Structural Analysis of Alkaline β-Mannanase from Alkaliphilic Bacillus sp. N16-5: Implications for Adaptation to Alkaline Conditions

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)8-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)8-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pKa calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism

Type One Protein Phosphatase 1 and Its Regulatory Protein Inhibitor 2 Negatively Regulate ABA Signaling

The phytohormone abscisic acid (ABA) regulates plant growth, development and responses to biotic and abiotic stresses. The core ABA signaling pathway consists of three major components: ABA receptor (PYR1/PYLs), type 2C Protein Phosphatase (PP2C) and SNF1-related protein kinase 2 (SnRK2). Nevertheless, the complexity of ABA signaling remains to be explored. To uncover new components of ABA signal transduction pathways, we performed a yeast two-hybrid screen for SnRK2-interacting proteins. We found that Type One Protein Phosphatase 1 (TOPP1) and its regulatory protein, At Inhibitor-2 (AtI-2), physically interact with SnRK2s and also with PYLs. TOPP1 inhibited the kinase activity of SnRK2.6, and this inhibition could be enhanced by AtI-2. Transactivation assays showed that TOPP1 and AtI-2 negatively regulated the SnRK2.2/3/6-mediated activation of the ABA responsive reporter gene RD29B, supporting a negative role of TOPP1 and AtI-2 in ABA signaling. Consistent with these findings, topp1 and ati-2 mutant plants displayed hypersensitivities to ABA and salt treatments, and transcriptome analysis of TOPP1 and AtI-2 knockout plants revealed an increased expression of multiple ABA-responsive genes in the mutants. Taken together, our results uncover TOPP1 and AtI-2 as negative regulators of ABA signaling. © 2016 Hou et al

Novel decontamination approaches and their potential application for post-harvest aflatoxin control

Background: Aflatoxin is considered to be the most important mycotoxin in the world for human food and animal feed. Current strategies for the reduction of mycotoxins in food and feed includes both prevention and removal. It is clear that the development and implementation of novel decontamination methods is critical for the protection of human and animal health. Scope and approach: This review focuses on post-harvest- biological, chemical and physical processes that could potentially be applied to aflatoxin decontamination. The application of novel technologies are reviewed in detail, as well as the advantages, disadvantages and limitations of these methods. This review investigates the potential for novel approaches to achieve aflatoxin decontamination. Key findings and conclusion: The limitations that are associated with conventional methods of mycotoxin removal have led to ongoing research into alternative decontamination methods using novel technologies. The combination of fluorescence-based sorting to remove highly contaminated produce, paired with a secondary decontamination process is believed to have great potential to deliver effective reduction in aflatoxin contamination, whilst retaining the organoleptic and nutritional profile, and preventing significant food waste. Novel decontamination approaches when applied to aflatoxin decontamination are of huge interest and a growing need for global food security

Toxin YafQ Reduces Escherichia coli Growth at Low Temperatures.

Toxin/antitoxin (TA) systems reduce metabolism under stress; for example, toxin YafQ of the YafQ/DinJ Escherichia coli TA system reduces growth by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites, and antitoxin DinJ is a global regulator that represses its locus as well as controls levels of the stationary sigma factor RpoS. Here we investigated the influence on cell growth at various temperatures and found that deletion of the antitoxin gene, dinJ, resulted in both reduced metabolism and slower growth at 18°C but not at 37°C. The reduction in growth could be complemented by producing DinJ from a plasmid. Using a transposon screen to reverse the effect of the absence of DinJ, two mutations were found that inactivated the toxin YafQ; hence, the toxin caused the slower growth only at low temperatures rather than DinJ acting as a global regulator. Corroborating this result, a clean deletion of yafQ in the ΔdinJ ΔKmR strain restored both metabolism and growth at 18°C. In addition, production of YafQ was more toxic at 18°C compared to 37°C. Furthermore, by overproducing all the E. coli proteins, the global transcription repressor Mlc was found that counteracts YafQ toxicity only at 18°C. Therefore, YafQ is more effective at reducing metabolism at low temperatures, and Mlc is its putative target

Δ<i>dinJ</i> reduces growth only at 18°C.

<p>Comparison of growth in LB medium for BW25113 wild-type (■), Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i> (●), Δ<i>dinJ</i> Δ<i>yafQ ΔKm</i>^<i>R</i> (▲), and Δ<i>yafQ</i> Δ<i>Km</i>^<i>R</i> (▼) at 18°C (<b>A</b>) and 37°C (<b>B</b>). Data are the average of three independent cultures and one standard deviation is shown.</p

Δ<i>dinJ</i> reduces metabolism only at 18°C.

<p>Comparison of metabolic activity via the Biolog assay for BW25113 wild-type (■), Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i> (●) and Δ<i>dinJ</i> Δ<i>yafQ</i> Δ<i>Km</i>^<i>R</i> (▲) at 18°C (<b>A</b>), 30°C (<b>B</b>), and 37°C (<b>C</b>). Data are the average of two independent cultures and one standard deviation is shown.</p

<i>E</i>. <i>coli</i> bacterial strains and plasmids used in this study.

<p><i>E</i>. <i>coli</i> bacterial strains and plasmids used in this study.</p

Complementation of the Δ<i>dinJ</i> lower growth phenotype at 18°C.

<p>Comparison of growth in LB medium for BW25113/pCA24N (■), BW25113 Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i>/pCA24N (●) and BW25113 Δ<i>dinJ</i> Δ<i>Km</i>^<i>R</i>/pCA24N-<i>dinJ</i> (▲)at 18°C with IPTG induction of 0 mM IPTG (<b>A</b>), 0.05 mM IPTG (<b>B</b>), and 1 mM IPTG (<b>C</b>). Data are averaged from three independent cultures and one standard deviation is shown.</p

Mlc suppresses YafQ toxicity.

<p>Comparison of growth in LB medium for BW25113 <i>ΔdinJ ΔKm</i>^<i>R</i>/pCA24N (●) and BW25113 <i>ΔdinJ ΔKm</i>^<i>R</i>/pCA24N-<i>mlc</i> (♦) with 200 μM IPTG induction at 18°C (<b>A</b>) and 37°C (<b>B</b>). Data are averaged from three independent cultures, and one standard deviation is shown.</p

Gene inactivations that restore growth at 18°C.

<p>Gene inactivations that restore growth at 18°C.</p