Heterologous Production of Glycine Betaine Using Synechocystis sp. PCC 6803-Based Chassis Lacking Native Compatible Solutes.

Among compatible solutes, glycine betaine has various applications in the fields of nutrition, pharmaceuticals, and cosmetics. Currently, this compound can be extracted from sugar beet plants or obtained by chemical synthesis, resulting in low yields or high carbon footprint, respectively. Hence, in this work we aimed at exploring the production of glycine betaine using the unicellular cyanobacterium Synechocystis sp. PCC 6803 as a photoautotrophic chassis. Synechocystis mutants lacking the native compatible solutes sucrose or/and glucosylglycerol-∆sps, ∆ggpS, and ∆sps∆ggpS-were generated and characterized. Under salt stress conditions, the growth was impaired and accumulation of glycogen decreased by ∼50% whereas the production of compatible solutes and extracellular polymeric substances (capsular and released ones) increased with salinity. These mutants were used as chassis for the implementation of a synthetic device based on the metabolic pathway described for the halophilic cyanobacterium Aphanothece halophytica for the production of the compatible solute glycine betaine. Transcription of ORFs comprising the device was shown to be stable and insulated from Synechocystis' native regulatory network. Production of glycine betaine was achieved in all chassis tested, and was shown to increase with salinity. The introduction of the glycine betaine synthetic device into the ∆ggpS background improved its growth and enabled survival under 5% NaCl, which was not observed in the absence of the device. The maximum glycine betaine production [64.29 µmol/gDW (1.89 µmol/mg protein)] was reached in the ∆ggpS chassis grown under 3% NaCl. Taking into consideration this production under seawater-like salinity, and the identification of main key players involved in the carbon fluxes, this work paves the way for a feasible production of this, or other compatible solutes, using optimized Synechocystis chassis in a pilot-scale.

Securing a furan-based biorefinery: disclosing the genetic basis of the degradation of hydroxymethylfurfural and its derivatives in the model fungus Aspergillus nidulans.

Hydroxymethylfurfural (HMF) is a promising lignocellulosic-derived source for the generation of diverse chemical building blocks constituting an alternative to fossil fuels. However, it remains unanswered if ubiquitous fungi can ensure their efficient decay, similar to that observed in highly specialised fungi. To disclose the genetic basis of HMF degradation in aspergilli, we performed a comprehensive analysis of Aspergillus nidulans ability to tolerate and to degrade HMF and its derivatives (including an HMF-dimer). We identified the degradation pathway using a suite of metabolomics methods and showed that HMF was modified throughout sequential reactions, ultimately yielding derivatives subsequently channelled to the TCA cycle. Based on the previously revealed hmfFGH gene cluster of Cupriavidus basilensis, we combined gene expression of homologous genes in Aspergillus nidulans and functional analyses in single-deletion mutants. Results were complemented with orthology analyses across the genomes of twenty-five fungal species. Our results support high functional redundancy for the initial steps of the HMF degradation pathway in the majority of the analysed fungal genomes and the assignment of a single-copy furan-2,5-dicarboxylic acid decarboxylase gene in A. nidulans. Collectively our data made apparent the superior capacity of aspergilli to mineralise HMF, furthering the environmental sustainability of a furan-based chemistry.

Trehalose-6-phosphate-mediated phenotypic change in Acinetobacter baumannii.

The stress protectant trehalose is synthesized in Acinetobacter baumannii from UPD-glucose and glucose-6-phosphase via the OtsA/OtsB pathway. Previous studies proved that deletion of otsB led to a decreased virulence, the inability to grow at 45°C and a slight reduction of growth at high salinities indicating that trehalose is the cause of these phenotypes. We have questioned this conclusion by producing ∆otsA and ∆otsBA mutants and studying their phenotypes. Only deletion of otsB, but not deletion of otsA or otsBA, led to growth impairments at high salt and high temperature. The intracellular concentrations of trehalose and trehalose-6-phosphate were measured by NMR or enzymatic assay. Interestingly, none of the mutants accumulated trehalose any more but the ∆otsB mutant with its defect in trehalose-6-phosphate phosphatase activity accumulated trehalose-6-phosphate. Moreover, expression of otsA in a ∆otsB background under conditions where trehalose synthesis is not induced led to growth inhibition and the accumulation of trehalose-6-phosphate. Our results demonstrate that trehalose-6-phosphate affects multiple physiological activities in A. baumannii ATCC 19606.

Phosphate and polyphosphate anion recognition by a dinuclear copper(ii) complex of an unsymmetrical squaramide.

In the search for receptors suitable for the recognition of phosphate or polyphosphate anions, a new unsymmetrical squaramide-based ligand bearing dipicolylamine (dpa) and ethylpiperazine units (L) was designed and prepared. The acid-base reactions of L, its copper(ii) complexation behaviour and the binding of phosphate and polyphosphate anions by the copper(ii) complexes used as receptors were evaluated. 1H and 13C NMR titrations of L performed in D2O allowed the determination of its protonation sequence. The ligand L is able to coordinate two copper(ii) cations forming thermodynamically stable dinuclear complexes likely having two water molecules bound to each metal centre, as supported by DFT calculations. Coordinated water molecules can be replaced by the O-donors of the phosphate/polyphosphate anions. The potentiometric studies showed that at 2 : 1 Cu2+ : L ratio the dinuclear [Cu2LH-1]3+ species predominates from pH ∼ 5 to ∼7, and hydroxodinuclear species prevail at pH > 7. 1H NMR experiments in both H2O/D2O 9 : 1 v/v and in DMSO proved that copper(ii) coordination provokes deprotonation of the squaramide NH bound to the ethylpiperazine moiety, resulting in [Cu2LH-1]3+ species. The dicopper(ii) complexes of L, [Cu2LH-i]4-i, were used as the receptor for the uptake of some phosphate and polyphosphate anions. The receptor presents very high association constants with HPPi3- and ATP4- and the determined Keff showed that at physiological pH ATP4- is selectively taken from an aqueous solution containing phenylphosphate (PhPO42-), aminoethylphosphate (Haep-), AMP2- and ADP3-, but HPPi3- strongly interferes. DFT calculations suggest that the strong interaction with HPPi3- and ATP4- is related to the simultaneous coordination of the polyphosphate unit to the two copper(ii) centres.

Expanding the toolbox for Synechocystis sp. PCC 6803: validation of replicative vectors and characterization of a novel set of promoters.

Cyanobacteria are promising 'low-cost' cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the 'green' Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference P rnpB , three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study.

Trehalose, a temperature- and salt-induced solute with implications in pathobiology of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions worldwide. A major factor contributing to success of this bacterium is its outstanding ability to survive on dry surfaces. The molecular basis for desiccation resistance is not completely understood. This study focused on growth under osmotic stress and aimed to identify the pool of compatible solutes synthesized in response to these low water activity conditions. A. baumannii produced mannitol as compatible solute, but in contrast to Acinetobacter baylyi, also trehalose was accumulated in response to increasing NaCl concentrations. The genome of A. baumannii encodes a trehalose-6-phosphate phosphatase (OtsB) and a trehalose-6-phosphate synthase (OtsA). Deletion of otsB abolished trehalose formation, demonstrating that otsB is essential for trehalose biosynthesis. Growth of the mutant was neither impaired at low salt nor at 500 mM NaCl, but it did not grow at high temperatures, indicating a dual function of trehalose in osmo- and thermoprotection. This led us to analyse temperature dependence of trehalose formation. Indeed, expression of otsB was not only induced by high osmolarity but also by high temperature. Concurrently, trehalose was accumulated in cells grown at high temperature. Taken together, these data point to an important role of trehalose in A. baumannii beyond osmoprotection.

Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase.

Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2 and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-ß-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a µ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2 or NO permeation were found. The present data represent the first structure for a NO-selective FDP.

Comparison of the compatible solute pool of two slightly halophilic planctomycetes species, Gimesia maris and Rubinisphaera brasiliensis.

Gimesia maris and Rubinisphaera brasiliensis are slightly halophilic representatives of the deep-branching phylum Planctomycetes. For osmoadaptation both species accumulated α-glutamate, sucrose, ectoine and hydroxyectoine. A major role was found for ectoine, hydroxyectoine as well as sucrose under hyper-osmotic shock conditions. Nevertheless, the levels of sucrose were up-regulated by the increased salinity levels and also by low nitrogen availability. Additionally, G. maris accumulated glucosylglycerate (GG) as major solute specifically under low nitrogen levels, which prompted us to analyse the transcript abundance of two homologues genes known for the biosynthesis of GG, namely glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP). By qPCR using a suitable reference gene selected in this study, the transcript abundance of the biosynthetic genes was quantified in G. maris cells under hyper-osmotic shock or under low nitrogen conditions. The gpgS gene was induced under nitrogen-limiting conditions suggesting that GG synthesis is regulated primarily at the transcription level. Moreover, the expression of a gene coding for a putative sucrose-phosphorylase (Spase) located upstream the gpgS and gpgP genes was up-regulated, predicting a metabolic role of Spase probably related to GG synthesis.

Inhibition of the STAT3 Protein by a Dinuclear Macrocyclic Complex.

A new diethylenetriamine-derived macrocycle bearing 2-methylpyridyl arms and containing m-xylyl spacers, L, was prepared, and its dinuclear copper(II) and zinc(II) complexes were used as receptors for the recognition in aqueous solution of a phosphorylated peptide derived from a sequence of the STAT3 protein. A detailed study of the acid-base behavior of L and of its complexation properties as well as of the association of the phosphorylated peptide to the receptor was carried out by potentiometry in aqueous solution at 298.2 K and I = 0.10 M in KNO3. The data revealed that the receptor forms stable associations with several protonated forms of the substrate, with constant values ranging from 3.32 to 4.25 log units. The affinity of the receptor for the phosphorylated substrate studied is higher at a pH value where the receptor is mainly in the [Cu2L](4+) form and the pY residue of the substrate is in the dianionic form (pH 6.55). These results, also supported by (31)P NMR studies, showed that the phosphopeptide is bound through the phosphoryl group in a bridging mode. Additionally, the receptor inhibited binding between active (phosphorylated) STAT3 and its target DNA sequence in a dose-dependent manner (IC50 63 ± 3.4 µM) in human nuclear extracts in vitro. Treatment of whole cells with the inhibitor revealed that it is bioactive in living cells and has oncostatic properties that could be interesting for the fight against cancer and other pathologies involving the STAT3 protein.

Partial depolymerization of genetically modified potato tuber periderm reveals intermolecular linkages in suberin polyester.

Suberin is a biopolyester found in specialized plant tissues, both internal and external, with key frontier physiological functions. The information gathered so far from its monomer and oligomer composition, and in situ studies made by solid state techniques, haven't solved the enigma of how the suberin polyester is assembled as a macromolecule. To investigate how monomers are linked in suberin, we analyzed oligomer fragments solubilized by the partial depolymerization of suberin from potato (Solanum tuberosum) tuber periderms. The structure of the suberin oligomers, namely which monomers they included, and the type and frequency of the inter-monomer ester linkages, was assessed by ESI-MS/MS and high resolution NMR analysis. The analyzed potato periderms included the one from wild type (cv. Desirée) and from plants where suberin-biosynthesis genes were downregulated in chain elongation (StKCS6), ω-hydroxylation (CYP86A33) and feruloylation (FHT). Two building blocks were identified as possible key structures in the macromolecular development of the potato periderm suberin: glycerol - α,ω-diacid - glycerol, as the core of a continuous suberin aliphatic polyester; and glycerol - ω-hydroxyacid - ferulic acid, anchoring this polyaliphatic matrix at its periphery to the vicinal polyaromatics, through linking to ferulic acid. The silencing of the StKCS6 gene led to non-significant alterations in suberin structure, showing the relatively minor role of the very-long chain (>C28) fatty acids in potato suberin composition. The silencing of CYP86A33 gene impaired significantly suberin production and disrupted the biosynthesis of acylglycerol structures, proving the relevance of the latter and thus of the glycerol - α,ω-diacid - glycerol unit for the typical suberin lamellar organization. The silencing of the FHT gene led to a lower frequency of ferulate linkages in suberin polyester but to more polyphenolic guaiacyl units as seen by FTIR analyses in the intact polymer.

A unique glyceryl diglycoside identified in the thermophilic, radiation-resistant bacterium Rubrobacter xylanophilus.

The solute pool of the actinobacterium Rubrobacter xylanophilus has been investigated as a function of the growth temperature and concentration of NaCl in the medium (Empadinhas et al. Extremophiles 11: 667-673, 2007). Changing the carbon source from glucose to maltose in a minimal growth medium led to the accumulation of an unknown organic compound whose structure was investigated by NMR and confirmed by chemical synthesis in the present study as: (2R)-2-(1-O-α-D-mannopyranosyl)-3-(1-O-α-D-glucopyranosyl)-D-glycerate (MGlyG). In addition to this newly identified diglycoside, the solute pool of R. xylanophilus included trehalose, mannosylglycerate, di-myo-inositol phosphate and di-N-acetyl-glucosamine phosphate. The structure of MGlyG was established by NMR and confirmed by chemical synthesis. The availability of g-amounts of the synthetic material allowed us to perform stabilization tests on three model enzymes (malate dehydrogenase, staphylococcal nuclease, and lysozyme), and compare the efficacy of MGlyG with other natural glyceryl glycosides, such as α-D-mannosyl-D-glycerate, α-D-glucosyl-D-glycerate and α-D-glucosyl-(1 → 6)-α-D-glucosyl-(1 → 2)-D-glycerate.

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via ß-alanine.

Microbial fermentation of renewable feedstocks into plastic monomers can decrease our fossil dependence and reduce global CO2 emissions. 3-Hydroxypropionic acid (3HP) is a potential chemical building block for sustainable production of superabsorbent polymers and acrylic plastics. With the objective of developing Saccharomyces cerevisiae as an efficient cell factory for high-level production of 3HP, we identified the ß-alanine biosynthetic route as the most economically attractive according to the metabolic modeling. We engineered and optimized a synthetic pathway for de novo biosynthesis of ß-alanine and its subsequent conversion into 3HP using a novel ß-alanine-pyruvate aminotransferase discovered in Bacillus cereus. The final strain produced 3HP at a titer of 13.7±0.3gL(-1) with a 0.14±0.0C-molC-mol(-1) yield on glucose in 80h in controlled fed-batch fermentation in mineral medium at pH 5, and this work therefore lays the basis for developing a process for biological 3HP production.

LsrF, a coenzyme A-dependent thiolase, catalyzes the terminal step in processing the quorum sensing signal autoinducer-2.

The quorum sensing signal autoinducer-2 (AI-2) regulates important bacterial behaviors, including biofilm formation and the production of virulence factors. Some bacteria, such as Escherichia coli, can quench the AI-2 signal produced by a variety of species present in the environment, and thus can influence AI-2-dependent bacterial behaviors. This process involves uptake of AI-2 via the Lsr transporter, followed by phosphorylation and consequent intracellular sequestration. Here we determine the metabolic fate of intracellular AI-2 by characterizing LsrF, the terminal protein in the Lsr AI-2 processing pathway. We identify the substrates of LsrF as 3-hydroxy-2,4-pentadione-5-phosphate (P-HPD, an isomer of AI-2-phosphate) and coenzyme A, determine the crystal structure of an LsrF catalytic mutant bound to P-HPD, and identify the reaction products. We show that LsrF catalyzes the transfer of an acetyl group from P-HPD to coenzyme A yielding dihydroxyacetone phosphate and acetyl-CoA, two key central metabolites. We further propose that LsrF, despite strong structural homology to aldolases, acts as a thiolase, an activity previously undescribed for this family of enzymes. With this work, we have fully characterized the biological pathway for AI-2 processing in E. coli, a pathway that can be used to quench AI-2 and control quorum-sensing-regulated bacterial behaviors.

Mannosylglucosylglycerate biosynthesis in the deep-branching phylum Planctomycetes: characterization of the uncommon enzymes from Rhodopirellula baltica.

The biosynthetic pathway for the rare compatible solute mannosylglucosylglycerate (MGG) accumulated by Rhodopirellula baltica, a marine member of the phylum Planctomycetes, has been elucidated. Like one of the pathways used in the thermophilic bacterium Petrotoga mobilis, it has genes coding for glucosyl-3-phosphoglycerate synthase (GpgS) and mannosylglucosyl-3-phosphoglycerate (MGPG) synthase (MggA). However, unlike Ptg. mobilis, the mesophilic R. baltica uses a novel and very specific MGPG phosphatase (MggB). It also lacks a key enzyme of the alternative pathway in Ptg. mobilis - the mannosylglucosylglycerate synthase (MggS) that catalyses the condensation of glucosylglycerate with GDP-mannose to produce MGG. The R. baltica enzymes GpgS, MggA, and MggB were expressed in E. coli and characterized in terms of kinetic parameters, substrate specificity, temperature and pH dependence. This is the first characterization of genes and enzymes for the synthesis of compatible solutes in the phylum Planctomycetes and for the synthesis of MGG in a mesophile.

A Unique Pool of Compatible Solutes on Rhodopirellula baltica, Member of the Deep-Branching Phylum Planctomycetes.

The intracellular accumulation of small organic solutes was described in the marine bacterium Rhodopirellula baltica, which belongs to the globally distributed phylum Planctomycetes whose members exhibit an intriguing lifestyle and cell morphology. Sucrose, α-glutamate, trehalose and mannosylglucosylglycerate (MGG) are the main solutes involved in the osmoadaptation of R. baltica. The ratio and total intracellular organic solutes varied significantly in response to an increase in salinity, temperature and nitrogen content. R. baltica displayed an initial response to both osmotic and thermal stresses that includes α-glutamate accumulation. This trend was followed by a rather unique and complex osmoadaptation mechanism characterized by a dual response to sub-optimal and supra-optimal salinities. A reduction in the salinity to sub-optimal conditions led primarily to the accumulation of trehalose. In contrast, R. baltica responded to salt stress mostly by increasing the intracellular levels of sucrose. The switch between the accumulation of trehalose and sucrose was by far the most significant effect caused by increasing the salt levels of the medium. Additionally, MGG accumulation was found to be salt- as well as nitrogen-dependent. MGG accumulation was regulated by nitrogen levels replacing α-glutamate as a K(+) counterion in nitrogen-poor environments. This is the first report of the accumulation of compatible solutes in the phylum Planctomycetes and of the MGG accumulation in a mesophilic organism.

Remarkable inertness of copper(II) chelates of cyclen-based macrobicycles with two trans-N-acetate arms.

Two cross-bridged cyclen-based macrocycles with two trans-N-acetic acid arms, one having a dibenzofuran (DBF) moiety as the bridge, H2L1, and the other a diphenyl ether (DPE) one, H2L2, were synthesized. Both compounds behave as "proton sponges." The thermodynamic stability constants for the Cu(2+), Zn(2+), Al(3+), and Ga(3+) complexes of both compounds were determined. They exhibit an excellent thermodynamic selectivity for copper(II), ensuring that metal ions largely present in the human body do not interfere with the copper(II) chelates. All complexes are very slow to form, and [CuL2] and [CuL1] are extremely inert to demetallate, especially [CuL2]. The acid-assisted dissociation of [CuL1] led to a half-life of 4.28 h in 5 M HCl at 363.2 K, while [CuL2] needed harsher conditions of 12 M HCl at 363.2 K with a half-life of 30.8 days. To the best of our knowledge, [CuL2] exhibits the highest half-life value for a copper(II) complex of a polyazamacrocycle derivative reported in the literature until now. Single crystal X-ray diffraction determined for [Cu(H2L1)](ClO4)2 showed the copper center in a distorted octahedral environment bound to the N4O donors of the macrobicycle and one oxygen atom from a carboxylic arm, while for [CuL2] it showed the copper center in a trigonal bipyramid geometry only bound to the donors of the macrobicycle and leaving the carboxylate arms away from the coordination sphere. UV-vis-NIR and X-band EPR spectra showed that in [CuL1] the copper center adopts a distorted compressed octahedral environment, which is the only structure found in solution for this complex, while in [CuL2] a similar environment was found in the first stages of its slow formation but reached a square-pyramidal geometry upon stabilization. The acetate arms play therefore an important role during the formation of the complex, as revealed by the comparison of its complexation behavior with the corresponding parent compounds.

Harnessing the flexibility of peptidic scaffolds to control their copper(II)-coordination properties: a potentiometric and spectroscopic study.

Designing small peptides that are capable of binding Cu(2+) ions mainly through the side-chain functionalities is a hard task because the amide nitrogen atoms strongly compete for Cu(2+) ion coordination. However, the design of such peptides is important for obtaining biomimetic small systems of metalloenyzmes as well as for the development of artificial systems. With this in mind, a cyclic decapeptide, C-Asp, which contained three His residues and one Asp residue, and its linear derivative, O-Asp, were synthesized. The C-Asp peptide has two Pro-Gly ß-turn-inducer units and, as a result of cyclization, and as shown by CD spectroscopy, its backbone is constrained into a more defined conformation than O-Asp, which is linear and contains a single Pro-Gly unit. A detailed potentiometric, mass spectrometric, and spectroscopic study (UV/Vis, CD, and EPR spectroscopy) showed that at a 1:1 Cu(2+)/peptide ratio, both peptides formed a major [CuHL](2+) species in the pH range 5.0-7.5 (C-Asp) and 5.5-7.0 (O-Asp). The corrected stability constants of the protonated species (log K*(CuH(O-Asp))=9.28 and log K*(CuH(C-Asp))=10.79) indicate that the cyclic peptide binds Cu(2+) ions with higher affinity. In addition, the calculated value of K(eff) shows that this higher affinity for Cu(2+) ions prevails at all pH values, not only for a 1:1 ratio but even for a 2:1 ratio. The spectroscopic data of both [CuHL](2+) species are consistent with the exclusive coordination of Cu(2+) ions by the side-chain functionalities of the three His residues and the Asp residue in a square-planar or square-pyramidal geometry. Nonetheless, although these data show that, upon metal coordination, both peptides adopt a similar fold, the larger conformational constraints that are present in the cyclic scaffold results in different behaviour for both [CuHL](2+) species. CD and NMR analysis revealed the formation of a more rigid structure and a slower Cu(2+)-exchange rate for [CuH(C-Asp)](2+) compared to [CuH(O-Asp](2+). This detailed comparative study shows that cyclization has a remarkable effect on the Cu(2+)-coordination properties of the C-Asp peptide, which binds Cu(2+) ions with higher affinity at all pH values, stabilizes the [CuHL](2+) species in a wider pH range, and has a slower Cu(2+)-exchange rate compared to O-Asp.

Organic solutes in the deepest phylogenetic branches of the Bacteria: identification of α(1-6)glucosyl-α(1-2)glucosylglycerate in Persephonella marina.

The accumulation of organic solutes was investigated in the thermophilic bacteria Persephonella marina and Marinitoga piezophila, two representatives of the deepest lineages in the domain Bacteria. These organisms grow optimally at around 70 °C in medium containing 3 % NaCl. A new disaccharide, accumulating in Persephonella marina, was identified as α(1-6)glucosyl-α(1-2)glucosylglycerate (GGG), by nuclear magnetic resonance. This identification was validated by comparison with the spectra of the compound obtained by chemical synthesis. Besides GGG, the solute pool of Persephonella marina comprised ß-glutamate, di-myo-inositol-1,3'-phosphate and 2-O-α-glucosylglycerate. In contrast, amino acids such as α-glutamate, proline and alanine were the dominant components of the solute pool of Marinitoga piezophila and sugar derivatives were absent. The ability of GGG to protect protein structure against heat denaturation was assessed using model proteins. A genomic search for the biosynthetic pathways of known ionic solutes in Aquificales and Thermotogales shows the inability of this analysis to predict the nature of compatible solutes and underlines the need for efficient cultivation techniques.

Mannosylglycerate stabilizes staphylococcal nuclease with restriction of slow ß-sheet motions.

Mannosylglycerate is a compatible solute typical of thermophilic marine microorganisms that has a remarkable ability to protect proteins from thermal denaturation. This ionic solute appears to be a universal stabilizing agent, but the extent of protection depends on the specific protein examined. To understand how mannosylglycerate confers protection, we have been studying its influence on the internal motions of a hyperstable staphylococcal nuclease (SNase). Previously, we found a correlation between the magnitude of protein stabilization and the restriction of fast backbone motions. We now report the effect of mannosylglycerate on the fast motions of side-chains and on the slower unfolding motions of the protein. Side-chain motions were assessed by (13)CH(3) relaxation measurements and model-free analysis while slower unfolding motions were probed by H/D exchange measurements at increasing concentrations of urea. Side-chain motions were little affected by the presence of different concentrations of mannosylglycerate or even by the presence of urea (0.25M), and show no correlation with changes in the thermodynamic stability of SNase. Native hydrogen exchange experiments showed that, contrary to reports on other stabilizing solutes, mannosylglycerate restricts local motions in addition to the global motions of the protein. The protein unfolding/folding pathway remained undisturbed in the presence of mannosylglycerate but the solute showed a specific effect on the local motions of ß-sheet residues. This work reinforces the link between solute-induced stabilization and restriction of protein motions at different timescales, and shows that the solute preferentially affects specific structural elements of SNase.

Processing the interspecies quorum-sensing signal autoinducer-2 (AI-2): characterization of phospho-(S)-4,5-dihydroxy-2,3-pentanedione isomerization by LsrG protein.

The molecule (S)-4,5-dihydroxy-2,3-pentanedione (DPD) is produced by many different species of bacteria and is the precursor of the signal molecule autoinducer-2 (AI-2). AI-2 mediates interspecies communication and facilitates regulation of bacterial behaviors such as biofilm formation and virulence. A variety of bacterial species have the ability to sequester and process the AI-2 present in their environment, thereby interfering with the cell-cell communication of other bacteria. This process involves the AI-2-regulated lsr operon, comprised of the Lsr transport system that facilitates uptake of the signal, a kinase that phosphorylates the signal to phospho-DPD (P-DPD), and enzymes (like LsrG) that are responsible for processing the phosphorylated signal. Because P-DPD is the intracellular inducer of the lsr operon, enzymes involved in P-DPD processing impact the levels of Lsr expression. Here we show that LsrG catalyzes isomerization of P-DPD into 3,4,4-trihydroxy-2-pentanone-5-phosphate. We present the crystal structure of LsrG, identify potential catalytic residues, and determine which of these residues affects P-DPD processing in vivo and in vitro. We also show that an lsrG deletion mutant accumulates at least 10 times more P-DPD than wild type cells. Consistent with this result, we find that the lsrG mutant has increased expression of the lsr operon and an altered profile of AI-2 accumulation and removal. Understanding of the biochemical mechanisms employed by bacteria to quench signaling of other species can be of great utility in the development of therapies to control bacterial behavior.