ThiN as a Versatile Domain of Transcriptional Repressors and Catalytic Enzymes of Thiamine Biosynthesis.

Thiamine biosynthesis is commonly regulated by a riboswitch mechanism; however, the enzymatic steps and regulation of this pathway in archaea are poorly understood. Haloferax volcanii, one of the representative archaea, uses a eukaryote-like Thi4 (thiamine thiazole synthase) for the production of the thiazole ring and condenses this ring with a pyrimidine moiety synthesized by an apparent bacterium-like ThiC (2-methyl-4-amino-5-hydroxymethylpyrimidine [HMP] phosphate synthase) branch. Here we found that archaeal Thi4 and ThiC were encoded by leaderless transcripts, ruling out a riboswitch mechanism. Instead, a novel ThiR transcription factor that harbored an N-terminal helix-turn-helix (HTH) DNA binding domain and C-terminal ThiN (TMP synthase) domain was identified. In the presence of thiamine, ThiR was found to repress the expression of thi4 and thiC by a DNA operator sequence that was conserved across archaeal phyla. Despite having a ThiN domain, ThiR was found to be catalytically inactive in compensating for the loss of ThiE (TMP synthase) function. In contrast, bifunctional ThiDN, in which the ThiN domain is fused to an N-terminal ThiD (HMP/HMP phosphate [HMP-P] kinase) domain, was found to be interchangeable for ThiE function and, thus, active in thiamine biosynthesis. A conserved Met residue of an extended α-helix near the active-site His of the ThiN domain was found to be important for ThiDN catalytic activity, whereas the corresponding Met residue was absent and the α-helix was shorter in ThiR homologs. Thus, we provide new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveal that thiamine biosynthesis in archaea is regulated by a transcriptional repressor, ThiR, and not by a riboswitch.IMPORTANCE Thiamine pyrophosphate (TPP) is a cofactor needed for the enzymatic activity of many cellular processes, including central metabolism. In archaea, thiamine biosynthesis is an apparent chimera of eukaryote- and bacterium-type pathways that is not well defined at the level of enzymatic steps or regulatory mechanisms. Here we find that ThiN is a versatile domain of transcriptional repressors and catalytic enzymes of thiamine biosynthesis in archaea. Our study provides new insight into residues that distinguish catalytic from noncatalytic ThiN domains and reveals that archaeal thiamine biosynthesis is regulated by a ThiN domain transcriptional repressor, ThiR, and not by a riboswitch.

Conserved active site cysteine residue of archaeal THI4 homolog is essential for thiamine biosynthesis in Haloferax volcanii.

BACKGROUND: Thiamine (vitamin B1) is synthesized de novo by certain yeast, fungi, plants, protozoans, bacteria and archaea. The pathway of thiamine biosynthesis by archaea is poorly understood, particularly the route of sulfur relay to form the thiazole ring. Archaea harbor structural homologs of both the bacterial (ThiS-ThiF) and eukaryotic (THI4) proteins that mobilize sulfur to thiazole ring precursors by distinct mechanisms. RESULTS: Based on comparative genome analysis, halophilic archaea are predicted to synthesize the pyrimidine moiety of thiamine by the bacterial pathway, initially suggesting that also a bacterial ThiS-ThiF type mechanism for synthesis of the thiazole ring is used in which the sulfur carrier ThiS is first activated by ThiF-catalyzed adenylation. The only ThiF homolog of Haloferax volcanii (UbaA) was deleted but this had no effect on growth in the absence of thiamine. Usage of the eukaryotic THI4-type sulfur relay was initially considered less likely for thiamine biosynthesis in archaea, since the active-site cysteine residue of yeast THI4p that donates the sulfur to the thiazole ring by a suicide mechanism is replaced by a histidine residue in many archaeal THI4 homologs and these are described as D-ribose-1,5-bisphosphate isomerases. The THI4 homolog of the halophilic archaea, including Hfx. volcanii (HVO_0665, HvThi4) was found to differ from that of methanogens and thermococci by having a cysteine residue (Cys165) corresponding to the conserved active site cysteine of yeast THI4p (Cys205). Deletion of HVO_0665 generated a thiamine auxotroph that was trans-complemented by a wild-type copy of HVO_0665, but not the modified gene encoding an HvThi4 C165A variant. CONCLUSIONS: Based on our results, we conclude that the archaeon Hfx. volcanii uses a yeast THI4-type mechanism for sulfur relay to form the thiazole ring of thiamine. We extend this finding to a relatively large group of archaea, including haloarchaea, ammonium oxidizing archaea, and some methanogen and Pyrococcus species, by observing that these organisms code for THI4 homologs that have a conserved active site cysteine residue which is likely used in thiamine biosynthesis. Thus, archaeal members of IPR002922 THI4 family that have a conserved cysteine active site should be reexamined for a function in thiamine biosynthesis.

Xanthation of alginate for heavy metal ions removal. Characterization of xanthate-modified alginates and its metal derivatives.

Xanthates are widely used in mining industry as collectors for its high affinity towards metal sulfides and precious metal ores. The possibility of using alginate for xanthation has not been explored yet despite the feasibility by the presence of hydroxyl groups alongside the polymeric chains. Therefore, this work aims to evaluate the alginate as a matrix for xanthation and its application on heavy metal ions removal. In order to obtain green materials, important pararmeter were explored such as the effect of reaction time (4-12 h), type of base (NaOH/KOH) and amount of carbon disulfide (2-10%v/v). Xanthated alginates were analyzed by NMR techniques and evidence of ß-elimination was detected at 5.45 ppm. Furthermore, the presence of S element was confirmed by EDS mapping technique, while XRD showed a semi-crystalline structure. On the other hand, the chemical shifts of δ(C=S) and ν(C=S) bands were found around 863-805 cm-1 and 662-602 cm-1 respectively. Also, a shoulder at 182 ppm is appreciated by NMR in solid state attributed to CS group. According to FESEM analyses, morphology of xanthated alginates is affected by interaction with heavy metal ions. Finally, suitable materials for the removal of heavy metal ions were established at optimum pH values.

Functionalization of an Alginate-Based Material by Oxidation and Reductive Amination.

This research focused on the synthesis of a functional alginate-based material via chemical modification processes with two steps: oxidation and reductive amination. In previous alginate functionalization with a target molecule such as cysteine, the starting material was purified and characterized by UV-Vis, 1H-NMR and HSQC. Additionally, the application of FT-IR techniques during each step of alginate functionalization was very useful, since new bands and spiked signals around the pyranose ring (1200-1000 cm-1) and anomeric region (1000-750 cm-1) region were identified by a second derivative. Additionally, the presence of C1-H1 of ß-D-mannuronic acid residue as well as C1-H1 of α-L-guluronic acid residue was observed in the FT-IR spectra, including a band at 858 cm-1 with characteristics of the N-H moiety from cysteine. The possibility of attaching cysteine molecules to an alginate backbone by oxidation and post-reductive amination processes was confirmed through 13C-NMR in solid state; a new peak at 99.2 ppm was observed, owing to a hemiacetal group formed in oxidation alginate. Further, the peak at 31.2 ppm demonstrates the presence of carbon -CH2-SH in functionalized alginate-clear evidence that cysteine was successfully attached to the alginate backbone, with 185 µmol of thiol groups per gram polymer estimated in alginate-based material by UV-Visible. Finally, it was observed that guluronic acid residue of alginate are preferentially more affected than mannuronic acid residue in the functionalization.

Xanthate-modified alginates for the removal of Pb(II) and Ni(II) from aqueous solutions: A brief analysis of alginate xanthation.

Mining is the most common activity that introduces heavy metal ions into aquatic ecosystems, especially in low income-developing nations where governments are implementing stricter regulations for industrial wastewater. In this context, this work is focused on the application of xanthate-modified alginates for the removal of Pb(II) and Ni(II) from aqueous solutions. In order to confirm the presence of xanthate groups alongside alginate chains, characterization by second-derivative FT-IR was carried out and significance evidence attributed to xanthate groups was found at around 1062-1079 cm-1, 829-845 cm-1 and 620-602 cm-1. In addition to this, thermogravimetric analysis and differential scanning calorimetry were employed to explore thermal properties of modified alginates. According to these results, enthalpy changes (∆H) characteristic of dehydration and collapse of biopolymeric structure were estimated as +11.41 J/g and -6.83 J/g, respectively. Furthermore, the presence of S element was confirmed by EDS mapping technique, whereas FESEM image showed a cracked and homogeneous surface distribution. On the other hand, the effect of important parameters such as pH, dosage, initial concentration as well as Langmuir and Freundlich isotherm were deeply discussed. Finally, rheological measurements were performed aiming to investigate the gel-like viscoelastic features associated to nickel xanthate compound.

CoproID predicts the source of coprolites and paleofeces using microbiome composition and host DNA content.

Shotgun metagenomics applied to archaeological feces (paleofeces) can bring new insights into the composition and functions of human and animal gut microbiota from the past. However, paleofeces often undergo physical distortions in archaeological sediments, making their source species difficult to identify on the basis of fecal morphology or microscopic features alone. Here we present a reproducible and scalable pipeline using both host and microbial DNA to infer the host source of fecal material. We apply this pipeline to newly sequenced archaeological specimens and show that we are able to distinguish morphologically similar human and canine paleofeces, as well as non-fecal sediments, from a range of archaeological contexts.

Chemical modification of sodium alginate with thiosemicarbazide for the removal of Pb(II) and Cd(II) from aqueous solutions.

A new material (AlgOx-TSC), based on alginate (Alg) chemically modified with thiosemicarbazide (TSC), has been synthesized and tested as an effective biomaterial to remove Pb(II) and Cd(II) ions in aqueous solutions. The synthesis was carried out by controlling the following steps, i/partial oxidation process of alginate in NaIO4 to obtain AlgOx, ii/reacting of AlgOx, at 40-45 °C, with TSC in NaBH4. AlgOx-TSC has been characterized by Field Emission Scanning Electron Microscopy (FESEM/EDS), Fourier Transform Infrared Spectroscopy (ATR-IR), solid state 13C NMR spectroscopy and Point of Zero Charge (pHPZC) measuremenmts. In order to enhance the sorption process, the effect of contact time, sorbent dosage, initial concentration and reusability of the novel sorbent were investigated becoming the AlgOx-TSC a promising material capable of removing high concentrations of heavy metal ions such as Pb(II) (up to 950 mg/g at pH 3) and Cd(II) (up to 300 mg/g at pH 7) in aqueous solutions.