FISH-TAMB, a Fixation-Free mRNA Fluorescent Labeling Technique to Target Transcriptionally Active Members in Microbial Communities.

Keystone species or ecological engineers are vital to the health of an ecosystem; however, often, their low abundance or biomass present challenges for their discovery, identification, visualization and selection. We report the development of fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB), a fixation-free protocol that is applicable to archaea and bacteria. The FISH-TAMB method differs from existing FISH methods by the absence of fixatives or surfactants in buffers, the fast hybridization time of as short as 15 min at target cells' growth temperature, and the omission of washing steps. Polyarginine cell-penetrating peptides are employed to deliver molecular beacons (MBs) across prokaryotic cell walls and membranes, fluorescently labeling cells when MBs hybridize to target mRNA sequences. Here, the detailed protocol of the preparation and application of FISH-TAMB is presented. To demonstrate FISH-TAMB's ability to label intracellular mRNA targets, differentiate transcriptional states, detect active and rare taxa, and keep cell viability, labeling experiments were performed that targeted the messenger RNA (mRNA) of methyl-coenzyme M reductase A (mcrA) expressed in (1) Escherichia coli containing a plasmid with a partial mcrA gene of the methanogen Methanosarcina barkeri (E. coli mcrA+); (2) M. barkeri; and (3) an anaerobic methanotrophic (ANME) enrichment from a deep continental borehole. Although FISH-TAMB was initially envisioned for mRNA of any functional gene of interest without a requirement of prior knowledge of 16S ribosomal RNA (rRNA)-based taxonomy, FISH-TAMB has the potential for multiplexing and going beyond mRNA and thus is a versatile addition to the molecular ecologist's toolkit, with potentially widespread application in the field of environmental microbiology.

Evolutionary stasis of a deep subsurface microbial lineage.

Sulfate-reducing bacteria Candidatus Desulforudis audaxviator (CDA) were originally discovered in deep fracture fluids accessed via South African gold mines and have since been found in geographically widespread deep subsurface locations. In order to constrain models for subsurface microbial evolution, we compared CDA genomes from Africa, North America and Eurasia using single cell genomics. Unexpectedly, 126 partial single amplified genomes from the three continents, a complete genome from of an isolate from Eurasia, and metagenome-assembled genomes from Africa and Eurasia shared >99.2% average nucleotide identity, low frequency of SNP's, and near-perfectly conserved prophages and CRISPRs. Our analyses reject sample cross-contamination, recent natural dispersal, and unusually strong purifying selection as likely explanations for these unexpected results. We therefore conclude that the analyzed CDA populations underwent only minimal evolution since their physical separation, potentially as far back as the breakup of Pangea between 165 and 55 Ma ago. High-fidelity DNA replication and repair mechanisms are the most plausible explanation for the highly conserved genome of CDA. CDA presents a stark contrast to the current model organisms in microbial evolutionary studies, which often develop adaptive traits over far shorter periods of time.

Correction to: Characterization of the inhibition mechanism of a tissue factor inhibiting single-chain variable fragment: a combined computational approach.

One of the co-author's details (Leon du Preez-lategaan) was printed incorrectly in the above publication. The correct details are provided below.

Characterization of the inhibition mechanism of a tissuefactor inhibiting single-chain variable fragment: a combined computational approach.

The interaction of a single-chain variable fragment (scFv) directed against human tissue factor (TF) was predicted using an in silico approach with the aim to establish a most likely mechanism of inhibition. The structure of the TF inhibiting scFv (TFI-scFv) was predicted using homology modeling, and complementarity-determining regions (CDRs) were identified. The CDR was utilized to direct molecular docking between the homology model of TFI-scFv and the crystal structure of the extracellular domains of human tissue factor. The rigid-body docking model was refined by means of molecular dynamic (MD) simulations, and the most prevalent cluster was identified. MD simulations predicted improved interaction between TFI-scFv and TF and propose the formation of stable complex for duration of the 600-ns simulation. Analysis of the refined docking model suggests that the interactions between TFI-scFv would interfere with the allosterical activation of coagulation factor VII (FVII) by TF. This interaction would prevent the formation of the active TF:VIIa complex and in so doing inhibit the initiation phase of blood coagulation as observers during in vitro testing.

The genome of a subterrestrial nematode reveals adaptations to heat.

The nematode Halicephalobus mephisto was originally discovered inhabiting a deep terrestrial aquifer 1.3 km underground. H. mephisto can thrive under conditions of abiotic stress including heat and minimal oxygen, where it feeds on a community of both chemolithotrophic and heterotrophic prokaryotes in an unusual ecosystem isolated from the surface biosphere. Here we report the comprehensive genome and transcriptome of this organism, identifying a signature of adaptation: an expanded repertoire of 70 kilodalton heat-shock proteins (Hsp70) and avrRpt2 induced gene 1 (AIG1) proteins. The expanded Hsp70 genes are transcriptionally induced upon growth under heat stress, and we find that positive selection is detectable in several members of this family. We further show that AIG1 may have been acquired by horizontal gene transfer (HGT) from a rhizobial fungus. Over one-third of the genes of H. mephisto are novel, highlighting the divergence of this nematode from other sequenced organisms. This work sheds light on the genomic basis of heat tolerance in a complete subterrestrial eukaryotic genome.

Anaerobic reduction of europium by a Clostridium strain as a strategy for rare earth biorecovery.

The biorecovery of europium (Eu) from primary (mineral deposits) and secondary (mining wastes) resources is of interest due to its remarkable luminescence properties, important for modern technological applications. In this study, we explored the tolerance levels, reduction and intracellular bioaccumulation of Eu by a site-specific bacterium, Clostridium sp. 2611 isolated from Phalaborwa carbonatite complex. Clostridium sp. 2611 was able to grow in minimal medium containing 0.5 mM Eu3+. SEM-EDX analysis confirmed an association between Eu precipitates and the bacterium, while TEM-EDX analysis indicated intracellular accumulation of Eu. According to the HR-XPS analysis, the bacterium was able to reduce Eu3+ to Eu2+ under growth and non-growth conditions. Preliminary protein characterization seems to indicate that a cytoplasmic pyruvate oxidoreductase is responsible for Eu bioreduction. These findings suggest the bioreduction of Eu3+ by Clostridium sp. as a resistance mechanism, can be exploited for the biorecovery of this metal.

Biomineralization and Bioaccumulation of Europium by a Thermophilic Metal Resistant Bacterium.

Rare earth metals are widely used in the production of many modern technologies. However, there is concern that supply cannot meet the growing demand in the near future. The extraction from low-grade sources such as geothermal fluids could contribute to address the increasing demand for these compounds. Here we investigated the interaction and eventual bioaccumulation of europium (Eu) by a thermophilic bacterium, Thermus scotoductus SA-01. We demonstrated that this bacterial strain can survive in high levels (up to 1 mM) of Eu, which is hundred times higher than typical concentrations found in the environment. Furthermore, Eu seems to stimulate the growth of T. scotoductus SA-01 at low (0.01-0.1 mM) concentrations. We also found, using TEM-EDX analysis, that the bacterium can accumulate Eu both intracellularly and extracellularly. FT-IR results confirmed that carbonyl and carboxyl groups were involved in the biosorption of Eu. Infrared and HR-XPS analysis demonstrated that Eu can be biomineralized by T. scotoductus SA-01 as Eu2(CO3)3. This suggests that T. scotoductus SA-01 can potentially be used for the biorecovery of rare earth metals from geothermal fluids.

Aerobic and anaerobic enrichment cultures highlight the pivotal role of facultative anaerobes in soil hydrocarbon degradation.

Aliphatic and aromatic hydrocarbons are ubiquitous in the environment due to natural and anthropogenic processes. Under aerobic conditions hydrocarbons can be rapidly biodegraded but oxygenated environments often quickly become anaerobic when microbial respiration is coupled to contaminant oxidation. Most studies in literature usually focus on the initial microbial diversity of the hydrocarbon impacted environment and examine either aerobic or anaerobic conditions for enrichment. Hence, the aim of the present study was to enrich bacterial consortiums from two diesel impacted soil samples under both these conditions to assess the enrichment diversities and hydrocarbon degradation potentials. This would shed light upon how an environmental population shift would correlate to oxygen intrusion and depletion and still continue hydrocarbon degradation. Analysis of the 16S rRNA gene sequences showcases the different microbial populations that could emerge as the environmental factors change, resulting in different populations that are still capable of hydrocarbon degradation. Microbial diversity analysis also highlights the role of facultative anaerobic bacteria like Pseudomonas spp. and Citrobacter spp. in maintaining hydrocarbon degradation. This study shows that microorganisms capable of surviving under both oxic and anoxic (aerobic and anaerobic) conditions are the most crucial to the long term degradation of hydrocarbons in the environment.

Draft Genome Sequence of "Candidatus Bathyarchaeota" Archaeon BE326-BA-RLH, an Uncultured Denitrifier and Putative Anaerobic Methanotroph from South Africa's Deep Continental Biosphere.

Metagenomic sequencing of fracture fluid from South Africa recovered a nearly complete "Candidatus Bathyarchaeota" archaeon genome. The metagenome-assembled genome of BE326-BA-RLH contains genes involved in methane metabolism and dissimilatory nitrate reduction. This study presents the first genomic evidence for potential anaerobic methane oxidation in the phylum "Ca. Bathyarchaeota."

Complex Effects of Cytochrome P450 Monooxygenase on Purple Membrane and Bacterioruberin Production in an Extremely Halophilic Archaeon: Genetic, Phenotypic, and Transcriptomic Analyses.

Halophilic archaea are known to produce a diverse array of pigments for phototrophy and photoprotection. The aim of this paper was to determine the role of a Halobacterium gene encoding the predicted cytochrome P450 monooxygenase (CYP174A1) in pigment synthesis through a combined genetic, phenotypic, and transcriptomic approach. We report on the observed phenotype changes [increased bacterioruberin levels and the loss of purple membrane (PM)] between the Halobacterium salinarum R1 and its CYP174A1-deletion mutant. In addition, we report on the whole-genome DNA microarray analysis, which supports the phenotype of PM loss. This work expands our understanding of the bop-gene regulon, and its relation to carotenoid biosynthesis, and sheds light on our broader understanding of the role (s) of CYP174A1 in archaeal pigment synthesis. To date, this is the first study in which the physiological role of any cytochrome P450 monooxygenase (CYP450) in extremely halophilic archaea has been reported.

Evaluation of in vitro refolding vs cold shock expression: Production of a low yielding single chain variable fragment.

The development of therapeutic antibodies in their various forms has been a constant challenge since the development of the first monoclonal antibodies in 1975. This is especially true for the development of therapeutic single chain variable (scFv) fragments in Escherichia coli. In a previous study the selection of a tissue factor inhibiting single chain variable fragment (TFI-scFv) isolated from the Thomlinson I + J phage libraries was described. Although the initial findings were promising, additional characterization of the antibody fragment and subsequent application was hampered due low protein yield. This study reports on: i) the improved expression of a previously low yielding TFI-scFv in the cytoplasm of E. coli BL21 (DE3) through modifications to the expression systems in conjunction with codon optimization ii) evaluation of two commercial methods of protein recovery: in vitro refolding and the utilization of cold shock expression systems in conjunction with E. coli SHuffle. Results showed that TFI-scFv could be expressed at higher levels in the cytoplasm of E. coli than previously achieved in the periplasm. Both the in vitro refolding and cold shock strategies were capable of producing functional TFI-scFv with varying degrees of success. These procedures could be applied to improve the production of other problematic low yielding scFv isolated from phage display repositories in order to facilitate their characterization.

Flocculating performance of a bioflocculant produced by Arthrobacter humicola in sewage waste water treatment.

BACKGROUND: The discharge of poorly treated effluents into the environment has far reaching, consequential impacts on human and aquatic life forms. Thus, we evaluated the flocculating efficiency of our test bioflocculant and we report for the first time the ability of the biopolymeric flocculant produced by Arthrobacter humicola in the treatment of sewage wastewater. This strain was isolated from sediment soil sample at Sterkfontein dam in the Eastern Free State province of South Africa. RESULTS: Basic Local Alignment Search Tool (BLAST) analysis of the nucleotide sequence of the 16S rDNA revealed the bacteria to have 99% similarity to Arthrobacter humicola strain R1 and the sequence was deposited in the Gene bank as Arthrobacter humicola with accession number KC816574.1. Flocculating activity was enhanced with the aid of divalent cations, pH 12, at a dosage concentration of 0.8 mg/mL. The purified bioflocculant was heat stable and could retain more than 78% of its flocculating activity after heating at 100 °C for 25 min. Fourier Transform Infrared Spectroscopy analysis demonstrated the presence of hydroxyl and carboxyl moieties as the functional groups. The thermogravimetric analysis was used to monitor the pyrolysis profile of the purified bioflocculant and elemental composition revealed C: O: Na: P: K with 13.90: 41.96: 26.79: 16.61: 0.74 weight percentage respectively. The purified bioflocculant was able to remove chemical oxygen demand, biological oxygen demand, suspended solids, nitrate and turbidity from sewage waste water at efficiencies of 65.7%, 63.5%, 55.7%, 71.4% and 81.3% respectively. CONCLUSIONS: The results of this study indicate the possibility of using the bioflocculant produced by Arthrobacter humicola as a potential alternative to synthesized chemical flocculants in sewage waste water treatment and other industrial waste water.

Hexavalent chromium bioreduction and chemical precipitation of sulphate as a treatment of site-specific fly ash leachates.

Most of the power generation globally is by coal-fired power plants resulting in large stockpiles of fly ash. The trace elements associated with the ash particles are subjected to the leaching effects of precipitation which may lead to the subsequent contamination of surface and groundwater systems. In this study, we successfully demonstrate an efficient and sustainable dual treatment remediation strategy for the removal of high levels of Cr6+ and SO42- introduced by fly ash leachate generated by a power station situation in Mpumalanga, South Africa. The treatment consisted of a primary fixed-bed bioreactor kept at a reduction potential for Cr6+ reduction. Metagenome sequencing clearly indicated a diverse bacterial community containing various bacteria, predominantly of the phylum Proteobacteria which includes numerous species known for their ability to detoxify metals such as Cr6+. This was followed by a secondary BaCO3/dispersed alkaline substrate column for SO42- removal. The combination of these two systems resulted in the removal of 99% Cr6+ and 90% SO42-. This is the first effective demonstration of an integrated system combining a biological and chemical strategy for the remediation of multi-contaminants present in fly ash leachate in South Africa.

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2 Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic ß-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Whole Genome Comparison of Thermus sp. NMX2.A1 Reveals Principle Carbon Metabolism Differences with Closest Relation Thermus scotoductus SA-01.

Genome sequencing of the yellow-pigmented, thermophilic bacterium Thermus sp. NMX2.A1 resulted in a 2.29 Mb draft genome that encodes for 2312 proteins. The genetic relationship between various strains from the genus Thermus was assessed based on phylogenomic analyses using a concatenated set of conserved proteins. The resulting phylogenetic tree illustrated that Thermus sp. NMX2 A.1 clusters together with Thermus scotoductus SA-01, despite being isolated from vastly different geographical locations. The close evolutionary relationship and metabolic parallels between the two strains has previously been recognized; however, neither strain's genome data were available at that point in time. Genomic comparison of the Thermus sp. NMX2.A1 and T. scotoductus SA-01, as well as other closely related Thermus strains, revealed a high degree of synteny at both the genomic and proteomic level, with processes such as denitrification and natural cell competence appearing to be conserved. However, despite this high level of similarity, analysis revealed a complete, putative Calvin-Benson-Bassham (CBB) cycle in NMX2.A1 that is absent in SA-01. Analysis of horizontally transferred gene islands provide evidence that NMX2 selected these genes due to pressure from its HCO3 (-) rich environment, which is in stark contrast to that of the deep subsurface isolated SA-01.

A metagenomic window into carbon metabolism at 3 km depth in Precambrian continental crust.

Subsurface microbial communities comprise a significant fraction of the global prokaryotic biomass; however, the carbon metabolisms that support the deep biosphere have been relatively unexplored. In order to determine the predominant carbon metabolisms within a 3-km deep fracture fluid system accessed via the Tau Tona gold mine (Witwatersrand Basin, South Africa), metagenomic and thermodynamic analyses were combined. Within our system of study, the energy-conserving reductive acetyl-CoA (Wood-Ljungdahl) pathway was found to be the most abundant carbon fixation pathway identified in the metagenome. Carbon monoxide dehydrogenase genes that have the potential to participate in (1) both autotrophic and heterotrophic metabolisms through the reversible oxidization of CO and subsequent transfer of electrons for sulfate reduction, (2) direct utilization of H2 and (3) methanogenesis were identified. The most abundant members of the metagenome belonged to Euryarchaeota (22%) and Firmicutes (57%)-by far, the highest relative abundance of Euryarchaeota yet reported from deep fracture fluids in South Africa and one of only five Firmicutes-dominated deep fracture fluids identified in the region. Importantly, by combining the metagenomics data and thermodynamic modeling of this study with previously published isotopic and community composition data from the South African subsurface, we are able to demonstrate that Firmicutes-dominated communities are associated with a particular hydrogeologic environment, specifically the older, more saline and more reducing waters.

Deep subsurface mine stalactites trap endemic fissure fluid Archaea, Bacteria, and Nematoda possibly originating from ancient seas.

Stalactites (CaCO3 and salt) from water seeps are frequently encountered in ceilings of mine tunnels whenever they intersect water-bearing faults or fractures. To determine whether stalactites could be mineralized traps for indigenous fracture water microorganisms, we analyzed stalactites collected from three different mines ranging in depth from 1.3 to 3.1 km. During sampling in Beatrix gold mine (1.4 km beneath the surface), central South Africa, CaCO3 stalactites growing on the mine tunnel ceiling were collected and observed, in two cases, to contain a living obligate brackish water/marine nematode species, Monhystrella parvella. After sterilization of the outer surface, mineral layers were physically removed from the outside to the interior, and DNA extracted. Based upon 16S and 18S rRNA gene sequencing, Archaea, Bacteria, and Eukarya in different combinations were detected for each layer. Using CT scan and electron microscopy the inner structure of CaCO3 and salt stalactites were analyzed. CaCO3 stalactites show a complex pattern of lamellae carrying bacterially precipitated mineral structures. Nematoda were clearly identified between these layers confirming that bacteria and nematodes live inside the stalactites and not only in the central straw. Salt stalactites exhibit a more uniform internal structure. Surprisingly, several Bacteria showing highest sequence identities to marine species were identified. This, together with the observation that the nematode M. parvella recovered from Beatrix gold mine stalactite can only survive in a salty environment makes the origin of the deep subsurface colonization enigmatic. The possibility of a Permian origin of fracture fluids is discussed. Our results indicate stalactites are suitable for biodiversity recovery and act as natural traps for microorganisms in the fissure water long after the water that formed the stalactite stopped flowing.

Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population.

A major fraction of Earth's prokaryotic biomass dwells in the deep subsurface, where cellular abundances per volume of sample are lower, metabolism is slower, and generation times are longer than those in surface terrestrial and marine environments. How these conditions impact biotic interactions and evolutionary processes is largely unknown. Here we employed single cell genomics to analyze cell-to-cell genome content variability and signatures of horizontal gene transfer (HGT) and viral infections in five cells of Candidatus Desulforudis audaxviator, which were collected from a 3 km-deep fracture water in the 2.9 Ga-old Witwatersrand Basin of South Africa. Between 0 and 32% of genes recovered from single cells were not present in the original, metagenomic assembly of Desulforudis, which was obtained from a neighboring subsurface fracture. We found a transposable prophage, a retron, multiple clustered regularly interspaced short palindromic repeats (CRISPRs) and restriction-modification systems, and an unusually high frequency of transposases in the analyzed single cell genomes. This indicates that recombination, HGT and viral infections are prevalent evolutionary events in the studied population of microorganisms inhabiting a highly stable deep subsurface environment.

Optimization of a bioremediation system of soluble uranium based on the biostimulation of an indigenous bacterial community.

High concentrations of uranium(VI) in the Witwatersrand Basin, South Africa from mining leachate is a serious environmental concern. Treatment systems are often ineffective. Therefore, optimization of a bioremediation system that facilitates the bioreduction of U(VI) based on biostimulation of indigenous bacterial communities can be a viable alternative. Tolerance of the indigenous bacteria to high concentrations of U and the amount of citric acid required for U removal was optimized. Two bioreactor studies which showed effective U(VI) removal more than 99 % from low (0.0037 mg L(-1)) and high (10 mg L(-1)) concentrations of U to below the limit allowed by South African National Standards for drinking water (0.0015 mg L(-1)). The second bioreactor was able to successfully adapt even with increasing levels of U(VI) feed water up to 10 mg L(-1), provided that enough electron donor was available. Molecular biology analyses identified Desulfovibrio sp. and Geobacter sp. among known species, which are known to reduce U(VI). The mineralogical analysis determined that part of the uranium precipitated intracellularly, which meant that the remaining U(VI) was precipitated as U(IV) oxides and TEM-EDS also confirmed this analysis. This was predicted with the geochemical model from the chemical data, which demonstrated that the treated drainage was supersaturated with respect to uraninite > U4O9 > U3O8 > UO2(am). Therefore, the tolerance of the indigenous bacterial community could be optimized to remediate up to 10 mg L(-1), and the system can thus be upscaled and employed for remediation of U(VI) impacted sites.

Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov., thermophilic anaerobic representatives of the phylum Planctomycetes.

Two novel strains of thermophilic planctomycetes were recovered from terrestrial and subterranean habitats. Strain R1(T) was isolated from a hot spring (Kunashir Island, Russia) and strain SBP2(T) was isolated from a deep gold mine (South Africa). Both isolates grew in the temperature range 30-60 °C and pH range 5.0-8.0. Strain R1(T) grew optimally at 60 °C and pH 6.0-6.5; for SBP2(T) optimal conditions were at 52 °C and pH 7.5-8.0. Both strains were capable of anaerobic respiration with nitrate and nitrite as electron acceptors as well as of microaerobic growth. They also could grow by fermentation of mono-, di- and polysaccharides. Based on their phylogenetic position and phenotypic features we suggest that the new isolates represent two novel species belonging to a new genus in the order Planctomycetales, for which the names Thermogutta terrifontis gen. nov., sp. nov. and Thermogutta hypogea sp. nov. are proposed. The type strain of Thermogutta terrifontis, the type species of the genus, is R1(T) ( = DSM 26237(T) = VKM B-2805(T)), and the type strain of Thermogutta hypogea is SBP2(T) ( = JCM 19991(T) = VKM B-2782(T)).