Structural Characterization of an ACP from Thermotoga maritima: Insights into Hyperthermal Adaptation.

Thermotoga maritima, a deep-branching hyperthermophilic bacterium, expresses an extraordinarily stable Thermotoga maritima acyl carrier protein (Tm-ACP) that functions as a carrier in the fatty acid synthesis system at near-boiling aqueous environments. Here, to understand the hyperthermal adaptation of Tm-ACP, we investigated the structure and dynamics of Tm-ACP by nuclear magnetic resonance (NMR) spectroscopy. The melting temperature of Tm-ACP (101.4 °C) far exceeds that of other ACPs, owing to extensive ionic interactions and tight hydrophobic packing. The D59 residue, which replaces Pro/Ser of other ACPs, mediates ionic clustering between helices III and IV. This creates a wide pocket entrance to facilitate the accommodation of long acyl chains required for hyperthermal adaptation of the T. maritima cell membrane. Tm-ACP is revealed to be the first ACP that harbor an amide proton hyperprotected against hydrogen/deuterium exchange for I15. The hydrophobic interactions mediated by I15 appear to be the key driving forces of the global folding process of Tm-ACP. Our findings provide insights into the structural basis of the hyperthermal adaptation of ACP, which might have allowed T. maritima to survive in hot ancient oceans.

Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C-ring assembly in T3SS.

Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic-ring (C-ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non-flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF-T3SS C-ring component in Shigella flexneri is alternatively translated to produce both full-length (Spa33-FL) and a short variant (Spa33-C), with both required for secretion. They associate in a 1:2 complex (Spa33-FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33-C2 and identification of an unexpected intramolecular pseudodimer in Spa33-FL reveal a molecular model for their higher order assembly within NF-T3SS. Spa33-FL and Spa33-C are identified as functional counterparts of a FliM-FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritima FliM and FliN form a 1:3 complex structurally equivalent to Spa33-FL/C2 , allowing us to propose a unified model for C-ring assembly by NF-T3SS and flagellar-T3SS.

Adaptive Evolution of Thermotoga maritima Reveals Plasticity of the ABC Transporter Network.

Thermotoga maritima is a hyperthermophilic anaerobe that utilizes a vast network of ABC transporters to efficiently metabolize a variety of carbon sources to produce hydrogen. For unknown reasons, this organism does not metabolize glucose as readily as it does glucose di- and polysaccharides. The leading hypothesis implicates the thermolability of glucose at the physiological temperatures at which T. maritima lives. After a 25-day laboratory evolution, phenotypes were observed with growth rates up to 1.4 times higher than and glucose utilization rates exceeding 50% those of the wild type. Genome resequencing revealed mutations in evolved cultures related to glucose-responsive ABC transporters. The native glucose ABC transporter, GluEFK, has more abundant transcripts either as a result of gene duplication-amplification or through mutations to the operator sequence regulating this operon. Conversely, BglEFGKL, a transporter of beta-glucosides, is substantially downregulated due to a nonsense mutation to the solute binding protein or due to a deletion of the upstream promoter. Analysis of the ABC2 uptake porter families for carbohydrate and peptide transport revealed that the solute binding protein, often among the transcripts detected at the highest levels, is predominantly downregulated in the evolved cultures, while the membrane-spanning domain and nucleotide binding components are less varied. Similar trends were observed in evolved strains grown on glycerol, a substrate that is not dependent on ABC transporters. Therefore, improved growth on glucose is achieved through mutations favoring GluEFK expression over BglEFGKL, and in lieu of carbon catabolite repression, the ABC transporter network is modulated to achieve improved growth fitness.

Structure and mechanism of an intramembrane liponucleotide synthetase central for phospholipid biosynthesis.

Phospholipids are elemental building-block molecules for biological membranes. Biosynthesis of phosphatidylinositol, phosphatidylglycerol and phosphatidylserine requires a central liponucleotide intermediate named cytidine-diphosphate diacylglycerol (CDP-DAG). The CDP-DAG synthetase (Cds) is an integral membrane enzyme catalysing the formation of CDP-DAG, an essential step for phosphoinositide recycling during signal transduction. Here we report the structure of the Cds from Thermotoga maritima (TmCdsA) at 3.4 Å resolution. TmCdsA forms a homodimer and each monomer contains nine transmembrane helices arranged into a novel fold with three domains. An unusual funnel-shaped cavity penetrates half way into the membrane, allowing the enzyme to simultaneously accept hydrophilic substrate (cytidine 5'-triphosphate (CTP)/deoxy-CTP) from cytoplasm and hydrophobic substrate (phosphatidic acid) from membrane. Located at the bottom of the cavity, a Mg(2+)-K(+) hetero-di-metal centre coordinated by an Asp-Asp dyad serves as the cofactor of TmCdsA. The results suggest a two-metal-ion catalytic mechanism for the Cds-mediated synthesis of CDP-DAG at the membrane-cytoplasm interface.

The receptor-CheW binding interface in bacterial chemotaxis.

The basic structural unit of the signaling complex in bacterial chemotaxis consists of the chemotaxis kinase CheA, the coupling protein CheW, and chemoreceptors. These complexes play an important role in regulating the kinase activity of CheA and in turn controlling the rotational bias of the flagellar motor. Although individual three-dimensional structures of CheA, CheW, and chemoreceptors have been determined, the interaction between chemoreceptor and CheW is still unclear. We used nuclear magnetic resonance to characterize the interaction modes of chemoreceptor and CheW from Thermotoga maritima. We find that chemoreceptor binding surface is located near the highly conserved tip region of the N-terminal helix of the receptor, whereas the binding interface of CheW is placed between the ß-strand 8 of domain 1 and the ß-strands 1 and 3 of domain 2. The receptor-CheW complex shares a similar binding interface to that found in the "trimer-of-dimers" oligomer interface seen in the crystal structure of cytoplasmic domains of chemoreceptors from Escherichia coli. Based on the association constants inferred from fast exchange chemical shifts associated with receptor-CheW titrations, we estimate that CheW binds about four times tighter to its first binding site of the receptor dimer than to its second binding site. This apparent anticooperativity in binding may reflect the close proximity of the two CheW binding surfaces near the receptor tip or further, complicating the events at this highly conserved region of the receptor. This work describes the first direct observation of the interaction between chemoreceptor and CheW.

The genus Thermotoga: recent developments.

The genus Thermotoga comprises extremely thermophilic (Topt > or = 70 degrees C) and hyperthermophilic (Topt > or = 80 degrees C) bacteria, which have been extensively studied for insights into the basis for life at elevated temperatures and for biotechnological opportunities (e.g. biohydrogen production, biocatalysis). Over the past decade, genome sequences have become available for a number of Thermotoga species, leading to functional genomics efforts to understand growth physiology as well as genomics-based identification and characterization of novel high-temperature biocatalysts. Discussed here are recent developments along these lines for this group of microorganisms.

TM0486 from the hyperthermophilic anaerobe Thermotoga maritima is a thiamin-binding protein involved in response of the cell to oxidative conditions.

The COG database was used for a comparative genome analysis with genomes from anaerobic and aerobic microorganisms with the aim of identifying proteins specific to the anaerobic way of life. A total of 33 COGs were identified, five of which correspond to proteins of unknown function. We focused our study on TM0486 from Thermotoga maritima, which belongs to one of these COGs of unknown function, namely COG0011. The crystal structure of the protein was determined at 2 A resolution. The structure adopts a beta alpha beta beta alpha beta ferredoxin-like fold and assembles as a homotetramer. The structure also revealed the presence of a pocket in each monomer that bound an unidentified ligand. NMR and calorimetry revealed that TM0486 specifically bound thiamin with a K(d) of 1.58 microM, but not hydroxymethyl pyrimidine (HMP), which has been implicated as a potential ligand. We demonstrated that the TM0486 gene belongs to the same multicistronic unit as TM0483, TM0484 and TM0485. Although these three genes have been assigned to the transport of HMP, with TM0484 being the periplasmic thiamin/HMP-binding protein and TM0485 and TM0483 the transmembrane and the ATPase components, respectively, our results led us to conclude that this operon encodes an ABC transporter dedicated to thiamin, with TM0486 transporting charged thiamin in the cytoplasm. Given that this transcriptional unit was up-regulated when T. maritima was exposed to oxidative conditions, we propose that, by chelating cytoplasmic thiamin, TM0486 and, by extension, proteins belonging to COG0011 are involved in the response mechanism to stress that could arise during aerobic conditions.

The hyperthermophilic anaerobe Thermotoga Maritima is able to cope with limited amount of oxygen: insights into its defence strategies.

Thermotoga maritima, an anaerobic hyperthermophilic bacterium, was found able to grow in the presence of low concentrations of oxygen of up to 0.5% (v/v). Differential proteomics and transcripts analysis by qRT-PCR were used to identify the defence strategies used by T. maritima to protect itself against oxygen. A flavoprotein, homologous to rubredoxin oxygen oxidoreductase was found to be overproduced when cells were cultured in oxidative conditions. The recombinant protein, produced in Escherichia coli, exhibited an oxygen reductase activity, which could account for the observed decrease in oxygen concentration during growth. The gene encoding this oxygen reductase belongs to a multicistronic unit that includes genes encoding proteins involved in exopolysaccharide biosynthesis, which may be related to a biofilm formation induced by the presence of oxygen. Enzymes involved in reactive oxygen species detoxification, iron-sulfur centre synthesis/repair and the cysteine biosynthesis pathway were also overproduced. All these enzymatic systems together contribute to the defence strategy of T. maritima against oxygen. Because of the position of T. maritima in deep branches of the phylogenetic tree, we suggest that these strategies can be considered as ancestral mechanisms first developed by anaerobic microorganisms on the early Earth to protect themselves against primary abiotic or biotic oxygen production.

Rates of spontaneous disintegration of DNA and the rate enhancements produced by DNA glycosylases and deaminases.

To estimate the relative importance of alternate routes of spontaneous degradation of DNA and the rate enhancements produced by enzymes catalyzing these reactions, rate constants and thermodynamic activation parameters for the degradation of 2'-deoxynucleosides at 25 degrees C were determined by extrapolation of rates observed in the temperature range between 90 and 200 degrees C in neutral phosphate buffer. Rates of deamination of 2'-deoxycytidine, 1-methylcytosine, and cytidine were found to be identical within experimental error (t1/2 approximately 20 years, 37 degrees C). Rate constants for deamination of 2'-deoxyadenosine and 2'-deoxyguanosine, which could not be determined directly because of rapid glycoside cleavage, were estimated by assuming that methyl replacement should generate reasonable model substrates. The rates of deamination of 9-methyladenine and 9-methylguanine were found to be similar to each other (t1/2 approximately 6000 years, 37 degrees C) and approximately 10(2)-fold slower than the rates of glycoside cleavage in 2'-deoxyadenosine and 2'-deoxyguanosine. The deamination of 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine led to accelerated rates of glycoside cleavage. In the exceptional case of 2'-deoxycytidine, deamination and glycoside hydrolysis proceed at very similar rates at all temperatures. Glycoside cleavage proceeds with half-times ranging from 4 years for 2'-deoxyinosine to 40 years for 2'-deoxycytidine (37 degrees C). The rate enhancements produced by DNA glycosylases, estimated by comparison with the rates of these uncatalyzed reactions, are found to be substantially smaller than those produced by deaminases and staphylococcal nuclease.

Microbial biochemistry, physiology, and biotechnology of hyperthermophilic Thermotoga species.

High-throughput sequencing of microbial genomes has allowed the application of functional genomics methods to species lacking well-developed genetic systems. For the model hyperthermophile Thermotoga maritima, microarrays have been used in comparative genomic hybridization studies to investigate diversity among Thermotoga species. Transcriptional data have assisted in prediction of pathways for carbohydrate utilization, iron-sulfur cluster synthesis and repair, expolysaccharide formation, and quorum sensing. Structural genomics efforts aimed at the T. maritima proteome have yielded hundreds of high-resolution datasets and predicted functions for uncharacterized proteins. The information gained from genomics studies will be particularly useful for developing new biotechnology applications for T. maritima enzymes.

Insights into the LexA regulon of Thermotogales.

The lexA genes of Thermotoga maritima and Petrotoga miotherma, both members of the Order Thermotogales, have been cloned and their transcriptional organization, as well as the functional characteristics of their encoded products, analyzed. In both bacterial species, the lexA gene was found to be co-transcribed together with another four (T. maritima) or three (P. miotherma) upstream open-reading frames. The P. miotherma LexA was able to bind promoters of both the cognate lexA encoding operon and the uvrA gene but not to that of the recA. Conversely, LexA protein and crude cell extracts from T. maritima were unable to bind promoters governing the expression of either its lexA or recA genes. In agreement with these observations, no functional copy of the P. miotherma LexA box, corresponding to the GANTN(6)GANNAC motif, seems to be present in the T. maritima genome. Giving support to the proposal that the evolutionary branching order of the Order Thermotogales is very close to that of Gram-positive bacteria, the P. miotherma LexA protein was still able to recognize the previously described LexA-binding sequence for Gram-positive bacteria.

Identification of methylation sites in Thermotoga maritima chemotaxis receptors.

Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in Salmonella enterica and Escherichia coli chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from Thermotoga maritima, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different T. maritima chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in T. maritima that is distinct from the previously identified consensus sequence for E. coli and S. enterica. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.

GTPase activity, structure, and mechanical properties of filaments assembled from bacterial cytoskeleton protein MreB.

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of Thermotoga maritima MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers, T. maritima MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of T. maritima MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.

Recombination in Thermotoga: implications for species concepts and biogeography.

Here we characterize regions of the genomes of eight members of the hyperthermophilic genus Thermotoga. These bacteria differ from each other physiologically and by 3-20% in gene content and occupy physically distinct environments in widely disparate regions of the globe. Among the four different lineages (represented by nine different strains) that we compare, no two are closer than 96% in the average sequences of their genes. By most accepted recent definitions these are different "ecotypes" and different "species." And yet we find compelling evidence for recombination between them. We suggest that no single prokaryotic species concept can accommodate such uncoupling of ecotypic and genetic aspects of cohesion and diversity, and that without a single concept, the question of whether or not prokaryotic species might in general be cosmopolitan cannot be sensibly addressed. We can, however, recast biogeographical questions in terms of the distribution of genes and their alleles.

Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA.

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phosphotransfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. 15N-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored N(delta1)H tautomer of His-45, thereby rendering the N(epsilon2) imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.

Permeability and reactivity of Thermotoga maritima in latex bimodal blend coatings at 80 degrees C: a model high temperature biocatalytic coating.

Thermostable polymers cast as thin, porous coatings or membranes may be useful for concentrating and stabilizing hyperthermophilic microorganisms as biocatalysts. Hydrogel matrices can be unstable above 65 degrees C. Therefore a 55-microm thick, two layer (cell coat + polymer top coat) bimodal, adhesive latex coating of partially coalesced polystyrene particles was investigated at 80 degrees C using Thermotoga maritima as a model hyperthermophile. Coating permeability (pore structure) was critical for maintaining T. maritima viability. The permeability of bimodal coatings generated from 0.8 v/v of a suspension of non-film-forming 800 nm polystyrene particles with high glass transition temperature (T(g) = 94 degrees C, 26.9% total solids) blended with 0.2 v/v of a suspension of film-forming 158 nm polyacrylate/styrene particles (T(g) approximately -5 degrees C, 40.9% total solids) with 0.3 g sucrose/g latex was measured in a KNO3 diffusion cell. Diffusivity ratio remained above 0.04 (D(eff)/D) when incubated at 80 degrees C in artificial seawater (ASW) for 5 days. KNO3 permeability was corroborated by cryogenic-SEM images of the pore structure. In contrast, the permeability of a mono-dispersed acrylate/vinyl acetate latex Rovace SF091 (T(g) approximately 10 degrees C) rapidly decreased and became impermeable after 2 days incubation in ASW at 80 degrees C. Thermotoga maritima were entrapped in these coatings at a cell density of 49 g cell wet weight/liter of coating volume, 25-fold higher than the density in liquid culture. Viable T. maritima were released from single-layer coatings at 80 degrees C but accurate measurement of the percentage of viable entrapped cells by plate counting was not successful. Metabolic activity could be measured in bilayer coatings by utilization of glucose and maltose, which was identical for latex-entrapped and suspended cells. Starch was hydrolyzed for 200 h by latex-entrapped cells due to the slow diffusion of starch through the polymer top coat compared to only 24 h by suspended T. maritima. The observed reactivity and stability of these coatings was surprising since cryo-SEM images suggested that the smaller low T(g) polyacrylate/styrene particles preferentially bound to the T. maritima toga-sheath during coat formation. This model system may be useful for concentrating, entrapment and stabilization of metabolically active hyperthermophiles at 80 degrees C.

Transcriptional analysis of biofilm formation processes in the anaerobic, hyperthermophilic bacterium Thermotoga maritima.

Thermotoga maritima, a fermentative, anaerobic, hyperthermophilic bacterium, was found to attach to bioreactor glass walls, nylon mesh, and polycarbonate filters during chemostat cultivation on maltose-based media at 80 degrees C. A whole-genome cDNA microarray was used to examine differential expression patterns between biofilm and planktonic populations. Mixed-model statistical analysis revealed differential expression (twofold or more) of 114 open reading frames in sessile cells (6% of the genome), over a third of which were initially annotated as hypothetical proteins in the T. maritima genome. Among the previously annotated genes in the T. maritima genome, which showed expression changes during biofilm growth, were several that corresponded to biofilm formation genes identified in mesophilic bacteria (i.e., Pseudomonas species, Escherichia coli, and Staphylococcus epidermidis). Most notably, T. maritima biofilm-bound cells exhibited increased transcription of genes involved in iron and sulfur transport, as well as in biosynthesis of cysteine, thiamine, NAD, and isoprenoid side chains of quinones. These findings were all consistent with the up-regulation of iron-sulfur cluster assembly and repair functions in biofilm cells. Significant up-regulation of several beta-specific glycosidases was also noted in biofilm cells, despite the fact that maltose was the primary carbon source fed to the chemostat. The reasons for increased beta-glycosidase levels are unclear but are likely related to the processing of biofilm-based polysaccharides. In addition to revealing insights into the phenotype of sessile T. maritima communities, the methodology developed here can be extended to study other anaerobic biofilm formation processes as well as to examine aspects of microbial ecology in hydrothermal environments.

Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima.

The 1,860,725-base-pair genome of Thermotoga maritima MSB8 contains 1,877 predicted coding regions, 1,014 (54%) of which have functional assignments and 863 (46%) of which are of unknown function. Genome analysis reveals numerous pathways involved in degradation of sugars and plant polysaccharides, and 108 genes that have orthologues only in the genomes of other thermophilic Eubacteria and Archaea. Of the Eubacteria sequenced to date, T. maritima has the highest percentage (24%) of genes that are most similar to archaeal genes. Eighty-one archaeal-like genes are clustered in 15 regions of the T. maritima genome that range in size from 4 to 20 kilobases. Conservation of gene order between T. maritima and Archaea in many of the clustered regions suggests that lateral gene transfer may have occurred between thermophilic Eubacteria and Archaea.

Stabilization of the phospho-aspartyl residue in a two-component signal transduction system in Thermotoga maritima.

The central signaling pathway in many bacterial regulatory systems involves phosphotransfer between two conserved proteins, a histidine protein kinase, and a response regulator. The occurrence of two-component signaling systems in thermophilic bacteria raises questions of how both the proteins and the labile acyl phosphate of the response regulator are adapted to function at elevated temperatures. Thermotoga maritima HpkA is a transmembrane histidine kinase, and DrrA is its cognate response regulator. Both DrrA and the cytoplasmic region of HpkA (HpkA57) have been expressed in Escherichia coli, purified, and characterized. HpkA57 and DrrA have apparent Tm's of 75 and 90 degreesC, respectively. HpkA57 exhibits ATP-dependent autophosphorylation activity similar to that of histidine kinases from mesophiles, with maximum activity at 70 degreesC. DrrA catalyzes transfer of phosphoryl groups from HpkA57 and exhibits Mg2+-dependent autophosphatase activity, with maximum activity at approximately 80 degreesC. At this temperature, the half-life for phospho-DrrA is approximately 3 min. In the absence of Mg2+, the half-life is 26 min, significantly greater than the half-life of a typical acyl phosphate at 80 degreesC. In the absence of Mg2+, at all temperatures examined, phospho-DrrA exhibits much greater stability than acetyl phosphate. This suggests that the active site of this hyperthermophilic response regulator is designed to protect the phospho-aspartyl residue from hydrolysis.