copies of 16S rRNA genes per micromole of chloride released suggested growth by dehalorespiration with dibenzo-p-dioxins. For the analysis of carbon isotope fractionation, the dioxin congeners were isolated by solid-phase microextraction (SPME) from the headspace of the cultures. The delta13C composition of 1,2,4-TrCDD did not change remarkably during the course of reductive dehalogenation; however, the intermediate 1,3-DiCDD became enriched, and the final product 2-MCDD significantly depleted in 13C with a discrimination of 2.5-3.6 per thousand between 1,3-DiCDD and 2-MCDD. 1,2,3-TrCDD and its main product 2,3-DiCDD became slightly enriched in 13C, whereas the formed low concentrations of 2-MCDD were depleted in 13C by 5.5-4.8 per thousand. This study demonstrates carbon isotope fractionation during sequential reductive dehalogenation of chlorinated dibenzo-p-dioxins, whereby isotope fractionation upon dehalogenation of the intermediate was substantial. This can provide a basis for the development of a new method to monitor the fate of dioxins in the environment using compound specific stable isotope analyses.

Aquincola tertiaricarbonis gen. nov., sp. nov., a tertiary butyl moiety-degrading bacterium.

Strains L10(T), L108 and CIP I-2052 were originally obtained from methyl tert-butyl ether (MTBE)-contaminated groundwater and from a wastewater treatment plant, respectively. All share the ability to grow on tert-butanol, an intermediate of MTBE degradation. Cells are strictly aerobic, motile by a polar flagellum and exhibit strong pili formation. Poly beta-hydroxybutyrate (PHB) granules are formed. The DNA G+C content is 69-70.5 mol% and the main ubiquinone is Q-8. The major cellular fatty acids are 16 : 1 cis-9 and 16 : 0 and the only hydroxy fatty acid is 10 : 0 3-OH. The major phospholipids are phosphatidylethanolamine (PE) 16 : 1/16 : 1 and phosphatidylglycerol 16 : 0/16 : 1. A significant amount of PE 17 : 0/16 : 1 is present. The 16S rRNA gene sequences of these strains are almost identical and form a separate line of descent in the Rubrivivax-Roseateles-Leptothrix-Ideonella-Aquabacterium branch of the Betaproteobacteria with 97 % similarity to 16S rRNA genes of the type strains of Rubrivivax gelatinosus, Leptothrix mobilis and Ideonella dechloratans. However, physiological properties, DNA-DNA relatedness values and the phospholipid and cellular fatty acid profiles distinguish the novel isolates from the three closely related genera. Therefore, it is concluded that strains L10(T), L108 and CIP I-2052 represent a new genus and novel species for which the name Aquincola tertiaricarbonis gen. nov., sp. nov., is proposed. The type strain is strain L10(T) (=DSM 18512(T)=CIP 109243(T)).

Biological Activity in a Heavily Organohalogen-Contaminated River Sediment (8 pp).

BACKGROUND, AIMS AND SCOPE: Sediments of the Spittelwasser creek are highly polluted with organic compounds and heavy metals due to the discharge of untreated waste waters from the industrial region of Bitterfeld-Wolfen, Germany over the course of more than one century. However, relatively few data have been published about the chloroorganic contamination of the sediment. This paper reports on the content of different (chloro)organic compounds with special emphasis on polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F), and chlorobenzenes. Existing concepts for the remediation of Spittelwasser sediment include the investigation of natural attenuation processes, which largely depend on the presence of an intact microbial food web. In order to gain more insight in terms of biological activity, we analyzed the capacity of sediment microflora to degrade organic matter by measuring the activities of extracellular hydrolytic enzymes involved in the biogeochemical cycling of carbon, nitrogen, phosphorus and sulfur. Furthermore, the detection of physiologically active bacteria in the sediment, particularly of those known for their capability to reductively dehalogenate organochlorine compounds, illustrates the potential for intrinsic bioremediation processes. METHODS: PCDD/F and chlorobenzenes were analyzed by gas chromatography(GC)/mass spectrometry and GC/flame ionization detection, respectively. The activities of hydrolytic enzymes were determined from freshly sampled sediment layers using 4-methylumbelliferyl (MUF) or 7-amino-4-methylcoumarin-conjugated model compounds and kinetic fluorescence measurements. Physiologically active bacteria from different sediment layers were microscopically visualized by fluorescence in situ hybridization (FISH). Specific bacteria were identified by 16S rRNA gene amplification and sequencing. RESULTS AND DISCUSSION: The PCDD/F congener profile was dominated by dibenzofurans. In addition, the presence of specific tetra and pentachlorinated dibenzofurans supported the assumption that extensive magnesium production was one possible source for the high contamination. A range of other chloroorganic compounds, including several isomers of chlorobenzenes, hexachlorocyclohexane and 1,1,1-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT), was present in the sediment. Activities of extracellular hydrolytic enzymes showed a strong decrease in those sediment layers that were characterized by high contents of absorbable organic halogen (AOX), indicating disturbed organic matter decay. Interestingly, an abnormal increase of cellulolytic enzyme activities below the organochlorine-rich layers was observed, possibly caused by residual cellulose from discharges of sulfite pulping wastes. FISH revealed physiologically active bacteria in most sediment layers from the surface down to the depth of about 60 cm, including members of Desulfitobacterium (D.) and Sulfurospirillum. The presence of D. dehalogenans was confirmed by its partial 16S rRNA gene sequence. CONCLUSION: Results of chemical sediment analyses demonstrated high loads of organochlorine compounds, particularly of PCDD/F. Several years after stopping the waste water discharge to Spittelwasser creek, this sediment remains a main source for pollution of the downstream river system by way of the ongoing mobilization of sediment during high floods. As indicated by our enzyme activity measurements, the decomposition potential for organic matter is low in organochlorine-rich sediment layers. In contrast, the comparably higher enzyme activities in less organochlorine-polluted sediment layers as well as the presence of physiologically active bacteria suggest a considerable potential for natural attenuation. RECOMMENDATIONS AND PERSPECTIVE: . From our data we strongly recommend to explore the degradative capacity of sediment microorganisms and the limits for in situ activity towards specific sediment pollutants in more detail. This will give a sound basis for the integration of bioremediation approaches into general concepts to reduce the risk that permanently radiates from this highly contaminated sediment.

The alkyl tert-butyl ether intermediate 2-hydroxyisobutyrate is degraded via a novel cobalamin-dependent mutase pathway.

Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated beta-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed.