Enzymatic properties and physiological function of glutamate racemase from Thermus thermophilus.

d-Amino acids are physiologically important components of peptidoglycan in the bacterial cell wall, maintaining cell structure and aiding adaptation to environmental changes through peptidoglycan remodelling. Therefore, the biosynthesis of d-amino acids is essential for bacteria to adapt to different environmental conditions. The peptidoglycan of the extremely thermophilic bacterium Thermus thermophilus contains d-alanine (d-Ala) and d-glutamate (d-Glu), but its d-amino acid metabolism remains poorly understood. Here, we investigated the enzyme activity and function of the product of the TTHA1643 gene, which is annotated to be a Glu racemase in the T. thermophilus HB8 genome. Among 21 amino acids tested, TTHA1643 showed highly specific activity toward Glu as the substrate. The catalytic efficiency (kcat/Km) of TTHA1643 toward d- and l-Glu was comparable; however, the kcat value was 18-fold higher for l-Glu than for d-Glu. Temperature and pH profiles showed that the racemase activity of TTHA1643 is high under physiological conditions for T. thermophilus growth. To assess physiological relevance, we constructed a TTHA1643-deficient strain (∆TTHA1643) by replacing the TTHA1643 gene with the thermostable hygromycin resistance gene. Growth of the ∆TTHA1643 strain in synthetic medium without d-Glu was clearly diminished relative to wild type, although the TTHA1643 deletion was not lethal, suggesting that alternative d-Glu biosynthetic pathways may exist. The deterioration in growth was restored by adding d-Glu to the culture medium, showing that d-Glu is required for normal growth of T. thermophilus. Collectively, our findings show that TTHA1643 is a Glu racemase and has the physiological function of d-Glu production in T. thermophilus.

Identification and characterization of ABC transporters for carbohydrate uptake in Thermus thermophilus HB8.

Organisms use a variety of carbohydrates and metabolic pathways in order to capitalize in their specific environments. Depending upon their habitat, organism employs different types of transporters to maintain the cellular nutritional balance via central metabolism. A major contributor in this process in bacteria is a carbohydrate ABC transporter. The focus of this study is to get an insight into the carbohydrate transport and metabolism of a hot-spring-dwelling bacterium Thermus thermophilus HB8. We applied high-throughput data-mining approaches for identification and characterization of carbohydrate ABC transporters in T. thermophilus HB8. This enabled the identification of 11 putative carbohydrate ABC transport systems. To identify the cognate ligands for these transporters, functional annotation was performed. However, scarcity of homologous-protein's function hinders the process of functional annotation. Thus, to overcome this limitation, we integrated the functional annotation of carbohydrate ABC transporters with their metabolic analysis. Our results demonstrate that out of 11 putative carbohydrate ABC transporters, six are involved in the sugar (four for monosaccharides and polysaccharides-degraded products and two for osmotic regulation), four in phospholipid precursor (namely UgpABCE) and the remaining one in purine uptake. Further, analysis suggests the existence of sharing mechanism of transmembrane domains (TMDs) and/or nucleotide-binding domains (NBDs) among the 11 carbohydrate ABC transporters.

Ribosome Structure, Function, and Early Evolution.

Ribosomes are among the largest and most dynamic molecular motors. The structure and dynamics of translation initiation and elongation are reviewed. Three ribosome motions have been identified for initiation and translocation. A swivel motion between the head/beak and the body of the 30S subunit was observed. A tilting dynamic of the head/beak versus the body of the 30S subunit was detected using simulations. A reversible ratcheting motion was seen between the 30S and the 50S subunits that slide relative to one another. The 30S⁻50S intersubunit contacts regulate translocation. IF2, EF-Tu, and EF-G are homologous G-protein GTPases that cycle on and off the same site on the ribosome. The ribosome, aminoacyl-tRNA synthetase (aaRS) enzymes, transfer ribonucleic acid (tRNA), and messenger ribonucleic acid (mRNA) form the core of information processing in cells and are coevolved. Surprisingly, class I and class II aaRS enzymes, with distinct and incompatible folds, are homologs. Divergence of class I and class II aaRS enzymes and coevolution of the genetic code are described by analysis of ancient archaeal species.

Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.

Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 µM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

Identification of a VapBC toxin-antitoxin system in a thermophilic bacterium Thermus thermophilus HB27.

There are 12 putative toxin-antitoxin (TA) loci in the Thermus thermophilus HB27 genome, including four VapBC and three HicBA families. Expression of these seven putative toxin genes in Escherichia coli demonstrated that one putative VapC toxin TTC0125 and two putative HicA toxins, TTC1395 and TTC1705, inhibited cell growth, and co-expression with cognate antitoxin genes rescued growth, indicating that these genes function as TA loci. In vitro analysis with the purified TTC0125 and total RNA/mRNA from E. coli and T. thermophilus showed that TTC0125 has RNase activity to rRNA and mRNA; this activity was inhibited by the addition of the purified TTC0126. Translation inhibition assays showed that TTC0125 inhibited protein synthesis by degrading mRNA but not by inactivating ribosomes. Amino acid substitutions of 14 predicted catalytic and conserved residues in VapC toxins to Ala or Asp in TTC0125 indicated that nine residues are important for its in vivo toxin activity and in vitro RNase activity. These data demonstrate that TTC0125-TTC0126 functions as a VapBC TA module and causes growth inhibition by degrading free RNA. This is the first study to identify the function of TA systems in T. thermophilus.

Role of the semi-conserved histidine residue in the light-sensing domain of LitR, a MerR-type photosensory transcriptional regulator.

The LitR/CarH protein family transcriptional regulator is a new type of photoreceptor based on the function of adenosyl B12 (AdoB12) as a light-sensitive ligand. Here, we studied a semi-conserved histidine residue (His132) in the light-sensing (AdoB12-binding) domain at the C-terminus of LitR from a thermophilic Gram-negative bacterium, Thermus thermophilus HB27. The in vivo mutation of His132 within LitR caused a reduction in the rate of carotenoid production in response to illumination. BIAcore analysis revealed that the illuminated-LitRH132A possesses high DNA-binding activity compared to the wild-type protein. The subunit structure analysis showed that LitRH132A performed an incomplete subunit dissociation. The ability of LitRH132A to associate with AdoB12 was reduced compared with that of the wild-type protein in an equilibration dialysis experiment. Overall, these results suggest that His132 of LitR is involved in the association with AdoB12 as well as the light-sensitive DNA-binding activity based on oligomer dissociation.

Different effects of MglA and MglB on pilus-mediated functions and natural competence in Thermus thermophilus.

The thermophilic bacterium Thermus thermophilus is known for its high natural competence. Uptake of DNA is mediated by a DNA translocator that shares components with type IV pili. Localization and function of type IV pili in other bacteria depend on the cellular localization at the poles of the bacterium, a process that involves MglA and MglB. T. thermophilus contains homologs of MglA and MglB. The genes encoding MglA and MglB were deleted and the physiology of the mutants was studied. Deletion of the genes individually or in tandem had no effect on pili formation but pili lost their localization at the poles. The mutants abolished pilus-mediated functions such as twitching motility and adherence but had no effect on uptake of DNA by natural competence. These data demonstrate that MglA and MglB are dispensable for natural transformation and are consistent with the hypothesis that uptake of DNA does not depend on type IV pili or their cellular localization.

A comparison of structural and evolutionary attributes of Escherichia coli and Thermus thermophilus small ribosomal subunits: signatures of thermal adaptation.

Here we compare the structural and evolutionary attributes of Thermus thermophilus and Escherichia coli small ribosomal subunits (SSU). Our results indicate that with few exceptions, thermophilic 16S ribosomal RNA (16S rRNA) is densely packed compared to that of mesophilic at most of the analogous spatial regions. In addition, we have located species-specific cavity clusters (SSCCs) in both species. E. coli SSCCs are numerous and larger compared to T. thermophilus SSCCs, which again indicates densely packed thermophilic 16S rRNA. Thermophilic ribosomal proteins (r-proteins) have longer disordered regions than their mesophilic homologs and they experience larger disorder-to-order transitions during SSU-assembly. This is reflected in the predicted higher conformational changes of thermophilic r-proteins compared to their mesophilic homologs during SSU-assembly. This high conformational change of thermophilic r-proteins may help them to associate with the 16S ribosomal RNA with high complementary interfaces, larger interface areas, and denser molecular contacts, compared to those of mesophilic. Thus, thermophilic protein-rRNA interfaces are tightly associated with 16S rRNA than their mesophilic homologs. Densely packed 16S rRNA interior and tight protein-rRNA binding of T. thermophilus (compared to those of E. coli) are likely the signatures of its thermal adaptation. We have found a linear correlation between the free energy of protein-RNA interface formation, interface size, and square of conformational changes, which is followed in both prokaryotic and eukaryotic SSU. Disorder is associated with high protein-RNA interface polarity. We have found an evolutionary tendency to maintain high polarity (thereby disorder) at protein-rRNA interfaces, than that at rest of the protein structures. However, some proteins exhibit exceptions to this general trend.

Phosphoproteomic analysis reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27.

Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili-related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.

Flagellin gene (fliC) of Thermus thermophilus HB8: characterization of its product and involvement to flagella assembly and microbial motility.

Thermus thermophilus HB8 flagellin protein (FliC) is encoded by the TTHC004 (fliC) gene, which is located in the pTT8 plasmid of the bacterium. Flagellin monomer and flagella fibres were isolated from a culture of T. thermophilus grown in rich medium, or in mineral salt medium with sodium gluconate as the carbon source. Western blot immunodetection with anti-FliC revealed a stable complex (FliC)(1)(FliS)(2) of flagellin (FliC, 27.7 kDa) with a homodimer of FliS (FliS, 18.2 kDa) that are encoded by TTHC004 and TTHC003 genes, respectively. The complex is dissociable at low pHs and/or by heat treatment. Glycan staining of purified flagella and treatment with N-glycosidase F suggested that flagellin of T. thermophilus is a glycosylated protein. Size exclusion chromatography revealed that flagellar filaments (FliC) have a molecular mass higher than 200 kDa. The formation of flagella is enhanced after prolonged cultivation time where phosphate and other nutrient were depleted, giving in the bacterium considerable swimming motility in low viscosity media.

Identification of novel genes regulated by the oxidative stress-responsive transcriptional activator SdrP in Thermus thermophilus HB8.

The stationary phase-dependent regulatory protein (SdrP) from the extremely thermophilic bacterium, Thermus thermophilus HB8, a CRP/FNR family protein, is a transcription activator, whose expression increases in the stationary phase of growth. SdrP positively regulates the expression of several genes involved in nutrient and energy supply, redox control, and nucleic acid metabolism. We found that sdrP mRNA showed an increased response to various environmental or chemical stresses in the logarithmic growth phase, the most effective stress being oxidative stress. From genome-wide expression pattern analysis using 306 DNA microarray datasets from 117 experimental conditions, eight new SdrP-regulated genes were identified among the genes whose expression was highly correlated with that of sdrP. The gene products included manganese superoxide dismutase, catalase, and excinuclease ABC subunit B (UvrB), which plays a central role in the nucleotide excision repair of damaged DNA. Expression of these genes also tended to increase upon entry into stationary phase, as in the case of the previously identified SdrP-regulated genes. These results indicate that the main function of SdrP is in the oxidative stress response.

Modification of 16S ribosomal RNA by the KsgA methyltransferase restructures the 30S subunit to optimize ribosome function.

All organisms incorporate post-transcriptional modifications into ribosomal RNA, influencing ribosome assembly and function in ways that are poorly understood. The most highly conserved modification is the dimethylation of two adenosines near the 3' end of the small subunit rRNA. Lack of these methylations due to deficiency in the KsgA methyltransferase stimulates translational errors during both the initiation and elongation phases of protein synthesis and confers resistance to the antibiotic kasugamycin. Here, we present the X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit lacking these dimethylations. Our data indicate that the KsgA-directed methylations facilitate structural rearrangements in order to establish a functionally optimum subunit conformation during the final stages of ribosome assembly.

Exploring research frontiers in microbiology: recent advances in halophilic and thermophilic extremophiles.

Extremophilic prokaryotes inhabit ecosystems that are, from a human perspective, extreme, and life in these environments requires far-reaching cellular adaptations. Here, we will describe, for two examples (Thermus thermophilus, Halobacillus halophilus), how thermophilic or halophilic bacteria adapt to their environment; we will describe the molecular basis of sensing and responding to hypersalinity and we will analyze the impact and basis of natural competence for survival in hot environments.

Thermus thermophilus proteins that are differentially expressed in response to growth temperature and their implication in thermoadaptation.

As a kind of important extremophiles to realize the adaptation of life at high temperatures, thermophiles have attracted extensive studies. However, the pathways of thermophile proteins related to thermoadaptation remain to be addressed. Our study showed that there existed two types of protein profiles for the thermophile Thermus thermophilus wl in response to temperature change. One of them came from cultures growing below 65 degrees C, which was close to the optimal growth temperature, and another from cultures at or above 65 degrees C. These protein profiles were confirmed by Northern blots. On the basis of the proteomic and computational analyses, it was found that the thermophile proteins related to thermoadaptation might be involved in metabolic pathways as well as the stabilities and modifications of DNA and proteins. Interestingly, for the basic metabolism glycolysis, the phosphoglucomutase was up-regulated at below-optimum temperature, while the glyceraldehyde-3-phosphate dehydrogenase was up-regulated at above-optimum temperature, suggesting that different regulations might be used for basic metabolism at different temperatures. To characterize the proteins in response to high temperatures, superoxide dismutase (SOD), an important enzyme in organism to remove free radical produced in stress environment such as high temperature, was selected as a target protein for this investigation. SOD was inactivated to construct a SOD mutant. The results showed that the SOD protein was essential in thermoadaptation of T. thermophilus. Our study, therefore, presented the thermophile proteins required for thermoadaptation and their possible pathways in thermoadaptation.

Role of galE on biofilm formation by Thermus spp.

Thermus thermophilus and Thermus aquaticus are thermophilic bacteria that are frequently found to attach to solid surfaces in hot springs to form biofilms. Uridine diphosphate (UDP)-galactose-4'-epimerase (GalE) is an enzyme that catalyzes the conversion of UDP-galactose to UDP-glucose, an important biochemical step in exopolysaccharide synthesis. We expressed GalE obtained from T. thermophilus HB8 in Escherichia coli and found that the enzyme is stable at 80 degrees C and can epimerize UDP-galactose to UDP-glucose and UDP-N-acetylgalactosamine (UDP-GalNAc) to UDP-N-acetylglucosamine (UDP-GlcNAc). Enzyme overexpression in T. thermophilus HB27 led to an increased capacity of biofilm production. Therefore, the galE gene is important to biofilm formation because of its involvement in epimerizing UDP-galactose and UDP-N-acetylgalactosamine for exopolysaccharide biosynthesis.

Shuffling genes around in hot environments: the unique DNA transporter of Thermus thermophilus.

Natural transformation permits the transport of DNA through bacterial membranes and represents a dominant mode for the transfer of genetic information between bacteria and between microorganisms of distant evolutionary lineages and even between members of different domains. This phenomenon, known as horizontal, or lateral, gene transfer, has been a major force for genome plasticity over evolutionary history, and is largely responsible for the spread of fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Here, we present a survey of the natural transformation machinery of the thermophile Thermus thermophilus HB27. A tentative model of the transformation machinery comprising of components similar to proteins of type IV pili and type II secretion systems is presented. A comparative discussion of the subunits and the structure of the DNA translocator and the underlying mechanism of transfer of free DNA in T. thermophilus highlights conserved and unique features of the DNA translocator in T. thermophilus. We hypothesize that the extraordinary broad substrate specificity and the high efficiency of the T. thermophilus DNA uptake system is of major importance for thermoadaptation and interdomain DNA transfer in hot environments.

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.

Correlating the transcriptome, proteome, and metabolome in the environmental adaptation of a hyperthermophile.

We have performed a comprehensive characterization of global molecular changes for a model organism Pyrococcus furiosus using transcriptomic (DNA microarray), proteomic, and metabolomic analysis as it undergoes a cold adaptation response from its optimal 95 to 72 degrees C. Metabolic profiling on the same set of samples shows the down-regulation of many metabolites. However, some metabolites are found to be strongly up-regulated. An approach using accurate mass, isotopic pattern, database searching, and retention time is used to putatively identify several metabolites of interest. Many of the up-regulated metabolites are part of an alternative polyamine biosynthesis pathway previously established in a thermophilic bacterium Thermus thermophilus. Arginine, agmatine, spermidine, and branched polyamines N4-aminopropylspermidine and N4-( N-acetylaminopropyl)spermidine were unambiguously identified based on their accurate mass, isotopic pattern, and matching of MS/MS data acquired under identical conditions for the natural metabolite and a high purity standard. Both DNA microarray and semiquantitative proteomic analysis using a label-free spectral counting approach indicate the down-regulation of a large majority of genes with diverse predicted functions related to growth such as transcription, amino acid biosynthesis, and translation. Some genes are, however, found to be up-regulated through the measurement of their relative mRNA and protein levels. The complimentary information obtained by the various "omics" techniques is used to catalogue and correlate the overall molecular changes.

Control of the respiratory metabolism of Thermus thermophilus by the nitrate respiration conjugative element NCE.

The strains of Thermus thermophilus that contain the nitrate respiration conjugative element (NCE) replace their aerobic respiratory chain by an anaerobic counterpart made of the Nrc-NADH dehydrogenase and the Nar-nitrate reductase in response to nitrate and oxygen depletion. This replacement depends on DnrS and DnrT, two homologues to sensory transcription factors encoded in a bicistronic operon by the NCE. DnrS is an oxygen-sensitive protein required in vivo to activate transcription on its own dnr promoter and on that of the nar operon, but not required for the expression of the nrc operon. In contrast, DnrT is required for the transcription of these three operons and also for the repression of nqo, the operon that encodes the major respiratory NADH dehydrogenase expressed during aerobic growth. Thermophilic in vitro assays revealed that low DnrT concentrations allows the recruitment of the T. thermophilus RNA polymerase sigma(A) holoenzyme to the nrc promoter and its transcription, whereas higher DnrT concentrations are required to repress transcription on the nqo promoter. In conclusion, our data show a complex autoinducible mechanism by which DnrT functions as the transcriptional switch that allows the NCE to take the control of the respiratory metabolism of its host during adaptation to anaerobic growth.

Diversity and biosynthesis of compatible solutes in hyper/thermophiles.

The accumulation of compatible solutes, either by uptake from the medium or by de novo synthesis, is a general response of microorganisms to osmotic stress. The diversity of compatible solutes is large but falls into a few major chemical categories, such as carbohydrates or their derivatives and amino acids or their derivatives. This review deals with compatible solutes found in thermophilic or hyperthermophilic bacteria and archaea that have not been commonly identified in microorganisms growing at low and moderate temperatures. The response to NaCl stress of Thermus thermophilus is an example of how a thermophilic bacterium responds to osmotic stress by compatible solute accumulation. Emphasis is made on the pathways leading to the synthesis of mannosylglycerate and glucosylglycerate that have been recently elucidated in several hyper/thermophilic microorganisms. The role of compatible solutes in the thermoprotection of these fascinating microorganisms is also discussed.