Errol Duncan Cason

Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water.

South Africa has numerous thermal springs that represent topographically driven meteoric water migrating along major fracture zones. The temperature (40–70°C) and pH (8–9) of the thermal springs in the Limpopo Province are very similar to those of the low salinity fracture water encountered in the South African mines at depths ranging from 1.0 to 3.1 km. The major cation and anion composition of these thermal springs are very similar to that of the deep fracture water with the exception of the dissolved inorganic carbon and dissolved O2, both of which are typically higher in the springs than in the deep fracture water. The in situ biological relatedness of such thermal springs and the subsurface fracture fluids that feed them has not previously been evaluated. In this study, we evaluated the microbial diversity of six thermal spring and six subsurface sites in South Africa using high-throughput sequencing of 16S rRNA gene hypervariable regions. Proteobacteria were identified as the dominant phylum within both subsurface and thermal spring environments, but only one genera, Rheinheimera, was identified among all samples. Using Morisita similarity indices as a metric for pairwise comparisons between sites, we found that the communities of thermal springs are highly distinct from subsurface datasets. Although the Limpopo thermal springs do not appear to provide a new window for viewing subsurface bacterial communities, we report that the taxonomic compositions of the subsurface sites studied are more similar than previous results would indicate and provide evidence that the microbial communities sampled at depth are more correlated to subsurface conditions than geographical distance.

Reduction of U(VI) by the deep subsurface bacterium, Thermus scotoductus SA-01, and the involvement of the ABC transporter protein

In this study we investigated the effect of uranium on the growth of the bacterium Thermus scotoductus strain SA-01 as well as the whole cell U(VI) reduction capabilities of the organism. Also, site-directed mutagenesis confirmed the identity of a protein capable of a possible alternative mechanism of U(VI) reduction. SA-01 can grow aerobically in up to 1.25 mM uranium and has the capability to reduce low levels of U(VI) in under 20 h. TEM analysis performed on cells exposed to uranium showed extracellular and membrane-bound accumulation of uranium. The reductase-like protein was surprisingly identified as a peptide ABC transporter, peptide-binding protein. This study showcases the concept of protein promiscuity, where this protein with a distinct function in situ can also have the unintended function of a reactant for the reduction of U(VI).

Gold nanoparticle synthesis using the thermophilic bacterium Thermus scotoductus SA-01 and the purification and characterization of its unusual gold reducing protein

Nanoparticles are very important materials for implementing nanotechnology in diverse areas and are abundant in nature as living organisms operate at a nanoscale. As nanoparticles exhibit interesting size- and shape-dependent physical and chemical properties, the synthesis of uniform nanoparticles with controlled sizes and shapes is of great importance. Nanoparticles are the end products of a wide variety of physical, chemical and biological processes, some of which are novel and radically different and others of which are quite commonplace. The ability to produce nanoparticles with specific shapes and controlled sizes could result in interesting new applications that can potentially be utilized in areas such as optics, electronics and the biomedical field. In the present study, we have demonstrated the ability of the thermophilic bacterium Thermus scotoductus SA-01 to synthesize gold nanoparticles and determined the effect of the physico-chemical parameters on particle synthesis. Furthermore, a protein purified from this bacterium is shown to be capable of reducing HAuCl4 to form elemental nanoparticles in vitro. The protein was purified to homogeneity and identified through N-terminal sequencing as an ABC transporter, peptide-binding protein. It is speculated that this protein reduces Au(III) through an electron shuttle mechanism involving a cysteine disulphide bridge. Through manipulation of physico-chemical parameters, it was possible to vary nanoparticles in terms of number, shape and size. This is the first report of a transporter protein from a thermophile with the ability to produce nanoparticles in vitro thus expanding the limited knowledge around biological gold nanoparticle synthesis.

Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa.

Following the discovery of the first Eukarya in the deep subsurface, intense interest has developed to understand the diversity of eukaryotes living in these extreme environments. We identified that Platyhelminthes, Rotifera, Annelida and Arthropoda are thriving at 1.4 km depths in palaeometeoric fissure water up to 12,300 yr old in South African mines. Protozoa and Fungi have also been identified; however, they are present in low numbers. Characterization of the different species reveals that many are opportunistic organisms with an origin due to recharge from surface waters rather than soil leaching. This is the first known study to demonstrate the in situ distribution of biofilms on fissure rock faces using video documentation. Calculations suggest that food, not dissolved oxygen is the limiting factor for eukaryal population growth. The discovery of a group of Eukarya underground has important implications for the search for life on other planets in our solar system.

Metabolic promiscuity from the deep subsurface: a story of survival or superiority

The Witwatersrand Supergroup is a 2.9-billion-year-old formation of low permeability sandstone and shale with minor volcanic units and conglomerates with an ambient rock temperature of approximately 60°C. Thermus scotoductus SA-01 was isolated from fissure water at a depth of 3.2 kmbls in a South African gold mine and it shows the ability to reduce a variety of heavy metals under anaerobic conditions. It has been postulated that such microorganisms could play an important role in nutrient and metal cycling within the subsurface. Recently, our studies indicate that the cycling of metals could also occur under aerobic conditions and not only by the action of redox active enzymes, but other diverse metabolic proteins as well. In this study the capability of specific proteins to interact with metals is elucidated. Using Thermus SA-01 and its now completed genome sequence, metal reduction is studied through classic proteomic- and genomic methods. Finally we identify thermostable enzymes responsible for the transformation of various metals (Iron, Chrome, Uranium, Gold, etc) and discuss that reduction occurs via the serendipitous action of enzymes with other primary physiological functions, some of which are classical catabolic enzymes and anabolic proteins. This paper discusses the use of a ubiquitous enzyme/protein performing more than one function, possibly detoxifying the environment and using moonlighting as resource to decrease cellular energy requirements rather than elaborate metabolism in the subsurface.

Tepidibacillus infernus sp. nov., a moderately thermophilic, selenate- and arsenate-respiring hydrolytic bacterium isolated from a gold mine, and emended description of the genus Tepidibacillus.

A novel aerotolerant anaerobic, moderately thermophilic, organotrophic bacterium, strain MBL-TLPT, was isolated from a sample of microbial mat, developed under the flow of subsurface water in TauTona gold mine, South Africa. Cells of the new isolate were flagellated, spore-forming rods, 0.25-0.5 µm in width and 3-15 µm in length. Strain MBL-TLPT grew in the temperature range from 25 to 58 °C, pH range from 5.6 to 8.8 and at NaCl concentration from 0 to 85 g l-1. The isolate was able to ferment yeast extract and mono-, oligo- and polysaccharides, including starch and xanthan gum. The G+C content of the DNA was 35 mol%. Phylogenetic analysis of 16S rRNA gene sequences of strain MBL-TLPT and relatives showed its affiliation to the genus Tepidibacillus. Tepidibacillus fermentans STGHT was its closest relative (97.1 % identity of 16S rRNA gene sequences). Based on phylogenetic analysis and the physiological properties of the novel isolate, we propose a novel species, Tepidibacillus infernus sp. nov., with MBL-TLPT(=DSM 28123T=VKM В-2949T) as the type strain.

Labeling of prokaryotic mRNA in live cells using fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB)

High-throughput sequencing and cellular imaging have expanded our knowledge of microbial diversity and expression of cellular activity. However, it remains challenging to characterize low-abundance, slow-growing microorganisms that play key roles in biogeochemical cycling. With the goal of isolating transcriptionally active cells of these microorganisms from environmental samples, we developed fluorescent in situ hybridization of transcript-annealing molecular beacons (FISH-TAMB) to label living prokaryotic cells. FISH-TAMB utilizes polyarginine cell-penetrating peptides to deliver molecular beacons across cell walls and membranes. Target cells are fluorescently labeled via hybridization between molecular beacons and messenger RNA of targeted functional genes. FISH-TAMB’s target specificity and deliverance into both bacterial and archaeal cells were demonstrated by labeling intracellular methyl-coenzyme M reductase A (mcrA) transcripts expressed by Escherichia coli mcrA+, Methanosarcina barkeri, and a methanogenic enrichment of deep continental fracture fluid. Growth curve analysis supported sustained cellular viability following FISH-TAMB treatment. Flow cytometry and confocal microscopy detected labeled single cells and single cells in aggregates with unlabeled cells. As FISH-TAMB is amenable to target any functional gene of interest, when coupled with cell sorting, imaging, and sequencing techniques, FISH-TAMB will enable characterization of key uncharacterized rare biosphere microorganisms and of the syntrophically activated metabolic pathways between physically associated microorganisms.

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.

Sporosalibacterium tautonense sp. nov., a thermotolerant, halophilic, hydrolytic bacterium isolated from a gold mine, and emended description of the genus Sporosalibacterium

A novel strictly anaerobic, thermotolerant, moderately halophilic, organotrophic bacterium, strain MRo-4T, was isolated from a sample of a microbial mat, developed under the flow of subsurface water in TauTona gold mine, South Africa. Cells of the novel isolate stained Gram-positive and were motile, spore-forming rods, 0.2-0.3 µm in width and 5-20 µm in length. Strain MRo-4T grew at 25-50 °C, at pH 7.0-8.8 and at an NaCl concentration of 5-100 g l-1. The isolate was able to ferment yeast extract, peptone and mono-, oligo- and polysaccharides, including cellulose and chitin. Elemental sulfur, thiosulfate, sulfate, sulfite, nitrate, nitrite, fumarate and arsenate were not reduced. The major fatty acids were iso-C15 : 0, iso-C15 : 0 dimethyl acetyl and anteiso-C15 : 0. The G+C content of the DNA was 32.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences of strain MRo-4T and its nearest relatives showed its affiliation to the genus Sporosalibacterium. Sporosalibacteriumfaouarense SOL3f37T, the only valid published representative of the genus, appeared to be its closest relative (96.8 % 16S rRNA gene sequence similarity). However, strains MRo-4T and S. faouarense SOL3f37T differed in temperature, pH and salinity ranges for growth, requirement for yeast extract and substrate profiles. Based on the phylogenetic analysis and physiological properties of the novel isolate, we propose a novel species, Sporosalibacterium tautonense sp. nov. The type strain is MRo-4T (=DSM 28179T=VKM B-2948T).

Whole genome comparison of Thermus sp. NMX2.A1 reveals principal 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.