Kenneth M. Noll

On the chimeric nature, thermophilic origin, and phylogenetic placement of the Thermotogales

Since publication of the first Thermotogales genome, Thermotoga maritima strain MSB8, single- and multi-gene analyses have disagreed on the phylogenetic position of this order of Bacteria. Here we present the genome sequences of 4 additional members of the Thermotogales (Tt. petrophila, Tt. lettingae, Thermosipho melanesiensis, and Fervidobacterium nodosum) and a comprehensive comparative analysis including the original T. maritima genome. While ribosomal protein genes strongly place Thermotogales as a sister group to Aquificales, the majority of genes with sufficient phylogenetic signal show affinities to Archaea and Firmicutes, especially Clostridia. Indeed, on the basis of the majority of genes in their genomes (including genes that are also found in Aquificales), Thermotogales should be considered members of the Firmicutes. This result highlights the conflict between the taxonomic goal of assigning every species to a unique position in an inclusive Linnaean hierarchy and the evolutionary goal of understanding phylogenesis in the presence of pervasive horizontal gene transfer (HGT) within prokaryotes. Amino acid compositions of reconstructed ancestral sequences from 423 gene families suggest an origin of this gene pool even more thermophilic than extant members of this order, followed by adaptation to lower growth temperatures within the Thermotogales.

Kosmotoga olearia gen. nov., sp. nov., a thermophilic, anaerobic heterotroph isolated from an oil production fluid

A novel thermophilic, heterotrophic bacterium, strain TBF 19.5.1(T), was isolated from oil production fluid at the Troll B oil platform in the North Sea. Cells of strain TBF 19.5.1(T) were non-motile rods with a sheath-like structure, or toga. The strain was Gram-negative and grew at 20-80 degrees C (optimum 65 degrees C), pH 5.5-8.0 (optimum pH 6.8) and NaCl concentrations of 10-60 g l(-1) (optimum 25-30 g l(-1)). For a member of the order Thermotogales, the novel isolate is capable of unprecedented growth at low temperatures, with an optimal doubling time of 175 min (specific growth rate 0.24 h(-1)) and a final optical density of >1.4 when grown on pyruvate at 37 degrees C. Various carbohydrates, proteinaceous compounds and pyruvate served as growth substrates. Thiosulfate, but not elemental sulfur, enhanced growth of the isolate. Sulfate also enhanced growth, but sulfide was not produced. The strain grew in the presence of up to approximately 15 % oxygen, but only if cysteine was included in the medium. Growth of the isolate was inhibited by acetate, lactate and propionate, while butanol and malate prevented growth. The major fermentation products formed on maltose were hydrogen, carbon dioxide and acetic acid, with traces of ethanol and propionic acid. The G+C content of the genomic DNA was 42.5 mol%. Phylogenetic analyses of the 16S and 23S rRNA gene sequences as well as 29 protein-coding ORFs placed the strain within the bacterial order Thermotogales. Based on the phylogenetic analyses and the possession of a variety of physiological characteristics not previously found in any species of this order, it is proposed that the strain represents a novel species of a new genus within the family Thermotogaceae, order Thermotogales. The name Kosmotoga olearia gen. nov., sp. nov. is proposed. The type strain of Kosmotoga olearia is TBF 19.5.1(T) (=DSM 21960(T) =ATCC BAA-1733(T)).

Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded by Thermotoga maritima Bind Sugars

ABSTRACT The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the β-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), β-1,4-mannobiose; and TM1226 (ManD), β-1,4-mannobiose, β-1,4-mannotriose, β-1,4-mannotetraose, β-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), α-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.

Recent advances in genetic analyses of hyperthermophilic Archaea and Bacteria

Abstract Hyperthermophilic Archaea and Bacteria are an extraordinarily important class of organisms for which genetic tools remain to be developed. Unique technological obstacles to this goal are posed by the thermophilic and, in some cases, strictly anaerobic nature of these organisms. However, recent advances in the cultivation of hyperthermophiles, in the discovery of genetic elements for vector development, and in the construction of genetic markers point toward the achievement of this goal in the near future. Transformation protocols have already been reported for Sulfolobus and Pyrococcus, and plasmid-mediated conjugation was recently found in Sulfolobus. Plasmids are available for Sulfolobus, Pyrococcus, and the bacterial hyperthermophile Thermotoga, and these provide the bases for vector construction in these hosts. A Desulfurococcus mobile intron may provide a novel means to introduce genes into a variety of archaeal hosts. With full genome sequences of several hyperthermophiles available soon, genetic tools will allow full exploitation of this information to study these organisms in depth and to utilize their unique properties in biotechnological applications.

Whole-Genome Expression Profiling of Thermotoga maritima in Response to Growth on Sugars in a Chemostat

To provide data necessary to study catabolite-linked transcriptional networks in Thermotoga maritima, we used full-genome DNA microarray analysis of global transcriptional responses to growth on glucose, lactose, and maltose in a chemostat. A much larger number of genes changed expression in cells grown on lactose than on maltose, each relative to genes expressed in cells grown on glucose. Genes encoding putative oligopeptide transporters were often coregulated with adjacent glycosidase-encoding genes. Genes encoding enzymes catalyzing NADH oxidation were up-regulated on both lactose and maltose. Genes involved in iron and sulfur metabolism were differentially expressed in response to lactose. These data help define the sets of coregulated genes and suggest possible functions for their encoded products.

Substrate Specificities and Expression Patterns Reflect the Evolutionary Divergence of Maltose ABC Transporters in Thermotoga maritima

Duplication of transporter genes is apparent in the genome sequence of the hyperthermophilic bacterium Thermotoga maritima. The physiological impacts of these duplications are not well understood, so we used the bacteriums two putative maltose transporters to begin a study of the evolutionary relationship between a transporters function and the control of expression of its genes. We show that the substrate binding proteins encoded by these operons, MalE1 and MalE2, have different substrate specificities and affinities and that they are expressed under different growth conditions. MalE1 binds maltose (dissociation constant [KD], 24 +/- 1 microM), maltotriose (KD, 8 +/- 0.5 nM), and beta-(1-->4)-mannotetraose (KD, 38 +/- 1 microM). In contrast, MalE2 binds maltose (KD, 8.4 +/- 1 microM), maltotriose (KD, 11.5 +/- 1.5 microM), and trehalose (KD, 9.5 +/- 1.0 microM) confirming the findings of Wassenberg et al. (J. Mol. Biol. 295:279-288, 2000). Neither protein binds lactose. We examined the expression of these operons at both the transcriptional and translational levels and found that MalE1 is expressed in cells grown on lactose or guar gum and that MalE2 is highly expressed in starch- and trehalose-grown cells. Evidence is provided that malE1, malF1, and perhaps malG1 are cotranscribed and so constitute an operon. An open reading frame encoding a putative transcriptional regulatory protein adjacent to this operon (TM1200) is also up-regulated in response to growth on lactose. These evolutionarily related transporter operons have diverged both in function and expression to assume apparently different physiological roles.

Periplasmic maltose- and glucose-binding protein activities in cell-free extracts of Thermotoga maritima

In this study, high-affinity maltose- and glucose-binding activities in cell-free extracts of Thermotoga maritima were detected; these activities were distinct and specific. At the gross level, the expression of binding-protein activities was repressed by growth of T. maritima in the presence of the cognate sugar. Growth of the organism in the presence of maltose reduced maltose-binding activity but not glucose-binding activity, while growth in the presence of glucose reduced glucose-binding activity but not maltose-binding activity. In competition assays, these binding activities showed distinct patterns of substrate specificity: whereas the maltose-binding activity showed specificity for alpha-linked glucosides, the glucose-binding activity showed a broader specificity. All maltose- and glucose-binding activity was found in the supernatant retrieved following centrifugation (100,000 g) of the cell-free extracts prepared by French-pressure-cell treatment; no activity was found in an octyl-glucoside-treated extract of the membrane fraction. The maltose-binding-protein activity was recovered from the periplasmic fraction by selective release of the periplasmic contents of T. maritima cells using a newly developed freeze-thaw procedure. Annotation of the complete genome sequence of T. maritima suggests that there may be at least two maltose-binding proteins, MalE1 and MalE2, encoded in the genome. The maltose-binding activity corresponded to a protein of 43 kDa, which was consistent in size with either of the putative proteins. These data demonstrate that the hyperthermophilic bacterium T. maritima possesses separate maltose- and glucose-binding-protein activities that are freely soluble in its periplasm, in contrast to the membrane-bound sugar-binding proteins found in archaeal hyperthermophiles.

Catabolite repression in the hyperthermophilic bacterium Thermotoga neapolitana is independent of cAMP

Thermotoga neapolitana is a hyperthermophilic bacterium whose phylogenetic lineage includes the most primitive of the bacterial heterotrophs. It is not known whether Thermotoga exhibits preferences for growth substrates or regulates the synthesis of degradative enzymes. We have found that T. neapolitana exhibits diauxic growth in medium containing 300 microM glucose and 1 mM lactose. We measured the activity of beta-galactosidase and beta-glucosidase in extracts prepared from cells grown on defined media and found that cells grown on 0.5% lactose, galactose or cellobiose contained beta-galactosidase specific activities of 1.19, 1.78 and 1.34 U (mg protein)-1, respectively. Cells grown on 0.5% glucose, maltose, fructose, sucrose, xylose, ribose or starch had no measurable beta-galactosidase activity. beta-Glucosidase activity was found only in cells grown on cellobiose. Cells grown on the combination of 0.5% lactose or galactose and 0.05% glucose had no detectable beta-galactosidase activity, whereas up to 0.5% glucose did not prevent expression of beta-galactosidase or beta-glucosidase activity in cells induced with 0.5% cellobiose. These activities are catalysed by separate enzymes as determined by resolution of their activities on 6% native polyacrylamide gels. Therefore, only beta-galactosidase synthesis induced by lactose is subject to catabolite repression. To determine the mechanism of catabolite repression, the levels of cAMP were measured in T. neapolitana cells grown on various defined media using an enzyme-immunoassay. The cAMP levels ranged from 44 to 280 fmol (mg protein)-1 irrespective of the carbon source used. By comparison, Escherichia coli grown on lactose contained 5.1 pmol (mg protein)-1. Like Gram-positive bacteria, T. neapolitana displays a cAMP-independent mechanism for catabolite repression and this may represent the more ancient mode of regulation.

Reconstructed ancestral Myo-inositol-3-phosphate synthases indicate that ancestors of the Thermococcales and Thermotoga species were more thermophilic than their descendants.

The bacterial genomes of Thermotoga species show evidence of significant interdomain horizontal gene transfer from the Archaea. Members of this genus acquired many genes from the Thermococcales, which grow at higher temperatures than Thermotoga species. In order to study the functional history of an interdomain horizontally acquired gene we used ancestral sequence reconstruction to examine the thermal characteristics of reconstructed ancestral proteins of the Thermotoga lineage and its archaeal donors. Several ancestral sequence reconstruction methods were used to determine the possible sequences of the ancestral Thermotoga and Archaea myo-inositol-3-phosphate synthase (MIPS). These sequences were predicted to be more thermostable than the extant proteins using an established sequence composition method. We verified these computational predictions by measuring the activities and thermostabilities of purified proteins from the Thermotoga and the Thermococcales species, and eight ancestral reconstructed proteins. We found that the ancestral proteins from both the archaeal donor and the Thermotoga most recent common ancestor recipient were more thermostable than their descendants. We show that there is a correlation between the thermostability of MIPS protein and the optimal growth temperature (OGT) of its host, which suggests that the OGT of the ancestors of these species of Archaea and the Thermotoga grew at higher OGTs than their descendants.

Genome Sequence of Kosmotoga olearia Strain TBF 19.5.1, a Thermophilic Bacterium with a Wide Growth Temperature Range, Isolated from the Troll B Oil Platform in the North Sea

Kosmotoga olearia strain TBF 19.5.1 is a member of the Thermotogales that grows best at 65°C and very well even at 37°C. Information about this organism is important for understanding the evolution of mesophiles from thermophiles. Its genome sequence reveals extensive gene gains and a large content of mobile genetic elements. It also contains putative hydrogenase genes that have no homologs in the other member of the Thermotogales.