A Microbial Biorealm page on the genus Thermotoga neapolitana
Higher order taxa
Bacteria; Thermotogae; Thermotogae; Thermotogales; Thermotogaceae; Thermotoga
Description and significance
Thermotoga neapolitana cells are gram-negative rods, approximately 1.5 to 11 ~tm long and 0.6 Ixm wide, and can occur singly and in pairs. They have an unusually thick periplasmic cell wall layer, which, when compared with characteristic gram-negative cell walls, is covered with a more electron-dense outer layer. Cells are immotile, possessing no flagella, and are also non-sporulating. They are surrounded by a sheath-like outer structure that usually balloons over the ends; a "toga," as it were, from which the genus name is derived. Its species name is in reference to the location of its original isolation in 1986: a shallow marine sediment in a volcanic region near Lucrino, Bay of Naples, Italy. As one would expect from its location of origin, T. neapolitana is an extremely thermophilic bacteria, growing between 55 and 90 degrees Celsius, with an optimum growth temperature of 77 degrees Celsius. It is viable between pH 5.5 and 9; at pH 7 its growth is at a maximum (6).
There are three lines of reasoning that outline the significance of T. neapolitana having its genome sequenced. Firstly, due to its requiring only minimal anoxic precautions, and its ability to be grown on both solid and liquid media via materials that are readily available, the utilization of T. neapolitana is a preferable method for understanding the basic molecular biology of extreme thermophiles (4). Secondly, as it is closely related to the type species Thermotoga maratima, comparison of these two genomes will enable researchers to identify chromosomal segments that have undergone DNA rearrangements after the lineages diverged from a common ancestor (5). Lastly, current research has implicated T. neapolitana as a potentially viable source of hydrogen production. Current methods of hydrogen production are both costly and environmentally unfriendly; in a market in which hydrogen demands are expected to only increase, a new biological method for the generation of hydrogen gas is very desirable. Sequencing T. neapolitana's genome will further these ends by allowing for a deeper understanding of its genetic makeup (10, 11).
Thermotoga neapolitana has one circular chromosome, and a genome that is 1800kb in size. The molar ratio of guanine plus cytosine in the DNA has been calculated to be 41.3 mol%. Its DNA homology with Thermotoga maritima has been found to be approximately 24% (6). Thermotoga neopolitana strain ATCC 49045 (NS-E) is currently having its genome sequenced by the TIGR Institute for the purposes of comparative genome analysis. Its project ID is: 12534. (2).
Cell structure and metabolism
This organism is a hyperthermophile, and as such has adopted certain strategies to cope with the extreme conditions found in its natural habitat. Thermotoga neapolitana contains unusually high melting-point lipids within its cell membrane, meant to help maintain its structure. These in turn incorporate long-chain dicarboxylic fatty acids, which are thought to create a tightly bound polar layer within the membrane itself. In addition to this, T. neapolitana minimizes the amount of free water within its cells, and also utilizes reverse DNA gyrases, which cause its DNA to supercoil. Both of these methods help to inhibit DNA denaturing under the extreme thermal conditions to which it is exposed. Finally, T. neapolitana 's RNAs contain no extra sequences, as all bulges, mismatches, and other irregularities are cut out to minimize size. Shorter sequences are associated with fewer nonviable folding possibilities, which also serves to inhibit thermal denaturation (8,9).
Thermotoga neapolitana survives by scavenging biomolecules. Of all of the members of its order, T. neapolitana is thought to be the most robust, being adaptable to varying conditions and to different primary carbon sources. This is one of the characteristics that makes it such a desirable target for developmental commercial and industrial research. It catabolizes both mono- and polysaccharides, including galactose, glucose, lactose, maltose, ribose, starch, sucrose, and xylose. In contrast to other Thermotogales, amino acids do not support its growth, and it can therefore grow on a medium lacking proteins (4).
Rather than using oxygen as its final electron acceptor in metabolism, T. neapolitana uses sulfur. Therefore, instead of converting oxgyen to water, T. neapolitana reduces elemental sulfur to hydrogen sulfide (H2S). If the available sulfur is organically bound, considerable hydrogen gas can also be produced in this proccess. An interesting characteristic of T. neapolitana is that, despite its usual active particapation in the above process, it is not dependent on sulfur reduction for growth. For example, cystine and dimethyl disulfide can also be used as its electron sinks. While it has been shown that final cell yields are doubled by the inclusion of sulfur within the growth medium of T. neapolitana , their overall growth rates are unaffected (4).
Although it was once thought that T. neapolitana was an obligate anaerobe, recent research has shown that it is in fact a microaerophile, capable of utilizing reduced levels of oxygen to produce hydrogen as an endproduct (10,11).
Thermotoga neapolitana was found in a black smoker in the bay of Naples, Italy. Organisms in this environment are subjected to extremely high temperatures, yet relatively neutral pHs (3). At this time there exists no extensive research on the interactions of this organism with other organisms coexisting in its natural habitat.
Phylogenetic analyses have shown T. neapolitana to be a member of the deepest branch of the bacterial lineage. It's primary role in nature is to reduce sulfur to hydrogen sulfide via the oxidation of organic molecules (4). In this way it actively participates in the biogeochemical cycle of sulfur on Earth. This sulfur cycle involves the reduction of sulfur to hydrogen sulfide by certain sulfur-reducing bacteria for energy (of which T. neapolitana is one), and then the use of hydrogen sulfide as an energy source for a different set of bacteria, which thus oxidizes it back to elemental sulfur (7).
The ecology of T. neapolitana has been largely unstudied. However, of some interest is the fact that it, and sulfur-reducing bacteria like it (especially those that, like T. neapolitana exist in the marine layer), are implicated in the Permian mass extinction which occured 251.4 million years ago. This phenomenon involved the build-up of atmospheric hydrogen sulfide due to sulfate-reducing bacteria becoming a dominant force in oceanic ecosystems. This weakened the ozone layer and exposed the earth to high levels of UV radiation. It is theorized that these cascading events are what proved fatal to animal and plant life, with estimates of up to 70 percent of terrestrial vertabrate species and 96 percent of all marine species becoming extinct (7).
As an extremely thermophilic eubacteria, Thermotoga neapolitana is not viable under the conditions that support most life. It therefore has no known diseases associated with it, and at present has not been found to be pathogenic.
Application to Biotechnology
An advantage of a bacterium that grows in an environment of extreme temperatures is that its associated enzymes must also have adapted to these conditions. Thermostable enzymes have several commercial applications because of their inherent stability. They have been used in the starch industry, the food industry, and the petroleum industry, to name but a few. As T. neapolitana is an extreme thermophile, its thermostable enzymes (such as alkaline phosphatase) may also be of use for industrial purposes. Its ability to grow in temperatures of up to 90 degrees Celsius could be incredibly useful in harsh industrial and biotechnological conditions (3).
8.1 Hydrogen Production
A majority of the members of the order Thermotogales have the ability to produce hydrogen to varying degrees. Thermotoga neapolitana however, outstrips all of its fellow members in this ability. Its hydrogen gas generation, as theorized in the equation: C6H12O6 + 6H20 -> 6CO2 + 12H2 is consistently 25-30%, with 12-15% carbon dioxide as the only other significant product. Also, most members of this order were shown to not only tolerate but utilize moderate amounts of oxygen, with no decrease in hydrogen generation, which contradicts past research on this topic. Lastly, the idea that the accumulation of hydrogen minimizes growth of the bacterium appears to only apply at very high hydrogen tensions, as log phase bacterial roads were observed in 25-35% hydrogen concentrations. To minimize the production of hydrogen sulfide and maximize the generation of hydrogen, inorganic sulfur donors were avoided and the cysteine concentration in the medium increased (9).
8.2 H2 Production and Carbon Utilization: Anaerobic and Microaerobic Growth Conditions
Contrary to previous studies reporting that the strains Petrotoga miotherma, Thermosipho africanus, Thermotoga elfii, Fervidobacterium pennavorans, and Thermotoga neapolitana were strict anaerobes, all of these strains grew and generated hydrogen in the presence of micromolar levels of oxygen. The highest production of hydrogen was observed with T. neapolitana. The amount of hydrogen generation and overall growth was found to be limited by an accompanying and rapid decrease in pH. Itaconic acid was found to be a carbon source that could best serve as a non-toxic and simple physiological buffer to overcome this pH limitation. Agitating the cultures at 75 rpm was also found to improve the generation of hydrogen. It was in this way shown the pH control and moderate agitation are important factors to consider in the production of hydrogen by T. neapolitana (10).
8.3 Chromosome Evolution in the Thermotogales: T. maritima vs. T. neapolitana.
Comparing genomes that are closely related enables us to identify chromosomal segments that have experienced DNA rearrangements sometime after the lineages diverged from a shared ancestor. A technique used to make this comparison is to calculate the pair-wise alignment of the two microbial genomes' translated peptide sequences, and/or their DNA sequences. A whole-genome alignment of T. maritima and T. neapolitana showed several large-scale DNA rearrangments. The majority of these rearrangments were associated with CRISPR DNA repeats and/or tRNA genes (CRISPRs = Clustered Regularly Interspaced Short Palindrome Repeats). PCR amplification and sequence analysis of the DNA joints associated with the major rearrangements revealed that the overall chromosome identity was conserved at most DNA joints for other strains of T. neapolitana. This indicates that these chromosomal rearrangements in the Thermotogales occured by successive inversions after their inital divergence from their shared ancestor, yet before strain diversification. Also, the size polymorphisms in the DNA joints associated with CRISPRs, via sequence analysis, can be attributed to expansion and/or contraction of the DNA spacer and repeat unit. This method can provide a handy tool for discerning the relatedness of strains from different geographical locales (4).
(5) Deboy, R.T., Mongodin, E.F., Emerson, J.B., and Nelson, K.E. (2006). Chromsome evolution in the Thermotogales: large-scale inversions and strain diversification of CRISPR sequences. Journal of Bacteriology. v. 188, 7, p.2364-2374
(9) Nesbo, C.L., L'Haridon, S., Stetter, K.O., and Doolittle, W.F. (2001). Phylogenetic analyses of two "Archaeal" genes in Thermotoga maritima reveal multiple transfers between Archaea and Bacteria. Mol. Bio. Evol. 18: 362-375.
(11) Van Ooteghem, S.A., Jones, A., van der Lelie, D., Dong, B., and Mahajan, D. (2004). H2 production and carbon utilization by Thermotoga neapolitana under anaerobic and microaerobic growth conditions. Biotechnology Letters 26: 1223-1232.
Edited by Jamie Tarouilly, student of Rachel Larsen