A Microbial Biorealm page on the genus Dictyoglomus thermophilum
Higher order taxa
Bacteria; Dictyoglomi; Dictyoglomi; Dictyoglomales; Dictyoglomaceae
Description and significance
Dictyoglomus thermophilum is light grey, anaerobic, extremely thermophilic, rod-shaped bacterium first isolated from a slightly alkaline hot spring (pH 7.2, 100% N2) in Japan, 1985. Since then, the organism has been found in hot spring beds located in New Zealand and Russia as well. Like other thermophiles, D. thermophilum, thrives in environments of high temperature (between 50 and 80˚C, with an optimal value of 78˚C) [7, 10, 12].
This bacterium is unique in that it is the only member of the phylum Dictyoglomi. The properties of this strain do not fit those of any previously described genus, warranting its very own phylum. D. thermophilum only lives in aquatic environments and is not known to have any form of motility . It is a chemoorganotroph, meaning it derives energy by metabolizing organic molecules. This mysterious and elusive bacterium has generated interest because it has the ability to express xylanase, an enzyme involved in the digestion of xylan (a heteropolymer of the pentose sugar xylose). By treating wood pulp with this enzyme, manufacturers can give paper its characteristic “whiteness” without the use of chlorine bleach [4, 8]. Scientists have attempted to sequence and study the genes which encode this unusual protein and many others, with the intention of understanding how this organism is able to thrive under such extreme conditions.
Dictyoglomus thermophilum is a fairly young organism, only discovered in the year 1985 by Saiki et al. in a tsuetate hot spring of Japan [3, 12].
The complete genome for D. thermophilum has yet to be fully sequenced. Since the year 2006, geneticists at The Institute for Genomic Research (TIGR), have been conducting “random” shotgun sequencing in an attempt to construct the organism’s entire genome. The lack of information has made it difficult for microbiologists to isolate numerous proteins and enzymes synthesized by this organism [10, 14].
The G+C content of D. thermophilum is around 29 mol% . Thirteen nucleotide regions, which code for a total of 17 proteins, have been sequenced from the genome of Dictyoglomus thermophilum . These numbers are miniscule when compared to the thousands of coding regions that have been identified from genomes in other organisms. Of the 17 discovered coding regions, many of them code for amylase proteins, xylanse enzymes, DNA polymerase, multiple unnamed proteins, and various kinase and transferase proteins.
A total of three DNA sequences have been identified encoding three different Alpha-amylase enzymes (amyA, amyB, and amyC). Each of the sequences are 2649, 2496, and 2226 base pairs long. These enzymes are primarily involved in starch breakdown, providing the microbe with nutrients [6, 7, 9]. A 2639 base pair sequence was found to encode for DNA polymerase I . Another internal 1507-bp fragment was sequenced and shown to code for a small, 352 amino acid long xylanase protein (XynA) . This enzyme is involved in the degradation of heteropolymer, xylan. Another xylanase, xynB, is also coded in the genome of D. thermophilum. XynB also is involved in the breakdown of xylan; however, it is of greater interest than xynA because of its ability to bleach paper. Various nucleotide sequences such as these have been isolated and found to encode proteins primarily involved in hydrolysis of organic molecules. The exact positioning of these genes on the chromosome is not yet known. Once the complete genome has been constructed, a lot more can be inferred about its genomic structure.
Cell structure and metabolism
D. thermophilum is a Gram negative (-) bacterium, meaning that it contains both an inner and outer cell membrane. Living in extremely warm environments, its cell membrane is fairly rigid, containing many saturated fatty acid chains, while its proteins contain many charged amino acids to prevent denaturation when subject to extreme heat. D. thermophilum typically resides in slightly alkaline mediums, with a pH around 7.2 . Oddly enough, its amylase proteins function best at a pH between 5 and 5.5. It is possible that the internal environment of the bacterium is slightly acidic when compared to the medium in which it resides.
Being an organotroph, D. thermophilum typically makes use of enzymes to degrade organic macromolecules for nutrients. The major food sources consumed are starch, galactomannan, and xylan. Due to its elusive nature, only a handful of enzymes involved in catabolic and anabolic pathways have been isolated from D. thermophilum. Three amylase enzymes have been purified from this strain, all of them thought to be involved in the hydrolysis of starch as a nutrient source. It was recently proved that the enzyme amylase A, has transglycosylation properties as well. Given this new development, it is possible that D. thermophilum also makes use of other saccharides as a nutrient source (see “Current Research”). In addition, it uses a beta-mannose enzyme to hydrolyze galactomannase into mannose, mannobiose and mannotriose . D. thermophilum has generated significant interest given its ability to solubilize the heteropolymer xylan. Two xylanase enzymes, xynA and xynB, are thought to be involved in this process. Both enzymes function at similar temperatures and pH ranges. XynA, related to the family F group of xylanases, is capable of hydrolyzing xylan to xylotriose and xylobiose. Only xynB has proven useful in pulp bleaching .
The pfp gene from D. thermophilum encodes a pyrophosphate-dependent phosphofructokinase (PPi-PFK). A phylogenetic analysis of this enzyme indicates that it is closely related to another from the organism Thermoproteus tenax. It is known that ATP-dependent phosphofructokinase (PFK) is a vital enzyme in the glycolysis pathway. It is possible that PPi-PFK plays a role in the metabolism of glucose for energy in D. thermophilum. However, it was previously suggested that PPi-PFK represented an ancestral form of PFK during a time when pyrophosphate was the primary source of metabolic energy (before the advent of ATP) . This sheds light on the idea that D. thermophilum may in fact be a fairly old organism, partially explaining its elusive nature.
D. thermophilum resides in dilute mediums with a slightly basic pH (7.2) at temperatures around 78˚C. D. thermophilum is of the Bacterial domain, whereas most extremophiles are of the domain Archaea. It lives exclusively in aquatic environments and does not have any known method of locomotion. The medium from which D. thermophilum was discovered contained a mixture small quantities of ionic compounds such as Na2HPO4 x 12 H2O, ZnSO4, CaCl2 x 2 H2O, NH4Cl, KH2PO4, and Fe(NH4)2(SO4)2 x 6 H2O . The medium also contained starch, glucose, and other polysaccharides. D. thermophilum is a rod-shaped bacterium and can arrange itself in singles, pairs, chains and spherical bodies .
A thermophilic, chemoorganotrophic, and anaerobic strain of bacterium, similar to that of D. thermophilum was isolated from hot springs in New Zealand, 1996 . Since its discovery, D. thermophilum, has been isolated in a few countries around the globe, including New Zealand. This indicates that the two bacteria might share similar characteristics in their genome. The strain discovered in New Zealand had a DNA base composition of 29.5% G+C , while D. thermophilum has a 29 mol% G+C composition. This new organism could be the second to join the Dictyoglomi phylum, which was created solely for D. thermophilum, upon its discovery.
At this time, there are no known diseases caused by this organism. It has not been found to be pathogenic either .
Application to Biotechnology
D. thermophilum has been of great interest to the paper-making business. The endo-1,4-beta-D-xylanases synthesized by this organism, are responsible for cleavage of the xylan backbone and has industrial significance because it may be possible to use them in manufacturing bleached pulp without the use of chemicals .
Multiple tests have been conducted to test the bleachability of the two main xylanases synthesized in D. thermophilum, xynA and xynB. XynA is effective at cleaving the xylan polymer; however, it has a negligible effect on the bleachability of kraft pulp. However, xynB proved to be highly successful in bleaching multiple kinds of kraft pulp. With respect to a reference pulp, xynB increased brightness by 1.5 percentage units (from 88.5 to 90%) in elemental chlorine-free bleaching (ECF). In ECF bleaching, the xylanase is added with a small fraction of chlorine dioxide. In total chlorine-free bleaching (TCF), the xylanase increased the brightness of the pulp from 82.2 to 86.6%. In general, by using xylanases as an agent in bleaching, the paper industry can save up to 15% in chlorine-dioxide and other chemical agents .
The crystal structure of xynB was determined by a newly developed software suite called Crystallography and NMR system, or CNS. CNS is an advanced and highly flexible system allowing for integration from other techniques such as electron microscopy (EM) and NMR spectroscopy. It allows scientists to generate electron-density maps and atomic properties of protein crystals. Overall, it is an easy software to use and has been of great help in the field of biology when attempting to map the structure of proteins .
At this point, geneticists at TIGR are in the process of constructing the entire genome for D. thermophilum. Upon its completion, microbiologists will be able to sequence and isolate thousands of other proteins. This would provide much needed information concerning the lifestyle of the organism. By studying the entire genome, the many mysterious surrounding D. thermophilum, could be solved .
DNA Polymerase Reverse-Transcriptase Activity and Possible Relatives:
Recent studies have shown that thermophilic bacterial DNA polymerases have reverse-transcriptase activity. Conserved motifs in known polymerase I DNA sequences from many strains of bacteria, D. thermophilum included, have been identified. A total of 13 polymerase I genes were PCR amplified and indicate similarity in structure across different species. Several of the polymerases exhibit reverse-transcriptase activity when exposed to Mg2+. The strains Clostridium stercorarium, Bacillus caldolyticus EA1, and Caldibacillus cellovorans CompA.2 all exhibit this behavior. Although no such activity is thought to exist in D. thermophilum, the same homologous polymerase sequences have been identified from its still incomplete genome. This new information may lead to an eventual phylogenetic connection between D. thermophilum, and the species listed . Another fact to take into account is that only a few bacteria have been discovered to live in thermophilic environments: most extremophilic organisms are of the domain Archaea. However, D. thermophilum and the strains listed, are all bacteria found in extreme environments; this is more evidence that the organisms might be related.
Additional Activity from Amylase A:
It was discovered that the Amylase A (amyA) enzyme from D. thermophilum shared around 40% identity with a 4-alpha-glucanotransferase (GTase for short) from the species Thermococcus litoralis. The GTase from this species has been well-studied, and was found to be involved in transglycosylation activity. This meant that it has the ability to add saccharides to proteins and lipids. It was reported that amylase A from D. thermophilum hydrolyzes starch; however, recent studies have shown that the enzyme is also able to use long chain amylase and maltooligosaccharides as substrates and has transglysoylation activity. When cultured in a medium containing maltooligosaccharides, long chain amylose as well as starch, amylase A hydrolyzed starch to a small extent and primarily used the other saccharides as substrates in adding them to lipids and proteins . This new information may shed light as to what substances D. thermophilum uses as a nutrient source. Much more experimentation must be conducted to fully understand the metabolic activity that D. thermophilum undergoes as a means for survival.
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4. Gibbs, MD.; Reeves, RA.; Bergguist, PL. “Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate.” Appl. Environ. Microbiol., 1995; 61 (12): 4403-4408.
5. Gibbs, MD.; Reeves, RA; Sunna A.; Bergguist, PL. “Sequencing and expression of a beta-mannanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1, and characteristics of the recombinant enzyme.” Curr. Microbiol., 1999; 39 (6): 351-357.
6. Horinouchi, S.; Fukusumi, S.; Ohshim, T.; Beppu, T. “ Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents.” Eur. J. Biochem., 1988; 176 (2): 243-253.
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8. Morris, DD., et al. “Cloning of the xynB gene from Dictyoglomus thermophilum Rt46B.1 and action of the gene product on kraft pulp.” Appl. Enviorn. Microbiol., 1998; 64 (5): 1759/1765.
9. Nakajima, Masahiro; Imamura, Hiromi; Hirofumi, Shoun; Horinouchi, Sueharu; Wakagi, Takayoshi. “Transglycosylation Activity of Dictyoglomus thermophilum Amylase A.” Biosci. Biotechnol. Biochem., 2004; 68 (11): 2367-2373.
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13. Shandilya, H, et al. “Thermophilic bacterial DNA polymerases with reverse-transcriptase activity.” Extremophiles, 2004; 243-251.
14. TIGR Microbial Database. Accessed August 24, 2007. <http://www.tigr.org/tdb/mdb/mdbinprogress.html>
Edited by Nevin Murthy, student of Rachel Larsen
Edited by KLB