Australia: The Land Where Time Began

A biography of the Australian continent 

A Single-Species Ecosystem Deep Within the Earth Revealed by Environmental Genomics

A single complete genome has been sequenced and assembled from DNA recovered from low-biodiversity fracture water that was collected at a depth of 2.8 km in a gold mine in South Africa. A bacterium, Candidatus Desulforudis audaxviator, comprises more than 99.9 % of organisms that inhabit the fluid phase of this particular fracture. It is indicated by its genome to be a motile, sporulating, sulphate-reducing chemoautotrophic thermophile that has the ability to fix its own nitrogen and carbon by using mechanisms shared with archaea. Candidatus Desulforudis audaxviator has an independent lifestyle deep within the crust of the Earth that is well adapted to long-term isolation from the photosphere and offers an example of a natural ecosystem that apparently has its biological component encoded entirely with a single genome.

Recently a more complete picture of life within, as well as on the Earth, has been made possible by the extraction and sequencing of DNA from an environmental sample, the process being called environmental genomics or metagenomics (Deutchbauer, Chivian & Arkin, 2006; Beja et al., 2000; Rondon et al., 2000; Venter, 2004; Tyson et al., 2004; Tringe, 2004; Strous et al., 2006; Rusch et al., 2007). The identification of members of microbial communities has been made possible by this approach, as well as characterisation of abilities of the dominant members of the community, even when isolation of those members has proven to be intractable. The complexity of natural microbial communities has, however, hampered the assembly of complete or even near-complete genomes for a substantial portion of the member species.

As well as elevated temperatures and anoxic conditions within the crust of the Earth at depths of more than 1 km are fundamentally different from those of the surface and deep ocean environments. Cell-doubling times of from 100s to 1,000s of years (Phelps et al., 1994; Jørgensen & D’Hondt, 2006; Lin et al., 2006), are believed to result from severe nutrient limitation, and therefore the subsurface microorganisms might be expected to reduce their reproductive burden and exhibit genomes that are streamlined as is found in specialists or spend more time in semi-senescence as they wait for a return of favourable conditions. As such organisms permit insight into a mode of life that is independent of the photosphere they are of particular interest.

A bacterium, named by Chivian et al. Candidatus Desulforudis audaxviator, phylum Firmicutes, is prominent in small subunit (SSU of 16S) ribosomal RNA (rRNA) gene clone libraries (Lin et al., 2006; 11-14) from almost all fracture fluids sampled to date from deposits greater than 1.5 km in depth throughout the Witwatersrand Basin, which covers 150 km by 300 km near Johannesburg, South Africa. It has been shown in a previous geochemical and 16S rRNA study (Lin et al., 2006) that this bacterium dominates the indigenous microorganisms found in a fracture zone at 2.8 km below the surface of the land at level 104 in the Mponeng mine (MP104). Chivian et al. were surprised with the results of the environmental study reported in this paper that showed that a single species was present within the fracture fluid, though it had been discovered (Lin et al., 2006) that the least diverse natural free-living microbial community that had been reported at that time, which exceeded the approximate 80 % dominance by the methanogenic archaeon IUA5/6 of a shallow subsurface community in Idaho (Chapelle et al., 2002). Also, it was found by Chivian et al. that this organism apparently possessed all the metabolic abilities that were necessary to live independently. This gene component was consistent with geochemical and thermodynamic analyses that had been carried out previously at the ambient temperature of about 60^oC and 9.3 pH, which indicated that the energy and nutrients to the system (Lin et al., 2006) was provided by radiolytically generated chemical species, with formate and H₂ possessing the greatest potential among candidate electron donors, and the dominant electron-accepting process (Lin et al., 2006) being sulphate (SO₄^2-).

Discussion

According to Chivian et al. if D. audaxviator proves to be the only inhabitant of this habitat, then the complete genetic complement for the maintenance of the biological component of the ecosystem must be present, and this would prohibit the extreme reduction of its genome. At 2.35 Mbp the genome was smaller than the 3 Mbp of its nearest relative to be sequenced, Pelotomaculum thermopropionicum. The genome of D. audaxviator contained 2,157 predicted coding genes; which is more than are present in streamlined free-living microorganisms, which typically have fewer than 2,000 genes (Giovannoni et al., 2005). It was found in this study that all the genes coding for necessary processes for life were present in the genome, and this included energy metabolism, carbon fixation, and nitrogen fixation.

The genome has the capability for dissimilarity sulphate reduction  (DSR), having a gene repertoire like that of other microorganisms (Mussmann et al., 2005) that can reduce sulphate (SO₄^2-), which is consistent with the thermodynamic evaluation  (Lin et al., 2006) that SO₄^2- offers the most energetically favourable electron acceptor. These genes are present in a set of operons, including an extra copy of an archaeal-type sulphate adenylyltransferase (Sat), both appearing to be consequent upon horizontal gene transfer (HGT). Chivian et al. suggest it is probable that high potential electrons enter primarily by the activity of a variety of hydrogenases which act upon H₂.

Assimilation of carbon may be from a number of sources depending on local conditions. Transporters of sugar and amino acid are present in the genome, which suggests that at locations where there is high biodiversity, heterotrophic sources could be used, which includes recycling of dead cells. Where biodiversity is low, as occurs at MP104, carbon from inorganic sources is fixed. D. audaxviator doesn’t appear to use the reverse TCA cycle, though it did have all the machinery of the acetyl-coenzyme A (CoA) synthesis (Wood-Ljungdahl) pathway (Drake, 2005; Wu et al., 2005), which uses carbon monoxide dehydrogenase (CODH) to assimilate inorganic carbon. Its anionic species may accomplish the entry into the cell of CO₂ substrate through its putative carbonate adenosine triphosphate (ATP)-binding cassette transporter or a putative bicarbonate/Na⁺ symporter. Formate and carbon monoxide may serve as alternate carbon sources that are more direct in other fractures when sufficiently abundant. In the fracture water the ambient concentration of ammonia ([NH₃] + NH₄⁺] = ~100 μM) (Lin et al., 2006) appears to be sufficient for D. audaxviator (which has an ammonium transporter as well as a glutamine synthetase) to obtain its nitrogen from ammonia without needing to resort to a nitrogenase conversion of N₂ to ammonia which is more costly. A nitrogenase is nonetheless present in the genome with a nifH subunit that is more similar to archaeal types, which includes high temperature variants (Mehta & Baross, 2006), than to the nitrogenase of Desulfotomaculum reducens. Chivian et al. suggests D. audaxviator is not always present with sufficient amounts of ammonia; therefore the versatility that is provided by nitrogenase that is horizontally acquired may have contributed substantially to the success of D. audaxviator in colonising such habitats. There are other genes that are shared between D. audaxviator and archaea that are suggested to possibly confer benefits in extreme environments. As well as the unusual nitrogenase and sulphate adenylyltransferase, acquisitions by ancestors of D. audaxviator include a second CODH system, cobalamin biosynthesis protein CobN, and genes that code for gas vesicles. Also, it has 2 clustered regularly interspaced shot palindromic repeat (CRISPR) regions that are used for viral defence (Barrangou et al., 2007) and which occur in the genome with genes that are CRISPR-associated (CAS, some of which are horizontally shared between D. audaxviator and archaea.

The possession by D. audaxviator of all the amino acid synthesis pathways assists its ability to colonise independently. The ability to form endospores and the potential for it to grow in deeper, hotter conditions than provided by MP104 are other factors that may confer fitness in this environment. It appears that D. audaxviator is capable of sensing nutrients in its environment and has flagellar genes to permit motility along chemical gradients, such as those occurring at the mineral surfaces of the fracture (Wanger, Onstott & Southam, 2006). A complete system for oxygen resistance is an ability that D. audaxviator lacks, which suggests long term isolation from oxygen.

Contained within the MP104 fracture is the simplest environmental microbial community that has been described to date and has yielded a single complete genome of a microorganism that wasn’t cultured with the use of environmental genomics. The ability of D. audaxviator to reduce SO₄^2- allows access to the electron acceptor that is energetically favourable in the fracture zone of the Witwatersrand basin (Onstott et al., 2006). Also, characteristics inherited by D. audaxviator, such as motility, sporulation and carbon fixation, have been complemented by systems that are frequently present in archaea that have been acquired horizontally. D. audaxviator has been enabled by these abilities to colonise the deep subsurface, a process which differs from surface habitats, permitting more immediate access, has required fitness throughout the history of colonisation. This audaxviator (‘bold traveller’) has revealed a mode of life that is isolated from the photosphere, which captures all the roles that are necessary for a life-style that is independent and shows the possibility of encoding the entire biological component of a simple ecosystem within a single genome.

Chivian, D., E. L. Brodie, E. J. Alm, D. E. Culley, P. S. Dehal, T. Z. DeSantis, T. M. Gihring, A. Lapidus, L.-H. Lin, S. R. Lowry, D. P. Moser, P. M. Richardson, G. Southam, G. Wanger, L. M. Pratt, G. L. Andersen, T. C. Hazen, F. J. Brockman, A. P. Arkin and T. C. Onstott (2008). "Environmental Genomics Reveals a Single-Species Ecosystem Deep Within Earth." Science 322(5899): 275-278.