Dehalococcoides strains reductively dechlorinate a wide variety of halogenated compounds including chlorinated benzenes, biphenyls, naphthalenes, dioxins, and ethenes. Recent genome sequencing of the two Dehalococcoides strains CBDB1 and 195 revealed the presence of 32 and 18 reductive dehalogenase homologous genes, respectively, and therefore suggested an even higher dechlorinating potential than previously anticipated. Here, we demonstrate reductive dehalogenation of chlorophenol congeners by Dehalococcoides strains CBDB1 and 195. Strain CBDB1 completely converted 2,3-dichlorophenol, all six trichlorophenols, all three tetrachlorophenols, and pentachlorophenol to lower chlorinated phenols. Observed dechlorination rates in batch cultures with cell numbers of 10(7) mL(-1) amounted up to 35 microM day(-1). Chlorophenols were preferentially dechlorinated in the ortho position, but also doubly flanked and singly flanked meta- or para-chlorine substituents were removed. We used a newly designed computer-assisted direct cell counting protocol and quantitative PCR to demonstrate that strain CBDB1 uses chlorophenols as electron acceptors for respiratory growth. The growth yield of strain CBDB1 with 2,3-dichlorophenol was 7.6 x 10(13) cells per mol of Cl- released, and the growth rate was 0.41 day(-1). For strain 195, fast ortho dechlorination of 2,3-dichlorophenol, 2,3,4-trichlorophenol, and 2,3,6-trichlorophenol was detected, with only the ortho chlorine removed. Because chlorinated phenolic compounds are widely distributed as natural components in anaerobic environments, our results reveal one mode in which the Dehalococcoides species could have survived through earth history.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.Teredinidae | endosymbionts | symbiosis | xylotrophy | carbohydrate-active enzymes

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 genomic studies have revealed that many of the phenotypic differences observed among closely related microbial species in nature are due to genetic islands of diversity that are frequently copied, rearranged, and laterally transferred. Pathogenicity islands, which are mobile genetic elements that confer virulence, are well known in host-associated microorganisms (8). A recent comparative genomic study of the marine photoautotroph Prochlorococcus suggests that natural populations of microorganisms may also contain genetic islands that confer unique phenotypic traits on closely related strains (3). A comparative genomic study of representatives from the Dehalococcoides lineage within the Chloroflexi phylum of bacteria, a group which reductively dehalogenates chlorinated organic pollutants (25), suggests that reductive dehalogenases (RDases) are key enzymes conferring functional differences on closely related strains of Dehalococcoides (14).Sequenced Dehalococcoides genomes (strains 195, CBDB1, and BAV1 [unfinished]) share a high degree of genomic similarity and synteny in nearly all "housekeeping" genes. There are, however, differences in the total numbers and types of RDases that these strains harbor and in their corresponding substrate ranges (1, 2, 14, 17). In Dehalococcoides ethenogenes (strain 195), RDases are responsible for reductive dechlorination of chlorinated organic compounds, such as the common groundwater contaminants and suspected human carcinogens tetrachloroethene (PCE) and trichloroethene (TCE). In strains 195 and CBDB1, the majority of putative RDase genes are in clusters located near the predicted origin of replication, and while the locations of the clusters are similar, the RDase gene contents are different. The complete genome sequence of strain 195 revealed 19 potential RDase genes, 4 of which were contained within putative integrated mobile genetic elements that may have been acquired recently or are marked for dissemination (23). Thirty...

Reductive dehalogenase (RD) gene transcript levels in Dehalococcoides ethenogenes strain 195 were investigated using reverse transcriptase quantitative PCR during growth and reductive dechlorination of tetrachloroethene (PCE), trichloroethene (TCE), or 2,3-dichlorophenol (2,3-DCP). Cells grown with PCE or TCE had high transcript levels (greater than that for rpoB) for tceA, which encodes the TCE RD, pceA, which encodes the PCE RD, and DET0162, which contains a predicted stop codon and is considered nonfunctional. In cells grown with 2,3-DCP, tceA mRNA was less than 1% of that for rpoB, indicating that its transcription was regulated. pceA and DET0162 were the only RD genes with high transcript levels in cells grown with 2,3-DCP. Proteomic analysis of PCE-grown cells detected both PceA and TceA with high peptide coverage but not DET0162, and analysis of 2,3-DCP-grown cells detected PceA with high coverage but not TceA, DET0162, or any other potential RD. Cells grown with PCE or 2,3-DCP were tested for the ability to dechlorinate PCE, TCE, or 2,3-DCP with H 2 as the electron donor. 2,3-DCP-grown cells were unable to dechlorinate TCE but dechlorinated PCE to TCE without a lag, and PCE-grown cells dechlorinated 2,3-DCP without a lag. These results show that 2,3-DCP-grown cells do not produce TceA and that DET0162 is transcribed but its translation product is not detectable in cells and are consistent with PceA's being bifunctional, also serving as the 2,3-DCP RD. Chlorophenols naturally occur in soils and are good candidates for the original substrates for PceA.Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride (VC) and ethene (21,32). In addition to chlorinated ethenes, strain 195 has been found to reductively dechlorinate chlorobenzenes and other chloroaromatics (7) and more recently 2,3-dichlorophenol (2,3-DCP) and 2,3,4-trichlorophenol in the ortho position to 3-monochlorophenol or 3,4-dichlorophenol, respectively (1). The reduction of halogenated compounds by Dehalococcoides is carried out by membranebound respiratory reductive dehalogenases (RDs) (12,19,26,27), and although more than 90 RD-homologous genes have been identified in this genus (11,15,26,30), little is known about their specific functions. PCE-RD (PceA) and TCE-RD (TceA) were first characterized in mixed dechlorinating enrichment cultures containing strain 195 and were found to reductively dehalogenate PCE to TCE and TCE to VC and ethene, respectively (19). The gene encoding TCE-RD was subsequently cloned, sequenced, and designated tceA (18).The genome sequence of strain 195 (30) revealed 17 RDhomologous genes in addition to tceA (designated DET0079) and pceA (designated DET0318; J. Magnuson, personal communication). Common features of RDs include the presence of a putative twin-arginine transport signal sequence used for transport into the periplasm of folded proteins that can contain prosthetic groups, iron-sulfur cluster-binding motifs, and an adjacent "B" RD gene...

Marine bivalves of the family Teredinidae (shipworms) are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes) of the gills (ctenidia). These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH) and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis). These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose.

Previously, we demonstrated the reductive dehalogenation of dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and MCB to benzene in sediment microcosms derived from a chlorobenzene-contaminated site. In this study, enrichment cultures were established for each DCB isomer and each produced MCB and trace amounts of benzene as end products. MCB dehalogenation activity could only be transferred in sediment microcosms. The 1,2-DCB-dehalogenating culture was studied the most intensively. Whereas Dehalococcoides spp. were not detected in any of the microcosms or cultures, Dehalobacter spp. were detected in 16S rRNA gene clone libraries from 1,2-DCB enrichment cultures, and by PCR using Dehalobacter-specific primers in 1,3-DCB and 1,4-DCB enrichments and MCB-dehalogenating microcosms. Quantitative PCR showed Dehalobacter 16S rRNA gene copies increased up to 3 orders of magnitude upon dehalogenation of DCBs or MCB, and that nearly all of bacterial 16S rRNA genes in a 1,2-DCB-dehalogenating culture belonged to Dehalobacter spp. Dehalobacter 16S rRNA genes from DCB enrichment cultures and MCB-dehalogenating microcosms showed considerable diversity, implying that 16S rRNA sequences do not predict dehalogenation-spectra of Dehalobacter spp. These studies support a role for Dehalobacter spp. in the reductive dehalogenation of DCBs and MCB, and this genus should be considered for its potential impact on chlorobenzene fate at contaminated sites.

Anaerobic microcosms were constructed using sediments from a historically chlorobenzene-contaminated site and were provided with yeast extract as an electron donor. In these methanogenic microcosms, all three isomers of dichlorobenzene (DCB) were reductively dehalogenated to monochlorobenzene (MCB) when added together or individually, with 1,2-DCB dehalogenation being the most rapid and 1,4-DCB the slowest. When nearly all of the DCBs were consumed, benzene was detected and its accumulation was concomitant with MCB disappearance. Small amounts of toluene were also detected along with benzene. Subsequent MCB doses were also converted to benzene, and benzene reached levels in excess of 5000 micromol/L in some microcosms. An initial DCB dose stimulated, and in some cases was necessary for, MCB dehalogenation. Subsequent doses of DCB or MCB were dehalogenated more rapidly than previous ones, consistent with a growth-related process. Addition of a ca. 4% inoculum from microcosms that had consumed DCBs or MCB stimulated DCB and MCB dehalogenation in fresh microcosms, also indicative of growth and suggests thatthe chlorobenzene-dehalogenating microorganisms in these microcosms are candidates for bioaugmentation at anaerobic DCB or MCB contaminated sites. These studies add to evidence that benzene production from chlorobenzenes needs to be considered when modeling processes at contaminated sites.

Teredinibacter turnerae is a cultivable intracellular endosymbiont of xylotrophic (wood-feeding) bivalves of the Family Teredinidae (shipworms). Although T. turnerae has been isolated from many shipworm taxa collected in many locations, no systematic effort has been made to explore genetic diversity within this symbiont species across the taxonomic and geographic range of its hosts. The mode of symbiont transmission is unknown. Here we examine sequence diversity in fragments of six genes (16S rRNA, gyrB, sseA, recA, rpoB, and celAB) among 25 isolates of T. turnerae cultured from 13 shipworm species collected in 15 locations in the Atlantic, Pacific and Indian Oceans. While 16S rRNA sequences are nearly invariant among all examined isolates (maximum pairwise difference <0.26%) variation among examined protein coding loci is greater (mean pairwise difference 2.2-5.9%). Phylogenetic analyses based on each protein-coding locus differentiate the 25 isolates into two distinct and well-supported clades. With five exceptions, clade assignments for each isolate were supported by analysis of alleles of each of the five protein coding loci. These exceptions include (1) putative recombinant alleles of the celAB and gyrB loci in two isolates (PMS-535T.S.1b.3 and T8510), suggesting homologous recombination between members of the two clades, and (2) evidence for a putative lateral gene transfer event affecting a second locus (recA) in three isolates (T8412, T8503 and T8513). These results demonstrate that T. turnerae isolates do not represent a homogeneous global population. Instead they indicate the emergence of two lineages that, although distinct, likely experience some level of genetic exchange with each other and with other bacterial species.