16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species

Members of the genera Desulfuromonas and Dehalococcoides reductively dechlorinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specific to hypervariable regions of the 16S rRNA genes of the Dehalococcoides group (comprising Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (comprising Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) were designed. The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with the Desulfuromonas dechlorinator-targeted primer pair was 1 x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR. All river sediment samples yielded positive signals with the BB1- and the Dehalococcoides-targeted primers. One chloroethene-contaminated aquifer tested positive with the Dehalococcoides-targeted primers, and another contaminated aquifer tested positive with the Desulfuromonas dechlorinator-targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from other known Desulfuromonas species. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the specific primers.     

Occurrence of several genes encoding putative reductive dehalogenases in Desulfitobacterium hafniense/frappieri and Dehalococcoides ethenogenes

Desulfitobacterium frappieri PCP-1 has the capacity to dehalogenate several halogenated aromatic compounds by reductive dehalogenation, however, the genes encoding the enzymes involved in such processes have not yet been identified. Using a degenerate oligonucleotide corresponding to a conserved sequence of CprA/PceA reductive dehalogenases, a cprA-like gene fragment was amplified by PCR from this bacterial strain. A Desulfitobacterium frappieri PCP-1 cosmid library was screened with the PCR product, allowing the cloning and sequencing of a 1.9-kb fragment. This fragment contains a nucleic acid sequence identical to one genomic contig of Desulfitobacterium hafniense, a bacterium closely related to Desulfitobacterium frappieri that is also involved in reductive dehalogenation. Other genes related to the Desulfitobacterium dehalogenans cpr locus were identified in this contig. Interestingly, the gene arrangement shows the presence of two copies of cprA-, cprB-, cprC-, cprD-, cprK-, and cprT-related genes, suggesting that gene duplication occurred within this chromosomic region. The screening of Delfitobacterium hafniense genomic contigs with a CprA-deduced amino acid sequence revealed two other cprA-like genes. Microbial genomes available in gene databases were also analyzed for sequences related to CprA/PceA. Two open reading frames encoding other putative reductive dehalogenases in Desulfitobacterium hafniense contigs were detected, along with 17 in the Dehalococcoides ethenogenes genome, a bacterium involved in the reductive dehalogenation of tetrachloroethene to ethene. The fact that several gene encoding putative reductive dehalogenases exist in Delfitobacterium hafniense, probably in other members of the genus Desulfitobacterium, and in Dehalococcoides ethenogenes suggests that these bacteria use distinct but related enzymes to achieve the dehalogenation of several chlorinated compounds [corrected].     

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.     

Comparative genomics of "Dehalococcoides ethenogenes" 195 and an enrichment culture containing unsequenced "Dehalococcoides" strains

Tetrachloroethene (PCE) and trichloroethene (TCE) are prevalent groundwater contaminants that can be completely reductively dehalogenated by some "Dehalococcoides" organisms. A Dehalococcoides-organism-containing microbial consortium (referred to as ANAS) with the ability to degrade TCE to ethene, an innocuous end product, was previously enriched from contaminated soil. A whole-genome photolithographic microarray was developed based on the genome of "Dehalococcoides ethenogenes" 195. This microarray contains probes designed to hybridize to >99% of the predicted protein-coding sequences in the strain 195 genome. DNA from ANAS was hybridized to the microarray to characterize the genomic content of the ANAS enrichment. The microarray results revealed that the genes associated with central metabolism, including an apparently incomplete carbon fixation pathway, cobalamin-salvaging system, nitrogen fixation pathway, and five hydrogenase complexes, are present in both strain 195 and ANAS. Although the gene encoding the TCE reductase, tceA, was detected, 13 of the 19 reductive dehalogenase genes present in strain 195 were not detected in ANAS. Additionally, 88% of the genes in predicted integrated genetic elements in strain 195 were not detected in ANAS, consistent with these elements being genetically mobile. Sections of the tryptophan operon and an operon encoding an ABC transporter in strain 195 were also not detected in ANAS. These insights into the diversity of Dehalococcoides genomes will improve our understanding of the physiology and evolution of these bacteria, which is essential in developing effective strategies for the bioremediation of PCE and TCE in the environment.     

Partial purification, stereospecificity and stoichiometry of three dehalogenases from a Rhizobium species

Abstract A fast-growing species of Rhizobium that utilized 2,2-dichloropropionate (2,2DCP) and d,l -2-chloropropionate ( dl -2CP) as sole sources of carbon and energy was shown to contain three inducible dehalogenases. These enzymes differed in their substrate specificities: dehalogenase II degraded 2,2DCP, d - and l -2CP, monochloroacetate (MCA) and dichloroacetate (DCA) whilst dehalogenase I showed activity only towards l -2CP and DCA. Dehalogenase III liberated halide from d -2CP and MCA. This is the first report of a dehalogenase acting solely on the d -isomer of a haloalkanoate. All three dehalogenases inverted the isomeric configuration during dehalogenation, forming d (−) and l (+) lactate from l - and d -2CP, respectively.     

Unusual codon bias in vinyl chloride reductase genes of Dehalococcoides species

Vinyl chloride reductases (VC-RDase) are the key enzymes for complete microbial reductive dehalogenation of chloroethenes, including the groundwater pollutants tetrachloroethene and trichloroethene. Analysis of the codon usage of the VC-RDase genes vcrA and bvcA showed that these genes are highly unusual and are characterized by a low G+C fraction at the third position. The third position of codons in VC-RDase genes is biased toward the nucleotide T, even though available Dehalococcoides genome sequences indicate the absence of any tRNAs matching codons that end in T. The comparatively high level of abnormality in the codon usage of VC-RDase genes suggests an evolutionary history that is different from that of most other Dehalococcoides genes.     

Multiple modes in a coral species abundance distribution

Species abundance distributions are an important measure of biodiversity and community structure. These distributions are affected by sampling, and alternative species-abundance models often make similar predictions for small sample sizes. Very large samples reveal the relative abundances of rare species, and thus provide information about species relative abundances that small samples cannot. Here, we present the species-abundance distribution for a sample of > 40,000 coral colonies at a single site, exceeding existing samples of coral local assemblages by over an order of magnitude. This abundance distribution is multimodal when examined on a logarithmic scale. Four different model selection procedures all indicate that the underlying community abundance distribution has at least three modes. We show that the multiple modes are not caused by mixtures of species with different habitat preferences. However, spatial aggregation partially explains our results. We inspect published work on species abundance distributions, and suggest that multimodality may be a common feature of large samples.     

River flow and fish abundance in a South African estuary

The ichthyofauna of the Thukela Estuary, a small (55 ha), shallow (<1·5 m) system on the KwaZulu-Natal coast (mean annual river runoff of 3865 × 10⁶ m³, from a large catchment of 29 000 km², is seasonal: peak inputs occurring between November and March), was dominated by the juveniles of marine taxa that used the estuary as a nursery area. A striking feature of the above community was the decline in fish abundance with increasing river input, with flow values >100 m³ s⁻¹ leading to increased loss of species from the system. This decline was linked to the lack of saline intrusion into the estuary and increased freshwater flooding through the system.     

Improved quantification of Dehalococcoides species by fluorescence in situ hybridization and catalyzed reporter deposition

Chlorinated ethenes contamination of soil and groundwater is a widespread problem in most industrialized countries. To date, there is a general consensus in the literature that members of the genus Dehalococcoides are required for complete dechlorination of these compounds. The availability of specific identification tools to track their distribution in the field is therefore a topic of particular relevance in environmental studies. These microorganisms have been successfully visualized by fluorescence in situ hybridization (FISH) in highly active dechlorinating cultures. However, FISH detection of Dehalococcoides under low activity conditions can be strongly hampered by their small cell size and low ribosome content. In this study, catalyzed reporter deposition (CARD)-FISH was employed as an alternative detection method. In a trichloroethene (TCE) dechlorinating enrichment culture, CARD-FISH, using proteinase K as a permeabilization pre-treatment, was found to be significantly superior to conventional FISH in terms of both microscopic visualization and quantification efficiency (about 30%). An application of this method on contaminated aquifer samples is also presented and discussed.     

Single-nucleotide primer extension assay for detection and sequence typing of "Dehalococcoides" spp

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.     

Estimating the number of species in a stochastic abundance model

Consider a stochastic abundance model in which the species arrive in the sample according to independent Poisson processes, where the abundance parameters of the processes follow a gamma distribution. We propose a new estimator of the number of species for this model. The estimator takes the form of the number of duplicated species (i.e., species represented by two or more individuals) divided by an estimated duplication fraction. The duplication fraction is estimated from all frequencies including singleton information. The new estimator is closely related to the sample coverage estimator presented by Chao and Lee (1992, Journal of the American Statistical Association 87, 210-217). We illustrate the procedure using the Malayan butterfly data discussed by Fisher, Corbet, and Williams (1943, Journal of Animal Ecology 12, 42-58) and a 1989 Christmas Bird Count dataset collected in Florida, U.S.A. Simulation studies show that this estimator compares well with maximum likelihood estimators (i.e., empirical Bayes estimators from the Bayesian viewpoint) for which an iterative numerical procedure is needed and may be infeasible.     

How species richness and total abundance constrain the distribution of abundance

The species abundance distribution (SAD) is one of the most intensively studied distributions in ecology and its hollow‐curve shape is one of ecology's most general patterns. We examine the SAD in the context of all possible forms having the same richness (S) and total abundance (N), i.e. the feasible set. We find that feasible sets are dominated by similarly shaped hollow curves, most of which are highly correlated with empirical SADs (most R² values > 75%), revealing a strong influence of N and S on the form of the SAD and an a priori explanation for the ubiquitous hollow curve. Empirical SADs are often more hollow and less variable than the majority of the feasible set, revealing exceptional unevenness and relatively low natural variability among ecological communities. We discuss the importance of the feasible set in understanding how general constraints determine observable variation and influence the forms of predicted and empirical patterns.     

Frequent concomitant presence of Desulfitobacterium spp. and "Dehalococcoides" spp. in chloroethene-dechlorinating microbial communities

The presence of chloroethene dechlorination activity as well as several bacterial genera containing mainly organohalide-respiring members was investigated in 34 environmental samples from 18 different sites. Cultures inoculated with these environmental samples on tetrachloroethene and amended weekly with a seven organic electron donor mixture resulted in 11 enrichments with cis-DCE, ten with VC, and 11 with ethene as dechlorination end product, and only two where no dechlorination was observed. “Dehalococcoides” spp. and Desulfitobacterium spp. were detected in the majority of the environmental samples independently of the dechlorination end product formed. The concomitant presence of Dehalococcoides spp. and Desulfitobacterium spp. in the majority of the enrichments suggested that chloroethene dechlorination was probably the result of catalysis by at least two organohalide-respiring genera either in parallel or by stepwise catalysis. A more detailed study of one enrichment on cis-DCE suggested that in this culture Desulfitobacterium spp. as well as Dehalococcoides spp. dechlorinated cis-DCE whereas dechlorination of VC was only catalyzed by the latter.