Formation of sporangiospores in Dictyobacter aurantiacus (class Ktedonobacteria in phylum Chloroflexi).

Currently, actinomycetes and myxobacteria are the only bacteria believed to form sporangia. Here, we describe a sporangium-forming process identified in Dictyobacter aurantiacus strain S27T belonging to the class Ktedonobacteria in the phylum Chloroflexi. Microscopic observations showed that strain S27T forms a substrate mycelium and subsequently produces globose or subglobose terminal sporangia arising from the vegetative mycelia through short stalk cells. This morphogenetic differentiation is similar to that seen in members of Actinoplanes belonging to the class Actinobacteria. However, unlike in Actinoplanes, motile spores could not be observed. This is the first report of the existence of a bacterium, other than actinomycetes and myxobacteira, with a complex morphogenetic differentiation that forms sporangia and is an important microbiological discovery.

'Candidatus Viridilinea mediisalina', a novel phototrophic Chloroflexi bacterium from a Siberian soda lake.

In this article, we present the description of a novel mesophilic phototrophic Chloroflexi bacterium, 'Candidatus Viridilinea mediisalina' Kir15-3F. We have isolated an anaerobic, highly enriched culture of this bacterium from the Kiran soda lake (Siberia) and optimized its cultivation. Metagenomic sequencing revealed that 'Ca. Viridilinea mediisalina' Kir15-3F is a bacteriochlorophyll-containing Chloroflexi bacterium in the enrichment culture. Fluorescent in situ hybridisation demonstrated a link between the phenotype described here and the 'Ca. Viridilinea mediisalina' Kir15-3F genome. Spectrophotometry and high-performance liquid chromatography analyses showed the presence of bacteriochlorophylls d, c and a, as well as lycopene, γ-carotene and ß-carotene. Transmission electron microscopy showed chlorosomes, gas vesicles, polyhydroxyalkanoate-like and polyphosphate-like granules. Our results illustrated that 'Ca. Viridilinea mediisalina' Kir15-3F is an alkaliphilic, salt-tolerant, obligately mesophilic, anaerobic, phototrophic bacterium. The genome sequences lack genes of the Calvin cycle and a sulphide:quinone reductase gene for sulphide oxidation. Owing to the lack of an axenic culture and based on the genomic and phenotypic data, we have presented the description of the bacterium in the Candidatus category.

Electron-acceptor loadings affect chloroform dechlorination in a hydrogen-based membrane biofilm reactor.

Chloroform (CF) can undergo reductive dechlorination to dichloromethane, chloromethane, and methane. However, competition for hydrogen (H2 ), the electron-donor substrate, may cause poor dechlorination when multiple electron acceptors are present. Common acceptors in anaerobic environments are nitrate (NO3- ), sulfate (SO42- ), and bicarbonate (HCO3- ). We evaluated CF dechlorination in the presence of HCO3- at 1.56 e- Eq/m2 -day, then NO3- at 0.04-0.15 e- Eq/m2 -day, and finally NO3- (0.04 e- Eq/m2 -day) along with SO42- at 0.33 e- Eq/m2 -day in an H2 -based membrane biofilm reactor (MBfR). When the biofilm was initiated with CF-dechlorination conditions (no NO3- or SO42- ), it yielded a CF flux of 0.14 e- Eq/m2 -day and acetate production via homoacetogenesis up to 0.26 e- eq/m2 -day. Subsequent addition of NO3- at 0.05 e- Eq/m2 -day maintained full CF dechlorination and homoacetogenesis, but NO3- input at 0.15 e- Eq/m2 -day caused CF to remain in the reactor's effluent and led to negligible acetate production. The addition of SO42- did not affect CF reduction, but SO42- reduction significantly altered the microbial community by introducing sulfate-reducing Desulfovibrio and more sulfur-oxidizing Arcobacter. Dechloromonas appeared to carry out CF dechlorination and denitrification, whereas Acetobacterium (homoacetogen) may have been involved with hydrolytic dechlorination. Modifications to the electron acceptors fed to the MBfR caused the microbial community to undergo changes in structure that reflected changes in the removal fluxes.

Microbial community composition of sediments influenced by intensive mariculture activity.

Marine aquaculture is a major industry that supports the economy in many countries, including the Philippines. However, excess feeds and fish waste generated by mariculture activities contribute an immense nutrient load to the environment that can affect the underlying sediment. To better understand these impacts, we compared the physicochemical characteristics and microbial community composition of sediments taken at a fish cage and an off cage site in Bolinao, Philippines. Sediments and pore water at the fish cage site showed evidence of greater organic enrichment relative to the off cage site. Under these conditions, we found lower relative abundance of dissimilatory sulfate reductase and nitrite reductase genes, suggesting shifts in prevalent nutrient cycling processes. This is further supported by 16S rRNA gene sequencing that revealed differences in the community composition between sites. Fish cage sediments favored the growth of taxa that thrive in anaerobic, organic carbon-enriched environments, such as members of class Anaerolineae, which can potentially serve as bioindicators of eutrophication in sediments. This study demonstrates that intensive mariculture activity can cause eutrophic sediment conditions that influence microbial community structure and function.

Genome Analysis, Metabolic Potential, and Predatory Capabilities of Herpetosiphon llansteffanense sp. nov.

Herpetosiphon spp. are ubiquitous, chemoheterotrophic, filamentous gliding bacteria with the ability to prey on other microbes through a "wolf pack" mechanism. The genus currently comprises four known species (H. aurantiacus, H. geysericola, H. giganteus, and H. gulosus), which produce antimicrobial secondary metabolites such as siphonazole. As part of a study isolating myxobacterial wolf pack predators, we serendipitously isolated a novel environmental strain (CA052B) from the edge of a stream at Llansteffan, United Kingdom, which was identified as a member of the Herpetosiphon genus. A lawn culture method was utilized to analyze the predatory activity of CA052B against 10 prey organisms of clinical relevance. CA052B was found to prey on Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus faecalis, Bacillus subtilis, and Candida albicans Purified CA052B outer membrane vesicles also exhibited killing activity against the prey organisms when tested by flow cytometry. 16S rRNA sequencing of CA052B showed 98 to 99% identity with other Herpetosiphon species members. Comparing the genome of CA052B with the publicly available genomes of H. aurantiacus and H. geysericola revealed average nucleotide identities of only 84% and 91%, respectively, whereas the genome-to-genome distance calculation showed sequence identities of 28.2% and 46.6%, respectively. Biochemical characterization also revealed distinctions between CA052B and both H. gulosus and H. giganteus Thus, strain CA052BT (= DSM 107618T = NBRC 113495T) is proposed to be the type strain of a novel species, Herpetosiphon llansteffanense sp. nov. The genome sequence of CA052B also revealed diverse secondary metabolite biosynthetic clusters, encouraging further exploration of its antibiotic production potential.IMPORTANCE Predatory bacteria are able to kill and consume other microbes and are therefore of interest as potential sources of new antimicrobial substances for applications in the clinic. "Wolf pack" predators kill prey by secreting antimicrobial substances into their surroundings, and those substances can kill prey organisms independently of the predatory cells. The genus Herpetosiphon exhibits wolf pack predation, yet its members are poorly described compared to other wolf pack predators, such as the myxobacteria. By providing a thorough characterization of a novel Herpetosiphon species, including its predatory, biochemical, and genomic features, this study increases our understanding of genomic variation within the Herpetosiphon genus and how that variation affects predatory activity. This will facilitate future rational exploitation of genus members (and other wolf pack predators) as sources of novel antimicrobials.

Agelas Wasting Syndrome Alters Prokaryotic Symbiont Communities of the Caribbean Brown Tube Sponge, Agelas tubulata.

The brown tube sponge Agelas tubulata (cf. Agelas conifera) is an abundant and long-lived sponge on Caribbean reefs. Recently, a disease-like condition, Agelas wasting syndrome (AWS), was described from A. tubulata in the Florida Keys, where prevalence of the syndrome increased from 7 to 35% of the sponge population between 2010 and 2015. In this study, we characterized the prokaryotic symbiont community of A. tubulata for the first time from individuals collected within the same monitoring plots where AWS was described. We also sampled tissue from A. tubulata exhibiting symptoms of AWS to determine its effect on the diversity and structure of prokaryotic symbiont communities. Bacteria from the phyla Chloroflexi and Proteobacteria, particularly the class Gammaproteobacteria, dominated the sponge microbiome in tissue samples of both healthy sponges and those exhibiting AWS. Prokaryotic community structure differed significantly between the diseased and healthy sponge samples, with greater variability among communities in diseased samples compared to healthy samples. These differences in prokaryotic community structure included a shift in relative abundance of the dominant, ammonia-oxidizing (Thaumarchaeota) symbionts present in diseased and healthy sponge samples. Further research is required to determine the functional consequences of this shift in microbial community structure and the causal relationship of dysbiosis and sponge disease in A. tubulata.

Biogas production from food waste via co-digestion and digestion- effects on performance and microbial ecology.

In this work, performance and microbial structure of a digestion (food waste-only) and a co-digestion process (mixture of cow manure and food waste) were studied at mesophilic (37 °C) and thermophilic (55 °C) temperatures. The highest methane yield (480 mL/g VS) was observed in the mesophilic digester (MDi) fed with food waste alone. The mesophilic co-digestion of food waste and manure (McoDi) yielded 26% more methane than the sum of individual digestions of manure and food waste. The main volatile fatty acid (VFA) in the mesophilic systems was acetate, averaging 93 and 172 mg/L for McoDi and MDi, respectively. Acetate (2150 mg/L) and propionate (833 mg/L) were the main VFAs in the thermophilic digester (TDi), while propionate (163 mg/L) was the major VFA in the thermophilic co-digester (TcoDi). The dominant bacteria in MDi was Chloroflexi (54%), while Firmicutes was dominant in McoDi (60%). For the mesophilic reactors, the dominant archaea was Methanosaeta in MDi, while Methanobacterium and Methanosaeta had similar abundance in McoDi. In the thermophilic systems, the dominant bacteria were Thermotogae, Firmicutes and Synergistetes in both digesters, however, the relative abundance of these phyla were different. For archaea, the genus Methanothermobacter were entirely dominant in both TDi and TcoDi.

Microbial community analysis of bulk sludge/cake layers and biofouling-causing microbial consortia in a full-scale aerobic membrane bioreactor.

Pyrosequencing was used to investigate biofouling-causing microbial consortia at the community level in bulk sludge and cake layers within a full-scale membrane bioreactor (MBR). The analysis revealed Chao's estimates of total operational taxonomic units (OTUs) of 1726, 1806, and 1362 for bulk sludge, cake outer layer, and cake inner layer, respectively. The bulk sludge and cake outer layer OTUs clustered together, whereas the cake inner layer OTUs formed a separate group, indicating that environmental conditions affected the microbial community composition within the MBR. Bacteroidetes, Proteobacteria, and Chloroflexi were the dominant phyla in both the bulk sludge and the cake layers. Comparison at the genus level showed twelve distinct genera in the cake layers that were absent in bulk sludge. Twenty distinct genera were recorded in the inner cake layer. Those genera are likely the microbial colonization pioneers in full-scale membrane bioreactors.

Efficient metabolic exchange and electron transfer within a syntrophic trichloroethene-degrading coculture of Dehalococcoides mccartyi 195 and Syntrophomonas wolfei.

Dehalococcoides mccartyi 195 (strain 195) and Syntrophomonas wolfei were grown in a sustainable syntrophic coculture using butyrate as an electron donor and carbon source and trichloroethene (TCE) as an electron acceptor. The maximum dechlorination rate (9.9 ± 0.1 µmol day(-1)) and cell yield [(1.1 ± 0.3) × 10(8) cells µmol(-1) Cl(-)] of strain 195 maintained in coculture were, respectively, 2.6 and 1.6 times higher than those measured in the pure culture. The strain 195 cell concentration was about 16 times higher than that of S. wolfei in the coculture. Aqueous H2 concentrations ranged from 24 to 180 nM during dechlorination and increased to 350 ± 20 nM when TCE was depleted, resulting in cessation of butyrate fermentation by S. wolfei with a theoretical Gibbs free energy of -13.7 ± 0.2 kJ mol(-1). Carbon monoxide in the coculture was around 0.06 µmol per bottle, which was lower than that observed for strain 195 in isolation. The minimum H2 threshold value for TCE dechlorination by strain 195 in the coculture was 0.6 ± 0.1 nM. Cell aggregates during syntrophic growth were observed by scanning electron microscopy. The interspecies distances to achieve H2 fluxes required to support the measured dechlorination rates were predicted using Fick's law and demonstrated the need for aggregation. Filamentous appendages and extracellular polymeric substance (EPS)-like structures were present in the intercellular spaces. The transcriptome of strain 195 during exponential growth in the coculture indicated increased ATP-binding cassette transporter activities compared to the pure culture, while the membrane-bound energy metabolism related genes were expressed at stable levels.

Reconstruction of bacteriochlorophyll biosynthesis pathways in the filamentous anoxygenic phototrophic bacterium Oscillochloris trichoides DG-6 and evolution of anoxygenic phototrophs of the order Chloroflexales.

It is commonly accepted that green filamentous anoxygenic phototrophic (FAP) bacteria are the most ancient representatives of phototrophic micro-organisms. Modern FAPs belonging to the order Chloroflexales are divided into two suborders: Chloroflexineae and Roseiflexineae. Representatives of Roseiflexineae lack chlorosomes and synthesize bacteriochlorophyll a, whereas those of Chloroflexineae synthesize bacteriochlorophylls a and c and utilize chlorosomes for light harvesting. Though they constitute a small number of species, FAPs are quite diverse in their physiology. This bacterial group includes autotrophs and heterotrophs, thermophiles and mesophiles, aerobes and anaerobes, occupying both freshwater and halophilic environments. The anaerobic mesophilic autotroph Oscillochloris trichoides DG-6 is still not well studied in its physiology, and its evolutionary origin remains unclear. The goals of this study included identification of the reaction centre type of O. trichoides DG-6, reconstruction of its bacteriochlorophyll biosynthesis pathways, and determination of its evolutionary relationships with other FAPs. By enzymic and genomic analysis, the presence of RCII in O. trichoides DG-6 was demonstrated and the complete gene set involved in biosynthesis of bacteriochlorophylls a and c was established. We found that the bacteriochlorophyll gene sets differed between aerobic and anaerobic FAPs. The aerobic FAP genomes code oxygen-dependent AcsF cyclases, but lack the bchQ/bchR genes, which have been associated with adaptation to low light conditions in the anaerobic FAPs. A scenario of evolution of FAPs belonging to the order Chloroflexales is proposed.

Genomic distribution of B-vitamin auxotrophy and uptake transporters in environmental bacteria from the Chloroflexi phylum.

Bacteria from the Chloroflexi phylum are dominant members of phototrophic microbial mat communities in terrestrial thermal environments. Vitamins of B group are key intermediates (precursors) in the biosynthesis of indispensable enzyme cofactors driving numerous metabolic processes in all forms of life. A genomics-based reconstruction and comparative analysis of respective biosynthetic and salvage pathways and riboswitch regulons in over 20 representative Chloroflexi genomes revealed a widespread auxotrophy for some of the vitamins. The most prominent predicted phenotypic signature, auxotrophy for vitamins B1 and B7 was experimentally confirmed for the best studied model organism Chloroflexus aurantiacus. These observations along with identified candidate genes for the respective uptake transporters pointed to B vitamin cross-feeding as an important aspect of syntrophic metabolism in microbial communities. Inferred specificities of homologous substrate-binding components of ABC transporters for vitamins B1 (ThiY) and B2 (RibY) were verified by thermofluorescent shift approach. A functional activity of the thiamine-specific transporter ThiXYZ from C. aurantiacus was experimentally verified by genetic complementation in E. coli. Expanding the integrative approach, which was applied here for a comprehensive analysis of B-vitamin metabolism in Chloroflexi would allow reconstruction of metabolic interdependencies in microbial communities.

A fresh look at the evolution and diversification of photochemical reaction centers.

In this review, I reexamine the origin and diversification of photochemical reaction centers based on the known phylogenetic relations of the core subunits, and with the aid of sequence and structural alignments. I show, for example, that the protein folds at the C-terminus of the D1 and D2 subunits of Photosystem II, which are essential for the coordination of the water-oxidizing complex, were already in place in the most ancestral Type II reaction center subunit. I then evaluate the evolution of reaction centers in the context of the rise and expansion of the different groups of bacteria based on recent large-scale phylogenetic analyses. I find that the Heliobacteriaceae family of Firmicutes appears to be the earliest branching of the known groups of phototrophic bacteria; however, the origin of photochemical reaction centers and chlorophyll synthesis cannot be placed in this group. Moreover, it becomes evident that the Acidobacteria and the Proteobacteria shared a more recent common phototrophic ancestor, and this is also likely for the Chloroflexi and the Cyanobacteria. Finally, I argue that the discrepancies among the phylogenies of the reaction center proteins, chlorophyll synthesis enzymes, and the species tree of bacteria are best explained if both types of photochemical reaction centers evolved before the diversification of the known phyla of phototrophic bacteria. The primordial phototrophic ancestor must have had both Type I and Type II reaction centers.

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).

Bacterial distribution along a 50 °C temperature gradient reveals a parceled out hot spring environment.

Understanding the distribution of bacteria is a major goal of microbial ecology which remains to be fully deciphered. In this study, a model 50 °C temperature gradient at a Northern Thailand hot spring was analyzed to determine how the bacterial communities were structured in the environment. Communities were examined through 16S rRNA gene amplification, denaturing gradient gel electrophoresis, and sequencing. The two major phyla, Cyanobacteria and Chloroflexi, showed characteristic distributions along the temperature gradient. Different clades were allocated at specific portions of the gradient. Comparisons of the bacterial communities along the temperature gradient showed sharp decreases of similarity at increasing temperature difference. Peaks of maximum richness were observed at 50 and 70 °C. This study contributes to explain how environmental conditions and microbial interactions can influence the distribution of specific bacterial clades and phyla shaping the structure of microbial communities in nature.

Enriching for microbial reductive dechlorination of polychlorinated dibenzo-p-dioxins and dibenzofurans.

Anaerobic enrichment cultures derived from contaminated Kymijoki River sediments dechlorinated 1,2,3,4-tetrachlorodibenzofuran (1,2,3,4-tetra-CDF), octachlorodibenzofuran (octa-CDF) and 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-tetra-CDD). 1,2,3,4-tetra-CDF was dechlorinated via 1,2,3-, 2,3,4-, and 1,3,4/1,2,4-tri-CDFs to 1,3-, 2,3-, and 2,4-di-CDFs and finally to 4-mono-CDF. The dechlorination rate of 1,2,3,4-tetra-CDF was generally slower than that of 1,2,3,4-tetra-CDD. The rate and extent of 1,2,3,4-tetra-CDD dechlorination was enhanced by addition of pentachloronitrobenzene (PCNB) as a co-substrate. Dechlorination of spiked octa-CDF was observed with the production of hepta-, hexa-, penta- and tetra-CDFs over 6 months. Two major phylotypes of the Chloroflexi community showed an increase, one of which was identical to the Dehalococcoides mccartyi Pinellas subgroup. A set of twelve putative reductive dehalogenase (rdh) genes increased in abundance with addition of 1,2,3,4-tetra-CDF, 1,2,3,4-tetra-CDD and/or PCNB. This information will aid in understanding how indigenous microbial communities impact the fate of PCDFs and in developing strategies for bioremediation of PCDD/F contaminated sediments.

Isolation and characterization of a thermophilic, obligately anaerobic and heterotrophic marine Chloroflexi bacterium from a Chloroflexi-dominated microbial community associated with a Japanese shallow hydrothermal system, and proposal for Thermomarinilinea lacunofontalis gen. nov., sp. nov.

A novel marine thermophilic and heterotrophic Anaerolineae bacterium in the phylum Chloroflexi, strain SW7(T), was isolated from an in situ colonization system deployed in the main hydrothermal vent of the Taketomi submarine hot spring field located on the southern part of Yaeyama Archipelago, Japan. The microbial community associated with the hydrothermal vent was predominated by thermophilic heterotrophs such as Thermococcaceae and Anaerolineae, and the next dominant population was thermophilic sulfur oxidizers. Both aerobic and anaerobic hydrogenotrophs including methanogens were detected as minor populations. During the culture-dependent viable count analysis in this study, an Anaerolineae strain SW7(T) was isolated from an enrichment culture at a high dilution rate. Strain SW7(T) was an obligately anaerobic heterotroph that grew with fermentation and had non-motile thin rods 3.5-16.5 µm in length and 0.2 µm in width constituting multicellular filaments. Growth was observed between 37-65°C (optimum 60°C), pH 5.5-7.3 (optimum pH 6.0), and 0.5-3.5% (w/v) NaCl concentration (optimum 1.0%). Based on the physiological and phylogenetic features of a new isolate, we propose a new species representing a novel genus Thermomarinilinea: the type strain of Thermomarinilinea lacunofontalis sp. nov., is SW7(T) (=JCM15506(T)=KCTC5908(T)).

Isolation of two new Dehalococcoides mccartyi strains with dissimilar dechlorination functions and their characterization by comparative genomics via microarray analysis.

Microbial reductive dechlorination of trichloroethene (TCE) in groundwater often results in the accumulation of dichloroethenes (DCEs). Dehalococcoides mccartyi (Dhc) are the only known bacteria capable of dechlorination beyond DCE to non-toxic ethene. In this study, two newly isolated Dhc strains (11a and 11a5) with dissimilar functional abilities are described. Strain 11a reductively dechlorinates TCE, 1,1-DCE, cis-DCE, trans-DCE, and vinyl chloride (VC) to ethene, while strain 11a5 dechlorinates TCE and all three DCE isomers only to VC. Each of these dechlorination reactions are coupled to growth by these strains. The VC dechlorination rate of strain 11a occurs at a rate of 258 nmol per min per mg of protein, about two times faster than previously reported stains. Strain 11a possesses the vcrA gene while strain 11a5 contains the tceA gene. Strains 11a and 11a5 share 100% 16S rRNA gene sequence identity with previously sequenced Dhc strains BAV1 and CBDB1, placing it within the Pinellas subgroup, and 85.4% and 89.5% of all genes present in the CBDB1 and BAV1 genomes were detected in strains 11a and 11a5, respectively, using a custom-designed microarray targeting four sequenced Dhc strains. Genes that were not detected in strains 11a and 11a5 are mostly within the high plasticity regions or integrated elements of the sequenced strains. This study reports the functional description and comparative genomics of two additional Dhc isolates and provides evidence that the observed functional incongruence between the activity and core genome phylogenies of Dhc strains is likely driven by the horizontal transfer of key reductive dehalogenase-encoding genes.

Organic cofactors in the metabolism of Dehalococcoides mccartyi strains.

Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.

Dechlorination and organohalide-respiring bacteria dynamics in sediment samples of the Yangtze Three Gorges Reservoir.

Several groups of bacteria such as Dehalococcoides spp., Dehalobacter spp., Desulfomonile spp., Desulfuromonas spp., or Desulfitobacterium spp. are able to dehalogenate chlorinated pollutants such as chloroethenes, chlorobenzenes, or polychlorinated biphenyls under anaerobic conditions. In order to assess the dechlorination potential in Yangtze sediment samples, the presence and activity of the reductively dechlorinating bacteria were studied in anaerobic batch tests. Eighteen sediment samples were taken in the Three Gorges Reservoir catchment area of the Yangtze River, including the tributaries Jialing River, Daning River, and Xiangxi River. Polymerase chain reaction analysis indicated the presence of dechlorinating bacteria in most samples, with varying dechlorinating microbial community compositions at different sampling locations. Subsequently, anaerobic reductive dechlorination of tetrachloroethene (PCE) was tested after the addition of electron donors. Most cultures dechlorinated PCE completely to ethene via cis-dichloroethene (cis-DCE) or trans-dichloroethene. Dehalogenating activity corresponded to increasing numbers of Dehalobacter spp., Desulfomonile spp., Desulfitobacterium spp., or Dehalococcoides spp. If no bacteria of the genus Dehalococcoides spp. were present in the sediment, reductive dechlorination stopped at cis-DCE. Our results demonstrate the presence of viable dechlorinating bacteria in Yangtze samples, indicating their relevance for pollutant turnover.

Methanospirillum respiratory mRNA biomarkers correlate with hydrogenotrophic methanogenesis rate during growth and competition for hydrogen in an organochlorine-respiring mixed culture.

Molecular biomarkers hold promise for inferring rates of key metabolic activities in complex microbial systems. However, few studies have assessed biomarker levels for simultaneously occurring (and potentially competing) respirations. In this study, methanogenesis biomarkers for Methanospirillum hungatei were developed, tested, and compared to Dehalococcoides mccartyi biomarkers in a well-characterized mixed culture. Proteomic analyses of mixed culture samples (n = 4) confirmed expression of many M. hungatei methanogenesis enzymes. The mRNAs for two oxidoreductases detected were explored as quantitative biomarkers of hydrogenotrophic methanogenesis: a coenzyme F(420)-reducing hydrogenase (FrcA) and an iron sulfur protein (MvrD). As shown previously in D. mccartyi, M. hungatei transcript levels correlated linearly with measured (R = 0.97 for FrcA, R = 0.91 for MvrD; n = 7) or calculated respiration rate (R = 0.81 for FrcA, R = 0.62 for MvrD; n = 35) across two orders of magnitude on a log-log scale. The average abundance of MvrD transcripts was consistently two orders of magnitude lower than FrcA, regardless of experimental condition. In experiments where M. hungatei was competing for hydrogen with D. mccartyi, transcripts for the key respiratory hydrogenase HupL were generally less abundant per mL than FrcA and more abundant than MvrD. With no chlorinated electron acceptor added, HupL transcripts fell below both targets. These biomarkers hold promise for the prediction of in situ rates of respiration for these microbes, even when growing in mixed culture and utilizing a shared substrate which has important implications for both engineered and environmental systems. However, the differences in overall biomarker abundances suggest that the strength of any particular mRNA biomarker relies upon empirically established quantitative trends under a range of pertinent conditions.