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.     

Quantitative PCR Targeting 16S rRNA and Reductive Dehalogenase Genes Simultaneously Monitors Multiple Dehalococcoides Strains

The 16S rRNA gene provides insufficient information to infer the range of chloroorganic electron acceptors used by different Dehalococcoides organisms. To overcome this limitation and provide enhanced diagnostic tools for growth measurements, site assessment, and bioremediation monitoring, a quantitative real-time PCR (qPCR) approach targeting 16S rRNA genes and three Dehalococcoides reductive dehalogenase (RDase) genes with assigned function (i.e., tceA, bvcA, and vcrA) was designed and evaluated. qPCR standard curves generated for the RDase genes by use of genomic DNA from Dehalococcoides pure cultures correlated with standard curves obtained for both Bacteria- and Dehalococcoides-targeted 16S rRNA genes, suggesting that the RDase genes are useful targets for quantitative assessment of Dehalococcoides organisms. RDase gene probe/primer pairs were specific for the Dehalococcoides strains known to carry the diagnostic RDase gene sequences, and the qPCR method allowed the detection of as few as 1 to 20 and quantification of as few as 50 to 100 tceA, bvcA, or vcrA gene targets per PCR volume. The qPCR approach was applied to dechlorinating enrichment cultures, microcosms, and samples from a contaminated site. In characterized enrichment cultures where known Dehalococcoides strains were enumerated, the sum of the three RDase genes equaled the total Dehalococcoides cell numbers. In site samples and chloroethane-dechlorinating microcosms, the sum of the three RDase genes was much less than that predicted by Dehalococcoides-targeted qPCR, totaling 10 to 30% of the total Dehalococcoides cell numbers. Hence, a large number of Dehalococcoides spp. contain as-yet-unidentified RDase genes, indicating that our current understanding of the dechlorinating Dehalococcoides community is incomplete.     

Complete Detoxification of Vinyl Chloride by an Anaerobic Enrichment Culture and Identification of the Reductively Dechlorinating Population as a Dehalococcoides Species

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 μmol liter⁻¹day⁻¹, and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K_S) for VC was 5.8 μM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30°C, and negligible dechlorination occurred at 4 and 35°C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H₂ as electron donor. VC-dechlorinating cultures consumed H₂ to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.     

Detection and Quantification of Dehalococcoides-Related Bacteria in a Chlorinated Ethene-Contaminated Aquifer Undergoing Natural Attenuation

Detection and quantification of bacteria related to Dehalococcoides is essential for the development of effective remediation strategies for tetrachloroethene (PCE)-contaminated sites. In this study, the authors applied three methods for quantifying Dehalococcoides-like bacteria in a PCE-contaminated aquifer undergoing natural attenuation in Grenchen, Switzerland: a catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) protocol, a competitive nested polymerase chain reaction (PCR) approach, and a direct PCR end point quantification with external standards. For the investigated aquifer, multiple lines of evidence indicated that reductive dechlorination (and likely dehalorespiration) was an active process. Both PCR-based quantification methods indicated that low numbers of mostly sediment-bound Dehalococcoides were present in the contaminated zone of the Grenchen aquifer. Estimates based on the quantitative PCR methods ranged from 2.1 × 10⁷ to 1.5 × 10⁸ sediment-bound Dehalococcoides 16S rRNA gene copies per liter of aquifer volume. In contrast, the liquid phase only contained between 8 and 80 copies per liter aquifer volume. CARD-FISH was not sensitive enough for the quantification of Dehalococcoides cell numbers in this aquifer. Cloning and sequencing of the PCR products revealed the presence of sequences closely related to Dehalococcoides isolates such as D. ethenogenes and Dehalococcoides sp. BAV1. An apparently abundant group (termed “Grenchen Cluster”) of sequences more distantly related to Dehalococcoides was also identified, so far without cultured representatives.     

Molecular Analysis of Dehalococcoides 16S Ribosomal DNA from Chloroethene-Contaminated Sites throughout North America and Europe

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.     

Development of a Highly Sensitive Nested-PCR Procedure Using a Single Closed Tube for Detection of Erwinia amylovora in Asymptomatic Plant Material

A novel method, which involves a nested PCR in a single closed tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was similar to that of a standard nested PCR in two tubes. The specificity and sensitivity were greater than those of standard PCR procedures that used a single primer pair. The presence of inhibitors in plant material, very common in E. amylovora hosts, is overcome with this system in combination with a simple DNA extraction protocol because it eliminates many of the inhibitory compounds. In addition, it needs a very small sample volume (1 μl of DNA extracted). With 83 samples of naturally infected material, this method achieved better results than any other PCR technique: standard PCR detected 55% of positive samples, two-tube nested PCR detected 71% of positive samples, and nested PCR in a single closed tube detected 78% of positive samples. When analyzing asymptomatic plant material, the number of positive samples detected by the developed nested PCR was also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method is proposed for the detection of endophytic and epiphytic populations of E. amylovora in epidemiological studies and for routine use in quarantine surveys, due to its high sensitivity, specificity, speed, and simplicity.     

Quantitative PCR Confirms Purity of Strain GT, a Novel Trichloroethene-to-Ethene-Respiring Dehalococcoides Isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 μmol liter⁻¹ day⁻¹, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76 ± 0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

A new diagnostic system for ultra-sensitive and specific detection and quantification of Candidatus Liberibacter asiaticus, the bacterium associated with citrus Huanglongbing

An ultra-sensitive and quantitative diagnostic system by combining nested PCR and TaqMan® PCR in a single tube was developed for detection of “Candidatus Liberibacter asiaticus”. The procedure involves two PCR steps using the species-specific outer and inner primer pairs. Different annealing temperatures allow both the first and the second rounds of PCR to be performed sequentially in the same closed tube. The first PCR with outer primers was performed at a higher annealing temperature and with limited amount of primers to prevent interference with the inner primers during the second round of PCR. The specificity of the dual primer TaqMan® is high because the fluorescent signal can only be generated from the TaqMan® probes that are homologous to the product amplified by the outer and inner primers. This new detection system can reliably detect as few as single copies of target DNA. The sensitivity of the dual primer system is comparable to the conventional two-tube nested PCR, but it eliminates the potential risk of cross contamination commonly associated with conventional nested PCR. This one-tube dual primer TaqMan® PCR method is gel-free with reduced handling time and is cost effective. At the same time, this system provides significantly increased sensitivity, improved reliability and high through-put capability suitable for routine, large scale diagnoses of clinical plant tissue and insect samples. The technique described here is generic and can be applied to the detection of other plant pathogenic bacteria.     

Detection and Quantification of Geobacter lovleyi Strain SZ: Implications for Bioremediation at Tetrachloroethene- and Uranium-Impacted Sites

Geobacter lovleyi strain SZ reduces hexavalent uranium, U(VI), to U(IV) and is the first member of the metal-reducing Geobacter group capable of using tetrachloroethene (PCE) as a growth-supporting electron acceptor. Direct and nested PCR with specific 16S rRNA gene-targeted primer pairs distinguished strain SZ from other known chlorinated ethene-dechlorinating bacteria and closely related Geobacter isolates, including its closest cultured relative, G. thiogenes. Detection limits for direct and nested PCR were approximately 1 × 10⁶ and 1 × 10⁴ 16S rRNA gene copies per μl of template DNA, respectively. A quantitative real-time PCR (qPCR) approach increased the sensitivity to as few as 30 16S rRNA gene copies per μl of template DNA but was less specific. Melting curve analysis and comparison of the shapes of amplification plots identified false-positive signals and distinguished strain SZ from G. thiogenes when analyzed separately. These indicators were less reliable when target (strain SZ) DNA and nontarget (G. thiogenes) DNA with high sequence similarity were mixed, indicating that the development of qPCR protocols should not only evaluate specificity but also explore the effects of nontarget DNA on the accuracy of quantification. Application of specific tools detected strain SZ-like amplicons in PCE-dechlorinating consortia, including the bioaugmentation consortium KB-1, and two chlorinated ethene-impacted groundwater samples. Strain SZ-like amplicons were also detected in 13 of 22 groundwater samples following biostimulation at the uranium- and chlorinated solvent-contaminated Integrated Field-Scale Subsurface Research Challenge (IFC) site in Oak Ridge, TN. The numbers of strain SZ-like cells increased from below detection to 2.3 × 10⁷ ± 0.1 × 10⁷ per liter groundwater, suggesting that strain SZ-like organisms contribute to contaminant transformation. The G. lovleyi strain SZ-specific tools will be useful for monitoring bioremediation efforts at uranium- and/or chlorinated solvent-impacted sites such as the Oak Ridge IFC site.     

Continuous Steady-State Method Using Tenax for Delivering Tetrachloroethene to Chloro-Respiring Bacteria

Tenax-TA, a solid-phase sorbent, was used as an alternative to hexadecane for continuous delivery of tetrachloroethene (PCE) to Desulfuromonas strain BB1, a chloro-respiring microorganism. In both batch and bioreactor configurations, Tenax not only maintained low, steady-state concentrations of PCE in an active culture for several months but also adsorbed the product of dechlorination, cis-1,2-dichloroethene, before it approached toxic levels.     

The Dehalococcoides Population in Sediment-Free Mixed Cultures Metabolically Dechlorinates the Commercial Polychlorinated Biphenyl Mixture Aroclor 1260

Microbial reductive dechlorination of commercial polychlorinated biphenyl (PCB) mixtures (e.g., Aroclors) in aquatic sediments is crucial to achieve detoxification. Despite extensive efforts over nearly two decades, the microorganisms responsible for Aroclor dechlorination remained elusive. Here we demonstrate that anaerobic bacteria of the Dehalococcoides group derived from sediment of the Housatonic River (Lenox, MA) simultaneously dechlorinate 64 PCB congeners carrying four to nine chlorines in Aroclor 1260 in the sediment-free JN cultures. Quantitative real-time PCR showed that the Dehalococcoides cell titer in JN cultures amended with acetate and hydrogen increased from 7.07 × 10⁶ ± 0.42 × 10⁶ to 1.67 × 10⁸ ± 0.04 × 10⁸ cells/ml, concomitant with a 64.2% decrease of the PCBs with six or more chlorines in Aroclor 1260. No Dehalococcoides growth occurred in parallel cultures without PCBs. Aroclor 1260 dechlorination supported the growth of 9.25 × 10⁸ ± 0.04 × 10⁸ Dehalococcoides cells per μmol of chlorine removed. 16S rRNA gene-targeted PCR analysis of known dechlorinators (i.e., Desulfitobacterium, Dehalobacter, Desulfuromonas, Sulfurospirillum, Anaeromyxobacter, Geobacter, and o-17/DF-1-type Chloroflexi organisms) ruled out any involvement of these bacterial groups in the dechlorination. Our results suggest that the Dehalococcoides population present in the JN cultures also catalyzes in situ dechlorination of Aroclor 1260 in the Housatonic River. The identification of Dehalococcoides organisms as catalysts of extensive Aroclor 1260 dechlorination and our ability to propagate the JN cultures under defined conditions offer opportunities to study the organisms' ecophysiology, elucidate nutritional requirements, identify reductive dehalogenase genes involved in PCB dechlorination, and design molecular tools required for bioremediation applications.     

Populations Implicated in Anaerobic Reductive Dechlorination of 1,2-Dichloropropane in Highly Enriched Bacterial Communities

1,2-Dichloropropane (1,2-D), a widespread groundwater contaminant, can be reductively dechlorinated to propene by anaerobic bacteria. To shed light on the populations involved in the detoxification process, a comprehensive 16S rRNA gene-based bacterial community analysis of two enrichment cultures derived from geographically distinct locations was performed. Analysis of terminal restriction fragments, amplicons obtained with dechlorinator-specific PCR primers, and enumeration with quantitative real-time PCR as well as screening clone libraries all implied that Dehalococcoides populations were involved in 1,2-D dechlorination in both enrichment cultures. Physiological traits (e.g., dechlorination in the presence of ampicillin and a requirement for hydrogen as the electron donor) supported the involvement of Dehalococcoides populations in the dechlorination process. These findings expand the spectrum of chloroorganic compounds used by Dehalococcoides species as growth-supporting electron acceptors. The combined molecular approach allowed a comparison between different 16S rRNA gene-based approaches for the detection of Dehalococcoides populations.     

Evaluation for the Clinical Diagnosis of Pythium insidiosum Using a Single-Tube Nested PCR

Pythiosis is a rare infectious disease caused by Pythium insidiosum, which typically occurs in tropical and subtropical regions. The high mortality rate may be in consequence of the lack of diagnosis. The objective of this study was to evaluate reliability of a new single-tube nested PCR for detection of P. insidiosum DNA. A total of 78 clinical isolates of various fungi and bacteria, 106 clinical specimens and 80 simulated positive blood samples were tested. The developed primer pairs CPL6–CPR8 and YTL1–YTR1 are located on 18S subunit of the rRNA gene of P. insidiosum. The specificity, negative and positive predictive values were 100, 100 and 87.5 %, respectively, as compared with direct microscopy and cultivation. The detection limit of the single-tube nested PCR was 21 zoospores corresponding to 2.7 pg of the DNA. The results demonstrate that the new single-tube nested PCR offers a highly sensitive, specific and rapid genetic method for detecting P. insidiosum.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Multiple Nonidentical Reductive-Dehalogenase-Homologous Genes Are Common in Dehalococcoides

Degenerate primers were used to amplify large fragments of reductive-dehalogenase-homologous (RDH) genes from genomic DNA of two Dehalococcoides populations, the chlorobenzene- and dioxin-dechlorinating strain CBDB1 and the trichloroethene-dechlorinating strain FL2. The amplicons (1,350 to 1,495 bp) corresponded to nearly complete open reading frames of known reductive dehalogenase genes and short fragments (approximately 90 bp) of genes encoding putative membrane-anchoring proteins. Cloning and restriction analysis revealed the presence of at least 14 different RDH genes in each strain. All amplified RDH genes showed sequence similarity with known reductive dehalogenase genes over the whole length of the sequence and shared all characteristics described for reductive dehalogenases. Deduced amino acid sequences of seven RDH genes from strain CBDB1 were 98.5 to 100% identical to seven different RDH genes from strain FL2, suggesting that both strains have an overlapping substrate range. All RDH genes identified in strains CBDB1 and FL2 were related to the RDH genes present in the genomes of Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain BAV1; however, sequence identity did not exceed 94.4 and 93.1%, respectively. The presence of RDH genes in strains CBDB1, FL2, and BAV1 that have no orthologs in strain 195 suggests that these strains possess dechlorination activities not present in strain 195. Comparative sequence analysis identified consensus sequences for cobalamin binding in deduced amino acid sequences of seven RDH genes. In conclusion, this study demonstrates that the presence of multiple nonidentical RDH genes is characteristic of Dehalococcoides strains.     

Isolation and Characterization of “Dehalococcoides” sp. Strain MB,Which Dechlorinates Tetrachloroethene to trans-1,2-Dichloroethene

In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture “Dehalococcoides” sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (±0.4):1. It utilizes H₂ as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 μm in diameter and ∼150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.Dehalorespiring bacteria play an important role in the transformation and detoxification of a wide range of halogenated compounds, e.g., chlorophenols, chloroethenes, chlorobenzenes, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) (2, 4, 9, 14, 16, 17, 32, 35, 38). Among these compounds, the organic solvents tetrachloroethene (PCE) and trichloroethene (TCE) are suspected carcinogens that are found in soil and groundwater due to their extensive usage and improper disposal (6). The widespread PCE and TCE in the subsurface environment have driven intensive studies of anaerobic microbes capable of reductive dechlorination of chloroethenes (40). Over the last decade, at least 18 isolates, which belong to the genera Desulfitobacterium, Sulfurospirillum, Desulfomonile, Desulfuromonas, Geobacter, “Dehalococcoides,” and Dehalobacter, show reductive dechlorination of chlorinated ethenes (16, 40). In particular, most of these microbes produce cis-1,2-dichloroethene (cis-DCE) as the end product in the chloroethene-contaminated sites, whereas complete detoxification of PCE or TCE to ethene has been restricted only to members of the genus Dehalococcoides. Thus, the Dehalococcoides species have received considerable attention from the bioremediation community in the past decade.Several strains of Dehalococcoides species (e.g., 195, CBDB1, BAV1, and VS) have been sequenced for their whole genomes (24, 39). Their dechlorinating capabilities have also been well addressed through identification and quantification of the known chloroethene reductive dehalogenase (RDase) genes or expression of specific RDase genes (18, 21, 25, 41). In chloroethene-contaminated sites, the natural activities of single or multiple Dehalococcoides strains can lead either to more-toxic, mobile intermediates (e.g., cis- or trans-DCEs and vinyl chloride [VC]) via partial dechlorination of PCE/TCE or to harmless ethene by complete detoxification (10, 13, 15, 41). Many mixed cultures and pure isolates have been reported to produce cis-DCE or VC during PCE/TCE dechlorination processes (15, 40, 43). However, trans-DCE has been detected in more than one-third of the U.S. Environmental Protection Agency (EPA) superfund sites (3a). The source of trans-DCE production was thought to be an abiotic process; however, recently both trans-DCE generation and cis-DCE generation were reported to occur via microbial dechlorination.To date, microbes from either Dehalococcoides- or DF-1-containing mixed cultures have been reported to produce more trans- than cis-DCE, with a ratio of 1.2:1 to 3.5:1 in laboratory-scale studies (8, 10, 22, 31). For example, in a recent report by Kittelmann and Friedrich (22), trans-/cis-DCE at a ratio of 3.5:1 was generated in tidal flat sediment-containing microcosms with microbes closely related to Dehalococcoides sp. or DF-1-like microbes. Additionally, Griffin et al. identified Dehalococcoides species of the Pinellas subgroup in several enrichment cultures, which dechlorinated TCE (∼0.25 mM) to trans-DCE and cis-DCE at a ratio of ∼3:1 (10). There is no information available on the Dehalococcoides isolates that generate trans-DCE as the main end product. This also means a lack of information on the genomic contents of trans-DCE-producing bacteria. Therefore, finding microorganisms that produce trans-DCE in pure culture will be useful for the comprehensive characterization of this group of bacteria.The aim of this study was to isolate a PCE-to-trans-DCE-dechlorinating culture to facilitate the elucidation of trans-DCE formation during reductive dechlorination processes. Microarray analysis was conducted to compare the whole-genome contents of the new isolate and the well-characterized Dehalococcoides ethenogenes strain 195 (30). In addition, a coculture which consisted of the new isolate and TCE-to-cis-DCE-to-VC-dechlorinating Dehalococcoides sp. strain ANAS1 was explored to study the interaction, distribution, and function of the dechlorinators in the dechlorinating process.     

NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions

Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.     

Genetic Diversity of nifH Gene Sequences in Paenibacillus azotofixans Strains and Soil Samples Analyzed by Denaturing Gradient Gel Electrophoresis of PCR-Amplified Gene Fragments

The diversity of dinitrogenase reductase gene (nifH) fragments in Paenibacillus azotofixans strains was investigated by using molecular methods. The partial nifH gene sequences of eight P. azotofixans strains, as well as one strain each of the close relatives Paenibacillus durum, Paenibacillus polymyxa, and Paenibacillus macerans, were amplified by PCR by using degenerate primers and were characterized by DNA sequencing. We found that there are two nifH sequence clusters, designated clusters I and II, in P. azotofixans. The data further indicated that there was sequence divergence among the nifH genes of P. azotofixans strains at the DNA level. However, the gene products were more conserved at the protein level. Phylogenetic analysis showed that all nifH cluster II sequences were similar to the alternative (anf) nitrogenase sequence. A nested PCR assay for the detection of nifH (cluster I) of P. azotofixans was developed by using the degenerate primers as outer primers and two specific primers, designed on the basis of the sequence information obtained, as inner primers. The specificity of the inner primers was tested with several diazotrophic bacteria, and PCR revealed that these primers are specific for the P. azotofixans nifH gene. A GC clamp was attached to one inner primer, and a denaturing gradient gel electrophoresis (DGGE) protocol was developed to study the genetic diversity of this region of nifH in P. azotofixans strains, as well as in soil and rhizosphere samples. The results revealed sequence heterogeneity among different nifH genes. Moreover, nifH is probably a multicopy gene in P. azotofixans. Both similarities and differences were detected in the P. azotofixans nifH DGGE profiles generated with soil and rhizosphere DNAs. The DGGE assay developed here is reproducible and provides a rapid way to assess the intraspecific genetic diversity of an important functional gene in pure cultures, as well as in environmental samples.     

Detection and Quantification of Dehalogenimonas and “Dehalococcoides” Populations via PCR-Based Protocols Targeting 16S rRNA Genes

Members of the haloalkane dechlorinating genus Dehalogenimonas are distantly related to “Dehalococcoides” but share high homology in some variable regions of their 16S rRNA gene sequences. In this study, primers and PCR protocols intended to uniquely target Dehalococcoides were reevaluated, and primers and PCR protocols intended to uniquely target Dehalogenimonas were developed and tested. Use of the genus-specific primers revealed the presence of both bacterial groups in groundwater at a Louisiana Superfund site.“Dehalococcoides” strains are the only bacteria presently known to reductively dehalogenate the carcinogen vinyl chloride (10-12, 17, 22), and DNA-based approaches have been widely applied to detect and quantify these bacteria in mixed cultures and environmental samples (1, 3, 4, 6, 7, 13, 15, 16, 20). As recently reported, Dehalococcoides strains are the closest previously cultured phylogenetic relatives of Dehalogenimonas lykanthroporepellens strains BL-DC-8 and BL-DC-9^T (18, 23). The newly isolated Dehalogenimonas strains, which can reductively dehalogenate a variety of polychlorinated alkanes (e.g., 1,2,3-trichloropropane and 1,2-dichloroethane) but not chlorinated ethenes (e.g., tetrachloroethene and vinyl chloride), however, are only distantly related to Dehalococcoides, with 90% identity in 16S rRNA gene sequences. Research reported here was aimed at (i) reevaluating PCR primers and protocols previously reported as allowing specific detection of Dehalococcoides 16S rRNA gene sequences in light of the 16S rRNA gene sequences of the recently isolated Dehalogenimonas strains and (ii) designing and testing PCR primers and protocols that allow detection and quantification of Dehalogenimonas strains.