Host cell factors involved in classical swine fever virus entry.

Classical swine fever virus (CSFV) is an ancient pathogen that continues to pose a threat to animal agriculture worldwide. The virus belongs to the genus Pestivirus and the family Flaviviridae. It causes a multisystemic disease that affects only pigs and is responsible for significant economic losses. CSFV infection is probably a multistep process that involves the proteins in the virus envelope and more than one receptor in the membrane of permissive cells. To date, the cellular receptors essential for CSFV entry and their detailed functions during this process remains unknown. All the viral envelope proteins Erns, E1 and E2 are involved in the entry process to some extent and the experimental approaches conducted until now have helped to unveil their contributions. This review aims to provide an overview of current knowledge on cellular molecules described to be involved in CSFV entry, including complement regulatory protein 46 (CD46), heparan sulphate (HS), Laminin receptor, Integrin ß3, Annexin II, MERKT and ADAM17. This knowledge would not only help to understand the molecular mechanisms involved in pestivirus infection, but also provide a rational basis for the development of nonvaccinal alternatives for CSFV control.

Effect of temperature and nucleotide on the binding of BiP chaperone to a protein substrate.

BiP (immunoglobulin heavy-chain binding protein) is a Hsp70 monomeric ATPase motor that plays broad and crucial roles in maintaining proteostasis inside the cell. Structurally, BiP is formed by two domains, a nucleotide-binding domain (NBD) with ATPase activity connected by a flexible hydrophobic linker to the substrate-binding domain. While the ATPase and substrate binding activities of BiP are allosterically coupled, the latter is also dependent on nucleotide binding. Recent structural studies have provided new insights into BiP's allostery; however, the influence of temperature on the coupling between substrate and nucleotide binding to BiP remains unexplored. Here, we study BiP's binding to its substrate at the single molecule level using thermo-regulated optical tweezers which allows us to mechanically unfold the client protein and explore the effect of temperature and different nucleotides on BiP binding. Our results confirm that the affinity of BiP for its protein substrate relies on nucleotide binding, by mainly regulating the binding kinetics between BiP and its substrate. Interestingly, our findings also showed that the apparent affinity of BiP for its protein substrate in the presence of nucleotides remains invariable over a wide range of temperatures, suggesting that BiP may interact with its client proteins with similar affinities even when the temperature is not optimal. Thus, BiP could play a role as a "thermal buffer" in proteostasis.

New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate.

Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. This enzyme has several analogies with agmatinase, which catalyzes the hydrolysis of agmatine into putrescine and urea. However, this contrasts with the highlighted specificity that each one presents for their respective substrate. A comparison of available crystal structures for arginases reveals an important difference in the extension of two loops located in the entrance of the active site. The first, denominated loop A (I129-L140) contains the residues that interact with the alpha carboxyl group or arginine of arginase, and the loop B (D181-P184) contains the residues that interact with the alpha amino group of arginine. In this work, to determine the importance of these loops in the specificity of arginase, single, double, and triple arginase mutants in these loops were constructed, as well as chimeras between type I human arginase and E. coli agmatinase. In previous studies, the substitution of N130D in arginase (in loop A) generated a species capable of hydrolyzing arginine and agmatine. Now, the specificity of arginase is completely altered, generating a chimeric species that is only active with agmatine as a substrate, by substituting I129T, N130Y, and T131A together with the elimination of residues P132, L133, and T134. In addition, Quantum Mechanic/Molecular Mechanic (QM/MM) calculations were carried out to study the accommodation of the substrates in in the active site of this chimera. With these results it is concluded that this loop is decisive to discriminate the type of substrate susceptible to be hydrolyzed by arginase. Evidence was also obtained to define the loop B as a structural determinant for substrate affinity. Concretely, the double mutation D181T and V182E generate an enzyme with an essentially unaltered kcat value, but with a significantly increased Km value for arginine and a significant decrease in affinity for its product ornithine.

First Identification of Reinfection by a Genetically Different Variant of SARS-CoV-2 in a Homeless Person from the Metropolitan Area of Santiago, Chile.

The identification and tracking of SARS-CoV-2 infected patients in the general population are essential components of the global strategy to limit the COVID-19 viral spread, specifically for maintaining traceability and suppressing the resurgence of local outbreaks. Public health programs that include continuous RT-qPCR testing for COVID-19 in the general population, viral sequencing, and genomic surveillance for highly contagious forms of the virus have allowed for the identification of SARS-CoV-2 infections and reinfections. This work identified SARS-CoV-2 reinfection in a homeless person, which occurred 58 days after the first COVID-19 diagnosis. Genomic sequencing identified a different Nextstrain classification clade (20A and 20B) and PANGO lineage, with a divergence of 4 single nucleotide variants (SNVs) in S and ORF1ab genes, suggesting reinfection by different viral variants. This study is the first from the great metropolitan area of Santiago, Chile, one of the top ten countries in the world to live during the COVID-19 pandemic. We support the importance of performing intensive genomic surveillance programs in the whole population and high-risk groups, such as homeless people, nearly 20 thousand people in Chile, and have limited access to health care services and poor viral traceability.

Analysis by real-time PCR of five transport and conservation mediums of nasopharyngeal swab samples to COVID-19 diagnosis in Santiago of Chile.

Due to the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit mediums, including DNA/RNA Shield™, NAT, VTM-N, Ezmedlab™, and phosphate-buffered saline (PBS), for NPSs from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield™ shows higher relative fluorescence units (RFUs) and lower Cq values for the reference gene. Collectively, our results suggest that the PBS medium could compromise the sample diagnosis because of its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N, and DNA/RNA Shield™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis.

Crystal Structure of Escherichia coli Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity.

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer's, Parkinson's, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.

The Rapid Antigen Detection Test for SARS-CoV-2 Underestimates the Identification of COVID-19 Positive Cases and Compromises the Diagnosis of the SARS-CoV-2 (K417N/T, E484K, and N501Y) Variants.

Timely detection of severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been the gold- strategy for identifying positive cases during the current pandemic. However, faster and less expensive methodologies are also applied for the massive diagnosis of COVID-19. In this way, the rapid antigen test (RAT) is widely used. However, it is necessary to evaluate its detection efficiency considering the current pandemic context with the circulation of new viral variants. In this study, we evaluated the sensitivity and specificity of RAT (SD BIOSENSOR, South Korea), widely used for testing and SARS-CoV-2 diagnosis in Santiago of Chile. The RAT showed a 90% (amplification range of 20 ≤ Cq <25) and 10% (amplification range of 25 ≤ Cq <30) of positive SARS-CoV-2 cases identified previously by RT-qPCR. Importantly, a 0% detection was obtained for samples within a Cq value>30. In SARS-CoV-2 variant detection, RAT had a 42.8% detection sensitivity in samples with RT-qPCR amplification range 20 ≤ Cq <25 containing the single nucleotide polymorphisms (SNP) K417N/T, N501Y and E484K, associated with beta or gamma SARS-CoV-2 variants. This study alerts for the special attention that must be paid for the use of RAT at a massive diagnosis level, especially in the current scenario of appearance of several new SARS-CoV-2 variants which could generate false negatives and the compromise of possible viral outbreaks.

Gabapentin Inhibits Multiple Steps in the Amyloid Beta Toxicity Cascade.

Oligomeric ß-amyloid peptide (Aß) is one of the main neurotoxic agents of Alzheimer's disease (AD). Oligomers associate to neuronal membranes, forming "pore-like" structures that cause intracellular calcium and neurotransmitter dyshomeostasis, leading to synaptic failure and death. Through molecular screening targeting the C terminal region of Aß, a region involved in the toxic properties of the peptide, we detected an FDA approved compound, gabapentin (GBP), with neuroprotective effects against Aß toxicity. At micromolar concentrations, GBP antagonized peptide aggregation over time and reduced the Aß absorbance plateau to 28% of control. In addition, GBP decreased Aß association to membranes by almost half, and the effects of Aß on intracellular calcium in hippocampal neurons were antagonized without causing effects on its own. Finally, we found that GBP was able to block the synaptotoxicity induced by Aß in hippocampal neurons, increasing post-synaptic currents from 1.7 ± 0.9 to 4.2 ± 0.7 fC and mean relative fluorescence intensity values of SV2, a synaptic protein, from 0.7 ± 0.09 to 1.00 ± 0.08. The results show that GBP can interfere with Aß-induced toxicity by blocking multiple steps, resulting in neuroprotection, which justifies advancing toward additional animal and human studies.

Insights into the Mn2+ Binding Site in the Agmatinase-Like Protein (ALP): A Critical Enzyme for the Regulation of Agmatine Levels in Mammals.

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn2+ for catalytic activity. However, the Mn2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn2+ binding site of the ALP and we propose new putative Mn2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the "central-ALP", which include the putative ligands of Mn2+. The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered Km for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn2+ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.

Insights on the participation of Glu256 and Asp204 in the oligomeric structure and cooperative effects of human arginase type I.

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of L-arginine to L-ornithine and urea, and requires a bivalent cation, especially Mn2+ for its catalytic activity. It is a component of the urea cycle and regulates the intracellular levels of l-arginine, which makes the arginase a target for treatment of vascular diseases and asthma. Mammalian arginases contain an unusual S-shaped motif located at the intermonomeric interface. Until now, the studies were limited to structural role of the motif. Then, our interest was focused on functional aspects and our hypothesis has been that the motif is essential for maintain the oligomeric state, having Arg308 as a central axis. Previously, we have shown that the R308A mutant is monomeric and re-associates to the trimeric-cooperative state in the presence of low concentrations of guanidine chloride. We have now mutated Asp204 that interacts with Arg308 in the neighbor subunit, and also we mutated Glu256, proposed as important for oligomerization. Concretely, the human arginase I mutants D204A, D204E, E256A, E256Q and E256D were generated and examined. No differences were observed in the kinetic parameters at pH 9.5 or in tryptophan fluorescence. However, the D204A and E256Q variants were monomeric. On the other hand, D204E and E256D proved to be trimeric and kinetically cooperative at pH 7.5, whereas hyperbolic kinetics was exhibited by E256A, also trimeric. The results obtained strongly support the importance of the interaction between Arg255 and Glu256 in the cooperative properties of arginase, and Asp204 would be relevant to maintain the oligomeric state through salt bridges with Arg255 and Arg308.

Biosynthesis and characterization of a recombinant eukaryotic allophycocyanin using prokaryotic accessory enzymes.

Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αß subunits that oligomerizes as a trimer (αß)3 . Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and ß subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λmaxAbs , λmaxEm , fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high-quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes.

Functional analysis of the Mn2+ requirement in the catalysis of ureohydrolases arginase and agmatinase - a historical perspective.

Ureohydrolases form a conserved family of enzymes with a strict requirement for divalent metal ions for catalytic activity. They catalyze the hydrolysis of the guanidino group and produce urea. In their active sites six highly conserved amino acid residues form a binding pocket for two catalytically essential metal ions that are needed to activate a water molecule to initiate the hydrolysis of the guanidino group in a nucleophilic attack. Focus in this review is on two members of the ureohydrolase family, the Mn2+-dependent arginase and agmatinase, which play important roles in functions related to replication and cell survival. We will focus in particular on Mn2+ binding interactions, and on how this metal ion contributes to the reaction catalyzed by these enzymes. We also include the agmatinase-like protein (ALP) because it is functionally closely related to agmatinase, also requires at least one Mn2+ ion for catalytic activity, but may possess an active site that differs significantly from all other known ureohydrolases.

Correction: The γ33 subunit of R-phycoerythrin from Gracilaria chilensis has a typical double linked phycourobilin similar to ß subunit.

[This corrects the article DOI: 10.1371/journal.pone.0195656.].

Synthesis and Antibacterial Activity of Metal(loid) Nanostructures by Environmental Multi-Metal(loid) Resistant Bacteria and Metal(loid)-Reducing Flavoproteins.

Microbes are suitable candidates to recover and decontaminate different environments from soluble metal ions, either via reduction or precipitation to generate insoluble, non-toxic derivatives. In general, microorganisms reduce toxic metal ions generating nanostructures (NS), which display great applicability in biotechnological processes. Since the molecular bases of bacterial reduction are still unknown, the search for new -environmentally safe and less expensive- methods to synthesize NS have made biological systems attractive candidates. Here, 47 microorganisms isolated from a number of environmental samples were analyzed for their tolerance or sensitivity to 19 metal(loid)s. Ten of them were highly tolerant to some of them and were assessed for their ability to reduce these toxicants in vitro. All isolates were analyzed by 16S rRNA gene sequencing, fatty acids composition, biochemical tests and electron microscopy. Results showed that they belong to the Enterobacter, Staphylococcus, Acinetobacter, and Exiguobacterium genera. Most strains displayed metal(loid)-reducing activity using either NADH or NADPH as cofactor. While Acinetobacter schindleri showed the highest tellurite ( TeO32- ) and tetrachloro aurate ( AuCl4- ) reducing activity, Staphylococcus sciuri and Exiguobacterium acetylicum exhibited selenite ( SeO32- ) and silver (Ag+) reducing activity, respectively. Based on these results, we used these bacteria to synthetize, in vivo and in vitro Te, Se, Au, and Ag-containing nanostructures. On the other hand, we also used purified E. cloacae glutathione reductase to synthesize in vitro Te-, Ag-, and Se-containing NS, whose morphology, size, composition, and chemical composition were evaluated. Finally, we assessed the putative anti-bacterial activity exhibited by the in vitro synthesized NS: Te-containing NS were more effective than Au-NS in inhibiting Escherichia coli and Listeria monocytogenes growth. Aerobically synthesized TeNS using MF09 crude extracts showed MICs of 45- and 66- µg/ml for E. coli and L. monocytogenes, respectively. Similar MIC values (40 and 82 µg/ml, respectively) were observed for TeNS generated using crude extracts from gorA-overexpressing E. coli. In turn, AuNS MICs for E. coli and L. monocytogenes were 64- and 68- µg/ml, respectively.

The γ33 subunit of R-phycoerythrin from Gracilaria chilensis has a typical double linked phycourobilin similar to ß subunit.

Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ33) from Gracilaria chilensis, is a chromophorylated rod linker associated to (αß)6 hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ33 was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and ß R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ33 subunit that were confirmed between Cys62 and Cys73 (DL-PUB62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB50/61 in the ß subunit of R-PE. The position of single linked PUB at Cys95 and a single linked PEB at Cys172 were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.

Comparative genomic analysis of a new tellurite-resistant Psychrobacter strain isolated from the Antarctic Peninsula.

The Psychrobacter genus is a cosmopolitan and diverse group of aerobic, cold-adapted, Gram-negative bacteria exhibiting biotechnological potential for low-temperature applications including bioremediation. Here, we present the draft genome sequence of a bacterium from the Psychrobacter genus isolated from a sediment sample from King George Island, Antarctica (3,490,622 bp; 18 scaffolds; G + C = 42.76%). Using phylogenetic analysis, biochemical properties and scanning electron microscopy the bacterium was identified as Psychrobacter glacincola BNF20, making it the first genome sequence reported for this species. P. glacincola BNF20 showed high tellurite (MIC 2.3 mM) and chromate (MIC 6.0 mM) resistance, respectively. Genome-wide nucleotide identity comparisons revealed that P. glacincola BNF20 is highly similar (>90%) to other uncharacterized Psychrobacter spp. such as JCM18903, JCM18902, and P11F6. Bayesian multi-locus phylogenetic analysis showed that P. glacincola BNF20 belongs to a polyphyletic clade with other bacteria isolated from polar regions. A high number of genes related to metal(loid) resistance were found, including tellurite resistance genetic determinants located in two contigs: Contig LIQB01000002.1 exhibited five ter genes, each showing putative promoter sequences (terACDEZ), whereas contig LIQB1000003.2 showed a variant of the terZ gene. Finally, investigating the presence and taxonomic distribution of ter genes in the NCBI's RefSeq bacterial database (5,398 genomes, as January 2017), revealed that 2,623 (48.59%) genomes showed at least one ter gene. At the family level, most (68.7%) genomes harbored one ter gene and 15.6% exhibited five (including P. glacincola BNF20). Overall, our results highlight the diverse nature (genetic and geographic diversity) of the Psychrobacter genus, provide insights into potential mechanisms of metal resistance, and exemplify the benefits of sampling remote locations for prospecting new molecular determinants.

Metabolic strategies for the degradation of the neuromodulator agmatine in mammals.

Agmatine (1-amino-4-guanidinobutane), a precursor for polyamine biosynthesis, has been identified as an important neuromodulator with anticonvulsant, antineurotoxic and antidepressant actions in the brain. In this context it has emerged as an important mediator of addiction/satiety pathways associated with alcohol misuse. Consequently, the regulation of the activity of key enzymes in agmatine metabolism is an attractive strategy to combat alcoholism and related addiction disorders. Agmatine results from the decarboxylation of L-arginine in a reaction catalyzed by arginine decarboxylase (ADC), and can be converted to either guanidine butyraldehyde by diamine oxidase (DAO) or putrescine and urea by the enzyme agmatinase (AGM) or the more recently identified AGM-like protein (ALP). In rat brain, agmatine, AGM and ALP are predominantly localised in areas associated with roles in appetitive and craving (drug-reinstatement) behaviors. Thus, inhibitors of AGM or ALP are promising agents for the treatment of addictions. In this review, the properties of DAO, AGM and ALP are discussed with a view to their role in the agmatine metabolism in mammals.

Reduction of Gold (III) and Tellurium (IV) by Enterobacter cloacae MF01 Results in Nanostructure Formation Both in Aerobic and Anaerobic Conditions.

Microorganism survival in the presence of toxic substances such as metal(loid)s lies chiefly on their ability to resist (or tolerate) such elements through specific resistance mechanisms. Among them, toxicant reduction has attracted the attention of researchers because metal(loid)-reducing bacteria are being used to recover and/or decontaminate polluted sites. Particularly, our interest is to analyze the toxicity of gold and tellurium compounds for the environmental microorganism Enterobacter cloacae MF01 and also to explore the generation of nanostructures to be used in future biotechnological processes. Resistance of E. cloacae MF01 to gold and tellurium salts as well as the putative mechanisms involved -both in aerobic and anaerobic growth conditions- was evaluated. These metal(loid)s were selected because of their potential application in biotechnology. Resistance to auric tetrachloride acid (HAuCl4) and potassium tellurite (K2TeO3) was assessed by determining areas of growth inhibition, minimum inhibitory concentrations, and growth curves as well as by viability tests. E. cloacae MF01 exhibited higher resistance to HAuCl4 and K2TeO3 under aerobic and anaerobic conditions, respectively. In general, their toxicity is mediated by the generation of reactive oxygen species and by a decrease of intracellular reduced thiols (RSH). To assess if resistance implies toxicant reduction, intra- and extra-cellular toxicant-reducing activities were evaluated. While E. cloacae MF01 exhibited intra- and extra-cellular HAuCl4-reducing activity, tellurite reduction was observed only intracellularly. Then, Au- and Te-containing nanostructures (AuNS and TeNS, respectively) were synthesized using crude extracts from E. cloacae MF01 and their size, morphology, and chemical composition was evaluated.

Structural models of the different trimers present in the core of phycobilisomes from Gracilaria chilensis based on crystal structures and sequences.

Phycobilisomes (PBS) are accessory light harvesting protein complexes that directionally transfer energy towards photosystems. Phycobilisomes are organized in a central core and rods radiating from it. Components of phycobilisomes in Gracilaria chilensis (Gch) are Phycobiliproteins (PBPs), Phycoerythrin (PE), and Phycocyanin (PC) in the rods, while Allophycocyanin (APC) is found in the core, and linker proteins (L). The function of such complexes depends on the structure of each component and their interaction. The core of PBS from cyanobacteria is mainly composed by cylinders of trimers of α and ß subunits forming heterodimers of Allophycocyanin, and other components of the core including subunits αII and ß18. As for the linkers, Linker core (LC) and Linker core membrane (LCM) are essential for the final emission towards photoreaction centers. Since we have previously focused our studies on the rods of the PBS, in the present article we investigated the components of the core in the phycobilisome from the eukaryotic algae, Gracilaria chilensis and their organization into trimers. Transmission electron microscopy provided the information for a three cylinders core, while the three dimensional structure of Allophycocyanin purified from Gch was determined by X-ray diffraction method and the biological unit was determined as a trimer by size exclusion chromatography. The protein sequences of all the components of the core were obtained by sequencing the corresponding genes and their expression confirmed by transcriptomic analysis. These subunits have seldom been reported in red algae, but not in Gracilaria chilensis. The subunits not present in the crystallographic structure were modeled to build the different composition of trimers. This article proposes structural models for the different types of trimers present in the core of phycobilisomes of Gch as a first step towards the final model for energy transfer in this system.

Biophysical characterization data of the artificial protein Octarellin V.1 and binding test with its X-ray helpers.

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016/S0022-2836(02)01206-8[2]). The protein has been characterized by circular dichroism and fluorescence techniques, in order to obtain data related to its thermo and chemical stability. Moreover, the data for the secondary structure content studied by circular dichroism and infra red techniques is reported for the Octarellin V and V.1. Two crystallization helpers, nanobodies (http://dx.doi.org/10.1038/nprot.2014.039[3]) and αRep (http://dx.doi.org/10.1016/j.jmb.2010.09.048[4]), have been used to create stable complexes. Here we present the data obtained of the binding characterization of the Octarellin V.1 with the crystallization helpers by isothermal titration calorimetry.