José Berenguer

Thermus thermophilus as biological model.

Thermus spp is one of the most wide spread genuses of thermophilic bacteria, with isolates found in natural as well as in man-made thermal environments. The high growth rates, cell yields of the cultures, and the constitutive expression of an impressively efficient natural competence apparatus, amongst other properties, make some strains of the genus excellent laboratory models to study the molecular basis of thermophilia. These properties, together with the fact that enzymes and protein complexes from extremophiles are easier to crystallize have led to the development of an ongoing structural biology program dedicated to T. thermophilus HB8, making this organism probably the best so far known from a protein structure point view. Furthermore, the availability of plasmids and up to four thermostable antibiotic selection markers allows its use in physiological studies as a model for ancient bacteria. Regarding biotechnological applications this genus continues to be a source of thermophilic enzymes of great biotechnological interest and, more recently, a tool for the over-expression of thermophilic enzymes or for the selection of thermostable mutants from mesophilic proteins by directed evolution. In this article, we review the properties of this organism as biological model and its biotechnological applications.

Coating of Soluble and Immobilized Enzymes with Ionic Polymers: Full Stabilization of the Quaternary Structure of Multimeric Enzymes

This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).

A thermophilic nitrate reductase is responsible for the strain specific anaerobic growth of Thermus thermophilus HB8.

T. thermophilus HB8 contains a nitrate reductase gene cluster which is absent from closely related strains. This cluster encodes 4 ORFs (a-d) similar in organization and protein sequence to those encoded by respiratory nitrate reductase operons (narGHJI) of Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens, and Thiosphaera pantothropha. The highest similarity is shown between the proteins encoded by the ORFa, ORFb and ORFd, and the structural components of the mesophilic nitrate reductases NarG (alpha), NarH (beta), and NarI (gamma) proteins, whilst ORFc encodes a protein which showed lower similarity to NarJ, a protein of unknown function encoded between narH and narI genes in all the nar cluster so far sequenced. This T. thermophilus HB8 narGHJI cluster is strongly induced by the combined effect of nitrate and low oxygen concentration, giving rise to the synthesis of an enzyme whose optimal temperature and pH was determined to be 80 degrees C, and pH 10, respectively. We also demonstrate that insertional inactivation of the narG and narH genes of this cluster results in strictly aerobic mutants, showing its sole responsibility in the strain specific ability of T. thermophilus HB8 to grow anaerobically.

Binding to pyruvylated compounds as an ancestral mechanism to anchor the outer envelope in primitive bacteria

Electron microscopy of isolated cell walls of the ancient bacterium Thermus thermophilus revealed that most of the peptidoglycan (PG) surface, apart from the septal region, was shielded against specific αPG antibodies. On the other hand, an antiserum raised against S‐layer‐attached cell wall fragments (αSAC) bound to most of the surface except for the septal regions. Treatments with α‐amylase and pronase E made the entire cell wall surface uniformly accessible to αPG and severely decreased the binding of αSAC. We concluded that a layer of strongly bound secondary cell wall polymers (SCWPs) covers most of the cell wall surface in this ancient bacterium. A preliminary analysis revealed that such SCWPs constitute 14% of the cell wall and are essentially composed of sugars. Enzyme treatments of the cell walls revealed that SCWP was required in vitro for the binding of the S‐layer protein through the S‐layer homology (SLH) motif. The csaB gene was necessary for the attachment of the S‐layer–outer membrane (OM) complex to the cell wall in growing cells of T. thermophilus. In vitro experiments confirmed that cell walls from a csaB mutant bound to the S‐layer with a much lower affinity (∼1/10) than that of the wild type. CsaB was found to be required for pyruvylation of components of the SCWP and for immunodetection with α‐SAC antiserum. Therefore, the S‐layer–OM complex of T. thermophilus binds to the cell wall through the SLH motif of the S‐layer protein via a strong interaction with a highly immunogenic pyruvylated component of the SCWP. Immuno‐cross‐reactive compounds were detected with αSAC on cell walls of other Thermus spp. and in the phylogenetically related microorganism Deinococcus radiodurans. These results imply that the interaction between the SLH motif and pyruvylated components of the cell wall arose early during bacterial evolution as an ancestral mechanism for anchoring proteins and outer membranes to the cell walls of primitive bacteria.

pH-dependent conformational switch activates the inhibitor of transcription elongation

Gfh1, a transcription factor from Thermus thermophilus, inhibits all catalytic activities of RNA polymerase (RNAP). We characterized the Gfh1 structure, function and possible mechanism of action and regulation. Gfh1 inhibits RNAP by competing with NTPs for coordinating the active site Mg2+ ion. This coordination requires at least two aspartates at the tip of the Gfh1 N‐terminal coiled‐coil domain (NTD). The overall structure of Gfh1 is similar to that of the Escherichia coli transcript cleavage factor GreA, except for the flipped orientation of the C‐terminal domain (CTD). We show that depending on pH, Gfh1‐CTD exists in two alternative orientations. At pH above 7, it assumes an inactive ‘flipped’ orientation seen in the structure, which prevents Gfh1 from binding to RNAP. At lower pH, Gfh1‐CTD switches to an active ‘Gre‐like’ orientation, which enables Gfh1 to bind to and inhibit RNAP.

Export of Thermus thermophilus alkaline phosphatase via the twin-arginine translocation pathway in Escherichia coli

The bacterial twin‐arginine translocation (Tat) pathway is distinct from the Sec system by its remarkable capacity to export folded enzymes. To address the question whether the two systems are capable of translocating homologous enzymes catalyzing the same reaction, we cloned the tap gene encoding Thermus thermophilus alkaline phosphatase (Tap) and expressed it in Escherichia coli. Unlike the alkaline phosphatase of E. coli, which is translocated through the Sec system and then activated in the periplasm, Tap was exported exclusively via the Tat pathway and active Tap precursor was observed in the cytoplasm. These results demonstrate that two sequence and functional related enzymes are exported by distinct protein transport systems, which may play an integral role in the bacterial adaptation to their environment during the evolution.

Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology

Superfolder GFP (sGFP) is a variant of the Green Fluorescent Protein that folds efficiently when fused to poorly folded proteins. In this study, we show that sGFP, but not enhanced GFP, is functional in vivo at 70 degrees C in the extreme thermophile Thermus thermophilus (Tth); thus, permitting the use of sGFP as a localization tag in vivo. We created a suite of plasmids that allow the expression of carboxy-terminal sGFP fusion proteins in both Escherichia coli and Tth. In order to demonstrate the facility of sGFP as an in vivo localization tag in Tth, we tagged GroES (the small subunit of the bacterial GroES/GroEL chaperone), NarC (a membrane component of the nitrate respiration apparatus) and PhoA (a TAT-secreted periplasmic protein), and visualized the distribution of the sGFP fusion proteins using confocal microscopy. Fusions to NarC and PhoA produced enzymatically active proteins that complemented both the narC and the phoA strains respectively. Observation of the distribution of the GroES-sGFP protein by confocal microscopy revealed a homogeneous fluorescence in the cells, which is in full agreement with the cytoplasmic nature of GroES, whereas the NarC-sGFP protein was localized to the membrane. Finally, a combination of confocal microscopy and biochemistry revealed that PhoA is localized in the periplasm. We suggest that sGFP will be broadly applicable in characterizing various extreme thermophile systems.

Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01

BackgroundMany strains of Thermus have been isolated from hot environments around the world. Thermus scotoductus SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different Thermus thermophilus strains have been completed. This paper represents the completed genome from a second Thermus species - T. scotoductus.ResultsThe genome of Thermus scotoductus SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the Thermus thermophilus genomes. The T. thermophilus megaplasmid genes are part of the T. scotoductus chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of T. scotoductus and T. thermophilus. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from Meiothermus ruber. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of Thermus scotoductus was confirmed experimentally as expected as most of the proteins of the natural transformation system of Thermus thermophilus are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle.ConclusionsThe genome of Thermus scotoductus SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to Thermus thermophilus. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of Thermus scotoductus illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.

The role of the nitrate respiration element of Thermus thermophilus in the control and activity of the denitrification apparatus

The nitrate conjugative element (NCE) encodes the ability to respire nitrate in the facultative Thermus thermophilus NAR1 strain. This process is carried out by two heterotetrameric enzymes that catalyse the oxidation of NADH (Nrc) and the reduction of nitrate (Nar), whose expression is activated by the NCE-encoded transcription factors DnrS and DnrT. We report the presence of NCE in other facultative strains of T. thermophilus and analyse its role in subsequent steps of the denitrification pathway. We encountered that nrc mutants of denitrifying strains show a decrease in anaerobic growth rates not only with nitrate, but also with nitrite, NO and N(2)O, which is concomitant to their lower NADH oxidation activities in vitro. We show that nitrate, nitrite and NO are activating signals for transcription of nrc in these strains. Finally, we demonstrate that DnrS and DnrT are required for anaerobic growth not only with nitrate, but also with nitrite, NO and N(2)O. These data allow us to conclude that: (i) Nrc constitutes the main electron donor for the four reductases of the denitrification pathway, and (ii) the NCE controls the expression of the whole denitrification pathway and the repression of the aerobic respiration through the transcription factors DnrS and DnrT.

Thermus thermophilus as a Cell Factory for the Production of a Thermophilic Mn-Dependent Catalase Which Fails To Be Synthesized in an Active Form in Escherichia coli

ABSTRACT Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H2O2-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.