Anaerobic Dehalogenation of Chloroanilines by Dehalococcoides mccartyi Strain CBDB1 and Dehalobacter Strain 14DCB1 via Different Pathways as Related to Molecular Electronic Structure.

Dehalococcoides mccartyi strain CBDB1 and Dehalobacter strain 14DCB1 are organohalide-respiring microbes of the phyla Chloroflexi and Firmicutes, respectively. Here, we report the transformation of chloroanilines by these two bacterial strains via dissimilar dehalogenation pathways and discuss the underlying mechanism with quantum chemically calculated net atomic charges of the substrate Cl, H, and C atoms. Strain CBDB1 preferentially removed Cl doubly flanked by two Cl or by one Cl and NH2, whereas strain 14DCB1 preferentially dechlorinated Cl that has an ortho H. For the CBDB1-mediated dechlorination, comparative analysis with Hirshfeld charges shows that the least-negative Cl discriminates active from nonactive substrates in 14 out of 15 cases and may represent the preferred site of primary attack through cob(I)alamin. For the latter trend, three of seven active substrates provide strong evidence, with partial support from three of the remaining four substrates. Regarding strain 14DCB1, the most positive carbon-attached H atom discriminates active from nonactive chloroanilines in again 14 out of 15 cases. Here, regioselectivity is governed for 10 of the 11 active substrates by the most positive H attached to the highest-charge (most positive or least negative) aromatic C carrying the Cl to be removed. These findings suggest the aromatic ring H as primary site of attack through the supernucleophile Co(I), converting an initial H bond to a full electron transfer as start of the reductive dehalogenation. For both mechanisms, one- and two-electron transfer to Cl (strain CBDB1) or H (strain 14DCB1) are compatible with the presently available data. Computational chemistry research into reaction intermediates and pathways may further aid in understanding the bacterial reductive dehalogenation at the molecular level.

Genome of Methanoregula boonei 6A8 reveals adaptations to oligotrophic peatland environments.

Analysis of the genome sequence of Methanoregula boonei strain 6A8, an acidophilic methanogen isolated from an ombrotrophic (rain-fed) peat bog, has revealed unique features that likely allow it to survive in acidic, nutrient-poor conditions. First, M. boonei is predicted to generate ATP using protons that are abundant in peat, rather than sodium ions that are scarce, and the sequence of a membrane-bound methyltransferase, believed to pump Na+ in all methanogens, shows differences in key amino acid residues. Further, perhaps reflecting the hypokalemic status of many peat bogs, M. boonei demonstrates redundancy in the predicted potassium uptake genes trk, kdp and kup, some of which may have been horizontally transferred to methanogens from bacteria, possibly Geobacter spp. Overall, the putative functions of the potassium uptake, ATPase and methyltransferase genes may, at least in part, explain the cosmopolitan success of group E1/E2 and related methanogenic archaea in acidic peat bogs.

Molecular characterization of the enzymes involved in the degradation of a brominated aromatic herbicide.

Dehalogenation is the key step in the degradation of halogenated aromatics, while reductive dehalogenation is originally thought to rarely occur in aerobes. In this study, an aerobic strain of Comamonas sp. 7D-2 was shown to degrade the brominated aromatic herbicide bromoxynil completely and release two equivalents of bromides under aerobic conditions. The enzymes involved in the degradation of bromoxynil to 4-carboxy-2-hydroxymuconate-6-semialdehyde, including nitrilase, reductive dehalogenase (BhbA), 4-hydroxybenzoate 3-monooxygenase and protocatechuate 4,5-dioxygenase, were molecularly characterized. The novel dehalogenase BhbA was shown to be a complex of a respiration-linked reductive dehalogenase (RdhA) domain and a NAD(P)H-dependent oxidoreductase domain and to have key features of anaerobic respiratory RdhAs, including two predicted binding motifs for [4Fe-4S] clusters and a close association with a hydrophobic membrane protein (BhbB). BhbB was confirmed to anchor BhbA to the membrane. BhbA was partially purified and found to use NAD(P)H as electron donors. Full-length bhbA homologues were found almost exclusively in marine aerobic proteobacteria, suggesting that reductive dehalogenation occurs extensively in aerobes and that bhbA is horizontally transferred from marine microorganisms. The discovery of a functional reductive dehalogenase and ring-cleavage oxygenases in an aerobe opens up possibilities for basic research as well as the potential application for bioremediation.

Microscale sulfur cycling in the phototrophic pink berry consortia of the Sippewissett Salt Marsh.

Microbial metabolism is the engine that drives global biogeochemical cycles, yet many key transformations are carried out by microbial consortia over short spatiotemporal scales that elude detection by traditional analytical approaches. We investigate syntrophic sulfur cycling in the 'pink berry' consortia of the Sippewissett Salt Marsh through an integrative study at the microbial scale. The pink berries are macroscopic, photosynthetic microbial aggregates composed primarily of two closely associated species: sulfide-oxidizing purple sulfur bacteria (PB-PSB1) and sulfate-reducing bacteria (PB-SRB1). Using metagenomic sequencing and (34) S-enriched sulfate stable isotope probing coupled with nanoSIMS, we demonstrate interspecies transfer of reduced sulfur metabolites from PB-SRB1 to PB-PSB1. The pink berries catalyse net sulfide oxidation and maintain internal sulfide concentrations of 0-500 µm. Sulfide within the berries, captured on silver wires and analysed using secondary ion mass spectrometer, increased in abundance towards the berry interior, while δ(34) S-sulfide decreased from 6‰ to -31‰ from the exterior to interior of the berry. These values correspond to sulfate-sulfide isotopic fractionations (15-53‰) consistent with either sulfate reduction or a mixture of reductive and oxidative metabolisms. Together this combined metagenomic and high-resolution isotopic analysis demonstrates active sulfur cycling at the microscale within well-structured macroscopic consortia consisting of sulfide-oxidizing anoxygenic phototrophs and sulfate-reducing bacteria.

Meta-analyses of Dehalococcoides mccartyi strain 195 transcriptomic profiles identify a respiration rate-related gene expression transition point and interoperon recruitment of a key oxidoreductase subunit.

A cDNA-microarray was designed and used to monitor the transcriptomic profile of Dehalococcoides mccartyi strain 195 (in a mixed community) respiring various chlorinated organics, including chloroethenes and 2,3-dichlorophenol. The cultures were continuously fed in order to establish steady-state respiration rates and substrate levels. The organization of array data into a clustered heat map revealed two major experimental partitions. This partitioning in the data set was further explored through principal component analysis. The first two principal components separated the experiments into those with slow (1.6±0.6 µM Cl-/h)- and fast (22.9±9.6 µM Cl-/h)-respiring cultures. Additionally, the transcripts with the highest loadings in these principal components were identified, suggesting that those transcripts were responsible for the partitioning of the experiments. By analyzing the transcriptomes (n=53) across experiments, relationships among transcripts were identified, and hypotheses about the relationships between electron transport chain members were proposed. One hypothesis, that the hydrogenases Hup and Hym and the formate dehydrogenase-like oxidoreductase (DET0186-DET0187) form a complex (as displayed by their tight clustering in the heat map analysis), was explored using a nondenaturing protein separation technique combined with proteomic sequencing. Although these proteins did not migrate as a single complex, DET0112 (an FdhB-like protein encoded in the Hup operon) was found to comigrate with DET0187 rather than with the catalytic Hup subunit DET0110. On closer inspection of the genome annotations of all Dehalococcoides strains, the DET0185-to-DET0187 operon was found to lack a key subunit, an FdhB-like protein. Therefore, on the basis of the transcriptomic, genomic, and proteomic evidence, the place of the missing subunit in the DET0185-to-DET0187 operon is likely filled by recruiting a subunit expressed from the Hup operon (DET0112).

Methanobacterium paludis sp. nov. and a novel strain of Methanobacterium lacus isolated from northern peatlands.

Two mesophilic, hydrogenotrophic methanogens, designated strains SWAN1T and AL-21, were isolated from two contrasting peatlands: a near circumneutral temperate minerotrophic fen in New York State, USA, and an acidic boreal poor fen site in Alaska, USA, respectively. Cells of the two strains were rod-shaped, non-motile, stained Gram-negative and resisted lysis with 0.1% SDS. Cell size was 0.6×1.5-2.8 µm for strain SWAN1T and 0.45-0.85×1.5-35 µm for strain AL-21. The strains used H2/CO2 but not formate or other substrates for methanogenesis, grew optimally around 32-37 °C, and their growth spanned through a slightly low to neutral pH range (4.7-7.1). Strain AL-21 grew optimally closer to neutrality at pH 6.2, whereas strain SWAN1T showed a lower optimal pH at 5.4-5.7. The two strains were sensitive to NaCl with a maximal tolerance at 160 mM for strain SWAN1T and 50 mM for strain AL-21. Na2S was toxic at very low concentrations (0.01-0.8 mM), resulting in growth inhibition above these values. The DNA G+C content of the genomes was 35.7 mol% for strain SWAN1T and 35.8 mol% for strain AL-21. Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains are members of the genus Methanobacterium. Strain SWAN1T shared 94-97% similarity with the type strains of recognized species of the genus Methanobacterium, whereas strain AL-21 shared 99% similarity with Methanobacterium lacus 17A1T. On the basis of phenotypic, genomic and phylogenetic characteristics, strain SWAN1T (=DSM 25820T=JCM 18151T) is proposed as the type strain of a novel species, Methanobacterium paludis sp. nov., while strain AL-21 is proposed as a second strain of Methanobacterium lacus.

Dehalogenation of chlorobenzenes, dichlorotoluenes, and tetrachloroethene by three Dehalobacter spp.

Three enrichment cultures containing Dehalobacter spp. were developed that dehalogenate each of the dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and the strains using 1,2-DCB (12DCB1) or 1,3-DCB (13DCB1) are now considered isolated, whereas the strain using 1,4-DCB (14DCB1) is considered highly enriched. In this study, we examined the dehalogenation capability of each strain to use chlorobenzenes with three or more chlorines, tetrachloroethene (PCE), or dichlorotoluene (DCT) isomers. Strain 12DCB1 preferentially dehalogenated singly flanked chlorines, but not doubly flanked or unflanked chlorines. It dehalogenated pentachlorobenzene to MCB with little buildup of intermediates. Strain 13DCB1, which could use either 1,3-DCB or 1,2-DCB, demonstrated the widest dehalogenation spectrum of electron acceptors tested, and dehalogenated every chlorobenzene isomer except 1,4-DCB. Notably, strain 13DCB1 dehalogenated the recalcitrant 1,3,5-trichlorobenzene isomer to MCB, and qPCR of 16S rRNA genes indicated that strain 13DCB1 grew. Strain 14DCB1 exhibited the narrowest range of substrate utilization, but was the only strain to dehalogenate para-substituted chlorines. Strains 12DCB1 and 13DCB1 dehalogenated PCE to cis-dichloroethene, and all strains dehalogenated 3,4-DCT to monochlorotoluene. These findings show that Dehalobacter spp., like Dehalococcoides spp., are versatile dehalogenators and should be considered when determining the fate of chlorinated organics at contaminated sites.

Distinct carbon isotope fractionation during anaerobic degradation of dichlorobenzene isomers.

Chlorinated benzenes are ubiquitous organic contaminants found in groundwater and soils. Compound specific isotope analysis (CSIA) has been increasingly used to assess natural attenuation of chlorinated contaminants, in which anaerobic reductive dechlorination plays an essential role. In this work, carbon isotope fractionation of the three dichlorobenzene (DCB) isomers was investigated during anaerobic reductive dehalogenation in methanogenic laboratory microcosms. Large isotope fractionation of 1,3-DCB and 1,4-DCB was observed while only a small isotope effect occurred for 1,2-DCB. Bulk enrichment factors (εbulk) were determined from a Rayleigh model: -0.8 ± 0.1 ‰ for 1,2-DCB, -5.4 ± 0.4 ‰ for 1,3-DCB, and -6.3 ± 0.2 ‰ for 1,4-DCB. εbulk values were converted to apparent kinetic isotope effects for carbon (AKIE) in order to characterize the carbon isotope effect at the reactive positions for the DCB isomers. AKIE values are 1.005 ± 0.001, 1.034 ± 0.003, and 1.039 ± 0.001 for 1,2-DCB, 1,3-DCB, and 1,4-DCB, respectively. The large difference in AKIE values between 1,2-DCB and 1,3-DCB (or 1,4-DCB) suggests distinct reaction pathways may be involved for different DCB isomers during microbial reductive dechlorination by the methanogenic cultures.

Isolation of an aerobic vinyl chloride oxidizer from anaerobic groundwater.

Vinyl chloride (VC) is a known human carcinogen and common groundwater contaminant. Reductive dechlorination of VC to non-toxic ethene under anaerobic conditions has been demonstrated at numerous hazardous waste sites. However, VC disappearance without stoichiometric production of ethene has also been observed at some sites and in microcosms. In this study we identify an organism responsible for this observation in presumably anaerobic microcosms and conclude that oxygen was not detectable based on a lack of color change from added resazurin. This organism, a Mycobacterium sp. closely related to known VC oxidizing strains, was present in high numbers in 16S rRNA gene clone libraries from a groundwater microcosm. Although the oxidation/reduction indicator resazurin remained in the clear reduced state in these studies, these results suggest inadvertent oxygen contamination occurred. This study helps to elucidate the dynamic behavior of chlorinated ethenes in contaminated groundwater, through the isolation of a strictly aerobic organism that may be responsible for at least some disappearance of VC without the concomitant production of ethene in groundwater considered anaerobic.

Global gene expression of Dehalococcoides within a robust dynamic TCE-dechlorinating community under conditions of periodic substrate supply.

A microarray targeting four sequenced strains in the Dehalococcoides (Dhc) genus was used to analyze gene expression in a robust long-term trichloroethene (TCE)-degrading microbial community (designated ANAS) during feeding cycles that involve conditions of periodic substrate supply. The Dhc transcriptome was examined at three time-points throughout a batch feeding cycle: T1 (27 h) when TCE, dichloroethene (DCE), and vinyl chloride (VC) were present; T2 (54 h) when only VC remained; and T3 (13 days) when Dhc had been starved of substrate for 9 days. Ninety percent of the Dhc open reading frames (ORFs) that were detected in the ANAS DNA were found to be expressed as RNA sometime during the time course, demonstrating extraordinary utilization of the streamlined genome. Ninety-seven percent of these transcripts were differentially expressed during the time course indicating efficiency of transcription through regulation in Dhc. Most Dhc genes were significantly down-regulated at T3 , responding to a lack of substrate as would be expected. The tceA and vcrA genes, which code for proteins with known chlorinated ethene reduction functions, were highly expressed at both T1 and T2 , whereas two other putative reductive dehalogenase genes (DET0173 and DET1545) were most highly expressed at T2 , likely in response to the presence of VC. Hydrogenases were most highly expressed at T1 , reflecting their important role in accumulating electrons used to initiate reductive dechlorination and other biosynthesis pathways. Cobalamin transport genes were preferentially expressed at T2 , reflecting an increase in corrinoid transport as chloroethenes were degraded and a decrease in activity of the transport system after dehalogenation was complete. This is the first application of a microarray targeting a known genus, including both core genomes and identified strain-specific genes, to improve our understanding of transcriptional dynamics within an undefined microbial community.

Dehalococcoides mccartyi gen. nov., sp. nov., obligately organohalide-respiring anaerobic bacteria relevant to halogen cycling and bioremediation, belong to a novel bacterial class, Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov., within the phylum Chloroflexi.

Six obligately anaerobic bacterial isolates (195(T), CBDB1, BAV1, VS, FL2 and GT) with strictly organohalide-respiring metabolisms were obtained from chlorinated solvent-contaminated aquifers, contaminated and uncontaminated river sediments or anoxic digester sludge. Cells were non-motile with a disc-shaped morphology, 0.3-1 µm in diameter and 0.1-0.2 µm thick, and characteristic indentations on opposite flat sides of the cell. Growth occurred in completely synthetic, reduced medium amended with a haloorganic electron acceptor (mostly chlorinated but also some brominated compounds), hydrogen as electron donor, acetate as carbon source, and vitamins. No other growth-supporting redox couples were identified. Aqueous hydrogen consumption threshold concentrations were <1 nM. Growth ceased when vitamin B(12) was omitted from the medium. Addition of sterile cell-free supernatant of Dehalococcoides-containing enrichment cultures enhanced dechlorination and growth of strains 195 and FL2, suggesting the existence of so-far unidentified stimulants. Dechlorination occurred between pH 6.5 and 8.0 and over a temperature range of 15-35 °C, with an optimum growth temperature between 25 and 30 °C. The major phospholipid fatty acids were 14 : 0 (15.7 mol%), br15 : 0 (6.2 mol%), 16 : 0 (22.7 mol%), 10-methyl 16 : 0 (25.8 mol%) and 18 : 0 (16.6 mol%). Unusual furan fatty acids including 9-(5-pentyl-2-furyl)-nonanoate and 8-(5-hexyl-2-furyl)-octanoate were detected in strains FL2, BAV1 and GT, but not in strains 195(T) and CBDB1. The 16S rRNA gene sequences of the six isolates shared more than 98 % identity, and phylogenetic analysis revealed an affiliation with the phylum Chloroflexi and more than 10 % sequence divergence from other described isolates. The genome sizes and G+C contents ranged from 1.34 to 1.47 Mbp and 47 to 48.9 mol% G+C, respectively. Based on 16S rRNA gene sequence comparisons, genome-wide average nucleotide identity and phenotypic characteristics, the organohalide-respiring isolates represent a new genus and species, for which the name Dehalococcoides mccartyi gen. nov., sp. nov. is proposed. Isolates BAV1 ( = ATCC BAA-2100  = JCM 16839  = KCTC 5957), FL2 ( = ATCC BAA-2098  = DSM 23585  = JCM 16840  = KCTC 5959), GT ( = ATCC BAA-2099  = JCM 16841  = KCTC 5958), CBDB1, 195(T) ( = ATCC BAA-2266(T)  = KCTC 15142(T)) and VS are considered strains of Dehalococcoides mccartyi, with strain 195(T) as the type strain. The new class Dehalococcoidia classis nov., order Dehalococcoidales ord. nov. and family Dehalococcoidaceae fam. nov. are described to accommodate the new taxon.

Anaerobic oxidation of ethene coupled to sulfate reduction in microcosms and enrichment cultures.

Ethene is considered recalcitrant under anaerobic conditions, but biological reduction to ethane and oxidation to CO2 have been reported; however, little is known about these processes or the organisms carrying them out. In this report we describe sulfate dependent ethene consumption in microcosms prepared with sediments from a freshwater canal. A first dose of 0.6 mmol/L ethene was consumed within 77 days, and a second dose was largely consumed twelve days later. Material from this microcosm was transferred into growth medium with ethene as the only electron donor (except for trace amounts of vitamins) and sulfate as the electron acceptor. Four doses of ethene were consumed at increasing rates, and the cultures have been transferred at least eight times in this medium. Conversion of [(14)C]ethene primarily to (14)CO2 was demonstrated in fifth and sixth generation cultures, as well as production of sulfide in other cultures, confirming the ethene/sulfate couple. Ovoid cells 1-2 µm in diameter were found in cultures containing ethene and sulfate, and quantitative PCR showed large increases in bacterial 16S rRNA gene copy number. Over half of a 16S rRNA gene clone library from a sixth-generation culture was a phylotype with a sequence ca. 90% identical with a clade of Deltaproteobacteria that includes Desulfovirga adipica and several Syntrophobacter spp. These studies have solidified the concept that deficits in mass balances for chloroethene fate in sulfate reducing zones of contaminated groundwater sites may be due to ethene oxidation, and suggest a unique phylotype is involved in this process.

Anaerobic conversion of chlorobenzene and benzene to CH4 and CO2 in bioaugmented microcosms.

Chlorobenzene is a widespread groundwater contaminant found at many industrial sites. Reductive dechlorination of chlorobenzene requires input of electron donor and results in problematic accumulation of benzene, which is more toxic than chlorobenzene. We hypothesized that coupling a culture capable of reductive dechlorination of chlorobenzene to benzene with a second benzene-degrading methanogenic culture would completely detoxify chlorobenzene. To this end, active chlorobenzene-dechlorinating microcosms that were producing benzene were inoculated with a previously described enriched methanogenic benzene-degrading consortium. The combination resulted in the transformation of chlorobenzene via benzene to the nontoxic degradation products, CO2 and CH4. Sustainable degradation of chlorobenzene and benzene was observed in the microcosms and was further confirmed by shifts in the carbon isotopic ratios of chlorobenzene and benzene during degradation. Moreover, we could show that benzene derived electrons fueled chlorobenzene dechlorination removing the need to provide exogenous electron donor. The results have promising implications for sustainable bioremediation of sites contaminated with chlorinated benzenes and benzene.

Global transcriptomic and proteomic responses of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation.

Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems-level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing cells of Dehalococcoides ethenogenes strain 195 to fixed nitrogen limitation (FNL), as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially upregulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and is implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly upregulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally downregulated in response to FNL, and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition, while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and posttranslational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number of genes coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

Methanolinea mesophila sp. nov., a hydrogenotrophic methanogen isolated from rice field soil, and proposal of the archaeal family Methanoregulaceae fam. nov. within the order Methanomicrobiales.

A novel mesophilic, hydrogenotrophic methanogen, designated strain TNR(T), was isolated from an anaerobic, propionate-degradation enrichment culture that was originally established from a rice field soil sample from Taiwan. Cells were non-motile rods, 2.0-6.5 µm long by 0.3 µm wide. Filamentous (up to about 100 µm) and coccoid (about 1 µm in diameter) cells were also observed in cultures in the late exponential phase of growth. Strain TNR(T) grew at 20-40 °C (optimally at 37 °C), at pH 6.5-7.4 (optimally at pH 7.0) and in the presence of 0-25 g NaCl l(-1) (optimally at 0 g NaCl l(-1)). The strain utilized H(2)/CO(2) and formate for growth and produced methane. The G+C content of the genomic DNA was 56.4 mol%. Based on sequences of both the 16S rRNA gene and the methanogen-specific marker gene mcrA, strain TNR(T) was related most closely to Methanolinea tarda NOBI-1(T); levels of sequence similarities were 94.8 and 86.4 %, respectively. The 16S rRNA gene sequence similarity indicates that strain TNR(T) and M. tarda NOBI-1(T) represent different species within the same genus. This is supported by shared phenotypic properties, including substrate usage and cell morphology, and differences in growth temperature. Based on these genetic and phenotypic properties, strain TNR(T) is considered to represent a novel species of the genus Methanolinea, for which the name Methanolinea mesophila sp. nov. is proposed; the type strain is TNR(T) ( = NBRC 105659(T) = DSM 23604(T)). In addition, we also suggest family status for the E1/E2 group within the order Methanomicrobiales, for which the name Methanoregulaceae fam. nov. is proposed; the type genus of family is Methanoregula.

Isolation of a novel acidiphilic methanogen from an acidic peat bog.

Acidic peatlands are among the largest natural sources of atmospheric methane and harbour a large diversity of methanogenic Archaea. Despite the ubiquity of methanogens in these peatlands, indigenous methanogens capable of growth at acidic pH values have resisted culture and isolation; these recalcitrant methanogens include members of an uncultured family-level clade in the Methanomicrobiales prevalent in many acidic peat bogs in the Northern Hemisphere. However, we recently succeeded in obtaining a mixed enrichment culture of a member of this clade. Here we describe its isolation and initial characterization. We demonstrate that the optimum pH for methanogenesis by this organism is lower than that of any previously described methanogen.

Color Catalogue of Life in Ice: Surface Biosignatures on Icy Worlds.

With thousands of discovered planets orbiting other stars and new missions that will explore our solar system, the search for life in the universe has entered a new era. However, a reference database to enable our search for life on the surface of icy exoplanets and exomoons by using records from Earth's icy biota is missing. Therefore, we developed a spectra catalogue of life in ice to facilitate the search for extraterrestrial signs of life. We measured the reflection spectra of 80 microorganisms-with a wide range of pigments-isolated from ice and water. We show that carotenoid signatures are wide-ranged and intriguing signs of life. Our measurements allow for the identification of such surface life on icy extraterrestrial environments in preparation for observations with the upcoming ground- and space-based telescopes. Dried samples reveal even higher reflectance, which suggests that signatures of surface biota could be more intense on exoplanets and moons that are drier than Earth or on environments like Titan where potential life-forms may use a different solvent. Our spectra library covers the visible to near-infrared and is available online. It provides a guide for the search for surface life on icy worlds based on biota from Earth's icy environments.

Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and its effects on growth and dechlorination activity.

Bacteria of the genus Dehalococcoides are important members of bioremediation communities because of their ability to detoxify chloroethenes to the benign end product ethene. Genome-enabled studies conducted with Dehalococcoides ethenogenes 195 have revealed that two ATP-binding cassette (ABC)-type amino acid transporters are expressed during its exponential growth stages. In light of previous findings that Casamino Acids enhanced its dechlorination activity, we hypothesized that strain 195 is capable of importing amino acids from its environment to facilitate dechlorination and growth. To test this hypothesis, we applied isotopomer-based dilution analysis with (13)C-labeled acetate to differentiate the amino acids that were taken up by strain 195 from those synthesized de novo and to determine the physiological changes caused by the significantly incorporated amino acids. Our results showed that glutamate/glutamine and aspartate/asparagine were almost exclusively synthesized by strain 195, even when provided in excess in the medium. In contrast, phenylalanine, isoleucine, leucine, and methionine were identified as the four most highly incorporated amino acids, at levels >30% of respective proteinogenic amino acids. When either phenylalanine or all four highly incorporated amino acids were added to the defined mineral medium, the growth rates, dechlorination activities, and yields of strain 195 were enhanced to levels similar to those observed with supplementation with 20 amino acids. However, genes for the putative ABC-type amino acids transporters and phenylalanine biosynthesis exhibited insignificant regulation in response to the imported amino acids. This study also demonstrates that using isotopomer-based metabolite analysis can be an efficient strategy for optimizing nutritional conditions for slow-growing microorganisms.

Methanoregula boonei gen. nov., sp. nov., an acidiphilic methanogen isolated from an acidic peat bog.

A novel acidiphilic, hydrogenotrophic methanogen, designated strain 6A8(T), was isolated from an acidic (pH 4.0-4.5) and ombrotrophic (rain-fed) bog located near Ithaca, NY, USA. Cultures were dimorphic, containing thin rods (0.2-0.3 µm in diameter and 0.8-3.0 µm long) and irregular cocci (0.2-0.8 µm in diameter). The culture utilized H(2)/CO(2) to produce methane but did not utilize formate, acetate, methanol, ethanol, 2-propanol, butanol or trimethylamine. Optimal growth conditions were near pH 5.1 and 35 °C. The culture grew in basal medium containing as little as 0.43 mM Na(+) and growth was inhibited completely by 50 mM NaCl. To our knowledge, strain 6A8(T) is one of the most acidiphilic (lowest pH optimum) and salt-sensitive methanogens in pure culture. Acetate, coenzyme M, vitamins and yeast extract were required for growth. It is proposed that a new genus and species be established for this organism, Methanoregula boonei gen. nov., sp. nov. The type strain of Methanoregula boonei is 6A8(T) (=DSM 21154(T) =JCM 14090(T)).