The Desulfitobacterium genus

Desulfitobacterium spp. are strictly anaerobic bacteria that were first isolated from environments contaminated by halogenated organic compounds. They are very versatile microorganisms that can use a wide variety of electron acceptors, such as nitrate, sulfite, metals, humic acids, and man-made or naturally occurring halogenated organic compounds. Most of the Desulfitobacterium strains can dehalogenate halogenated organic compounds by mechanisms of reductive dehalogenation, although the substrate spectrum of halogenated organic compounds varies substantially from one strain to another, even with strains belonging to the same species. A number of reductive dehalogenases and their corresponding gene loci have been isolated from these strains. Some of these loci are flanked by transposition sequences, suggesting that they can be transmitted by horizontal transfer via a catabolic transposon. Desulfitobacterium spp. can use H2 as electron donor below the threshold concentration that would allow sulfate reduction and methanogenesis. Furthermore, there is some evidence that syntrophic relationships occur between Desulfitobacterium spp. and sulfate-reducing bacteria, from which the Desulfitobacterium cells acquire their electrons by interspecies hydrogen transfer, and it is believed that this relationship also occurs in a methanogenic consortium. Because of their versatility, desulfitobacteria can be excellent candidates for the development of anaerobic bioremediation processes. The release of the complete genome of Desulfitobacterium hafniense strain Y51 and information from the partial genome sequence of D. hafniense strain DCB-2 will certainly help in predicting how desulfitobacteria interact with their environments and other microorganisms, and the mechanisms of actions related to reductive dehalogenation.     

Biological consequences of ancient gene acquisition and duplication in the large genome of Candidatus Solibacter usitatus Ellin6076

Members of the bacterial phylum Acidobacteria are widespread in soils and sediments worldwide, and are abundant in many soils. Acidobacteria are challenging to culture in vitro, and many basic features of their biology and functional roles in the soil have not been determined. Candidatus Solibacter usitatus strain Ellin6076 has a 9.9 Mb genome that is approximately 2-5 times as large as the other sequenced Acidobacteria genomes. Bacterial genome sizes typically range from 0.5 to 10 Mb and are influenced by gene duplication, horizontal gene transfer, gene loss and other evolutionary processes. Our comparative genome analyses indicate that the Ellin6076 large genome has arisen by horizontal gene transfer via ancient bacteriophage and/or plasmid-mediated transduction, and widespread small-scale gene duplications, resulting in an increased number of paralogs. Low amino acid sequence identities among functional group members, and lack of conserved gene order and orientation in regions containing similar groups of paralogs, suggest that most of the paralogs are not the result of recent duplication events. The genome sizes of additional cultured Acidobacteria strains were estimated using pulsed-field gel electrophoresis to determine the prevalence of the large genome trait within the phylum. Members of subdivision 3 had larger genomes than those of subdivision 1, but none were as large as the Ellin6076 genome. The large genome of Ellin6076 may not be typical of the phylum, and encodes traits that could provide a selective metabolic, defensive and regulatory advantage in the soil environment.     

Phenotypic and Genotypic Description of Sedimenticola selenatireducens Strain CUZ,a Marine (Per)Chlorate-Respiring Gammaproteobacterium,and Its Close Relative the Chlorate-Respiring Sedimenticola Strain NSS

Two (per)chlorate-reducing bacteria, strains CUZ and NSS, were isolated from marine sediments in Berkeley and San Diego, CA, respectively. Strain CUZ respired both perchlorate and chlorate [collectively designated (per)chlorate], while strain NSS respired only chlorate. Phylogenetic analysis classified both strains as close relatives of the gammaproteobacterium Sedimenticola selenatireducens. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) preparations showed the presence of rod-shaped, motile cells containing one polar flagellum. Optimum growth for strain CUZ was observed at 25 to 30°C, pH 7, and 4% NaCl, while strain NSS grew optimally at 37 to 42°C, pH 7.5 to 8, and 1.5 to 2.5% NaCl. Both strains oxidized hydrogen, sulfide, various organic acids, and aromatics, such as benzoate and phenylacetate, as electron donors coupled to oxygen, nitrate, and (per)chlorate or chlorate as electron acceptors. The draft genome of strain CUZ carried the requisite (per)chlorate reduction island (PRI) for (per)chlorate respiration, while that of strain NSS carried the composite chlorate reduction transposon responsible for chlorate metabolism. The PRI of strain CUZ encoded a perchlorate reductase (Pcr), which reduced both perchlorate and chlorate, while the genome of strain NSS included a gene for a distinct chlorate reductase (Clr) that reduced only chlorate. When both (per)chlorate and nitrate were present, (per)chlorate was preferentially utilized if the inoculum was pregrown on (per)chlorate. Historically, (per)chlorate-reducing bacteria (PRB) and chlorate-reducing bacteria (CRB) have been isolated primarily from freshwater, mesophilic environments. This study describes the isolation and characterization of two highly related marine halophiles, one a PRB and the other a CRB, and thus broadens the known phylogenetic and physiological diversity of these unusual metabolisms.     

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis -dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.     

Two distinct enzyme systems are responsible for tetrachloroethene and chlorophenol reductive dehalogenation in Desulfitobacterium strain PCE1

Desulfitobacterium strain PCE1 is able to use tetrachloroethene and chloroaromatics as terminal electron acceptors for growth. Cell extracts of Desulfitobacterium strain PCE1 grown with tetrachloroethene as electron acceptor showed no dehalogenase activity with 3-chloro-4-hydroxyphenylacetate (Cl-OH-phenylacetate) and other ortho-chlorophenolic compounds in an in vitro assay. Extracts of cells that were grown with Cl-OH-phenylacetate as electron acceptor dechlorinated tetrachloroethene at 10% of the dechlorination rate of Cl-OH-phenylacetate. In both cell extracts dechlorination was inhibited by the addition of 1-iodopropane and dinitrogen oxide, inhibitors of cobalamin-containing enzymes. The enzymes responsible for tetrachloroethene and Cl-OH-phenylacetate dechlorination were partially purified. A 100-fold enriched fraction of chlorophenol reductive dehalogenase was obtained that mainly contained a protein with a subunit size of 48 kDa. The characteristics of this enzyme are similar to that of the chlorophenol reductive dehalogenase of D. dehalogenans. After partial purification of the tetrachloroethene reductive dehalogenase, a fraction was obtained that also contained a 48-kDa protein, but the N-terminal sequence showed no similarity with that of the chlorophenol reductive dehalogenase sequence or with the N-terminal amino acid sequence of tetra- and trichloroethene reductive dehalogenase of Desulfitobacterium strain TCE1. These results provide strong evidence that two different enzymes are responsible for tetrachloroethene and chlorophenol dechlorination in Desulfitobacterium strain PCE1. Furthermore, the characterization of partially purified tetrachloroethene reductive dehalogenase indicated that this enzyme is a novel type of reductive dehalogenase.     

Physiological adaptation of Desulfitobacterium hafniense strain TCE1 to tetrachloroethene respiration

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the Wood-Ljungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.     

Sulfonates as terminal electron acceptors for growth of sulfite-reducing bacteria (Desulfitobacterium spp.) and sulfate-reducing bacteria: effects of inhibitors of sulfidogenesis.

This study demonstrates the ability of Desulfitobacterium spp. to utilize aliphatic sulfonates as terminal electron acceptors (TEA) for growth. Isethionate (2-hydroxyethanesulfonate) reduction by Desulfitobacterium hafniense resulted in acetate as well as sulfide accumulation in accordance with the expectation that the carbon portion of isethionate was oxidized to acetate and the sulfur was reduced to sulfide. The presence of a polypeptide, approximately 97 kDa, was evident in isethionate-grown cells of Desulfitobacterium hafniense, Desulfitobacterium sp. strain PCE 1, and the two sulfate-reducing bacteria (SRB)-Desulfovibrio desulfuricans IC1 (T. J. Lie, J. R. Leadbetter, and E. R. Leadbetter, Geomicrobiol. J. 15:135-149, 1998) and Desulfomicrobium norvegicum; this polypeptide was not detected when these bacteria were grown on TEA other than isethionate, suggesting involvement in its metabolism. The sulfate analogs molybdate and tungstate, effective in inhibiting sulfate reduction by SRB, were examined for their effects on sulfonate reduction. Molybdate effectively inhibited sulfonate reduction by strain IC1 and selectively inhibited isethionate (but not cysteate) reduction by Desulfitobacterium dehalogenans and Desulfitobacterium sp. strain PCE 1. Desulfitobacterium hafniense, however, grew with both isethionate and cysteate in the presence of molybdate. In contrast, tungstate only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium spp. Similarly, another inhibitor of sulfate reduction, 1,8-dihydroxyanthraquinone, effectively inhibited sulfate reduction by SRB but only partially inhibited sulfonate reduction by both SRB and Desulfitobacterium hafniense.     

Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroxybenzoate.

Strain Co23, an anaerobic spore-forming microorganism, was enriched and isolated from a compost soil on the basis of its ability to grow with 2,3-dichlorophenol (DCP) as its electron acceptor, ortho chlorines were removed from polysubstituted phenols but not from monohalophenols. Growth by chlororespiration was indicated by a growth yield of 3.24 g of cells per mol of reducing equivalents (as 2[H]) from lactate oxidation to acetate in the presence of 3-chloro-4-hydroxybenzoate but no growth in the absence of the halogenated electron acceptor. Other indicators of chlororespiration were the fraction of electrons from the electron donor used for dechlorination (0.67) and the H2 threshold concentration of < 1.0 ppm. Additional electron donors utilized for reductive dehalogenation were pyruvate, formate, butyrate, crotonate, and H2. Pyruvate supported homoacetogenic growth in the absence of an electron acceptor. Strain Co23 also used sulfite, thiosulfate, and sulfur as electron acceptors for growth, but it did not use sulfate, nitrate or fumarate. The temperature optimum for growth was 37 degrees C; however, the rates of dechlorination were optimum at 45 degrees C and activity persisted to temperatures as high as 55 degrees C. The 16S rRNA sequence was determined, and strain Co23 was found to be related to Desulfitobacterium dehalogenans JW/IU DC1 and Desulfitobacterium strain PCE1, with sequence similarities of 97.2 and 96.8%, respectively. The phylogenetic and physiological properties exhibited by strain Co23 place it into a new species designated Desulfitobacterium chlororespirans.     

A Desulfitobacterium strain isolated from human feces that does not dechlorinate chloroethenes or chlorophenols

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron acceptors such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. 13C-NMR labeling experiments showed homoacetogenic 13C-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 degrees C, respectively. The growth rate under these conditions was approximately 0.10 h(-1). Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor.     

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.     

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].     

Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.     

Genomic,Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1^T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.     

Comprehensive analysis of glycosyltransferases in eukaryotic genomes for structural and functional characterization of glycans

Glycosyltransferases comprise highly divergent groups of enzymes, which play a central role in the synthesis of complex glycans. Because the repertoire of glycosyltransferases in the genome determines the range of synthesizable glycans, and because the increasing amount of genome sequence data is now available, it is essential to examine these enzymes across organisms to explore possible structures and functions of the glycoconjugates. In this study, we systematically investigated 36 eukaryotic genomes and obtained 3426 glycosyltransferase homologs for biosynthesis of major glycans, classified into 53 families based on sequence similarity. The families were further grouped into six functional categories based on the biosynthetic pathways, which revealed characteristic patterns among organism groups in the degree of conservation and in the number of paralogs. The results also revealed a strong correlation between the number of glycosyltransferases and the number of coding genes in each genome. We then predicted the ability to synthesize major glycan structures including N-glycan precursors and GPI-anchors in each organism from the combination of the glycosyltransferase families. This indicates that not only parasitic protists but also some algae are likely to synthesize smaller structures than the structures known to be conserved among a wide range of eukaryotes. Finally we discuss the functions of two large families, sialyltransferases and β4-glycosyltransferases, by performing finer classifications into subfamilies. Our findings suggest that universality and diversity of glycans originate from two types of evolution of glycosyltransferase families, namely conserved families with few paralogs and diverged families with many paralogs.     

Phenyl methyl ethers: novel electron donors for respiratory growth of<Emphasis Type="Italic"> Desulfitobacterium hafniense</Emphasis> and<Emphasis Type="Italic"> Desulfitobacterium</Emphasis> sp. strain PCE-S

Desulfitobacterium hafniense and Desulfitobacterium sp. strain PCE-S grew under anoxic conditions with a variety of phenyl methyl ethers as electron donors in combination with fumarate as electron acceptor. The phenyl methyl ethers were O-demethylated to the corresponding phenol compounds. O-demethylation was strictly dependent on the presence of fumarate; no O-demethylation occurred with CO₂ as electron acceptor. One mol phenyl methyl ether R-O-CH₃ was O-demethylated to R-OH per 3 mol fumarate reduced to succinate. The growth yields with vanillate or syringate plus fumarate were approximately 15 g cells (dry weight) per mol methyl moiety converted. D. hafniense utilized vanillate or syringate as an electron donor for reductive dehalogenation of 3-Cl-4-hydroxyphenylacetate, whereas strain PCE-S was not able to dechlorinate tetrachloroethene with phenyl methyl ethers. Crude extracts of both organisms showed O-demethylase activity in the O-demethylase assay with vanillate or syringate as substrates when the organism was grown on syringate plus fumarate. Besides the homoacetogenic bacteria, only growing cells of Desulfitobacterium frappieri PCP-1 have thus far been reported to be capable of phenyl methyl ether O-demethylation. This present study is the first report of Desulfitobacteria utilizing phenyl methyl ethers as electron donors for fumarate reduction and for growth.Abbreviations PCE Tetrachloroethene - TCE Trichloroethene - DCE cis-1,2-Dichloroethene - ClOHPA 3-Cl-4-Hydroxyphenylacetate - OHPA 4-Hydroxyphenylacetate - FH₄ Tetrahydrofolate     

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.     

Genes and Processed Paralogs Co-exist in Plant Mitochondria

RNA-mediated gene duplication has been proposed to create processed paralogs in the plant mitochondrial genome. A processed paralog may retain signatures left by the maturation process of its RNA precursor, such as intron removal and no need of RNA editing. Whereas it is well documented that an RNA intermediary is involved in the transfer of mitochondrial genes to the nucleus, no direct evidence exists for insertion of processed paralogs in the mitochondria (i.e., processed and un-processed genes have never been found simultaneously in the mitochondrial genome). In this study, we sequenced a region of the mitochondrial gene nad1, and identified a number of taxa were two different copies of the region co-occur in the mitochondria. The two nad1 paralogs differed in their (a) presence or absence of a group II intron, and (b) number of edited sites. Thus, this work provides the first evidence of co-existence of processed paralogs and their precursors within the plant mitochondrial genome. In addition, mapping the presence/absence of the paralogs provides indirect evidence of RNA-mediated gene duplication as an essential process shaping the mitochondrial genome in plants.