Recombinant Cyclodextrinase from Thermococcus kodakarensis

A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the α-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0–10.0), with an optimal pH of 7.5 and a temperature optimum of 65°C. The purified enzyme preferred to hydrolyze β-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from β-CD was glucose. The Vmax and Km values were 3.13 ± 0.47 U mg−1 and 2.94 ± 0.16 mg mL−1 for β-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed

Structural insights into the mechanism of archaellar rotational switching

Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts with phosphorylated CheY. While the mechanism of CheY binding to the C-ring is well-studied in bacteria, the role of CheF in archaea remains enigmatic and mechanistic insights are absent. Here, we have determined the atomic structures of CheF alone and in complex with activated CheY by X-ray crystallography. CheF forms an elongated dimer with a twisted architecture. We show that CheY binds to the C-terminal tail domain of CheF leading to slight conformational changes within CheF. Our structural, biochemical and genetic analyses reveal the mechanistic basis for CheY binding to CheF and allow us to propose a model for rotational switching of the archaellum. Signal transduction via phosphorylated CheY is conserved in bacteria and archaea. In this study, the authors employ structural biochemistry combined with cell biology to delineate the mechanism of CheY recognition by the adaptor protein CheF

Start-Up and Aeration Strategies for a Completely Autotrophic Nitrogen Removal Process in an SBR

The start-up and performance of the completely autotrophic nitrogen removal via nitrite (CANON) process were examined in a sequencing batch reactor (SBR) with intermittent aeration. Initially, partial nitrification was established, and then the DO concentration was lowered further, surplus water in the SBR with high nitrite was replaced with tap water, and continuous aeration mode was turned into intermittent aeration mode, while the removal of total nitrogen was still weak. However, the total nitrogen (TN) removal efficiency and nitrogen removal loading reached 83.07% and 0.422 kgN/(m3·d), respectively, 14 days after inoculating 0.15 g of CANON biofilm biomass into the SBR. The aggregates formed in SBR were the mixture of activated sludge and granular sludge; the volume ratio of floc and granular sludge was 7 : 3. DNA analysis showed that Planctomycetes-like anammox bacteria and Nitrosomonas-like aerobic ammonium oxidization bacteria were dominant bacteria in the reactor. The influence of aeration strategies on CANON process was investigated using batch tests. The result showed that the strategy of alternating aeration (1 h) and nonaeration (1 h) was optimum, which can obtain almost the same TN removal efficiency as continuous aeration while reducing the energy consumption, inhibiting the activity of NOB, and enhancing the activity of AAOB

The switch complex ArlCDE connects the chemotaxis system and the archaellum

Cells require a sensory system and a motility structure to achieve directed movement. Bacteria and archaea possess rotating filamentous motility structures that work in concert with the sensory chemotaxis system. This allows microorganisms to move along chemical gradients. The central response regulator protein CheY can bind to the motor of the motility structure, the flagellum in bacteria, and the archaellum in archaea. Both motility structures have a fundamentally different protein composition and structural organization. Yet, both systems receive input from the chemotaxis system. So far, it was unknown how the signal is transferred from the archaeal CheY to the archaellum motor to initiate motor switching. We applied a fluorescent microscopy approach in the model euryarchaeon Haloferax volcanii and shed light on the sequence order in which signals are transferred from the chemotaxis system to the archaellum. Our findings indicate that the euryarchaeal-specific ArlCDE are part of the archaellum motor and that they directly receive input from the chemotaxis system via the adaptor protein CheF. Hence, ArlCDE are an important feature of the archaellum of euryarchaea, are essential for signal transduction during chemotaxis and represent the archaeal switch complex

The switch complex ArlCDE connects the chemotaxis system and the archaellum

Cells require a sensory system and a motility structure to achieve directed movement. Bacteria and archaea possess rotating filamentous motility structures that work in concert with the sensory chemotaxis system. This allows microorganisms to move along chemical gradients. The central response regulator protein CheY can bind to the motor of the motility structure, the flagellum in bacteria, and the archaellum in archaea. Both motility structures have a fundamentally different protein composition and structural organization. Yet, both systems receive input from the chemotaxis system. So far, it was unknown how the signal is transferred from the archaeal CheY to the archaellum motor to initiate motor switching. We applied a fluorescent microscopy approach in the model euryarchaeon Haloferax volcanii and shed light on the sequence order in which signals are transferred from the chemotaxis system to the archaellum. Our findings indicate that the euryarchaeal-specific ArlCDE are part of the archaellum motor and that they directly receive input from the chemotaxis system via the adaptor protein CheF. Hence, ArlCDE are an important feature of the archaellum of euryarchaea, are essential for signal transduction during chemotaxis and represent the archaeal switch complex

Effects of Fermented Bamboo Powder Supplementation on Serum Biochemical Parameters, Immune Indices, and Fecal Microbial Composition in Growing–Finishing Pigs

This experiment aimed to investigate the effects of fermented bamboo powder (FBP) on the growth performance, serum biochemical parameters, immunoglobulins and inflammatory cytokines, and fecal microbial composition of growing–finishing pigs. A total of 108 barrows (initial body weight, 56.30 ± 0.55 kg) were randomly allocated to three dietary treatments in a 75 d trial, including a control (CON) diet and two FBP supplementation diets. The CON diet was formulated to three-phase diets according to the body weight of pigs, and the FBP diets were formulated used 5.00% (FBP1) or 10.00% (FBP2) FBP to replace the wheat bran in the CON diet, respectively. The results showed that there were no influences on growth performances between the CON diet and FBP addition diets, whereas the 5% FBP addition decreased the feed:gain of pigs compared to the pigs fed the FBP2 diet from d 0–75 (p < 0.05). Meanwhile, the FBP addition increased the high-density lipoprotein cholesterol (HDLC) and immunoglobulin A (IgA) content in serum (linear, p < 0.05), and pigs fed the FBP1 diet had greater HDLC and IgA contents in serum than those in the pigs fed the CON diet (p < 0.05). Microbial analysis showed that the FBP addition diets decreased the abundance of Spirochaetes, and the FBP2 diet increased the abundance of Firmicutes more than the CON diet (p < 0.05). In addition, the pigs fed the FBP2 diet increased the abundance of uncultured_bacterium_f_Lachnospiraceae, Ruminococcaceae_UCG-005, Prevotellaceae_UCG-003, Lachnospiraceae_XPB1014_group, and Lactobacillus more than the CON group (p < 0.05). In conclusion, the FBP supplementation to the diet had no negative effects on the growth performance and exerted beneficial effects on promoting serum biochemical and immune indices, as well as modulating the fecal microbiota of pigs. Therefore, these results showed that the fermented bamboo powder could be one potential fiber-rich ingredient for growing–finishing pigs, and that the recommended addition proportion in the growing–finishing pigs’ diet is 5%

Crystal Structure of a Putative Modulator of Gyrase (TldE) from <i>Thermococcus kodakarensis</i>

TldD and TldE proteins interact and form a complex to degrade unfolded peptides. The gene Tk0499 from Thermococcus kodakarensis encoded a putative modulator of gyrase (TkTldE). Although TldE genes were common in bacteria and archaea, the structural basis on the evolution of proteins remained largely unknown. Here, the three-dimensional structure of TkTldE was determined by X-ray diffraction. Crystals were acquired by the sitting-drop vapor-diffusion method. X-ray diffraction data from crystals were collected at 2.35 &#197;. The space group and unit-cell parameters suggested that there were two molecules in the asymmetric unit. Our results showed that TkTldE forms a homodimer, which contained anti-parallel &#946;-strands and a pair of &#945;-helices. Comparison of the structures of TldE and TldD showed that despite their high sequence similarity, TldE lacked the conserved HExxxH and GxC motif in which two His and a Cys residues bound a metal ion. Taken together, these results provided insight into the structural information of this class of TldE/TldD

Influence of Lonicera japonica and Radix Puerariae Crude Extracts on the Fecal Microbiome and Nutrient Apparent Digestibility of Finishing Pigs

This study aims to investigate the influence of adding Lonicera japonica (L. japonica) and Radix Puerariae crude extracts and their mixture to the diet of finishing pigs on their fecal microbes and nutrient apparent digestibility. A total of 72 healthy Duroc &times; Landrace &times; Yorkshire crossbred barrows without significant differences in body weight (93 &plusmn; 2 kg) were selected and randomly divided into four groups (18 in each group). Three replicate pens per group (six pigs per pen) were used, and two pigs were evaluated for each pen. The groups were fed the following diets: control group (CON), basic diet; chlorogenic acid group (CGA group), basic diet + 1 kg/ton L. japonica crude extract; Pueraria flavonoid group (PF group), basic diet + 1 kg/ton Radix Puerariae crude extract; and mix group (Mix group), basic diet + 0.5 kg/ton L. japonica crude extract + 0.5 kg/ton Radix Puerariae crude extract. The following results were obtained: (1) At the phylum level, Bacteroidetes, Firmicutes, Spirochaetes, Proteobacteria, Fibrobaeteres, and Kiritimatiellaeota were the main components of the fecal microbiota (top 5); the relative abundance of bacteria from phyla Firmicutes significantly increased in the Mix group than in the CON group (p &lt; 0.05). At the genus level, Treponema_2, Rikenellaceae_RC9_gut_group, uncultured_bacterium_f_Lachnospiraceae, uncultured_bacterium_f_Prevotellaceae, and Prevotellaceae_NK3B31_group were the main components of the fecal microbiota (top 5); the relative abundance of bacteria from genus Lactobacillus significantly increased in the Mix group than in the CON group (p &lt; 0.05). Chao1 and Ace counts were significantly higher in group CGA than in the CON group and group Mix (p &lt; 0.05). The alpha and beta diversities and the relative abundance of fecal microbes were higher in all test groups than in the CON group. (2) The protein digestibility was significantly higher in the CGA and PF groups than in the CON group, and the TP digestibility was significantly higher in the CGA than in the CON and Mix groups (p &lt; 0.05). In conclusion, Lonicera japonica and Radix Puerariae crude extract supplementation in the diet significantly changed fecal microbiota and improved the protein and TP digestibility of finishing pigs

Prediction equations of the metabolizable energy in corn developed by chemical composition and enzymatic hydrolysate gross energy for roosters

ABSTRACT: Two experiments were conducted to establish the prediction equations for AME and TME of corn based on chemical composition and enzymatic hydrolysate gross energy (EHGE) in roosters. In experiment 1, eighty 32-wk-old Hy-line Brown roosters with an average body weight of 2.55 ± 0.21 kg were randomly assigned to 10 diet treatments in a completely randomized design to determine AME and TME by the force-feeding method. Each treatment had 8 replicates with 1 bird per replicate. The 10 test diets used in the experiment were formulated with corn (including 96.10%) as the sole source of energy. In experiment 2, the EHGE of 14 corn samples was measured by the computer-controlled simulated digestion system (CCSDS) with 5 replicates of each sample. The average AME and TME values of corn were 14.58 and 16.46 MJ/kg DM, respectively. The EHGE of 14 corn samples ranged from 14.66 to 15.89 (the mean was 15.24) MJ/kg DM. The best-fit equations for corn based on chemical composition were AME (MJ/kg DM) = 14.5504 + 0.1166 × ether extract (EE) + 0.5058 × Ash − 0.0957 × neutral detergent fiber (NDF) (R2 = 0.8194, residual standard deviation (RSD) = 0.0860, P < 0.01) and TME (MJ/kg DM) = 16.0625 + 0.1314 × EE + 0.4725 × Ash − 0.0872 × NDF (R2 = 0.7867, RSD = 0.0860, P < 0.01). The best-fit equations for corn based on EHGE were AME (MJ/kg DM) = 7.8883 + 0.4568 × EHGE (R2 = 0.8587, RSD = 0.0693, P < 0.01) and TME (MJ/kg DM) = 10.0099 + 0.4228 × EHGE (R2 = 0.8720, RSD = 0.0608, P < 0.01). The differences between determined and predicted values from equations established based on EHGE were lower than those observed from chemical composition equations. These results indicated that EHGE measured with CCSDS could predict the AME and TME of corn for roosters with high accuracy