Skip to Main contentScienceDirectJournals BooksRegisterSign in Sign inRegisterJournals BooksHelpThermotoga MaritimaRelated terms:MannitolXylanaseEnzymesProteinsAmino AcidsDNAArabinoseCelluloseBeta-GlucosidaseView all TopicsDownload as PDFSet alertAbout this pageThermostable DNA PolymerasesRichard D. Abramson, in PCR Strategies, 1995Tma DNA PolymeraseThermotoga maritima is an anaerobic hyperthermophilic eubacte-rium isolated from geothermally heated marine surfaces, growing at temperatures up to 90 ° C (Huber et al., 1986). Tma DNA polymerase is an 893-amino acid protein with an inferred molecular weight of 103,000 (Lawyer and Gelfand, 1992). Tma DNA polymerase is only 61% similar and 44% identical to Taq DNA polymerase, based on amino acid sequence. Like E. coli DNA polymerase I, Tma DNA polymerase contains both a 5′ to 3′ exonuclease (R. D. Abramson, unpublished data) and a 3′ to 5′ exonuclease (Lawyer and Gelfand, 1992). Similar to the Stoffel and Klenow fragments, an amino terminal truncated form of the enzyme, lacking 5′ to 3′ exonuclease activity (commercially available as Ultma DNA polymerase from Perkin-Elmer Corp., Norwalk, CT) has also been described (Lawyer and Gelfand, 1992; see also Table 1). This enzyme was produced by engineering translation to initiate at the Met284 codon, resulting in a 610-amino acid, 70-kDa protein which retains polymerase activity, with a specific activity of 115,000 U/mg, as well as 3′ to 5′ exonuclease activity. The enzyme is as thermoactive as Taq DNA polymerase; optimal polymerization activity is achieved at 75–80 ° C, with half-maximal activity at 55–60 ° C (D. Bost, S. Stoffel, and D. H. Gelfand, personal communication). Its thermostability, however, is considerably higher, with a half-life of activity of 40– 50 min at 97.5 ° C (P. Landre, personal communication). Maximal DNA polymerase activity is achieved in a Tris–HCl (25 mM), pH 8.3 buffer containing 10–35 mM KCl and 1.5–2.0 mM MgCl2 (D. Bost, S. Stoffel, and D. H. Gelfand, personal communication). Preliminary studies suggest that the inherent fidelity of the enzyme is such that nearly 100% of the PCR product is corrected by the proofreading activity in a 3′ mismatched primer correction assay (F. C. Lawyer, personal communication). In a standard PCR, amplification with Met284-Tma DNA polymerase is optimal in a Tris–HCl (10 mM), pH 8.8 buffer containing 10 mM KCl and 2.0 mM MgCl2 (F. C. Lawyer and D. H. Gelfand, personal communication).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B978012372182250006XBioprocess engineering approaches for the production of marine enzymesSreyashi Sarkar, ... Joydeep Mukherjee, in Marine Enzymes for Biocatalysis, 20136.4.8 DNA polymeraseRecombinant Tma (Thermotoga maritima) DNA polymerase was purified from E. coli strain DG116 containing plasmid Tma12–3 (Gelfand et al., 1997). The volume of seed culture inoculated into the fermenter was calculated such that the bacterial concentration was 0.5 mg dry weight/l. Foaming was controlled by the addition of propylene glycol. Airflow was maintained at 2 l/min. The culture was grown at 30 °C and then the growth temperature was shifted to 35 °C to induce the synthesis of recombinant Tma DNA polymerase. The temperature shift increased the copy number of the Tma12–3 plasmid and simultaneously derepressed the promoter controlling transcription of the modified Tma DNA polymerase gene.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9781907568800500065Methods in Protein DesignJosé Arcadio Farías-Rico, Birte Höcker, in Methods in Enzymology, 20132.1 Selection of the parental structuresThe starting structures for the construction of a novel chimeric protein should be selected based on the specific goal of the experiment. We designed CheYHisF in order to test whether evolution could have generated contemporary protein diversity using subdomain-sized fragments from different folds. Given that all proteins are built from the same secondary structure elements, local similarities can be mainly found among members of the same structural class (all α, all β, α/β, or α + β). Bioinformatic tools, such as PDBeFold (Krissinel Henrick, 2004), DALI (Holm Rosenström, 2010), or GANGSTA (Guerler Knapp, 2008) can be used to screen the structural repositories for structures with similarities to any desired query. In PDBeFold, for instance, it is possible to select the minimum aligned length of the query and the hit; another handy feature of the server is the possibility to sort the results based on sequence identity or P-value, among other scores. Using the program DALI, we originally detected the high structural similarity between the (βα)8-barrel HisF and the flavodoxin-like CheY (Höcker, Schmidt, Sterner, 2002). The resulting chimera CheYHisF turned out to be a well-folded and monomeric protein domain (albeit with an additional secondary structural element), which could be reengineered in successive rounds of rational design to become more compact and functional. This result indicates that our approach can be used to engineer new proteins. For example, we envision that in a similar manner, two complementary activities could be engineered into a single domain or a binding site could be transplanted onto a different scaffold protein.CheY and HisF from Thermotoga maritima were used as parental proteins to build the chimera CheYHisF. The following considerations were taken into account when these structures were chosen as starting points:1.Despite the fact that both protein structures belong to different folds, we detected a strong structural similarity that covered one β-strand and three consecutive βα elements (Fig. 18.2).Figure 18.2. Chimera design based on structural superposition. A structural superposition between CheY (top left; PDB-ID 1TMY) and HisF (top right; PDB-ID 1THF) was used to guide the design of the chimera CheYHisF. The superposed areas from both proteins are highlighted (in black). The superposition covered 77 Cα atoms with a Z-score of 6.6 and a RMSD of 2.3 Å. In the final chimera (bottom center; PDB-ID 2LLE), the black region from CheY replaced the black region from HisF.2.CheY and HisF are single domain proteins, which is probably an advantage. When using a fragment from a multidomain protein, one needs to consider interdomain interactions that might contribute to the stability of the single domains. To predict and reengineer such interactions in the novel chimeric environment is difficult. Therefore we favor the use of single domain proteins as starting structures, or we try to use domains with as little interface to other domains as possible.3.High protein stability is another desirable feature of the parental proteins. The fragments need to be both robust and plastic enough to accommodate the new environment. The proteins we used to build CheYHisF originated from the thermophile Thermotoga maritima. We reasoned that the enhanced stability of the parents would contribute to the stability of the new protein.4.In order to rationally design the chimera, high-quality atomic structures are needed. In an optimal case, one would like to have a set of structures from the same parental protein. Valuable information for the design can be gained from such data, for example, which parts of the protein are flexible or involved in certain functions.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123942920000187DNA Repair Enzymes: Structure, Biophysics, and MechanismMałgorzata Figiel, Marcin Nowotny, in Methods in Enzymology, 20175.1 Crystallization and Structure Determination of T. maritima RNase H2For the crystallization of Tm-RNase H2, we used a variant with a C-terminal truncation of 15 residues (Tm-RNase H2 ΔC). These residues are not visible in the previously published structure (PDB ID 2ETJ), meaning that they are likely disordered. The removal of unstructured regions from a protein often increases the likelihood of crystallization and improves the quality of the crystals. For cocrystallization with a nucleic acid substrate and Mg2+ and Mn2+ ions, the enzyme also contained a substitution at the active site (D107N). The protein at a final concentration of 2 mg/mL was mixed with the substrate at a 1:1.2 M ratio. The complexes were mixed with the reservoir solution at a 1:1 ratio and crystallized by the sitting drop vapor diffusion method at 18°C.The substrates that are cleaved by RNase H2 are RNA–DNA junctions, particularly single ribonucleotides that are incorporated into the genome. Therefore, as substrates for crystallization, we used 10–15 bp dsDNAs that contained a single ribonucleotide. These substrates were prepared by annealing the cleaved DNA strand that contained one ribonucleotide in the middle of the sequence to a complementary DNA strand of equal length. The high-performance liquid chromatography-purified oligonucleotides (Metabion) were ordered in a lyophilized form and suspended in ultrapure water with 0.5 mM EDTA. They were mixed at a 1:1 M ratio and annealed by 5 min incubation at 70°C followed by gradual cooling to room temperature. Complex crystals were obtained with a 12-bp substrate.Crystals of the complex that contained magnesium were obtained in a crystallization condition that consisted of 300 mM MgCl2, 23% PEG 3350, and 100 mM HEPES (pH 7.5); therefore, the crystallization solution provided the catalytic divalent metal ion. For cryoprotection, the crystals were transferred to a well solution that was supplemented with 35% PEG 3350 and flash-frozen in liquid nitrogen. The X-ray diffraction data for these crystals were collected at the Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung (BESSY) synchrotron at beamline MX-14.2 using a Mar225 CCD detector at 100K. Crystals that contained manganese were obtained by stepwise transfers of the magnesium-containing crystals to solutions with increasing MnCl2, NaCl, and PEG 3350 concentrations with a concomitant decrease in the MgCl2 concentration. The final solution contained 30 mM MnCl2, 200 mM NaCl, 35% PEG 3350, and 100 mM HEPES (pH 7.5). For data collection, the crystals were flash-frozen in liquid nitrogen. The dataset for these crystals was collected at beamline BW6 at the Deutsches Elektronen Synchrotron (DESY) using a Mar165 detector. For cocrystallization of the wild-type protein with the substrate, calcium was used as the divalent ion. Crystals were obtained with 300 mM CaCl2, 100 mM Tris–HCl (pH 8.5), and 20% PEG 3350. The cryoprotecting solution contained 35% PEG 3350, and the crystals were flash-frozen in liquid nitrogen. The X-ray diffraction data for these crystals were collected at beamline 14-4 at the European Synchrotron Radiation Facility (ESRF).All of the crystals listed earlier belonged to the C2 spacegroup and had very similar unit cell dimensions (for the magnesium-containing crystals: a = 105.06 Å, b = 48.57 Å, c = 78.39 Å; α = 90 degrees, β = 131.8 degrees, γ = 90 degrees). The datasets were processed and scaled using HKL2000 (Otwinowski Minor, 1997). In each of them, one complex was present in the asymmetric unit. The first complex structure was solved using the molecular replacement method in the Phaser program (McCoy, 2007) and with the structure of unliganded Tm-RNase H2 (PDB ID 2ETJ) as a search model. The model for nucleic acid was built manually in Coot (Emsley Cowtan, 2004) to complete the complex structure. This structure was next used as a search model for molecular replacement to solve the remaining structures. All of the structures were refined using phenix.refine (Adams et al., 2010) interspersed with manual building in Coot. The structures were deposited in the Protein Data Bank under the accession codes 3O3F (D107N variant with magnesium ions), 3O3G (wild-type protein with calcium ions), and 3O3H (D107N variant with manganese ions).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687917300903Actinomycetes from marine habitats and their enzymatic potentialSatya P. Singh, ... Kruti Dangar, in Marine Enzymes for Biocatalysis, 20138.7.8 The DNA polymerasesA thermostable DNA polymerase was derived from Thermotoga maritima, a hyperthermophilic eubacterium able to grow between 55 and 90 °C. Thermotoga isolated from geothermally heated sea floors in Italy and the Azores is a recently described genus with three species. These bacteria were originally isolated from geothermally heated marine sediments and hot springs. Slater et al., (2000) were awarded a US patent on the thermostable DNA polymerases derived from hyperthermophilic eubacteria Thermotoga neapolitana (Tne). The desired gene was cloned into E. coli strains. Tne polymerases were produced by the cells harboring the Tne expression vectors. The large scale culture was grown for 5 hours at 37 °C followed by induction by IPTG. However, the major disadvantage of the process is the use of an expensive inducer (IPTG).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9781907568800500089Thermophilic BacteriaK.M. Noll, in Brenner s Encyclopedia of Genetics (Second Edition), 2013Genome Sequences and the Evolutionary History of Thermophilic BacteriaThe first genome sequence of a thermophilic bacterium, Thermotoga maritima strain MSB8, was published in 1999. Since then, many more genome sequences have been elucidated and comparative sequence analyses have generally supported the hypothesis that the earliest ancestor of the bacteria was a hyperthermophile. Thermophiles are present in several bacterial lineages. Some of these clearly belong to sublineages ancestral to their mesophilic relatives, but the ancestral thermal phenotype of other lineages is less clear. Given the extensive contribution of horizontal gene transfer to the evolution of bacterial lineages, modern thermophiles are chimeras with genes derived from vertical inheritances mixed with those acquired from organisms with which they live.The genome sequence of T. maritima revealed that a substantial portion of its genes was inherited via horizontal or lateral gene transfer from the Archaea. Subsequent analyses of sequences derived from other members of the Thermotogales revealed that a significant fraction of Thermotogales genes have been inherited from anaerobic members of the Firmicutes, the low GC Gram-positive bacteria. Many of the genes shared with these other lineages encode catabolic and transport functions, often for use of sugars. This suggests that thermophiles share genes allowing organisms to occupy specialized niches while consuming common substrates. Perhaps the archaea, Thermotogales, and anaerobic Firmicutes thermophiles occupy different thermal niches while consuming a common pool of saccharides.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123749840015357Globins and Other Nitric Oxide-Reactive Proteins, Part BJoão B. Vicente, ... Carlos Frazão, in Methods in Enzymology, 20082 CRYSTALLIZATION OF FLAVODIIRON PROTEINSThe flavodiiron proteins for which X‐ray crystallographic structures have been solved were isolated as described in Vicente et al. (2007), except the FDP from Thermotoga maritima (Tm_FDP). Whereas D. gigas rubredoxin:oxygen oxidoreductase (Dg_ROO) was isolated from its source organism (Chen et al., 1993), the remainder of FDPs were overexpressed heterologously in E. coli (Seedorf et al., 2007; Silaghi‐Dumitrescu et al., 2005a). Tm_FDP was overexpressed in E. coli and purified by a high‐throughput automated method (DiDonato et al., 2004). Flavodiiron proteins were crystallized essentially by two methods: (i) the batch method, using Zinc acetate as precipitant, or (ii) the vapor diffusion method, using alcohol type precipitants (PEGs or MPD). When crystal cryostabilization was necessary for data collection, it was performed by incremental additions of the stabilizing agent.Dg_ROO crystals, obtained with PEG 1 to 6K as precipitating agents in a wide range of pH (5–9) (Frazão et al., 1999), appeared within 1 day at room temperature in large numbers, amidst a gelatinous precipitate. To prevent the formation of this gel and to control the number of crystals, crystallization trials were performed at 277K, and also adding the detergent SB12 (N‐dodecyl‐N,N‐dimethyl‐3‐ammonio‐1‐propansulfonate). Crystals obtained at both temperatures had similar parallelepiped shapes, although their diffraction quality changed significantly. Whereas the diffraction of crystals obtained at 277K exhibited twinning statistics, those obtained at room temperature developed as single crystals. Crystals that allowed solving the Dg_ROO structure were typically obtained as follows. A sitting drop composed by 3 μl of 10 mg/ml protein solution and an equal volume of precipitant solution [PEG 6K 10% (v/v), 100 mM Tris‐maleic acid, pH 6.0] was equilibrated against 500 μl of the same solution. Orange‐brown parallelepiped‐shaped crystals grew within approximately 3 weeks up to 0.2 to 0.4 mm in length and were cryostabilized by adding to 20‐μl drops of precipitant solution containing the crystals, small amounts of precipitant solution (initially ≈0.2 μl each) complemented with glycerol (25%, v/v), in the cold room, up to a final glycerol concentration of 25% (v/v). After stabilization, crystals were flash frozen in a nitrogen stream and diffraction data collected. Crystals belonged to space group P21212 (cell dimensions a = 98.2 Å, b = 101.2 Å, and c = 90.8 Å), diffracted to 2.5 Å resolution and contained two molecules (one dimer) in the asymmetric unit.The flavodiiron protein from Moorella thermoacetica (Mt_FDP) was crystallized (Silaghi‐Dumitrescu et al., 2005a) by the batch method in melting point capillaries (at room temperature) by mixing 10 μl of ≈1 mM (≈42 mg/ml) oxidized (as‐isolated) (Mt_FDP_ox) solution to an equal volume of precipitant solution. Two slightly different precipitant solutions yielded crystals, 200 mM zinc acetate, 5% 2‐propanol, 50 mM sodium cacodylate, pH 6.5, and 200 mM zinc acetate, 10% PEG 3000, 100 mM sodium acetate, pH 4.5. Relatively large crystals (0.2 × 0.2 × 0.5 mm) formed within 7 to 10 days, endowed with diffraction quality, were cryostabilized by soaking the crystals in mother liquor [1:1 (v/v) 25 mM MOPS (pH 7.3):precipitant] containing step increments of 5% ethylene glycol up to 20%, with each step taking ≈20 min. Crystals of reduced Mt_FDP (Mt_FDP_red) were obtained in an anaerobic glove box by the addition of sodium dithionite powder to as‐isolated Mt_FDP crystals [in 200 μl of mother liquor containing 20% (v/v) ethylene glycol and the pH 6.5 precipitant], resulting in crystals changing from orange‐brown to colorless within a few minutes. Crystals of Mt_FDP reoxidized with NO (Mt_FDP_NO) were obtained starting from the reduced crystals prepared as described earlier. These crystals were back‐soaked into the same mother liquor (though lacking sodium dithionite) and further incubated with the NO‐releasing compound DEA‐NONOate (in powder), leading to a fast color change back to the original orange‐brown, attesting the Mt_FDP reoxidation by NO. The crystals obtained in three states (oxidized, reduced, and reduced + NO reacted) were flash frozen in liquid nitrogen; all belonged to space group P43212. The cell dimensions and resolution of diffraction data were a = b = 159.6 Å, c = 276.7 Å, and 3.0 Å resolution for crystals of Mt_FDP_ox; a = b = 159.6 Å, c = 278.1 Å, and 2.8 Å resolution for Mt_FDP_red crystals; and a = b = 159.6 Å, c = 279.1 Å, and 2.8 Å resolution for Mt_FDP_NO crystals. Moreover, all the crystals were shown to contain four molecules (an assembly of two homodimers) in the asymmetric unit.Crystals of T. maritima FDP were obtained as part of a high‐throughput automated method for protein production, crystallization, and structure determination, focusing on the proteome of this organism (DiDonato et al., 2004). Because the structure of Tm_FDP was deposited in the PDB but not published, information on the crystallization procedure is still scarce (PDB entry 1VME). Crystals obtained at 277 K in sitting nanodrops, by the vapor diffusion method, using 35.0% MPD, 0.1 M acetate (pH 4.5), belonged to space group P21 with cell dimensions a = 55.24 Å, b = 95.83 Å, c = 90.13, and β = 95.43°, diffracted to 1.8 Å and contained two molecules (one dimer) in the asymmetric unit.Contrary to other FDPs for which the crystallographic structure was solved, the FprA from Methanothermobacter marburgensis (Mm_FprA) was purified and crystallized only in anaerobic conditions (in an anaerobic glove box) (Seedorf et al., 2007). Different crystallization conditions yielded three crystal forms, grown in drops up to 20 μl using 20 mg/ml Mm_FprA solution and precipitant solution, all three of them obtained by the hanging drop vapor diffusion method, at 283 K, and in the presence of 1 mM dithiothreitol. Crystals obtained with 0.2 M ammonium sulfate, 0.1 M MES/KOH, pH 6.5, and 16–22% PEG MME 5000 displayed two different monoclinic P21 crystal forms, one containing eight molecules in the asymmetric unit, with cell dimensions a = 97.8 Å, b = 123.1 Å, c = 135.9 Å, and β= 103.4°, diffracting to 2.25 Å resolution; and the second P21 form containing four molecules in the asymmetric unit, with cell dimensions a = 73.7 Å, b = 120.9 Å, c = 92.7 Å, and β= 110.4°, diffracting to 1.7 Å resolution. The third crystal form resulted from a lower concentration of PEG MME 5000 (8–16%), compensated with 15% glycerol, yielding tetragonal crystals in space group P43212, displaying four molecules in the asymmetric unit, with cell dimensions a = b = 88.7 Å, c = 450.4 Å, that diffracted to 2.25 Å resolution. Differences in data sets also resulted from the distinct ways the crystals were manipulated. The first crystal form (P21 with eight molecules in the a.u.) was measured after flash cooling the crystals inside the anaerobic chamber (grown in the presence of F420H2, the electron donor for Mm_FprA) in liquid nitrogen, corresponding to reduced Mm_FprA. The second and third crystal forms (P21 with four molecules in the a.u., and P43212) were flash frozen in a nitrogen gas stream after being exposed to air for minutes at 191 K, yielding presumably oxidized forms of Mm_Fpra.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687907370018Enzymes and Enzyme MechanismsOlga Khersonsky, Dan S. Tawfik, in Comprehensive Natural Products II, 20108.03.8.5 On- and Off-Pathway Evolutionary IntermediatesA notable case of large negative trade-offs was described for the directed evolution of HisA to yield TrpF activity (Table 3, entry 15; Figure 7). The gene was randomly mutated, and the Asp127Val mutant of Thermotoga maritima HisA isolated from the selection exhibited measurable TrpF activity (kcat/KM = 120 mol−1 l s−1) that was sufficient to complement the E. coli TrpF knockout strain used for the selection. The newly evolving TrpF activity led to a dramatic drop in the original HisA activity (∼104-fold). Perhaps this effect is not so surprising given that the starting point had no measurable TrpF activity, and that the mutation occurred in a key active-site residue: Asp127 is the putative acid catalyst in the Amadori rearrangement catalyzed by HisA.185Figure 7. The reactions catalyzed by HisA (isomerization of N′-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) and TrpF (isomerization of phosphoribosyl-anthranilate).Interestingly, few years later after this directed evolution experiment, a bi-functional enzyme dubbed PriA was discovered that performs both reactions with high efficiency, and within the very same active site 186, 187. In contrast to the laboratory selection for TrpF activity, in nature, a generalist enzyme evolved under selection to maintain both the HisA and TrpF functions. Thus, the ‘generalist’ intermediate originally proposed by Jurgens et al.152 exists in certain bacteria. However, it seems that the Asp127Val mutant isolated in the laboratory evolution experiment does not reflect the sequence and structural features of such a generalist intermediate.In a recent directed evolution experiment, a HisA Asp127Val mutant was further evolved for TrpF activity188. However, it led to a complete change in the reaction mechanism, and the evolved variants completely lost the original HisA activity. Thus, although this mutant provides a clear example of how TrpF activity could emerge in HisA, it seems to comprise an ‘off-pathway’ intermediate – namely, an intermediate that provides a temporary advantage but cannot lead to the eventual divergence of a proficient bifunctional TrpF-HisA enzyme. Off-pathway intermediates might be observed in nature (for example, see the E3 esterase example discussed in Section 8.03.8.6.3). They are as likely, if not more likely, to appear in laboratory evolution experiments, and even more so in rationally designed enzyme variants. It could may well be that some of the ‘generalist’ intermediates observed in laboratory evolution experiments are also ‘off-pathway’. Indeed, the ultimate proof for ‘on-pathway’ evolutionary intermediates lies in the ability to complete the divergence process and generate a new ‘specialist’ enzyme with native-like kinetic parameters. Completing the process, however, involves many rounds of mutation and selection, and numerous mutations. This is very rarely pursued, and when it is, the aim is the final enzyme product, and not the pathway leading to it 168,189,190. Nonetheless, these applicative engineering projects have the potential to provide interesting insights into the pathway, the role and order of individual mutations, and the nature of the intermediates.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780080453828001556Comparative Genomics in ProkaryotesT. RYAN GREGORY, ROB DESALLE, in The Evolution of the Genome, 2005IMPLICATIONS FOR PROKARYOTE EVOLUTION AND THE STUDY THEREOFLike many other issues in evolutionary biology, the question is not whether HGT occurs, but rather how frequently and with what implications. In some species, HGT has been suggested to be extensive, for example accounting for nearly ¼ of the Thermotoga maritima genome (Nelson et al., 1999). Using G + C content and codon bias information, Lawrence and Ochman (1998) estimated that about 18% of the E. coli genome had been derived from HGT events. According to their calculations, the E. coli genome has experienced at least 243 HGT events representing about 1600 kb of DNA (with ~550 kb still remaining after subsequent losses) since diverging from its common ancestor with Salmonella enterica about 100 million years ago—a mean transfer rate of 16 kb per million years. In a more general survey of G + C content distributions, codon and amino acid usage biases, and gene positions in 17 Bacteria and seven Archaea, Garcia-Vallvé et al. (2000) estimated that 1.5 to 14.5% represented a more typical contribution of HGT to prokaryote genomes. More recently, Koski et al. (2001) argued that codon bias and base pair composition are poor indicators of HGT, and provided additional evidence that horizontal transfer had indeed been very important in the evolution of the E. coli genome.Although the overall rate of HGT remains unclear, there are indications that not all genes are equally likely to be transferred horizontally. Kunin and Ouzounis (2003) estimated that only about 25–39% of protein families are involved in HGT. In particular, \"operational” genes (i.e., those involved in housekeeping) are much more likely to move via HGT than are \"informational” genes (i.e., those involved in transcription, translation, etc.) (Jain et al., 1999). One possible explanation for this is the \"complexity hypothesis,” which postulates that informational genes are less amenable to HGT because they are often part of complex networks of genes, whereas operational genes are not (Jain et al., 1999). However, this question, like many others in the study of HGT, still awaits a conclusive resolution (see also Doolittle, 1999; Lawrence, 1999; Eisen, 2000a).Most contentious of all is the question regarding the significance of HGT for understanding the evolutionary histories of prokaryote lineages. Some authors acknowledge the occurrence and relevance of HGT, but rank it well below other processes such as gene losses, de novo origins, and duplications as a factor in modern prokaryote genome evolution (e.g., Snel et al., 2002; Kunin and Ouzounis, 2003; Kurland et al., 2003). Intriguingly, it seems that genes transferred horizontally are more likely to be duplicated, which indicates that these processes are not entirely independent (Hooper and Berg, 2003). In any case, where losses and non-HGT gains are considered to predominate, the main phylogenetic signal in genome evolution is seen as vertical and Darwinian, with HGT representing only minor (albeit sometimes rather distracting) \"noise” (Kurland et al., 2003). Other authors point out that HGT appears to have contributed to the evolution of numerous important traits and to the large-scale diversification of lineages, making it a major force in prokaryotic genome evolution (e.g., Ochman et al., 2000; Boucher et al., 2003; Martin et al., 2004). Still others have gone so far as to claim that HGT has been sufficiently pervasive as to make the reconstruction of strictly branching phylogenetic trees a misguided procedure (Doolittle, 1999; Philippe and Douady, 2003), and perhaps that a \"circle of life” best represents the deepest evolutionary pattern (Rivera and Lake, 2004). Some have presented intermediate views on this latter question, suggesting that the construction of a global \"tree of life” is rendered difficult, but not impossible, by the occurrence of extensive HGT and gene loss (e.g., Wolf et al., 2002).Generally, there is more agreement regarding the contribution of HGT when considering the earliest stages of cellular evolution (Brown, 2003; Kurland et al., 2003). Woese (1998, 2000, 2002) has suggested that at the time of the universal ancestor of cellular life (i.e., before the divergence of the three domains), cells were simple, replication was inaccurate, and HGT was so rampant that this ancestor must be considered a diverse community of cells rather than a single type of organism. However, when cellular systems became more refined, the frequency of HGT declined and allowed the divergence of (mostly) separate evolutionary lineages. This transition from simple cells that primarily exchanged DNA horizontally to more sophisticated ones that transmit their genetic information vertically has been called the \"Darwinian threshold” (Woese, 2002). Such a scenario would not preclude the reconstruction of the subsequent evolutionary histories of the three domains, but it would have major implications for how the base of the tree of life must be envisioned (Rivera and Lake, 2004). On the other hand, some authors insist that this threshold has never been reached in prokaryotes, and favor the extreme view that there still exists but one giant, genetically interconnected bacterial \"species” (Margulis and Sagan, 2002).Whatever the ultimate contribution of HGT to prokaryote genome evolution proves to be, it has already clearly exerted a significant influence on the field of prokaryote genomics.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123014634500127Fructosyltransferases and Invertases: Useful Enzymes in the Food and Feed IndustriesLuis E. Trujillo Toledo, ... José M. Pais Chanfrau, in Enzymes in Food Biotechnology, 201926.5.2 Thermotoga maritima InvertaseAmong  50 different species of known prokaryotic hyperthermophiles (with optimal growth temperature of at least 80°C), only a few have been reported to belong to the bacteria domain (Counts et al., 2017). It is believed that these high temperature-resistant organisms retain ancestral characteristics in their biomolecules and metabolic pathways. T. marítima is by far the best source for thermostable invertase for industrial invert sugar syrup production (Martínez et al., 2014). T. maritima is a bacterium that does not form spores, in the form of bacillus, that are strictly anaerobic and heterotrophic. It was originally isolated from geothermal marine sediments and its optimal growth temperature is about 80°C. Phylogenetically, Thermotoga maritima seems to be one of the oldest lineages in Eubacteria. The T. maritima genome is a single circular chromosome of approximately 1.8 megabases in size, which codes for an estimated 1877 proteins. Because its genome has the highest percentage (24%) of genes similar to the archaea genes, T. maritima has become ideal for studying the organization of domain and the identification of new protein structures in Eubacteria and Archea, so that this microorganism has become a study model of hyperthermophilic bacteria. Thermotoga maritima invertase (BfrA) hydrolyzes sucrose, raffinose, inulin, and fructose polymers with a β-(1–2) terminal linkage to a glucose molecule, releasing fructose in each case. All substrates of BfrA have in common a fructosyl moiety bonded by β-(2–1) or β-(2–6) bond to the remaining parts of the saccharides. BfrA shows similar catalytic efficiency for the hydrolysis of sucrose and inulin with kcat/KM values around 4.1 × 104 M− 1 s− 1 and 3.1 × 104 M− 1 s− 1, respectively (at 75°C, pH 5.5) (Muñoz-Gutiérrez et al., 2009). BfrA has an optimum temperature of 90–95°C (in 10 min trials) and was extremely insensitive to thermoinactivation. For 5 h at temperatures up to 80°C and pH 7, the enzyme retained up to 85% of its initial activity. Thus, BfrA is the most thermostable β-fructosidase described to date (Liebl et al., 1998). A T. maritima invertase molecule is composed of two individual modules, a catalytic five-sheet β-propellant module (residues 1–295) bound by 10 residues to a β-sandwich C-terminal module (Menéndez et al., 2013).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780128132807000268Recommended publicationsInfo iconJournal of Theoretical BiologyJournalMolecular PlantJournalSystematic and Applied MicrobiologyJournalReference Module in Life SciencesReference work • 2016Browse books and journalsAbout ScienceDirectRemote accessShopping cartAdvertiseContact and supportTerms and conditionsPrivacy policyWe use cookies to help provide and enhance our service and tailor content and ads. 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