Effect of hydrogen sulfide (H2S) on the growth and development of tobacco seedlings in absence of stress.

BACKGROUND: Hydrogen sulfide (H2S) is a novel signaling molecule involved in the growth and development of plants and their response to stress. However, the involvement of H2S in promoting the growth and development of tobacco plants is still unclear. RESULTS: In this study, we explored the effect of pre-soaking or irrigating the roots of tobacco plants with 0.0, 2.0, 4.0, 6.0, and 8.0 mM of sodium hydrosulfide (NaHS) on endogenous H2S production, antioxidant enzymatic and cysteine desulfhydrase activities, seed germination, agronomic traits, photosynthetic pigments contents, and root vigor. The results revealed that exogenous NaHS treatment could significantly promote endogenous H2S production by inducing gene expression of D/L-CD and the activities of D/L-CD enzymes. Additionally, a significant increase in the agronomic traits and the contents of photosynthetic pigments, and no significant difference in carotenoid content among tobacco plants treated with 0.0 to 8.0 mM of NaHS was observed. Additionally, a significant increase in the germination speed, dry weight, and vigor of tobacco seeds, whereas no significant effect on the percentage of seed germination was observed on NaHS treatment. Furthermore, NaHS treatment could significantly increase the activity of superoxide dismutase (SOD) and peroxidase (POD) enzymes, which reduces damage due to oxidative stress by maintaining reactive oxygen species homeostasis. CONCLUSIONS: These results would aid in enhancing our understanding of the involvement of H2S, a novel signaling molecule to promote the growth and development of tobacco plants.

Improving the Quality of Wheat Flour Bread by a Thermophilic Xylanase with Ultra Activity and Stability Reconstructed by Ancestral Sequence and Computational-Aided Analysis.

Xylanase is an essential component used to hydrolyze the xylan in wheat flour to enhance the quality of bread. Presently, cold-activated xylanase is popularly utilized to aid in the development of dough. In this study, ancestral sequence reconstruction and molecular docking of xylanase and wheat xylan were used to enhance the activity and stability of a thermophilic xylanase. The results indicated that the ancestral enzyme TmxN3 exhibited significantly improved activity and thermal stability. The Vmax increased by 2.7 times, and the catalytic efficiency (Kcat/Km) increased by 1.7 times in comparison to TmxB. After being incubated at 100 °C for 120 min, it still retained 87.3% of its activity, and the half-life in 100 °C was 330 min, while the wild type xylanase was only 55 min. This resulted in an improved shelf life of bread, while adding TmxN3 considerably enhanced its quality with excellent volume and reduced hardness, chewiness, and gumminess. The results showed that the hardness was reduced by 55.2%, the chewiness was reduced by 40.11%, and the gumminess was reduced by 53.52%. To facilitate its industrial application, we further optimized the production conditions in a 5L bioreactor, and the xylanase activity reached 1.52 × 106 U/mL culture.

Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis.

BACKGROUND: Zymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis. RESULTS: We constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong Pgap promoter from a plasmid could render the Z. mobilis ZM4 cells sufficient sensitivity to 4-CP. The clmPheS-expressing cells were assayed to be extremely sensitive to 0.2 mM 4-CP. Subsequently, the clmPheS-assisted counterselection endowed fast curing of genome engineering plasmids immediately after obtaining the desired mutants, shortening the time of every two rounds of multiplex chromosome editing by at least 9 days, and enabled the development of a strategy for scarless modification of the native Z. mobilis ZM4 plasmids. CONCLUSIONS: This study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.

An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency.

Laccases are widely used in industrial production due to their broad substrate availability and environmentally friendly nature. However, the pursuit of laccases with superior stability and increased heterogeneous expression to meet industry demands appears to be an ongoing challenge. To address this challenge, we resurrected five ancestral sequences of laccase BsCotA and their homologues. All five variants were successfully expressed in soluble and functional forms with improved expression levels in Escherichia coli. Among the five variants, three exhibited higher catalytic rates, thermal stabilities, and acidic stabilities. Notably, AncCotA2, the best-performing variant, displayed a kcat/KM of 7.5 × 105 M-1·s-1, 5.2-fold higher than that of the wild-type BsCotA, an improved thermo- and acidic stability, and better dye decolorization ability. This study provides a laccase variant with high application potential and presents a new starting point for future enzyme engineering.

The Motion Paradigm of Pre-Dock Zearalenone Hydrolase Predictions with Molecular Dynamics and the Docking Phase with Umbrella Sampling.

Zearalenone (ZEN) is one of the most prevalent estrogenic mycotoxins, is produced mainly by the Fusarium family of fungi, and poses a risk to the health of animals. Zearalenone hydrolase (ZHD) is an important enzyme capable of degrading ZEN into a non-toxic compound. Although previous research has investigated the catalytic mechanism of ZHD, information on its dynamic interaction with ZEN remains unknown. This study aimed to develop a pipeline for identifying the allosteric pathway of ZHD. Using an identity analysis, we identified hub genes whose sequences can generalize a set of sequences in a protein family. We then utilized a neural relational inference (NRI) model to identify the allosteric pathway of the protein throughout the entire molecular dynamics simulation. The production run lasted 1 microsecond, and we analyzed residues 139-222 for the allosteric pathway using the NRI model. We found that the cap domain of the protein opened up during catalysis, resembling a hemostatic tape. We used umbrella sampling to simulate the dynamic docking phase of the ligand-protein complex and found that the protein took on a square sandwich shape. Our energy analysis, using both molecular mechanics/Poisson-Boltzmann (Generalized-Born) surface area (MMPBSA) and Potential Mean Force (PMF) analysis, showed discrepancies, with scores of -8.45 kcal/mol and -1.95 kcal/mol, respectively. MMPBSA, however, obtained a similar score to that of a previous report.

High-temperature behavior of hyperthermostable Thermotoga maritima xylanase XYN10B after designed and evolved mutations.

A hyperthermostable xylanase XYN10B from Thermotoga maritima (PDB code 1VBR, GenBank accession number KR078269) was subjected to site-directed and error-prone PCR mutagenesis. From the selected five mutants, the two site-directed mutants (F806H and F806V) showed a 3.3-3.5-fold improved enzyme half-life at 100 °C. The mutant XYNA generated by error-prone PCR showed slightly improved stability at 100 °C and a lower Km. In XYNB and XYNC, the additional mutations over XYNA decreased the thermostability and temperature optimum, while elevating the Km. In XYNC, two large side-chains were introduced into the protein's interior. Micro-differential scanning calorimetry (DSC) showed that the melting temperature (Tm) dropped in XYNB and XYNC from 104.9 °C to 93.7 °C and 78.6 °C, respectively. The detrimental mutations showed that extremely thermostable enzymes can tolerate quite radical mutations in the protein's interior and still retain high thermostability. The analysis of mutations (F806H and F806V) in a hydrophobic area lining the substrate-binding region indicated that active site hydrophobicity is important for high activity at extreme temperatures. Although polar His at 806 provided higher stability, the hydrophobic Phe at 806 provided higher activity than His. This study generates an understanding of how extreme thermostability and high activity are formed in GH10 xylanases. KEY POINTS: • Characterization and molecular dynamics simulations of TmXYN10B and its mutants • Explanation of structural stability of GH10 xylanase.

Molecular and biochemical characterizations of a new cold-active and mildly alkaline ß-Mannanase from Verrucomicrobiae DG1235.

Mannanases catalyze the cleavage of ß-1,4-mannosidic linkages in mannans and have various applications in different biotechnological industries. In this study, a new ß-mannanase from Verrucomicrobiae DG1235 (ManDG1235) was biochemically characterized and its enzymatic properties were revealed. Amino acid alignment indicated that ManDG1235 belonged to glycoside hydrolase family 26 and shared a low amino acid sequence identity to reported ß-mannanases (up to 50% for CjMan26C from Cellvibrio japonicus). ManDG1235 was expressed in Escherichia coli. Purified ManDG1235 (rManDG1235) exhibited the typical properties of cold-active enzymes, including high activity at low temperature (optimal at 20 °C) and thermal instability. The maximum activity of rManDG1235 was achieved at pH 8, suggesting that it is a mildly alkaline ß-mannanase. rManDG1235 was able to hydrolyze a variety of mannan substrates and was active toward certain types of glucans. A structural model that was built by homology modeling suggested that ManDG1235 had four mannose-binding subsites which were symmetrically arranged in the active-site cleft. A long loop linking ß2 and α2 as in CjMan26C creates a steric border in the glycone region of active-site cleft which probably leads to the exo-acting feature of ManDG1235, for specifically cleaving mannobiose from the non-reducing end of the substrate.

Gene dosage and coexpression with endoplasmic reticulum secretion-associated factors improved the secretory expression of α-galactosidase.

The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications.

Biochemical Characterization of a New ß-Agarase from Cellulophaga Algicola.

Cellulophaga algicola DSM 14237, isolated from the Eastern Antarctic coastal zone, was found to be able to hydrolyze several types of polysaccharide materials. In this study, a predicted ß-agarase (CaAga1) from C. algicola was heterologously expressed in Escherichia coli. The purified recombinant CaAga1 showed specific activities of 29.39, 20.20, 14.12, and 8.99 U/mg toward agarose, pure agar, and crude agars from Gracilaria lemaneiformis and Porphyra haitanensis, respectively. CaAga1 exhibited an optimal temperature and pH of 40 oC and 7, respectively. CaAga1 was stable over a wide pH range from 4 to 11. The recombinant enzyme showed an unusual thermostability, that is, it was stable at temperature below or equal to 40oC and around 70 oC, but was thermolabile at about 50 oC. With the agarose as the substrate, the Km and Vmax values for CaAga1 were 1.19 mg/mL and 36.21 U/mg, respectively. The reducing reagent (dithiothreitol) enhanced the activity of CaAga1 by more than one fold. In addition, CaAga1 was salt-tolerant given that it retained approximately 70% of the maximum activity in the presence of 2 M NaCl. The thin layer chromatography results indicated that CaAga1 is an endo-type ß-agarase and efficiently hydrolyzed agarose into neoagarotetraose (NA4) and neoagarohexaose (NA6). A structural model of CaAga1 in complex with neoagarooctaose (NA8) was built by homology modeling and explained the hydrolysis pattern of CaAga1.

High-level secretive expression of a novel achieved Talaromyces cellulolyticus endo-polygalacturonase in Pichia pastoris by improving gene dosage for hydrolysis of natural pectin.

Pectin is a type of complex hydrophilic polysaccharide widely distributed in plant resources. Thermal stable pectinase has its advantage in bioapplication in the fields of food processing, brewing, and papermaking, etc. In this study, we enzymatically characterized a putative endo-polygalacturonase TcPG from a Talaromyces cellulolyticus, realized its high-level expression in Pichia pastoris by in vitro constructing of a series of multi-copy expression cassettes and real time quantitative PCR screening. The secretive expression level of TcPG was nonlinear correlated to the gene dosage. Recombinants with five-copy TcPG gene in the host genome showed the highest expression. After cultivation in a bioreactor for about 96 h, the enzyme activity reached 7124.8 U/mL culture. TcPG has its optimal temperature of 70 °C. Under the optimized parameters, the pectin could be efficiently hydrolyzed into oligosaccharides.

Characterization of a novel cold-active xylanase from Luteimonas species.

Biotechnological application of xylanolytic enzymes is normally hindered by their temperature-dependent catalytic property. To satisfy the industrial demands, xylanases that can perform catalysis under cold condition are attracting attention. In this study, the biochemical properties of a predicted xylanase (laXynA) encoded in the genome of marine bacterium Luteimonas abyssi XH031T were characterized. Structure modeling and structure-based sequence alignment indicated that laXynA belongs to the glycoside hydrolase family 10, and it is 20-26% identical to other characterized cold-active xylanases in the same family. Recombinant laXynA was successfully produced in Escherichia coli system by autoinduction and purified by Ni-affinity chromatography. The isolated enzyme showed an optimum temperature of 30 °C toward beechwood xylan and retained important percentage of optimal activity at low temperatures (64, 55, and 29% at 10, 5, and 0 °C, respectively). A remarkable characteristic of laXynA was extreme halophilicity as demonstrated by fourfold enhancement on xylanase activity at 0.5 M NaCl and by maintaining nearly 100% activity at 4 M NaCl. Thin layer chromatography analysis demonstrated that laXynA is an endo xylanase. This study is the first to report the over-expression and characterization of a cold-active xylanase from Luteimonas species. The enzymatic property revealed the cold-active nature of laXynA. The enzyme is a promising candidate in saline food processing application.

Community composition and spatial variation of bacteria in the sediments of a eutrophic fresh water urban lake, East Lake, Wuhan, China.

Objective: Sediment bacteria are the important biological factors for remediating of eutrophic environments. To enrich our understanding of the bacteria communities in eutrophic urban lake sediments for better environment protection and pollution control in urban lake eco-systems, we resolved the composition of bacteria communities and their spatial variation in the sediments of a middle-size eutrophic urban lake, East Lake. Methods: We used 16S rRNA gene RFLP and sequencing methods to generate the phylogeny information of the bacteria community, used principal coordinates analysis (PCoA) and canonical correspondence analysis (CCA) methods to resolve the relationship between East Lake and other lakes, and the relationship between environmental factors and the bacteria communities. Results: Sediments inhabited 13 phyla and 2 unclassified clusters. PCoA further revealed that the bacteria communities in three sub-lakes of East Lake sediments were closely related to the communities in similar eutropic lake environments, and divergent from the hypereutrophic sub-lake Miao Lake, which was also found to inhabit a relative abundant amount of Thermogymnomonas-type archaea. CCA further revealed that the distribution of bacteria was closely correlated with the carbon, nitrogen and phosphate contents in the sediments. Conclusion: The environment factors regulated the bacteria community composition and distribution. The results of this study providereference to the research, protection and pollution control on urban lake eco-systems.

[Effect of environmental factors on bacterial community structure in petroleum contaminated soil of Karamay oil field].

Objective: This study aimed to study the phylogenetic diversity and community structure of bacteria in petroleum contaminated soils from Karamay oil field, and to analyze the relationship between the community variation and the environment parameters, to provide a reference for bioremediation of petroleum contaminated soils. Methods: We collected samples from petroleum contaminated soils in 5 cm, 20 cm and 50 cm depth layers, and measured the environment parameters subsequently. We constructed three 16S rRNA gene clone libraries of these soil samples, and then determined the operation taxonomy units (OTUs) restriction fragment length polymorphism method, and finally sequenced the representative clones of every OUT. The diversity, richness and evenness index of the bacteria communities were calculated by using Biodap software. Neighbor-Joining phylogenetic tree was constructed based on 16S rRNA gene sequences of bacteria from Karamay oil field and the references from related environments. Canonial correspondence analysis (CCA) was used to analyze the relationship between environment parameters and species by using CANOCO 4.5 software. Results: Environment parameters showed that 50 cm deep soil contained the highest amount of total nitrogen (TN) and total phosphorus (TP), whereas the 20 cm depth soil contained the lowest amount. The 5 cm depth soil contained the highest amount of total organic carbon (TOC), whereas the 50 cm depth soil contained the lowest amount. Among the 3 layers, 20 cm depth had the highest diversity and richness of bacteria, whereas the bacteria in 50 cm depth was the lowest. Phylogenic analyses suggested that the bacteria in Karamay oil field could be distributed into five groups at the level of phylum, Cluster I to V, respectively belong to Proteobacteria, Actinobacteria, Firmicute, Bacteroidetes, Planctomycetes. Cluster I accounts for 78.57% of all tested communities. CCA results showed that TN, TP, TOC significantly affected the bacteria community structure. Especially, TOC content is significantly related to the distribution of Pseudomonas. Conclusion: The petroleum-contaminated soil inhabited abundant of bacteria. The diversity index and spatial distribution of these communities were affected by the environment parameters in the soil.

Computational analysis of AnmK-like kinase: New insights into the cell wall metabolism of fungi.

1,6-Anhydro-N-acetylmuramic acid kinase (AnmK) is the unique enzyme that marks the recycling of the cell wall of Escherichia coli. Here, 81 fungal AnmK-like kinase sequences from 57 fungal species were searched in the NCBI database and a phylogenetic tree was constructed. The three-dimensional structure of an AnmK-like kinase, levoglucosan kinase (LGK) of the yeast Lipomyces starkeyi, was modeled; molecular docking revealed that AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively. Analysis suggests that 1,6-anhydro-N-acetylglucosamine (anhGlcNAc) and/or 1,6-anhydro-ß-d-glucosamine (anhGlcN) would be the appropriate substrates of AnmK-like kinases. Also, the counterparts of other characteristic enzymes of cell wall recycling of bacteria were found in fungi. Taken together, it is proposed that a putative recycling of anhGlcNAc/anhGlcN, which is associated with the hydrolysis of cell walls, exists in fungi. This computational analysis will provide new insights into the metabolism of fungal cell walls.

Molecular techniques revealed highly diverse microbial communities in natural marine biofilms on polystyrene dishes for invertebrate larval settlement.

Biofilm microbial communities play an important role in the larval settlement response of marine invertebrates. However, the underlying mechanism has yet to be resolved, mainly because of the uncertainties in characterizing members in the communities using traditional 16S rRNA gene-based molecular methods and in identifying the chemical signals involved. In this study, pyrosequencing was used to characterize the bacterial communities in intertidal and subtidal marine biofilms developed during two seasons. We revealed highly diverse biofilm bacterial communities that varied with season and tidal level. Over 3,000 operational taxonomic units with estimates of up to 8,000 species were recovered in a biofilm sample, which is by far the highest number recorded in subtropical marine biofilms. Nineteen phyla were found, of which Cyanobacteria and Proteobacteria were the most dominant one in the intertidal and subtidal biofilms, respectively. Apart from these, Actinobacteria, Bacteroidetes, and Planctomycetes were the major groups recovered in both intertidal and subtidal biofilms, although their relative abundance varied among samples. Full-length 16S rRNA gene clone libraries were constructed for the four biofilm samples and showed similar bacterial compositions at the phylum level to those revealed by pyrosequencing. Laboratory assays confirmed that cyrids of the barnacle Balanus amphitrite preferred to settle on the intertidal rather than subtidal biofilms. This preference was independent of the biofilm bacterial density or biomass but was probably related to the biofilm community structure, particularly, the Proteobacterial and Cyanobacterial groups.

Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees.

Cellulolytic bacteria in forest soil provide carbon sources to improve the soil fertility and sustain the nutrient balance of the forest ecological system through the decomposition of cellulosic remains. These bacteria can also be utilized for the biological conversion of biomass into renewable biofuels. In this study, the community compositions and activities of cellulolytic bacteria in the soils of forests planted with broad-leaved deciduous (Chang Qing Garden, CQG) and broad-leaved evergreen (Forest Park, FP) trees in Wuhan, China were resolved through restriction fragment length polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. All of the isolates exhibited 35 RFLP fingerprint patterns and were clustered into six groups at a similarity level of 50 %. The phylogeny analysis based on the 16S rRNA gene sequence revealed that these RFLP groups could be clustered into three phylogenetic groups and further divided into six subgroups at a higher resolution. Group I consists of isolates from Bacillus cereus, Bacillus subtilis complex (I-A) and from Paenibacillus amylolyticus-related complex (I-B) and exhibited the highest cellulase activity among all of the cellulolytic bacteria isolates. Cluster II consists of isolates belonging to Microbacterium testaceum (II-A), Chryseobacterium indoltheticum (II-B), and Flavobacterium pectinovorum and the related complex (II-C). Cluster III consists of isolates belonging to Pseudomonas putida-related species. The community shift with respect to the plant species and the soil properties was evidenced by the phylogenetic composition of the communities. Groups I-A and I-B, which account for 36.0 % of the cellulolytic communities in the CQG site, are the dominant groups (88.4 %) in the FP site. Alternatively, the ratio of the bacteria belonging to group III (P. putida-related isolates) shifted from 28.0 % in CQG to 4.0 % in FP. The soil nutrient analysis revealed that the CQG site planted with deciduous broad-leaved trees has a richer organic nutrient (total organic carbon and total nitrogen) than the FP site planted with evergreen broad-leaved trees. Against this background, the population density and the diversity of cellulolytic bacteria in the CQG site are clearly higher than those in the FP site, and the latter was dominated with high-cellulase-activity Bacillus- and Paenibacillus-related bacteria. The canonical correspondence analysis further indicated that the distribution of these groups is correlated with the FP site, whereas groups II and III are correlated with the organic nutrient-rich CQG site.

Community composition of nirS-type denitrifier in a shallow eutrophic lake.

Denitrification is a major biological process to reduce nitrate to molecular nitrogen (N2). In shallow eutrophic lakes, this process can remove the largest portion of fixed nitrogen and plays an important role in self-purification of this ecosystem. To understand the structure of denitrifying communities in a shallow eutrophic lake, denitrifier communities in four sub-lakes of East Lake in Wuhan, China, were explored by restriction fragment length polymorphisms (RFLP) analysis and sequencing of nirS gene clone libraries. nirS is a functional marker gene for denitrification encoding cytochrome cd 1-containing nitrite reductase, which catalyzes the reduction of nitrite to nitric oxide. Both RFLP fingerprints clustering analysis and phylogeny analysis based on the amino acid sequences of NirS revealed that NirS-type communities in East Lake sediment could be roughly divided into three clusters. Cluster I accounted for 74-82 % of clones from the moderately eutrophic sub-lakes Tuan, Tang Ling, and Guo Zheng. Cluster II accounted for 76 % of the communities in hypertrophic sub-lake Miao Lake and cluster III as a minor group (7 % of the total), mainly presented in Miao Lake. Phylogenetic analysis revealed that cluster I was related to the reference clones from a broad range of ecological environments, and clusters II and III were more phylogenetically related to the reference clones from entrophic environments. Canonical correspondence analysis indicated that total nitrogen, total phosphate, total organic carbon, and NH4-N and NO2-N were important environmental factors affecting the dispersion of NirS-type denitrifier in the sediments. Cluster I showed a weak relationship with the nutrient content, while cluster II and III were positively related with the nutrient content. Principal coordinates analysis indicated that NirS-type communities from Tuan Lake, Tang Ling Lake, and Guo Zheng Lake sediments were divergent from those found in river, estuary sediment, and forest soil but similar to communities in constructed wetland sediment despite large geographic distances. The communities from the hypertrophic sub-lake Miao Lake deviated from other sub-lakes and the reference communities and clustered independently. Our results support the argument that environmental factors regulate the composition and distribution of the functional bacterial groups.

Deep sequencing of Myxilla (Ectyomyxilla) methanophila, an epibiotic sponge on cold-seep tubeworms, reveals methylotrophic, thiotrophic, and putative hydrocarbon-degrading microbial associations.

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ(13)C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

Modifying the amino acids in conformational motion pathway of the α-amylase of Geobacillus stearothermophilus improved its activity and stability.

Amino acids along the conformational motion pathway of the enzyme molecule correlated to its flexibility and rigidity. To enhance the enzyme activity and thermal stability, the motion pathway of Geobacillus stearothermophilus α-amylase has been identified and molecularly modified by using the neural relational inference model and deep learning tool. The significant differences in substrate specificity, enzymatic kinetics, optimal temperature, and thermal stability were observed among the mutants with modified amino acids along the pathway. Mutants especially the P44E demonstrated enhanced hydrolytic activity and catalytic efficiency (kcat/KM) than the wild-type enzyme to 95.0% and 93.8% respectively, with the optimum temperature increased to 90°C. This mutation from proline to glutamic acid has increased the number and the radius of the bottleneck of the channels, which might facilitate transporting large starch substrates into the enzyme. The mutation could also optimize the hydrogen bonding network of the catalytic center, and diminish the spatial hindering to the substrate entry and exit from the catalytic center.

Spatial and species variations in bacterial communities associated with corals from the Red Sea as revealed by pyrosequencing.

Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which Gammaproteobacteria was the most dominant group, and Chloroflexi, Chlamydiae, and the candidate phylum WS3 were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of Vibrio, Pseudoalteromonas, Serratia, Stenotrophomonas, Pseudomonas, and Achromobacter in the corals. On the other hand, bacteria such as Chloracidobacterium and Endozoicomonas were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.