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PubMed2005

The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5'-translational operator mRNA.

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16S ribosomal RNA (rRNA) as well as a translational operator located in the 5'-untranslated region (5'-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5'-UTR of the BS15 gene and beta-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5'-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.

5' Untranslated Regions↗
PubMed2005

Crystal structure and structure-based mutational analyses of RNase HIII from Bacillus stearothermophilus: a new type 2 RNase H with TBP-like substrate-binding domain at the N terminus.

Ribonuclease HIII (Bst-RNase HIII) from the moderate thermophile Bacillus stearothermophilus is a type 2 RNase H but shows poor amino acid sequence identity with another type 2 RNase H, RNase HII. It is composed of 310 amino acid residues and acts as a monomer. Bst-RNase HIII has a large N-terminal extension with unknown function and a unique active-site motif (DEDE), both of which are characteristics common to RNases HIII. To understand the role of these N-terminal extension and active-site residues, the crystal structure of Bst-RNase HIII was determined in both metal-free and metal-bound forms at 2.1-2.6 angstroms resolutions. According to these structures, Bst-RNase HIII consists of the N-terminal domain and C-terminal RNase H domain. The structures of the N and C-terminal domains were similar to those of TATA-box binding proteins and archaeal RNases HII, respectively. The steric configurations of the four conserved active-site residues were very similar to those of other type 1 and type 2 RNases H. Single Mn and Mg ions were coordinated with Asp97, Glu98, and Asp202, which correspond to Asp10, Glu48, and Asp70 of Escherichia coli RNase HI, respectively. The mutational studies indicated that the replacement of either one of these residues with Ala resulted in a great reduction of the enzymatic activity. Overproduction, purification, and characterization of the Bst-RNase HIII derivatives with N and/or C-terminal truncations indicated that the N-terminal domain and C-terminal helix are involved in substrate binding, but the former contributes to substrate binding more greatly than the latter.

Amino Acid Sequence↗
PubMed2006

The denatured state under native conditions: a non-native-like collapsed state of N-PGK.

The guanidinium-denatured state of the N-domain of phosphoglycerate kinase (PGK) has been characterized using solution NMR. Rather than behaving as a homogenous ensemble of random coils, chemical shift changes for the majority of backbone amide resonances indicate that the denatured ensemble undergoes two definable equilibrium transitions upon titration with guanidinium, in addition to the major refolding event. (13)C and (15)N chemical shift changes indicate that both intermediary states have distinct helical character. At denaturant concentrations immediately above the mid-point of unfolding, size-exclusion chromatography shows N-PGK to have a compact, denatured form, suggesting that it forms a helical molten globule. Within this globule, the helices extend into some regions that become beta strands in the native state. This predisposition of the denatured state to extensive non-native-like conformation, illustrates that, rather than directing folding, conformational pre-organization in the denatured state can compete with the normal folding direction. The corresponding reduction in control of the direction of folding as proteins become larger, could thus constitute a restriction on the size of protein domains.

Geobacillus stearothermophilus↗
PubMed2006

Computational studies of tryptophanyl-tRNA synthetase: activation of ATP by induced-fit.

Catalysis of amino acid activation by Bacillus stearothermophilus tryptophanyl-tRNA synthetase involves three allosteric states: (1) Open; (2) closed pre-transition state (PreTS); and (3) closed products (Product). The interconversions of these states entail significant domain motions driven by ligand binding. We explore the application of molecular dynamics simulations to investigate ligand-linked conformational stability changes associated with this catalytic cycle. Multiple molecular dynamics trajectories (5 ns) for 11 distinct liganded and unliganded monomer configurations show that the PreTS conformation is unstable in the absence of ATP, reverting within approximately 600 ps nearly to the Open conformation. In contrast, Open and Product state trajectories were stable, even without ligands, confirming the previous suggestion that catalysis entails destabilization of the protein conformation, driven by ATP-binding energies developed as the PreTS state assembles during induced-fit. The simulations suggest novel mechanistic details associated with both induced-fit (Open-PreTS) and catalysis (PreTS-Product). Notably, Mg2+ -ATP interactions are coupled to interactions between ATP and active-site lysine side-chains via mechanisms that cannot be captured under the molecular mechanics approximations, and which therefore require restraining potentials for stable simulation. Simulations of Mg2+. ATP-bound PreTS complexes with restraining potentials and with a virtual K111A mutant confirm that these coupling interactions are necessary to sustain the PreTS conformation and, in turn, provide a new model for how the PreTS conformation activates ATP for catalysis. These results emphasize the central role of the PreTS state as a high-energy intermediate structure along the catalytic pathway and suggest that Mg2+ and the KMSKS loop function cooperatively during catalysis.

Adenosine Triphosphate↗
PubMed2006

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex.

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.

Amino Acid Motifs↗
PubMed2004

Visualization of alpha-helical features in a density map constructed using 9 molecular images of the 1.8 MDa icosahedral core of pyruvate dehydrogenase.

Strategies to achieve the highest resolutions in structures of protein complexes determined by cryo-electron microscopy generally involve averaging information from large numbers of individual molecular images. However, significant limitations are posed by heterogeneity in image quality and in protein conformation that are inherent to large data sets of images. Here, we demonstrate that the combination of iterative refinement and stringent molecular sorting is an effective method to obtain substantial improvements in map quality of the 1.8 MDa icosahedral catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus. From a starting set of 42,945 images of the core complex, we show that using only the best 139 particles in the data set produces a map that is superior to those constructed with greater numbers of images, and that the location of many of the alpha-helices in the structure can be unambiguously visualized in a map constructed from as few as 9 particles.

Cryoelectron Microscopy↗
PubMed2006

In vivo molecular evolution reveals biophysical origins of organismal fitness.

In nature, evolution occurs through the continuous adaptation of a population to its environment. At the molecular level, adaptive changes in protein sequence and expression impact organismal fitness and, consequently, dictate population dynamics. Here, we have used a "weak link" method to favor variations in one gene, allowing adaptation to thermostability to be studied in molecular detail as bacteria were grown continuously for approximately 1500 generations. Surprisingly, only six mutant alleles, representing less than 1% of the possible missense mutations, were observed, suggesting a highly constrained molecular landscape during protein evolution. The changes in organismal fitness were linked directly to incremental increases in enzyme stability and activity maxima and corresponded to the narrow temperature ranges where each mutant enjoyed success within the overall population. Thus, continuous evolution of a single gene permits a quantitative approach that extends from the phenotypes of the microbial populations to their underlying biophysical basis.

Adenylate Kinase↗
PubMed2006

[Bacterial DNA alteration by plasma generated atomic nitrogen. Real-time PCR detection contribution].

AIMS: Bacterial sterilization by the technology of plasma in post-discharge shows a growing interest. The main appeal of this new process resides in its action at dry and low temperature (60 degrees C). This technology would be therefore useful for the complex medical equipment, sensitive to the oxidization, humidity and/or requiring a temperature lower than 60 degrees C. The objective of this survey is to demonstrate the activity of an atomic flux emanating a plasma of pure molecular nitrogen on the bacterial DNA: does the plasma of nitrogen damage the genetic material? MATERIALS AND METHODS: The bacteria tested (Bacillus stearothermophilus, Staphylococcus aureus MRSA and Helicobacter pylori) are cultivated on suitable agar, and the bacterial DNA is extracted from every CFU by the technique of the columns (High Pure PCR Template, Roche). Every quantity of DNA extracted is diluted in 1 ml pure water. Then, 50 microl of each of these solutions of DNA are laid down in sterile Nunclon's plates holes, which undergo an advanced emptiness cycle during 60 minutes. The DNA residues will be then introduced during 40 minutes in a plasma sterilization surrounding wall (Plasmalyse), Satelec) where the debit of nitrogen, the pressure and the temperature are adjusted respectively to 1 L x min(-1), 5 Torrs and 60 degrees C. The DNA so ''plasmalysé'', is recovered then by aspiration in 500 mul pure water and processed to undergo an amplification/detection by Real-Time PCR (LightCycler2.0, Roche). The DNA ''plasmalysé'' will be compared to the intact DNA control(1), to the DNA control control(2) having undergone the cycle of emptiness solely, as well as to the DNA control(3) solely heated to 60 degrees C during 40 min. RESULTS: The amplification curves demonstrated that the only advanced emptiness and the only heat don't have any activity on the bacterial DNA. On the other hand, the DNA ''plasmalysé'' shows a deterioration of the amplified sequences. CONCLUSION: The genomic bacterial DNA, once extracted, is damaged by the gaseous flux of nitrogen plasma. A new sterilization process of the medical material will presumably impose itself in medium-term.

Base Sequence↗
PubMed2005

The propeptide is not required to produce catalytically active neutral protease from Bacillus stearothermophilus.

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste) is usually produced extracellularly in Bacillus subtilis, where it is expressed as preproenzyme and subsequently processed in an autocatalytic, intramolecular process. To create the basis for the production of inactive mutants of TLP-ste, which cannot be processed in B. subtilis, we studied the expression of TLP-ste and its propeptide in cis and in trans in Escherichia coli. In contrast to thermolysin, subtilisin and alpha-lytic protease, which could be obtained only in the presence of the corresponding propeptides, TLP-ste could be produced as an active mature enzyme in E. coli in the absence of its prosequence. Surprisingly, however, a much more effective access to active mature protease was found when TLP-ste (devoid of its prosequence) was expressed as protein with an N-terminal His6 tag which accumulated in the form of inclusion bodies. Completely unexpected, the protein could be renatured from the inclusion bodies after solubilization in guanidine hydrochloride solutions in high yields. Purification to homogeneity was possible by affinity chromatography on Bacitracin silica as well as by immobilized metal ion affinity chromatography. By addition of separately expressed propeptide to the renaturation mixture yields of renaturation could not be increased significantly, confirming that the propeptide is not essential for proper folding of the enzyme or its stabilization during the folding process. Also in vivo, the expression levels of active mature TLP-ste in Escherichia coli did not significantly differ when the mature sequence was expressed alone or coexpressed with the prosequence in cis or in trans.

Anti-Bacterial Agents↗
PubMed2004

Purification and characterization of thermostable xylanase and beta-xylosidase by the thermophilic bacterium Bacillus thermantarcticus.

Bacillus thermantarcticus, a thermophilic bacterium isolated from Antarctic geothermal soil near the crater of Mount Melbourne, produced extracellular xylanase (1,4-beta-D-xylan xylanohydrolase; E.C. 3.2.1.8) and beta-xylosidase (1,4-beta-D-xylan xylohydrolase; E.C. 3.2.1.37). Each extracellular enzyme was separated by gel filtration with Sephacryl S-200 and further purified to homogeneity (119-fold for xylanase and 160-fold for beta-xylosidase). The optimum temperatures were 80 degrees C for xylanase at pH 5.6 and 70 degrees C for beta-xylosidase at pH 6.0. The isoelectric points and molecular masses were 4.8 and 45 kDa for xylanase and 4.2 and 150 kDa for beta-xylosidase, respectively. Xylanase was stable at 60 degrees C for 24 h, whereas it showed a half life at 70 degrees C of 24 h and at 80 degrees C for 50 min. beta-xylosidase activity did not decrease after 1 h at 60 degrees C. Km of xylanase for xylan was 1.6 mg/ml, Km of beta-xylosidase for p-nitrophenyl-beta-D-xylopyranoside was 0.5 mM and for o-nitrophenyl-beta-D-xylopyranoside was 1.28 mM. The action of two enzymes on xylan gave only xylose.

Geobacillus stearothermophilus↗
PubMed2005

Studies on Bacillus stearothermophilus. Part IV. Influence of enhancers on biotransformation of testosterone.

The impact of chemical enhancers on the biotransformation of testosterone has been exploited. Application of crude cell concentrates to produce Bacillus stearothermophilus-mediated bioconversion of testosterone at 65 degrees C for 72 h has been examined. After incubation, the xenobiotic substrate was added to the concentrated whole cell suspensions. The enhancer molecules were included in the whole cell suspension. The resultant products, after extraction into an organic solvent, were purified by thin layer chromatography and identification was carried out through spectroscopic data. Five steroid metabolites 9,10-seco-4-androstene-3,9,17-trione, 5alpha-androstan-3,6,17-trione, 17beta-hydroxy-5alpha-androstan-3,6-dione, 3beta,17beta-dihydroxyandrost-4-ene-6-one and 17beta-hydroxyandrost-4,6-diene-3-one were identified as biotransformation products of testosterone. A possible biosynthetic route for these bioconversion products is postulated.

Biotransformation↗
PubMed2005

The bacterial helicase-primase interaction: a common structural/functional module.

The lack of a high-resolution structure for the bacterial helicase-primase complex and the fragmented structural information for the individual proteins have been hindering our detailed understanding of this crucial binary protein interaction. Two new structures for the helicase-interacting domain of the bacterial primases from Escherichia coli and Bacillus stearothermophilus have recently been solved and both revealed a unique and surprising structural similarity to the amino-terminal domain of the helicase itself. In this minireview, the current data are discussed and important new structural and functional aspects of the helicase-primase interaction are highlighted. An attractive structural model with direct biological significance for the function of this complex and also for the development of new antibacterial compounds is examined.

Amino Acid Sequence↗
PubMed2005

The molecular origins of specificity in the assembly of a multienzyme complex.

The pyruvate dehydrogenase (PDH) multienzyme complex is central to oxidative metabolism. We present the first crystal structure of a complex between pyruvate decarboxylase (E1) and the peripheral subunit binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2). The interface is dominated by a "charge zipper" of networked salt bridges. Remarkably, the PSBD uses essentially the same zipper to alternately recognize the dihydrolipoyl dehydrogenase (E3) component of the PDH assembly. The PSBD achieves this dual recognition largely through the addition of a network of interfacial water molecules unique to the E1-PSBD complex. These structural comparisons illuminate our observations that the formation of this water-rich E1-E2 interface is largely enthalpy driven, whereas that of the E3-PSBD complex (from which water is excluded) is entropy driven. Interfacial water molecules thus diversify surface complementarity and contribute to avidity, enthalpically. Additionally, the E1-PSBD structure provides insight into the organization and active site coupling within the approximately 9 MDa PDH complex.

Binding Sites↗
PubMed2006

Rounding up: Engineering 12-membered rings from the cyclic 11-mer TRAP.

The protein TRAP (trp RNA binding attenuation protein) forms a highly thermostable ring-shaped 11-mer. By linking in tandem two, three, or four DNA sequences encoding TRAP monomers, we have engineered new rings that consist of 12 TRAP subunits and bind 12 ligand molecules. The hydrogen bonding pattern and buried surface area within and between subunits are essentially identical between the 11-mer and 12-mer crystal structures. Why do the artificial proteins choose to make single 12-mer rings? The 12-mer rings are highly sterically strained by their peptide linkers and far from thermostable. That proteins choose to adopt a strained conformation of few subunits rather than an unstrained one with 11 subunits demonstrates the importance of entropic factors in controlling protein-protein interactions in general.

Bacterial Proteins↗
PubMed2005

Comparative study of tributyltin toxicity on two bacteria of the genus Bacillus.

Tributyltin is a potent biocide mainly used in marine anti-fouling paints. Owing to its widespread distribution in coast areas and its high toxicity to aquatic organisms, the use of this compound is generally restricted and under government regulation. Despite of that, it persists in the aquatic environment. Organotins used in industry have also been detected in terrestrial environments. The persistence and high lipophilicity explain bioaccumulation. The role of bacteria in recycling organic matter prompted us to study the interaction of tributyltin with two ubiquitous bacilli, B. stearothermophilus and B. subtilis, proposed as biological indicators of pollutants with ecological impact. These bacteria have been used as suitable models for the study of toxicity mechanisms of unselective lipophilic compounds (e.g., DDT and endosulfan). Drug effects on growth parameters, oxygen consumption and membrane organization were assessed. Bacteria growth in a liquid complex medium was disturbed by concentrations of TBT as low as 25 nM (8 microgL(-1)), close to the concentration in polluted environments. The respiratory activity is affected by TBT in both microorganisms. Membrane organization, assessed by fluorescence polarization of two fluidity probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and a propionic acid derivative (DPH-PA), was also perturbed by the xenobiotic. Alterations on growth, oxygen consumption and physical properties of membrane lipids are stronger in B. stearothermophilus as compared to B. subtilis. A putative relationship between growth inhibition and respiratory activity impairment induced by TBT and its effects on the physical behaviour of bacterial membrane lipids is suggested.

Bacillus subtilis↗
PubMed2005

Toxicity of methoprene as assessed by the use of a model microorganism.

Methoprene is an insect juvenile growth hormone mimic, commonly used as a pesticide. Although widely used for the control of several pests, toxic effects on organisms of different phyla have been reported. These events triggered studies to clarify the mechanisms of toxicity of this insecticide putatively involved in ecological issues. Here we show the effect of methoprene on the normal cell growth and viability of a strain of the thermophilic eubacterium Bacillus stearothermophilus, previously used as a model for toxicological evaluation of other environment pollutants. Respiration studies were also carried out attempting to identify a putative target for the cytotoxic action of methoprene. Cell growth was affected and a decrease of the number of viable cells was observed as a result of the addition of methoprene to the growth medium, an effect reverted by the presence of Ca(2+). Methoprene also inhibited the redox flow of B. stearothermophilus protoplasts before the cytochrome oxidase segment, an effect further studied by individually assessing the enzymatic activities of the respiratory complexes. This study suggests that methoprene membrane interaction and perturbation of cell bioenergetics may underlie the mechanism of toxicity of this compound in non-target organisms.

Calcium↗