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Bioelectrochemistry system (BES) provides a new strategy for the improvement of low temperature anaerobic digestion (AD) for methane production. In this study, the BES with carbon brushes as electrodes was operated at 15 °C to analyze the changes of microbial abundance and functional genes. The microbial community structure showed that the application of voltage had no effect to type and relative abundance of bacteria based on 16S-rRNA sequencing. Metatranscriptomic analyses revealed that significantly enhanced the abundance of fermentation bacteria in the fluid activated sludge, and the relative abundance of biosynthesis of other secondary metabolites (61.56%) was also highly expressed. More than that, the abundance of electricigens (Pseudomonas , Syntrophomonas) was also enriched in the BES system. These electricigens could interact with methanogens to promote electron transfer by direct interspecies electron transfer (DIET). The expression level of functional enzyme related to electron transport was increased at the anode (30.60%), but decreased at the cathode (−33.84%). Analysis of methanogens found that the application of voltage decreased the relative abundance of Methanosarcina and promoted the enrichment of Methanothrix. At 15 ℃, acetoclastic was the mainly methanogenic pathway. What's more, the application of voltage could enhance the acetoclastic and inhibited the hydrogenotrophic and the methylotrophic pathway. Further, the acetoclastic pathway was changed from acetate kinase (EC: 2.7.2.1) and phosphate acetyltransferase (EC: 2.3.1.8) to acetate-CoA ligase (EC: 6.2.1.1) by applied voltage. These results are conducive to the further development of low-temperature AD treatment technology. [Display omitted] • Methahnosarcina and Methanothrix were dominated in DNC and SC reactor, respectively. • The applied voltage increased the abundance of electroactive microorganisms. • The application of voltage promoted the acetoclastic pathway at low temperature. • The SC reactor achieved acetate to acetyl-CoA by acetate-CoA ligase. • The DNC reactor was through acetate kinase and phosphate acetyltransferase. [ABSTRACT FROM AUTHOR]

Candida albicans is an opportunistic pathogenic fungus which causes superficial and systemic infections in immunocompromised patients. It is important to characterize the roles of genes involved in its pathogenesis, virulence, and drug resistance. Several genetic manipulation toolkits have been developed for gene function research in C. albicans . Here, we describe efficient vector systems that allow economical and rapid C-terminal and N-terminal epitope-tagging, inducible and constitutive promoter replacements, and ectopic gene overexpression in C. albicans . These systems use modularized genetic elements (conventional and non-conventional selection markers, epitope tags and promoters) and universal primers. These advantages should greatly reduce laboratory work and costs of strain construction for C. albicans . [ABSTRACT FROM AUTHOR]

Sclerotium rolfsii (teleomorph Athelia rolfsii) is one of the plant pathogenic basidiomycetes, which causes severe stem-rot disease in hundreds of plants and produces important metabolites, such as scleroglucan and TF-specific lectin. However, further molecular biological research on this filamentous fungus is severely plateaued out due to the lack of genetic methods. In this study, the A. tumefaciens strain LBA4404 harboring a binary vector containing the basta resistance gene fused with three reporters (DsRed, tdTomato, and GUSPlus) respectively, driven by the SrGPD promoter, was used for genetic transformation of S. rolfsii. The results showed that the three reporter genes were all effectively expressed in S. rolfsii. This study also showed that the intron of the SrGPD promoter is not necessary for transgene expression in this fungus. Besides, we showed that these reporters' signals could be observed easily but in a short time window. The efficient Agrobacterium -mediated transformation system and the three reporter gene plasmids for S. rolfsii developed in this study are of significance in overcoming current limitations of no available transformation and genetic manipulation techniques in S. rolfsii , facilitating further genetic manipulations and gene function exploration. • An efficient Agrobacterium -mediated transformation for Sclerotium rolfsii. • Three reporter genes were efficiently expressed in S. rolfsii. • The SrGPD promoter without an intron is sufficient to drive transgene expression. [ABSTRACT FROM AUTHOR]

The laccase production by mycelial antagonistic interaction among white-rot fungi is a very important pathway for lignin degradation research. To gain a better understanding of competitive mechanisms under mycelial antagonistic interaction among three lignin-degrading white-rot basidiomycetes of Trametesversicolor (Tv), Pleurotusostreatus (Po) and Dichomitussqualens (Ds), mycelial morphology and proteins in three co-culture combinations TvPo (Tv cocultivated with Po), PoDs (Po cocultivated with Ds), TvDs (Tv cocultivated with Ds) were compared with corresponding each two mono-cultures. In this study, scanning electron microscopy detection of co-cultures indicated a highly close attachment of fungal hyphae with each other and conidiation could be inhibited under fungal interaction. In addition, a label-free proteomic analysis revealed changes on fungal proteomes existed in their counterpart competitors of co-culture. The maximum number of 1020 differentially expressed proteins (DEPs) were identified in PoDs relative to Po while the minimum number of 367 DEPs were identified in PoDs relative to Ds. Notably, we also found a large number of overexpressed proteins were oxidative stress-related proteins, followed by carbohydrate metabolism-related proteins and energy production-related proteins in all three co-culture combinations compared with control. These results were important for the future exploration of molecular mechanisms underlying lignin-degrading fungal interaction. [ABSTRACT FROM AUTHOR]

This study provided analysis of differentially expressed genes (DEGs) in Pleurotus ostreatus under the interaction with Dichomitus squalens and Trametes versicolor , which is valuable for exploration on the fungal defence system against stressful condition caused by interspecific antagonistic interaction. Our result showed significant upregulation of abundant defence-related genes encoding laccase, manganese peroxidase, aldo–keto reductase, and glutathione S-transferase, which all play important roles in oxidative stress-resistant response. Importantly, Lacc2 and Lacc10 were found to be dominantly induced laccase genes in P. ostreatus under interspecific interaction. Meanwhile, a large number of carbohydrate metabolism-related and energy production-related genes involved in nutrient and territory competition were also enhanced. These genes were annotated as glycoside hydrolase, citrate synthase, malate dehydrogenase, succinate dehydrogenase, succinyl-CoA synthetase, NADH dehydrogenase, cytochrome c reductase/oxidase, and ATP synthase. Also, 12 DEGs were selected for validation by quantitative real-time PCR (qRT-PCR), all these genes showed consistent expression between the result of qRT-PCR and RNA-seq. [ABSTRACT FROM AUTHOR]

Cyanobacterial blooms negatively affect aquatic ecosystems and human health. Algicidal bacteria can efficiently kill bloom-causing cyanobacteria. Bacillus altitudinis G3 isolated from Dianchi Lake shows high algicidal activity against Microcystis aeruginosa. In this study, we investigated its algicidal characteristics including attack mode, photosynthesis responses, and source and the contribution of reactive oxygen species (ROS). The results showed that G3 efficiently and specifically killed M. aeruginosa mainly by releasing both thermolabile and thermostable algicidal substances, which exhibited the highest algicidal activity (99.8%, 72 h) in bacterial mid-logarithmic growth phase. The algicidal ratio under full-light conditions (99.5%, 60 h) was significantly higher than under dark conditions (<20%, P < 0.001). G3 filtrate caused photosystem dysfunction by decreasing photosynthetic efficiency, as indicated by significantly decreased F v /F m and PI ABS (P < 0.001) values. It also inhibited photosynthetic electron transfer as indicated by significantly decreased rETR (P < 0.001), especially Q A − downstream, as revealed by significantly decreased φE o and ψ o , and increased M o (P < 0.001). These results indicated that the algicidal activity of G3 filtrate is light-dependent, and the cyanobacterial photosystem is an important target. Cyanobacterial ROS and malondialdehyde contents greatly increased by 37.1% and 208% at 36 h, respectively. ROS levels decreased by 49.2% (9 h) when diuron (3-(3-4-dichlorophenyl)-1,1-dimethylurea) partially blocked photosynthetic electron transport from Q A to Q B. Therefore, excessive ROS were produced from disrupted photosynthesis, especially the inhibited electron transport area in Q A − downstream, and caused severe lipid peroxidation with significantly increased MDA content and oxidative stress in cyanobacteria. The ROS scavenger N -acetyl- l -cysteine significantly decreased both cyanobacterial ROS levels (34%) and algicidal ratio (52%, P < 0.05) at 39 h. Thus, excessive ROS production due to G3 filtrate administration significantly contributed to its algicidal effect. G3 could be an excellent algicide to control M. aeruginosa blooms in waters under suitable light conditions. [Display omitted] • B. altitudinis G3 from Dianchi Lake bloom area is algicidal against M. aeruginosa. • Photosystem of M. aeruginosa as an important attack target of G3. • G3 decreased photosynthetic efficiency and inhibited electron transfer. • Dysfunctional photosystem results overproduction of reactive oxygen species. • Reactive oxygen species contributed to the algicidal effect of G3. [ABSTRACT FROM AUTHOR]

Anaerobic activated sludge is rich in humic substances and water, leading to significant differences in the stability of metagenomic DNA and metatranscriptomic RNA. Thus, it is of great difficulty to exact high-quality and high-yield DNA and RNA from them, especially those cultured at a wide range of temperatures. Here, we established fast and effective DNA and RNA extraction methods based on current commercial kits. The modified methods combined liquid nitrogen grinding with kits, achieving notable improvements in concentrations, yields, purity and integrities for both DNA and RNA. The ratios of OD260/280 of the metagenomic DNA were between 1.81 ± 0.03 and 1.83 ± 0.02, while OD260/280 and OD260/230 of the metatranscriptomic RNA ranged from 1.96 ± 0.01 to 2.13 ± 0.03 and from 1.94 ± 0.02 to 2.30 ± 0.03 respectively. Metagenomic DNA and metatranscriptomic RNA obtained by the modified methods perfectly met the requirements of second- and third-generation sequencing, providing valuable reference for extracting high-quality metagenomic DNA and metatranscriptomic RNA from environmental samples of high water content and humic substances under temperatures ranging from 18 °C to 52 °C. • Liquid nitrogen grinding combined with kits generated high-quality DNA/RNA. • DNA/RNA extracted improved greatly in concentration, yield, purity and integrity. • The methods are suitable for sludge at a wide range of temperatures. [ABSTRACT FROM AUTHOR]

Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent phosphoglycerate mutase (dPGM)-like phosphatases that have significant amino acid sequence similarity to dPGMs but lack phosphoglycerate mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate L-phosphoserine to find structural units important for its phosphatase activity toward L-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His85 and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases. [ABSTRACT FROM AUTHOR]

Abstract: A bioelectrochemical reactor (BER) containing carbon fiber fabric (CFF) (BER+CFF) enabled efficient methane fermentation from thickened sewage sludge. A cylindrical BER+CFF was proposed and scaled-up to a volume of 4.0-L. Thickened sewage sludge was treated using three types of methanogenic reactors. The working electrode potential in the BER+CFF was regulated at −0.8V (vs. Ag/AgCl). BER+CFF showed gas production of 3.57LL−1 day−1 at a hydraulic retention time (HRT) of 4.0days; however, non-BER+CFF showed a lower gas production rate (0.83LL−1 day−1) at this HRT, suggesting positive effects of electrochemical regulation. A stirred tank reactor (without CFF) deteriorated at an HRT of 10days, suggesting positive effects of CFF. 16S rRNA gene analysis showed that the BER+CFF included 3 kinds of hydrogenotrophic methanogens and 1 aceticlastic methanogen. These results demonstrate the effectiveness of the BER+CFF for scale-up and flexibility of this technology. [Copyright &y& Elsevier]

Wild-type Acetobacter aceti NBRC 14818 possesses genes encoding isocitrate lyase (aceA) and malate synthase (glcB), which constitute the glyoxylate pathway. In contrast, several acetic acid bacteria that are utilized for vinegar production lack these genes. Here, an aceA–glcB knockout mutant of NBRC 14818 was constructed and used for investigating the role of the glyoxylate pathway in acetate productivity. In medium containing ethanol as a carbon source, the mutant grew normally during ethanol oxidation to acetate, but exhibited slower growth than that of the wild-type strain as the accumulated acetate was oxidized. The mutant grew similarly to that of the wild-type strain in medium containing glucose as a carbon source, indicating that the glyoxylate pathway was not necessary for glucose utilization. However, in medium containing both ethanol and glucose, the mutant exhibited significantly poorer growth and lower glucose consumption compared to the wild-type strain. Notably, the mutant oxidized ethanol nearly stoichiometrically to acetate, which was retained in the medium for a longer period of time than the acetate produced by wild-type strain. The features of the aceA–glcB knockout mutant revealed here indicate that the lack of the glyoxylate pathway is advantageous for industrial vinegar production by A. aceti. [ABSTRACT FROM AUTHOR]

The aim of this study was to show the effectiveness of the membrane free bioelectrochemical system (BES) using three electrodes on inhibition of methanogenesis and construction of hydrogen fermentation from the artificial garbage slurry. The electrical redox-potential on the working electrode was adjusted to −1.0V (vs. Ag/AgCl) that has positive effect on methanogenesis. The redox-potential on the counter electrode was measured to be 1.6V. The pH in the effluents was 5.5–6.4. Hydrogen production rate at the cathode side was similar to that at the anode side and much higher than that calculated from current, and reached a maximum of 2445±815 (average±standard deviation) mL L−1 d−1 at an organic loading rate of 58.7g dichromate chemical oxygen demand per L d−1. Methane production was negligible throughout the experiment. Acetate and butyrate were the main products of the fermentation using a BES; these offered favorable conditions for hydrogen production. The bacterial community in the bioelectrochemical hydrogen fermentor differed from that in the methanogenic seed sludge and included hitherto unknown species. These results show that high redox-potential on the anodic electrode and acidic pH in the membrane free BES can be utilized for hydrogen fermentation from the artificial garbage slurry by avoiding methanogenesis. [ABSTRACT FROM AUTHOR]

Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. PSPs, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. However, certain organisms, particularly bacteria, lack a classical PSP gene, although they appear to possess a functional phosphoserine synthetic pathway. The apparent lack of a PSP ortholog in Hydrogenobacter thermophilus, an obligately chemolithoautotrophic and thermophilic bacterium, represented a missing link in serine anabolism because our previous study suggested that serine should be synthesized from phosphoserine. Here, we detected PSP activity in cell-free extracts of H. thermophilus and purified two proteins with PSP activity. Surprisingly, these proteins belonged to the histidine phosphatase superfamily and had been annotated as cofactor-dependent phosphoglycerate mutase (dPGM). However, because they possessed neither mutase activity nor the residues important for the activity, we defined these proteins as novel-type PSPs. Considering the strict substrate specificity toward L-phosphoserine, kinetic parameters, and PSP activity levels in cell-free extracts, these proteins were strongly suggested to function as PSPs in vivo. We also detected PSP activity from "dPGM-like" proteins of Thermus thermophilus and Arabidopsis thaliana, suggesting that PSP activity catalyzed by dPGM-like proteins may be distributed among a broad range of organisms. In fact, a number of bacterial genera, including Firmicutes and Cyanobacteria, were proposed to be strong candidates for possessing this novel type of PSP. These findings will help to identify the missing link in serine anabolism. [ABSTRACT FROM AUTHOR]

Abstract: Acetobacter aceti NBRC 14818 shows a diauxic growth profile and temporarily accumulates acetate when grown in medium containing ethanol. However, the mechanisms underlying the metabolic switching between the incomplete oxidation of ethanol and overoxidation of acetate, and the control of stress resistance systems in A. aceti cells grown on ethanol are not fully understood. In this study, time-dependent transcriptome changes in cells during growth on ethanol were analyzed by DNA microarray. In A. aceti, ethanol is oxidized to acetate via acetaldehyde by sequential reactions of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). We found that the genes encoding pyrroloquinoline quinone-dependent ADH, membrane-bound ALDH, and two NAD+-ADHs were expressed constitutively in cells throughout the culture period. In contrast, the expression levels of genes encoding tricarboxylic acid (TCA) cycle enzymes were low during acetate accumulation until ethanol was consumed, but were significantly upregulated after the accumulated acetate was started to be consumed. This result suggests that changes in the carbon metabolic flow through the TCA cycle are important for the metabolic switching from acetate accumulation to the overoxidation of acetate. In addition, the genes for glyoxylate pathway enzymes were significantly upregulated soon after the cells began oxidizing ethanol, indicating that this pathway is important for the utilization of ethanol as a carbon source. [Copyright &y& Elsevier]