Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties.     

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [¹⁴C]Gal from UDP-[¹⁴C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)₇ and anhydrous hydrogen fluoride-deglycosylated d(AO)₅₁. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [¹⁴C]Gal to (AO)₇, and the resulting product co-eluted with (AO)₇ by reverse-phase HPLC. Acid hydrolysis of the [¹⁴C]Gal-(AO)₇ product released ¹⁴C-radiolabel as Gal only. Base hydrolysis of the [¹⁴C]Gal-(AO)₇ product released a ¹⁴C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg²⁺ or Mn²⁺ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.     

Effects of X-rays and Colchicine on Pollen of Trichosanthes anguina L. (Cucurbitaceae)

Pollen grains of M, and C, generation plants of Trichosanthes anguina L. were studied after treatment of seeds with X-rays and colchicine respectively. Pollen sterility increased with increase in X-ray dose and colchicine concentration. The polar axis of X-irradiated populations was shorter than that of controls. But colchicine treatment resulted in larger pollen grains than the controls, although the exine did not increase as much. Ex-perimentally-produced tetraploids had larger pollen grains than diploids and triploids. The triploids were characterised by changes in pore morphology. Of 293 treated plants examined, 11 aberrant plants were isolated in the M, and C, generations. These plants had large and small fertile pollen grains, and also showed treatment effects in external morphology and chromosomal aberration during meiosis. The changes in polar diameter were not associated with variation in chromosomal material.     

Genome Sequencing Unveils a Novel Sea Enterotoxin-Carrying PVL Phage in Staphylococcus aureus ST772 from India

Staphylococcus aureus is a major human pathogen, first recognized as a leading cause of hospital-acquired infections. Community-associated S. aureus (CA-SA) pose a greater threat due to increase in severity of infection and disease among children and healthy adults. CA-SA strains in India are genetically diverse, among which is the sequence type (ST) 772, which has now spread to Australia, Europe and Japan. Towards understanding the genetic characteristics of ST772, we obtained draft genome sequences of five relevant clinical isolates and studied the properties of their PVL-carrying prophages, whose presence is a defining hallmark of CA-SA. We show that this is a novel prophage, which carries the structural genes of the hlb-carrying prophage and includes the sea enterotoxin. This architecture probably emerged early within the ST772 lineage, at least in India. The sea gene, unique to ST772 PVL, despite having promoter sequence characteristics typical of low expression, appears to be highly expressed during early phase of growth in laboratory conditions. We speculate that this might be a consequence of its novel sequence context. The crippled nature of the hlb-converting prophage in ST772 suggests that widespread mobility of the sea enterotoxin might be a selective force behind its ‘transfer’ to the PVL prophage. Wild type ST772 strains induced strong proliferative responses as well as high cytotoxic activity against neutrophils, likely mediated by superantigen SEA and the PVL toxin respectively. Both proliferation and cytotoxicity were markedly reduced in a cured ST772 strain indicating the impact of the phage on virulence. The presence of SEA alongside the genes for the immune system-modulating PVL toxin may contribute to the success and virulence of ST772.     

Production of 2,3-Butanediol from Sucrose by a <Emphasis Type="Italic">Klebsiella</Emphasis> Species

Chemical 2,3-butanediol is an important platform compound possessing diverse industrial applications. So far, it is mainly produced by using petrochemical feedstock which is associated with high cost and adverse environmental impacts. Hence, finding alternative routes (e.g., via fermentation using renewable carbon sources) to produce 2,3-butanediol are urgently needed. In this study, we report a wild-type Klebsiella sp. strain XRM21, which is capable of producing 2,3-butanediol from a wide variety of carbon sources including glucose, sucrose, xylose, and glycerol. Among them, fermentation of sucrose leads to the highest production of 2,3-butanediol. To maximize the production of 2,3-butanediol, fermentation conditions were first optimized for strain XMR21 by using response surface methodology (RSM) in batch reactors. Subsequently, a fed-batch fermentation strategy was designed based on the optimized parameters, where 91.2 g/L of 2,3-butanediol could be produced from substrate sucrose dosing in 100 g/L for three times. Moreover, random mutagenesis of stain XMR21 resulted in a highly productive mutant strain, capable of producing 119.4 and 22.5 g/L of 2,3-butanediol and ethanol under optimized fed-batch fermentation process within 65 h with a total productivity of 2.18 g/L/h, which is comparable to the reported highest 2,3-butanediol concentration produced by previous strains. This study provides a potential strategy to produce industrially important 2,3-butanediol from low-cost sucrose.     

Effect of Portal Glucose Sensing on Systemic Glucose Levels in SD and ZDF Rats

BackgroundThe global epidemic of Type-2-Diabetes (T2D) highlights the need for novel therapeutic targets and agents. Roux-en-Y-Gastric-Bypass (RYGB) is the most effective treatment. Studies investigating the mechanisms of RYGB suggest a role for post-operative changes in portal glucose levels. We investigate the impact of stimulating portal glucose sensors on systemic glucose levels in health and T2D, and evaluated the role of sodium-glucose-cotransporter-3 (SGLT3) as the possible sensor.MethodsSystemic glucose and hormone responses to portal stimulation were measured. In Sprague-Dawley (SD) rats, post-prandial state was simulated by infusing glucose into the portal vein. The SGLT3 agonist, alpha-methyl-glucopyranoside (αMG), was then added to further stimulate the portal sensor. To elucidate the neural pathway, vagotomy or portal denervation was followed by αMG+glucose co-infusion. The therapeutic potential of portal glucose sensor stimulation was investigated by αMG-only infusion (vs. saline) in SD and Zucker-Diabetic-Fatty (ZDF) rats. Hepatic mRNA expression was also measured.ResultsαMG+glucose co-infusion reduced peak systemic glucose (vs. glucose alone), and lowered hepatic G6Pase expression. Portal denervation, but not vagotomy, abolished this effect. αMG-only infusion lowered systemic glucose levels. This glucose-lowering effect was more pronounced in ZDF rats, where portal αMG infusion increased insulin, C-peptide and GIP levels compared to saline infusions.ConclusionsThe portal vein is capable of sensing its glucose levels, and responds by altering hepatic glucose handling. The enhanced effect in T2D, mediated through increased GIP and insulin, highlights a therapeutic target that could be amenable to pharmacological modulation or minimally-invasive surgery.