'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.

Preliminary analysis of Chloroflexi populations in full-scale UASB methanogenic reactors.

AIMS: The phylum Chloroflexi is frequently found in high abundance in methanogenic reactors, but their role is still unclear as most of them remain uncultured and understudied. Hence, a detailed analysis was performed in samples from five up-flow anaerobic sludge blanket (UASB) full-scale reactors fed different industrial wastewaters. METHODS AND RESULTS: Quantitative PCR show that the phylum Chloroflexi was abundant in all UASB methanogenic reactors, with higher abundance in the reactors operated for a long period of time, which presented granular biomass. Both terminal restriction fragment length polymorphism and 16S rRNA gene amplicon sequencing revealed diverse Chloroflexi populations apparently determined by the different inocula. According to the phylogenetic analysis, the sequences from the dominant Chloroflexi were positioned in branches where no sequences of the cultured representative strains were placed. Fluorescent in situ hybridization analysis performed in two of the reactors showed filamentous morphology of the hybridizing cells. CONCLUSIONS: While members of the Anaerolineae class within phylum Chloroflexi were predominant, their diversity is still poorly described in anaerobic reactors. Due to their filamentous morphology, Chloroflexi may have a key role in the granulation in methanogenic UASB reactors. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results bring new insights about the diversity, stability, dynamics and abundance of this phylum in full-scale UASB reactors which aid in understanding their function within the reactor biomass. However, new methodological approaches and analysis of bulking biomass are needed to completely unravel their role in these reactors. Combining all this knowledge with reactor operational parameters will allow to understand their participation in granulation and bulking episodes and design strategies to prevent Chloroflexi overgrowth.

Diversity of Ktedonobacteria with Actinomycetes-Like Morphology in Terrestrial Environments.

Bacteria with an actinomycetes-like morphology have recently been discovered, and the class Ktedonobacteria was created for these bacteria in the phylum Chloroflexi. They may prove to be a valuable resource with the potential to produce unprecedented secondary metabolites. However, our understanding of their diversity, richness, habitat, and ecological significance is very limited. We herein developed a 16S rRNA gene-targeted, Ktedonobacteria-specific primer and analyzed ktedonobacterial amplicons. We investigated abundance, diversity, and community structure in forest and garden soils, sand, bark, geothermal sediment, and compost. Forest soils had the highest diversity among the samples tested (1181-2934 operational taxonomic units [OTUs]; Chao 1 estimate, 2503-5613; Shannon index, 4.21-6.42). A phylogenetic analysis of representative OTUs revealed at least eight groups within unclassified Ktedonobacterales, expanding the known diversity of this order. Ktedonobacterial communities markedly varied among our samples. The common mesic environments (soil, sand, and bark) were dominated by diverse phylotypes within the eight groups. In contrast, compost and geothermal sediment samples were dominated by known ktedonobacterial families (Thermosporotrichaceae and Thermogemmatisporaceae, respectively). The relative abundance of Ktedonobacteria in the communities, based on universal primers, was ≤0.8%, but was 12.9% in the geothermal sediment. These results suggest that unknown diverse Ktedonobacteria inhabit common environments including forests, gardens, and sand at low abundances, as well as extreme environments such as geothermal areas.

Identification of a new diterpene biosynthetic gene cluster that produces O-methylkolavelool in Herpetosiphon aurantiacus.

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)-kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)-O-methylkolavelool from (+)-kolavelool, so this enzyme was an O-methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)-O-methylkolavelool biosynthetic gene cluster.

Development of a fluorescence-activated cell sorting method coupled with whole genome amplification to analyze minority and trace Dehalococcoides genomes in microbial communities.

Dehalococcoides mccartyi are functionally important bacteria that catalyze the reductive dechlorination of chlorinated ethenes. However, these anaerobic bacteria are fastidious to isolate, making downstream genomic characterization challenging. In order to facilitate genomic analysis, a fluorescence-activated cell sorting (FACS) method was developed in this study to separate D. mccartyi cells from a microbial community, and the DNA of the isolated cells was processed by whole genome amplification (WGA) and hybridized onto a D. mccartyi microarray for comparative genomics against four sequenced strains. First, FACS was successfully applied to a D. mccartyi isolate as positive control, and then microarray results verified that WGA from 10(6) cells or ∼1 ng of genomic DNA yielded high-quality coverage detecting nearly all genes across the genome. As expected, some inter- and intrasample variability in WGA was observed, but these biases were minimized by performing multiple parallel amplifications. Subsequent application of the FACS and WGA protocols to two enrichment cultures containing ∼10% and ∼1% D. mccartyi cells successfully enabled genomic analysis. As proof of concept, this study demonstrates that coupling FACS with WGA and microarrays is a promising tool to expedite genomic characterization of target strains in environmental communities where the relative concentrations are low.

Distribution of organohalide-respiring bacteria between solid and aqueous phases.

Contemporary microbial monitoring of aquifers relies on groundwater samples to enumerate nonattached cells of interest. One-dimensional column studies quantified the distribution of bacterial cells in solid and the aqueous phases as a function of microbial species, growth substrate availability and porous medium (i.e., Appling soil versus Federal Fine Ottawa sand with 0.75% and 0.01% [w/w] organic carbon, respectively). Without supplied growth substrates, effluent from columns inoculated with the tetrachloroethene- (PCE-) to-ethene-dechlorinating bacterial consortium BDI-SZ containing Dehalococcoides mccartyi (Dhc) strains and Geobacter lovleyi strain SZ (GeoSZ), or inoculated with Anaeromyxobacter dehalogenans strain W (AdehalW), captured 94-96, 81-99, and 73-84% of the Dhc, GeoSZ, and AdehalW cells, respectively. Cell retention was organism-specific and increased in the order Dhc < GeoSZ < AdehalW. When amended with 10 mM lactate and 0.11 mM PCE, aqueous samples accounted for 1.3-27 and 0.02-22% of the total Dhc and GeoSZ biomass, respectively. In Appling soil, up to three orders-of-magnitude more cells were associated with the solid phase, and attachment rate coefficients (katt) were consistently greater compared to Federal Fine sand. Cell-solid interaction energies ranged from -2.5 to 787 kT and were consistent with organism-specific deposition behavior, where GeoSZ and AdehalW exhibited greater attachment than Dhc cells. The observed disparities in microbial cell distributions between the aqueous and solid phases imply that groundwater analysis can underestimate the total cell abundance in the aquifer by orders-of-magnitude under conditions of growth and in porous media with elevated organic carbon content. The implications of these findings for monitoring chlorinated solvent sites are discussed.

Spectroscopic studies of carotenoid-to-bacteriochlorophyll energy transfer in LHRC photosynthetic complex from Roseiflexus castenholzii.

Carotenoids present in the photosynthetic light-harvesting reaction center (LHRC) complex from chlorosome lacking filamentous anoxygenic phototroph, Roseiflexus castenholzii were purified and characterized for their photochemical properties. The LHRC from anaerobically grown cells contains five different carotenoids, methoxy-keto-myxocoxanthin, gamma-carotene, and its three derivatives, whereas the LHRC from aerobically grown cells contains only three carotenoid pigments with methoxy-keto-myxocoxanthin being the dominant one. The spectroscopic properties and dynamics of excited singlet states of the carotenoids were studied by steady-state absorption, fluorescence and ultrafast time-resolved optical spectroscopy in organic solvent and in the intact LHRC complex. Time-resolved transient absorption spectroscopy performed in the near-infrared (NIR) on purified carotenoids combined with steady-state absorption spectroscopy led to the precise determination of values of the energies of the S(1)(2(1)A(g)(-)) excited state. Global and single wavelength fitting of the ultrafast spectral and temporal data sets of the carotenoids in solvents and in the LHRC revealed the pathways of de-excitation of the carotenoid excited states.

Isolation and characterization of "Dehalococcoides" sp. strain MB, which dechlorinates tetrachloroethene to trans-1,2-dichloroethene.

In an attempt to understand the microorganisms involved in the generation of trans-1,2-dichloroethene (trans-DCE), pure-culture "Dehalococcoides" sp. strain MB was isolated from environmental sediments. In contrast to currently known tetrachloroethene (PCE)- or trichloroethene (TCE)-dechlorinating pure cultures, which generate cis-DCE as the predominant product, Dehalococcoides sp. strain MB reductively dechlorinates PCE to trans-DCE and cis-DCE at a ratio of 7.3 (+/-0.4):1. It utilizes H2 as the sole electron donor and PCE or TCE as the electron acceptor during anaerobic respiration. Strain MB is a disc-shaped, nonmotile bacterium. Under an atomic force microscope, the cells appear singly or in pairs and are 1.0 microm in diameter and approximately 150 nm in depth. The purity was confirmed by culture-based approaches and 16S rRNA gene-based analysis and was corroborated further by putative reductive dehalogenase (RDase) gene-based, quantitative real-time PCR. Although strain MB shares 100% 16S rRNA gene sequence identity with Dehalococcoides ethenogenes strain 195, these two strains possess different dechlorinating pathways. Microarray analysis revealed that 10 putative RDase genes present in strain 195 were also detected in strain MB. Successful cultivation of strain MB indicates that the biotic process could contribute significantly to the generation of trans-DCE in chloroethene-contaminated sites. It also enhances our understanding of the evolution of this unusual microbial group, Dehalococcoides species.

Filamentous bacterium Eikelboom type 0092 in activated sludge plants in Australia is a member of the phylum Chloroflexi.

Molecular data show that the filamentous bacterium Eikelboom type 0092, frequently seen in Australian activated sludge plants, is a member of the phylum Chloroflexi. Fluorescence in situ hybridization (FISH) probes designed against cloned 16S rRNA sequences from a full-scale enhanced biological phosphate removal-activated sludge plant community, where this was a dominant filament morphotype, suggest that it can exist as two variants, differing in their trichome diameter. When applied to samples from several treatment plants in eastern Australia, each FISH probe targeted only the type 0092 filament morphotype against which it was designed. The patterns of FISH signals generated with both were consistent with the ribosomes not being evenly distributed but arranged as intracellular aggregates. The FISH survey data showed that these two variants appeared together in most but not all of the plants examined. None stained positively for intracellular presence of either poly-beta-hydroxyalkanoates or polyphosphate.

Quantification of cell specific uptake activity of microbial products by uncultured Chloroflexi by microautoradiography combined with fluorescence in situ hybridization.

We, for the first time, quantitatively determined cell specific uptake activities of microbial products (bacterial cell detritus and extracellular polymeric substances, EPS) by the member of uncultured Chloroflexiby using a microautoradiography combined with fluorescence in situ hybridization (MAR-FISH) technique. For this MAR-FISH analysis, we prepared [14C]-labeled microbial products from biomass sludge obtained and bacterial strains (Pseudomonas sp. and Acinetobacter sp.) isolated from our pilot-scale membrane bioreactor (MBR) as tracer substrates, which probably represent the more realistic food source in the MBR. The quantitative MAR-FISH analyses clearly showed that most of the uncultured Chloroflexi could indeed uptake the bacterial detritus of the two isolated strains with rates of 1.7-3.5 x 10(-7) g-C microm-2-surface area h(-1) (corresponding to 1.2-1.7 mg-C-bacterial detritus L(-1) h(-1)) in the cultures, which were, however, about 2 orders of magnitude lower than the uptake rates of simple monosaccharides (mannose, arabinose, fucose, and galactose). Based on these results and their high abundance (more than 20% of total bacteria detected with EUB338-mixed probes), it could be estimated that the uncultured Chloroflexi contributes 38-51% of the total degradation of microbial products occurred in the MAR-FISH cultures.

Bellilinea caldifistulae gen. nov., sp. nov. and Longilinea arvoryzae gen. nov., sp. nov., strictly anaerobic, filamentous bacteria of the phylum Chloroflexi isolated from methanogenic propionate-degrading consortia.

Thermophilic (strain GOMI-1(T)) and mesophilic (strain KOME-1(T)) strains were isolated from two different cultures of propionate-degrading consortia obtained from thermophilic digester sludge and rice paddy soil, respectively. The two strains were non-spore-forming, non-motile and Gram-negative. Both strains were obligately anaerobic micro-organisms, showing multicellular filamentous morphotypes more than 100 mum in length. The cell width for strain GOMI-1(T) was 0.2-0.4 mum and that of strain KOME-1(T) was 0.4-0.6 mum. Strain GOMI-1(T) could grow at 45-65 degrees C with a pH range of 6.0-7.5 (optimum growth at 55 degrees C, pH 7.0). The temperature range for growth of strain KOME-1(T) was 30-40 degrees C and the pH range was pH 5.0-8.5 (optimum growth around 37 degrees C, pH 7.0). Yeast extract was required for growth of both strains. Strain GOMI-1(T) was able to grow with a number of carbohydrates in the presence of yeast extract. In yeast extract-containing medium, strain KOME-1(T) could utilize proteins and a limited range of sugars for growth. The G+C contents of the DNA of strains GOMI-1(T) and KOME-1(T) were respectively 54.7 and 57.6 mol%. Major fatty acids of strain GOMI-1(T) were C(16 : 0), C(14 : 0) and iso-C(15 : 0), whereas those of strain KOME-1(T) were iso-C(15 : 0), anteiso-C(15 : 0) and C(14 : 0). Based on comparative analysis of 16S rRNA gene sequences of strains GOMI-1(T) and KOME-1(T), the strains were placed in different phylogenetic positions in the class Anaerolineae of the bacterial phylum Chloroflexi. Their phenotypic and genetic traits strongly supported the conclusion that the strains should be described as two independent taxa in the class Anaerolineae. Hence, we propose the names Bellilinea caldifistulae gen. nov., sp. nov., and Longilinea arvoryzae gen. nov., sp. nov., for strains GOMI-1(T) and KOME-1(T). The type strains of Bellilinea caldifistulae and Longilinea arvoryzae are respectively GOMI-1(T) (=JCM 13669(T) =DSM 17877(T)) and KOME-1(T) (=JCM 13670(T) =KTCC 5380(T)).

Recommended standards for the description of new species of anoxygenic phototrophic bacteria.

Recommended standards for the description of new species of the anoxygenic phototrophic bacteria are proposed in accordance with Recommendation 30b of the International Code of Nomenclature of Bacteria. These standards include information on the natural habitat, ecology and phenotypic properties including morphology, physiology and pigments and on genetic information and nucleic acid data. The recommended standards were supported by the Subcommittee on the taxonomy of phototrophic bacteria of the International Committee on Systematics of Prokaryotes. They are considered as guidelines for authors to prepare descriptions of new species.

Detection and quantitative estimation of Dehalococcoides spp. in a dechlorinating bioreactor by a combination of fluorescent in situ hybridisation (FISH) and kinetic analysis.

The unique capacity of Dehalococcoides ethenogenes of completely dechlorinating the common groundwater pollutant tetrachloroethene (PCE) to the harmless ethene makes this microorganism very attractive for application in natural or engineered bioremediation systems. In this study, the qualitative and quantitative determination of Dehalococcoides spp. in a lab-scale bioreactor was performed based on the combination of fluorescent in situ hybridisation (FISH) for specific detection, and kinetic batch tests at non-limiting hydrogen and PCE concentration for quantitative determination. The dechlorinating bioreactor was operated at a high and constant PCE loading rate of 255 micromol PCE [g volatile suspended solids (VSS)](-1) day(-1). Pale coccoid cells resembling the distinctive morphotype of D. ethenogenes were present in the microbial culture. These cocci hybridised with both eubacterial probes and the Dhe1259t probe recently designed for detecting Dehalococcoides spp. Positive hybridisation was also observed when the DHC1377 reverse primer was used as a specific probe and applied to the dechlorinating microbial consortium. The maximum dechlorination rate obtained under non-limiting hydrogen and PCE concentrations was 3.22 +/- 0.08 mmol Cl(-) l(-1 )day(-1). From the specific activity of D. ethenogenes [i.e. 0.055 +/- 0.008 mmol Cl(-) (mg VSS)(-1) day(-1)], as reported from pure culture study, this observed maximum rate corresponded to a concentration of this bacterium in the mixed liquor of the bioreactor of 59.0+/-10.4 mg VSS.l(-1) (41.5+/-11.2% of overall VSS). This calculated relative abundance of D. ethenogenes was in agreement with the percentage of methanol (in terms of reducing equivalents) channeled to reductive dechlorination (approximately 30%) supporting the assumption that most reductive dechlorination was actually due to this microorganism.