Expression of the recombinant xylose dehydrogenase and xylonolactonase from <i>C. crescentus</i>.

<p>Lane M, prestained protein ladder; lane 1, BL21ΔxylAB harboring pACYCduet-1; lane 2, BL21ΔxylAB harboring pA-xdh; lane 3, BL21ΔxylAB harboring pA-xylC; lane 4, BL21ΔxylAB harboring pA-xdhxylC.</p

Time profiles for cell density (OD₆₀₀), residual xylose, xylonate and xylonolactone concentrations in the culture broth during fed-batch culture of the finally engineered strain BL21ΔxylAB/pA-xdhxylC.

<p>Time profiles for cell density (OD₆₀₀), residual xylose, xylonate and xylonolactone concentrations in the culture broth during fed-batch culture of the finally engineered strain BL21ΔxylAB/pA-xdhxylC.</p

Comparison of xylonate and xylonolactone production of several different strains.

<p>Data were obtained after each strain was induced for 12 h in liquid LB medium supplemented with 1 g/L xylose. BL21/pA-xdh, strain BL21 star(DE3) expressing <i>C. crescentus</i> xylose dehydrogenase; BL21ΔxylAB/pA-xdh, knockout of native <i>xylA</i> and <i>xylB</i> while expressing <i>C. crescentus</i> xylose dehydrogenase; BL21ΔxylAB/pA-xdhxylC, knockout of native <i>xylA</i> and <i>xylB</i> while coexpressing <i>C. crescentus</i> xylose dehydrogenase and xylonolactonase.</p

Detection of xylose, xylonate and xylonolactone by ion chromatography.

<p>A, 1 ppm xylose, corresponding to the retention time of 2.75 min; B, 200 ppm xylonolactone, corresponding to the retention time of 2.61 min; C, 100 ppm xylonate, corresponding to the retention time of 3.43 min; D, detection of the enzymatic product of xylose dehydrogenase; E, detection of the enzymatic product of xylonolactonase; F, ion chromatogram of the extracellular metabolites of strain BL21ΔxylAB/pA-xdhxylC after being induced for 12 h. Both the enzymatic reaction mixtures and culture broth supernatant were appropriately diluted for ion chromatography analysis.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Tiacumicin Congeners with Improved Antibacterial Activity from a Halogenase-Inactivated Mutant

Tiacumicin B (<b>1</b>, also known as fidaxomicin or difimicin) is a marketed drug for the treatment of <i>Clostridium difficile</i> infections. The biosynthetic pathway of <b>1</b> has been studied in <i>Dactylosporangium aurantiacum</i> subsp. <i>hamdenensis</i> NRRL 18085 and has enabled the identification of TiaM as a tailoring dihalogenase. Herein we report the isolation, structure elucidation, and bioactivity evaluation of 14 tiacumicin congeners (including 11 new ones) from the <i>tiaM</i>-inactivated mutant. A new tiacumicin congener, <b>3</b>, with a propyl group at C-7‴ of the aromatic ring was found to exhibit improved antibacterial activity

Probing the interaction between NatA and the ribosome for co-translational protein acetylation

<div><p>N-terminal acetylation is among the most abundant protein modifications in eukaryotic cells. Over the last decade, significant progress has been made in elucidating the function of N-terminal acetylation for a number of diverse systems, involved in a wide variety of biological processes. The enzymes responsible for the modification are the N-terminal acetyltransferases (NATs). The NATs are a highly conserved group of enzymes in eukaryotes, which are responsible for acetylating over 80% of the soluble proteome in human cells. Importantly, many of these NATs act co-translationally; they interact with the ribosome near the exit tunnel and acetylate the nascent protein chain as it is being translated. While the structures of many of the NATs have been determined, the molecular basis for the interaction with ribosome is not known. Here, using purified ribosomes and NatA, a very well-studied NAT, we show that NatA forms a stable complex with the ribosome in the absence of other stabilizing factors and through two conserved regions; primarily through an N-terminal domain and an internal basic helix. These regions may orient the active site of the NatA to face the peptide emerging from the exit tunnel. This work provides a framework for understanding how NatA and potentially other NATs interact with the ribosome for co-translational protein acetylation and sets the foundation for future studies to decouple N-terminal acetyltransferase activity from ribosome association.</p></div

Toxicity effects of DnBP on biochemistry parameters of <i>E</i>. <i>fetida</i>.

<p>(a) Total protein content; (b) SOD activity; (c) POD activity; and (d) ROS activity were determined after treated for 7d, 14d, 21d and 28d in spiked natural soil CK, B-1, B-2, B-3, and B-4 (n = 4; error bars, SEM/mean values of standard errors). The spiked concentrations of DnBP were 0, 1, 2.5, 5, and 10 mg kg^-1 soil. Asterisk shows significant difference at <i>p</i><0.05 level compared to the control; double asterisks show significant difference at <i>p</i><0.01 level compared to the control.</p

Genotoxicity of DnBP analyzed by comet assay.

<p>(a) length of tail; (b) tail DNA ratio; (c) tail moment and (d) olive tail moment of coelomocyte in treated <i>E</i>. <i>fetida</i> after treated for 7d, 14d, 21d and 28d in spiked natural soil CK, B-1, B-2, B-3, and B-4 (n = 4; error bars, SEM). Length of tail (TL) means tail length in arbitrary units; tail DNA ratio means relative ratio of DNA in the comet tail; tail moment (TM) means integrated value of DNA density multiplied by the migration distance; and Olive tail moment (OTM) means the product of the distance between the center of gravity of the head and the center of gravity of the tail and percent tail DNA. Refer other annotates to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151128#pone.0151128.g001" target="_blank">Fig 1</a>.</p

Conservation analysis and electrostatic surface of NatA show two regions important for ribosome binding.

<p>(A) A cartoon representation of the NatA complex. Naa10 is shown in cyan, Naa15 in green, and the peptide substrate in magenta. The N-terminus and internal basic helix are indicated, as is the active site where N-termini are acetylated. (B) Conservation map of the NatA complex. Magenta areas represent regions of high sequence conservation and cyan areas represent regions of low sequence conservation. (C) Electrostatic potential map of NatA. Blue areas represented regions which are electropositive, and red areas represent regions which are electronegative. Electropositive region 1 (EPR1), and electropositive region 2 (EPR2) are indicated.</p