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.     

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.     

Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

Growth of Dehalobacter and Dehalococcoides spp. during degradation of chlorinated ethanes

Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.     

Comparative proteomics of Dehalococcoides spp. reveals strain-specific peptides associated with activity

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.     

Comparative Proteomics of Dehalococcoides spp. Reveals Strain-Specific Peptides Associated with Activity

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.     

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.     

Reductive dechlorination of hexachlorobenzene to tri- and dichlorobenzenes in anaerobic sewage sludge.

Hexachlorobenzene was dechlorinated to tri- and dichlorobenzenes in anaerobic sewage sludge. The complete biotransformation of 190 microM hexachlorobenzene (approximately 50 ppm) occurred within 3 weeks. The calculated rate of hexachlorobenzene dechlorination was 13.6 mumol liter-1 day-1. Hexachlorobenzene was dechlorinated via two routes, both involving the sequential removal of chlorine from the aromatic ring. The major route was hexachlorobenzene----pentachlorobenzene----1,2,3,5-tetrachlorobenzene--- -1,3,5- trichlorobenzene. Greater than 90% of the added hexachlorobenzene was recovered as 1,3,5-trichlorobenzene, and there was no evidence for further dechlorination of 1,3,5-trichlorobenzene. The minor route was hexachlorobenzene----pentachlorobenzene----1,2,4,5-tetrachlorobenzene--- -1,2,4- trichlorobenzene----dichlorobenzenes. These results extend reductive dechlorination to poorly water soluble aromatic hydrocarbons which could potentially include other important environmental pollutants like polychlorinated biphenyls.     

Unexpected Specificity of Interspecies Cobamide Transfer from Geobacter spp. to Organohalide-Respiring Dehalococcoides mccartyi Strains

Dehalococcoides mccartyi strains conserve energy from reductive dechlorination reactions catalyzed by corrinoid-dependent reductive dehalogenase enzyme systems. Dehalococcoides lacks the ability for de novo corrinoid synthesis, and pure cultures require the addition of cyanocobalamin (vitamin B(12)) for growth. In contrast, Geobacter lovleyi, which dechlorinates tetrachloroethene to cis-1,2-dichloroethene (cis-DCE), and the nondechlorinating species Geobacter sulfurreducens have complete sets of cobamide biosynthesis genes and produced 12.9 ± 2.4 and 24.2 ± 5.8 ng of extracellular cobamide per liter of culture suspension, respectively, during growth with acetate and fumarate in a completely synthetic medium. G. lovleyi-D. mccartyi strain BAV1 or strain FL2 cocultures provided evidence for interspecies corrinoid transfer, and cis-DCE was dechlorinated to vinyl chloride and ethene concomitant with Dehalococcoides growth. In contrast, negligible increase in Dehalococcoides 16S rRNA gene copies and insignificant dechlorination occurred in G. sulfurreducens-D. mccartyi strain BAV1 or strain FL2 cocultures. Apparently, G. lovleyi produces a cobamide that complements Dehalococcoides' nutritional requirements, whereas G. sulfurreducens does not. Interestingly, Dehalococcoides dechlorination activity and growth could be restored in G. sulfurreducens-Dehalococcoides cocultures by adding 10 μM 5',6'-dimethylbenzimidazole. Observations made with the G. sulfurreducens-Dehalococcoides cocultures suggest that the exchange of the lower ligand generated a cobalamin, which supported Dehalococcoides activity. These findings have implications for in situ bioremediation and suggest that the corrinoid metabolism of Dehalococcoides must be understood to faithfully predict, and possibly enhance, reductive dechlorination activities.     

Distribution and ecology of lampreys Lethenteron spp. in interior Alaskan rivers

This study describes phenetic characteristics and examines the life history of anadromous Arctic lamprey Lethenteron camtschaticum and freshwater‐resident Alaskan brook lamprey Lethenteron alaskense in two tributaries of the middle Yukon River, Alaska. Larval lampreys could not be identified to species using pigmentation density patterns or trunk myomere counts, but adults could be identified to species based on colouration, body size and oral‐disc dentition. Although larvae were patchily distributed in both rivers, there was a greater proportion of sample locations where Lethenteron spp. were absent in upper reaches than in middle and lower reaches. Relative abundance, density and median and maximum total length (L_T) of larvae were highly variable among sampling locations. Current velocity, substratum type and coarse woody debris were most strongly correlated with larval Lethenteron spp. density; velocity and substratum size was negatively correlated, whereas woody debris was positively correlated. Water depth, dissolved oxygen levels and specific conductance did not influence habitat selection. Length–frequency distributions, diet composition (organic detritus ≥ 98%), assimilation efficiency (>70%) and gut fullness (<0·2 mg diet ash‐free dry mass) were similar for larvae, regardless of river or sampling location. These results increase understanding of Lethenteron spp. biology and ecology in interior Alaska drainages and add to the existing literature on Lethenteron spp. life history.     

Role of the spleen in Bartonella spp. infection

Bartonella spp. are intra-erythrocytic pathogens of mammals. In this study, we investigated the role of the spleen, and other tissue and organs in Bartonella infection. Using an in vivo model of mice infection by Bartonella birtlesii, we detected accumulation of bacteria in the spleen, with transient infection of the liver, but failed to detect any bacteria in brain or lymph nodes. We then compared bacteraemia in normal Balb/C mice and in splenectomized mice. Bacteraemia in splenectomized mice was 10-fold higher than in normal mice and lasted 2?weeks longer. In conclusion, the spleen seems to retain and filter infected erythrocytes rather than to be a sanctuary for chronic Bartonella infection.     

Reductive dechlorination of hexachlorobenzene to tri- and dichlorobenzenes in anaerobic sewage sludge

Hexachlorobenzene was dechlorinated to tri- and dichlorobenzenes in anaerobic sewage sludge. The complete biotransformation of 190 microM hexachlorobenzene (approximately 50 ppm) occurred within 3 weeks. The calculated rate of hexachlorobenzene dechlorination was 13.6 mumol liter-1 day-1. Hexachlorobenzene was dechlorinated via two routes, both involving the sequential removal of chlorine from the aromatic ring. The major route was hexachlorobenzene----pentachlorobenzene----1,2,3,5-tetrachlorobenzene--- -1,3,5- trichlorobenzene. Greater than 90% of the added hexachlorobenzene was recovered as 1,3,5-trichlorobenzene, and there was no evidence for further dechlorination of 1,3,5-trichlorobenzene. The minor route was hexachlorobenzene----pentachlorobenzene----1,2,4,5-tetrachlorobenzene--- -1,2,4- trichlorobenzene----dichlorobenzenes. These results extend reductive dechlorination to poorly water soluble aromatic hydrocarbons which could potentially include other important environmental pollutants like polychlorinated biphenyls.     

Plasmid transformation of Bacteroides spp. by electroporation

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.     

Characterization and pathogenicity of three Phytophthora spp. recovered from rivers in Bulgaria

Many Phytophthora species are pathogens on fruit trees and may cause destructive diseases. In the current study, we examined six Phytophthora isolates recovered from rivers in Bulgaria, representatives of the following three species: Phytophthora chlamydospora, P. pseudocryptogea and P. syringae. Morphological traits, cardinal temperatures and growth rates of the isolates were described. We found considerable variation in the size of sporangia and significant difference in the mycelial growth rates of the two P. pseudocryptogea isolates, along with multiple polymorphic sites in the ITS region of one of them. In the cases of the other two Phytophthora species, no such differences were found between the isolates. Both P. chlamydospora isolates had a lower optimum growth temperature compared with the reported in the literature for this species. In pathogenicity tests against leaves and fruits of apple, pear, cherry, apricot and plum, the isolates proved to be capable of causing infections with varying severity. P. chlamydospora showed to be the most aggressive towards the leaves, while P. pseudocryptogea isolates induced the highest percentage of decay on the fruits of all tested tree species, which may suggest partial organ or tissue specificity. The demonstrated infection capacity of the described isolates points out the investigated Phytophthora species as a potential threat for the orchards in Bulgaria, if favourable conditions are available.     

Decreasing organic carbon bioreactivity in European rivers

1. European rivers experience increased loading of total organic carbon (TOC) from terrestrial sources due to factors involving changes in land use, climate and soil acidity. However, little is known about how increased TOC is linked to changes in the bioreactivity of organic matter in these rivers on a continental scale.
2. We compiled paired measurements of TOC and biological oxygen demand in 5-day 20°C dark incubations from 3,486 EU monitoring rivers. Assuming first-order decay and a fixed respiratory quotient, annual average TOC and biological oxygen demand values were used to calculate 11,060 values of the decay coefficient k.
3. The k decreased by two orders of magnitude as a power function of increasing TOC. This relationship could partly be explained by carbon quality, as the C:N ratio of the organic matter was the lowest in high-reactivity low-TOC rivers, and vice versa. A trend analysis showed that TOC increased by 18% from 1996 to 2012, while k decreased by as much as 50%. As a consequence, the biological oxygen demand in the water decreased over time in spite of the water browning trend (increased TOC).
4. Together, these results suggest that reactivity is low near terrestrial hot spots for TOC export, and low during years with high terrestrial TOC loading, but increases in rivers with low TOC concentrations where internal processes in the water have high relative influence on bulk TOC quality. Thus, browning of European freshwaters is linked to strong decreases in TOC reactivity on a continental scale, suggesting that the impacts of browning on microbial water deoxygenation and greenhouse gas production are less severe than previously thought.