Microbial Community Composition and Activity in Saline Soils of Coastal Agro-Ecosystems.

Soil salinity is a serious problem for agriculture in coastal regions. Nevertheless, the effects of soil salinity on microbial community composition and their metabolic activities are far from clear. To improve such understanding, we studied microbial diversity, community composition, and potential metabolic activity of agricultural soils covering non-, mild-, and severe-salinity. The results showed that salinity had no significant effect on bacterial richness; however, it was the major driver of a shift in bacterial community composition and it significantly reduced microbial activity. Abundant and diverse of microbial communities were detected in the severe-salinity soils with an enriched population of salt-tolerant species. Co-occurrence network analysis revealed stronger dependencies between species associated with severe salinity soils. Results of microcalorimetric technology indicated that, after glucose amendment, there was no significant difference in microbial potential activity among soils with the three salinity levels. Although the salt prolonged the lag time of microbial communities, the activated microorganisms had a higher growth rate. In conclusion, salinity shapes soil microbial community composition and reduces microbial activity. An addition of labile organic amendments can greatly alleviate salt restrictions on microbial activity, which provides new insight for enhancing microbial ecological functions in salt-affected soils.

Balanced stochastic versus deterministic assembly processes benefit diverse yet uneven ecosystem functions in representative agroecosystems.

Ecological assembly processes, by influencing community composition, determine ecosystem functions of microbiomes. However, debate remains on how stochastic versus deterministic assembly processes influence ecosystem functions such as carbon and nutrient cycling. Towards a better understanding, we investigated three types of agroecosystems (the upland, paddy, and flooded) that represent a gradient of stochastic versus deterministic assembly processes. Carbon and nutrient cycling multifunctionality, characterized by nine enzymes associated with soil carbon, nitrogen, phosphorous and sulfur cycling, was evaluated and then associated with microbial assembly processes and co-occurrence patterns of vital ecological groups. Our results suggest that strong deterministic processes favour microorganisms with convergent functions (as in the upland agroecosystem), while stochasticity-dominated processes lead to divergent functions (as in the flooded agroecosystem). To benefit agroecosystems services, we speculate that it is critical for a system to maintain balance between its stochastic and deterministic assembly processes (as in the paddy agroecosystem). By doing so, the system can preserve a diverse array of functional traits and also allow for particular traits to flourish. To further confirm this speculation, it is necessary to develop a systematic knowledge beyond merely characterizing general patterns towards the associations among community assembly, composition, and ecosystem functions.

Long-Term Stochasticity Combines With Short-Term Variability in Assembly Processes to Underlie Rice Paddy Sustainability.

Revealing temporal patterns of community assembly processes is important for understanding how microorganisms underlie the sustainability of agroecosystem. The ancient terraced rice paddies at Longji provide an ideal platform to study temporal dynamics of agroecosystem sustainability due to their chronosequential records of soil physicochemistry and well-archived microbial information along 630-year rice cultivation. We used statistical null models to evaluate microbial assembly processes along the soil chronosequences of Longji rice paddies through time. Stochastic and deterministic assembly processes jointly governed microbial community composition within successional eras (less than 250 years), and within-era determinism was mainly driven by soil fertility and redox conditions alone or in combination. Conversely, across successional eras (i.e., over 300 years), stochasticity linearly increased with increasing duration between eras and was eventually predominant for the whole 630 years. We suggest that the impact of stochasticity vs. determinism on assembly is timescale-dependent, and we propose that the importance of stochastic assembly of microbial community at longer timescales is due to the gradual changes in soil properties under long-term rice cultivation, which in turn contribute to the sustainability of paddy ecosystem by maintaining a diverse community of microorganisms with multi-functional traits. In total, our results indicate that knowledge on the timescales at which assembly processes govern microbial community composition is key to understanding the ecological mechanisms generating agroecosystem sustainability.

Balanced Fertilization Decreases Environmental Filtering on Soil Bacterial Community Assemblage in North China.

Although increasing evidences have emerged for responses of soil microorganisms to fertilizations, the knowledge regarding community assemblages that cause variations in composition is still lacking, as well as the possible feedback to soil fertility. Phylogenetic conservatism of species indicates their similar environmental preferences and/or function traits and phylogenetic signals further can infer community assemblages and influenced ecological processes. Here, we calculated the mean pairwise phylogenetic distance and nearest relative index, characterizing phylogenetic signal and the undergone ecological process to evaluate the community assembly of soil bacterial phylotypes in 20-year fertilized soils. The bacterial community assembly is structured by environmental filtering, regardless of fertilization regime. Soil phosphorous (P) availability imposes selection on community assemblage and influences their community turnover among fertilizations. When P nutrient lacks, the effect of environmental filtering becomes stronger, hence bacterial functional traits become more coherent; this process results into increased intraspecific interactions characterized by co-occurrence network analysis. In contrast, when P nutrient becomes abundant, the environmental selection is mitigated; function traits are evened. This process reduces intraspecific interactions and increases carbon sequestration efficiency, which is finally of great favor to the increases in soil fertility. This study has made the first attempt, at the bacterial level, to understand how fertilization affects agroecosystems. When more phylogenetic information on how nutrient cycling-related microbes respond to fertilization becomes available, the systematic knowledge will eventually provide guidance to optimal fertilization strategies.

Bioremediation of Wastewater by Iron Oxide-Biochar Nanocomposites Loaded with Photosynthetic Bacteria.

It has been reported that bacteria-mediated degradation of contaminants is a practical and innocuous wastewater treatment. In addition, iron oxide nanoparticles (NP) are wastewater remediation agents with great potentials due to their strong adsorption capacity, chemical inertness and superparamagnetism. Therefore, a combination of NPs and microbes could produce a very desirable alternative to conventional wastewater treatment. For this purpose, we first prepared Fe3O4/biochar nano-composites, followed by loading photosynthetic bacteria (PSB) onto them. It was found that Fe3O4/biochar nano-composites exhibited a high adsorption capacity for PSB (5.45 × 109 cells/g). The efficiency of wastewater pollutants removal by this PSB/Fe3O4/biochar agent was then analyzed. Our results indicated that when loaded onto Fe3O4/biochar nano-composites, PSB's nutrient removal capability was significantly enhanced (P < 0.05). This agent removed 83.1% of chemical oxygen demand, 87.5% of NH4+, and 92.1% of PO43- from the wastewater in our study. Our experiments also demonstrated that such composites are outstanding recyclable agents. Their nutrient removal capability remained effective even after five cycles. In conclusion, we found the PSB/Fe3O4/biochar composites as a very promising material for bioremediation in the wastewater treatment.

Geographical Distribution of Methanogenic Archaea in Nine Representative Paddy Soils in China.

Paddy field methanogenic archaea are responsible for methane (CH4) production and contribute significantly to climate change. The information regarding the spatial variations in the abundance, the diversity and the composition of such ecologically important microbes, however, is quite limited at large scale. In this investigation, we studied the abundance, alpha diversity and geographical distribution of methanogenic archaeal communities in nine representative paddy sites, along a large latitudinal gradient in China, using pyrosequencing and real-time quantitative PCR. It is found that all paddy soils harbor constant methanogenic archaeal constituents, which is dominated by family Methanocellaceae (37.3%), Methanobacteriaceae (22.1%), Methanosaetaceae (17.2%), and Methanosarcinaceae (9.8%). Methanogenic archaeal abundance is primarily influenced by soil C (R = 0.612, P = 0.001) and N (R = 0.673, P = 0.001) contents, as well as alpha diversity by soil pH (PD: R = -0.552, P = 0.006; Chao1: R = -0.615, P = 0.002). Further exploration revealed that both spatial distance (R = 0.3469, P = 0.001, partial mental test) and soil chemical variables mainly about soil C and N (R = 0.2847, P = 0.001) are the two major factors affecting methanogenic archaeal community composition distribution in paddy soils. This finding will allow us to develop a better picture of the biogeographic ranges of these ecologically important microbes and get deeper insights into their ecology.

Hydrogen-Bonding Network and OH Stretch Vibration of Cellulose: Comparison of Computational Modeling with Polarized IR and SFG Spectra.

Hydrogen bonds play critical roles in noncovalent directional interactions determining the crystal structure of cellulose. Although diffraction studies accurately determined the coordinates of carbon and oxygen atoms in crystalline cellulose, the structural information on hydrogen atoms involved in hydrogen-bonding is still elusive. This could be complemented by vibrational spectroscopy; but the assignment of the OH stretch peaks has been controversial. In this study, we performed calculations using density functional theory with dispersion corrections (DFT-D2) for the cellulose Iß crystal lattices with the experimentally determined carbon and oxygen coordinates. DFT-D2 calculations revealed that the OH stretch vibrations of cellulose are highly coupled and delocalized through intra- and interchain hydrogen bonds involving all OH groups in the crystal. Additionally, molecular dynamics (MD) simulations of a single cellulose microfibril showed that the conformations of OH groups exposed at the microfibril surface are not well-defined. Comparison of the computation results with the experimentally determined IR dichroism of uniaxially aligned cellulose microfibrils and the peak positions of various cellulose crystals allowed unambiguous identification of OH stretch modes observed in the vibrational spectra of cellulose.

Insight into structural rearrangements and interdomain interactions related to electron transfer between flavin mononucleotide and heme in nitric oxide synthase: A molecular dynamics study.

Calmodulin (CaM) binding to nitric oxide synthase (NOS) enables a conformational change, in which the FMN domain shuttles between the FAD and heme domains to deliver electrons to the active site heme center. A clear understanding of this large conformational change is critical, since this step is the rate-limiting in NOS catalysis. Herein molecular dynamics simulations were conducted on a model of an oxygenase/FMN (oxyFMN) construct of human inducible NOS (iNOS). This is to investigate the structural rearrangements and the domain interactions related to the FMN-heme interdomain electron transfer (IET). We carried out simulations on the iNOS oxyFMN·CaM complex models in [Fe(III)][FMNH(-)] and [Fe(II)][FMNH] oxidation states, the pre- and post-IET states. The comparison of the dynamics and conformations of the iNOS construct at the two oxidation states has allowed us to identify key factors related to facilitating the FMN-heme IET process. The computational results demonstrated, for the first time, that the conformational change is redox-dependent. Predictions of the key interacting sites in optimal interdomain FMN/heme docking are well supported by experimental data in the literature. An intra-subunit pivot region is predicted to modulate the FMN domain motion and correlate with existence of a bottleneck in the conformational sampling that leads to the electron transfer-competent state. Interactions of the residues identified in this work are proposed to ensure that the FMN domain moves with appropriate degrees of freedom and docks to proper positions at the heme domain, resulting in efficient IET and nitric oxide production.

Sensitivity-enhanced solid-state NMR detection of expansin's target in plant cell walls.

Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of (13)C spin diffusion from trace concentrations of (13)C, (15)N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short (13)C-(13)C distances of 4-6 Å between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D (13)C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets.

Cellulose microfibril twist, mechanics, and implication for cellulose biosynthesis.

All-atom molecular dynamics simulations with explicit water solvent were used to investigate the microstructure and conformational dynamics of cellulose Iß microfibrils as a function of microfibril length and cross-sectional size and shape. Cellulose microfibrils quickly develop a right-handed twist, which then remains stable over the entire 10 ns simulation time. The helical angle is independent of microfibril length and inversely proportional to its cross-sectional area, in accord with the expectations of continuum theory for an intrinsic chiral twist that is opposed by torsional shear. These calculations provide-to our knowledge-the first estimates of the shear modulus of a cellulose microfibril from MD simulations. The internal strains caused by this helical twist, propagated indefinitely along the microfibril axis, could be relaxed by periodic regions of amorphous structure along the axis of the cellulose microfibrils.

Dynamic multibody protein interactions suggest versatile pathways for copper trafficking.

As part of intracellular copper trafficking pathways, the human copper chaperone Hah1 delivers Cu(+) to the Wilson's Disease Protein (WDP) via weak and dynamic protein-protein interactions. WDP contains six homologous metal binding domains (MBDs) connected by flexible linkers, and these MBDs all can receive Cu(+) from Hah1. The functional roles of the MBD multiplicity in Cu(+) trafficking are not well understood. Building on our previous study of the dynamic interactions between Hah1 and the isolated fourth MBD of WDP, here we study how Hah1 interacts with MBD34, a double-domain WDP construct, using single-molecule fluorescence resonance energy transfer (smFRET) combined with vesicle trapping. By alternating the positions of the smFRET donor and acceptor, we systematically probed Hah1-MBD3, Hah1-MBD4, and MBD3-MBD4 interaction dynamics within the multidomain system. We found that the two interconverting interaction geometries were conserved in both intermolecular Hah1-MBD and intramolecular MBD-MBD interactions. The Hah1-MBD interactions within MBD34 are stabilized by an order of magnitude relative to the isolated single-MBDs, and thermodynamic and kinetic evidence suggest that Hah1 can interact with both MBDs simultaneously. The enhanced interaction stability of Hah1 with the multi-MBD system, the dynamic intramolecular MBD-MBD interactions, and the ability of Hah1 to interact with multiple MBDs simultaneously suggest an efficient and versatile mechanism for the Hah1-to-WDP pathway to transport Cu(+).

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of ß-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase.

Exposure of hydrophobic core in human prion protein pathogenic mutant H187R.

Pathogenesis studies have revealed that H187R mutation of human prion protein (huPrP) is related to GSS type of TSE diseases. Its pathogenic mechanism is still unclear. We here studied the globular domain of this mutant protein by molecular dynamics simulations. Compared to the wide-type protein, the mutant has similar dynamics and stability profiles in our simulation. Conformational rearrangements are concentrated around the mutation site, due to the introduction the positively charged side chain of Arg187. The strong electrostatic repulsion between Arg156 and Arg187 drives both side chains away from their original positions, leaving its hydrophobic core to be solvent accessible. Such a unfavorable conformational change may destabilize the mutant protein and make it more susceptible to unfolding.

Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Ibeta: the enzyme-substrate complex.

Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand cellobiohydrolase function, new simulations have been conducted using charmm of cellobiohydrolase I (CBH I) from T.reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.

Investigation of the effect of glycosylation on human prion protein by molecular dynamics.

Prion protein conformational isomerization, PrP(C)-->PrP(Sc), has been attributed as the cause of TSE diseases such as mad-cow disease. The mechanism of such isomerization, however, is little known due the experimental difficulties in studying the scrapie form. Among factors that affect PrP isomerization, the role which glycosylation plays remains vague. The number of innumerous glycan species, together with their high flexibility, leads to ineffective structural characterization. In this research, we studied the effect of chitobiose glycosylation on human PrP, in both monomeric (huPrP(mono)) and dimeric (huPrP(dimer)) forms, by molecular dynamics (MD) simulations. Our results show that this glycosylation has minimal impact on the structure of huPrP(mono). However, it affects the secondary structure of dimeric protein. An additional beta-sheet strand is found while the glycosylation is absent in the huPrP(dimer). Comparatively, when the protein is glycosylated with chitobiose, such beta-sheet addition is not observed.

Molecular modeling suggests induced fit of Family I carbohydrate-binding modules with a broken-chain cellulose surface.

Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, their molecular mode of action on cellulose is not well understood. These enzymes act to detach and hydrolyze cellodextrin chains from crystalline cellulose in a processive manner, and the carbohydrate-binding module (CBM) is thought to play an important role in this process. Understanding the interactions between the CBM and cellulose at the molecular level can assist greatly in formulating selective mutagenesis experiments to confirm the function of the CBM. Computational molecular dynamics was used to investigate the interaction of the CBM from Trichoderma reesei cellobiohydrolase I with a model of the (1,0,0) cellulose surface modified to display a broken chain. Initially, the CBM was located in different positions relative to the reducing end of this break, and during the simulations it appeared to translate freely and randomly across the cellulose surface, which is consistent with its role in processivity. Another important finding is that the reducing end of a cellulose chain appears to induce a conformational change in the CBM. Simulations show that the tyrosine residues on the hydrophobic surface of the CBM, Y5, Y31 and Y32 align with the cellulose chain adjacent to the reducing end and, importantly, that the fourth tyrosine residue in the CBM (Y13) moves from its internal position to form van der Waals interactions with the cellulose surface. As a consequence of this induced change near the surface, the CBM straddles the reducing end of the broken chain. Interestingly, all four aromatic residues are highly conserved in Family I CBM, and thus this recognition mechanism may be universal to this family.

Heme axial methionine fluxionality in Hydrogenobacter thermophilus cytochrome c552.

The heme group in paramagnetic (S = 1/2) ferricytochromes c typically displays a markedly asymmetric distribution of unpaired electron spin density among the heme pyrrole beta substituents. This asymmetry is determined by the orientations of the heme axial ligands, histidine and methionine. One exception to this is ferricytochrome c(552) from Hydrogenobacter thermophilus, which has similar amounts of unpaired electron spin density at the beta substituents on all four heme pyrroles. Here, determination of the orientation of the magnetic axes and analysis of NMR line shapes for H. thermophilus ferricytochrome c(552) is performed. These data reveal that the unusual electronic structure for this protein is a result of fluxionality of the heme axial methionine. It is proposed that the ligand undergoes inversion at the pyramidal sulfur, and the rapid interconversion between two diastereomeric forms results in the unusual heme electronic structure. Thus a fluxional process for a metal-bound amino acid side chain has now been identified.

Backbone dynamics and hydrogen exchange of Pseudomonas aeruginosa ferricytochrome c(551).

A model-free analysis of Pseudomonas aeruginosa ferricytochrome c(551) dynamics based on (15)N R(1), (15)N R(2), and [(1)H]-(15)N heteronuclear nuclear Overhauser effect data is reported. The protein backbone is highly rigid (< S(2)>=0.924+/-0.005) and displays little variation in picosecond-nanosecond time scale dynamics over the structure. The loop structure containing the axial methionine ligand (loop 3) displays anomalous rigidity, which is attributed to its high proline content. Also reported are protection factors calculated from hydrogen-exchange rates. These data reveal that loop 3 residues, including the axial methionine, are protected from exchange as a result of long-range hydrogen-bonding interactions. These results are contrasted with data reported for Saccharomyces cerevisiae iso-1-ferricytochrome c, which displays higher overall flexibility (< S(2)>=0.80+/-0.07), greater variation of dynamics as a function of structure, and low protection factors for loop 3. This analysis reveals that heme proteins with similar functions and topologies may display diverse dynamical properties.

Peptide mimotopes of pneumococcal capsular polysaccharide of 6B serotype: a peptide mimotope can bind to two unrelated antibodies.

Two groups of bacteriophage clones displaying the antigenic properties of serotype 6B pneumococcal capsular polysaccharide (PS) were obtained from different phage libraries expressing random heptameric peptides. One group, biopanned with a mouse mAb (Hyp6BM1), is comprised of 17 phage clones expressing 10 unique sequences of linear peptides. The other group, selected with another mAb (Hyp6BM8), contained six clones, all of which expressed the identical circular peptide. Phage clones expressing the linear peptides (e.g., PhaM1L3) bound only to Hyp6BM1, but not other 6B PS-specific mAb, and their binding could be inhibited with pneumococcal capsular type 6B PS only. In contrast, a phage clone expressing the circular peptide (PhaM8C1) cross-reacted with several other 6B PS-specific mAbs, and their binding could be inhibited with pneumococcal capsular PS of 6A and 6B serotypes. Two short peptides, PepM1L3 and PepM8C1, reflecting the peptide inserts of the corresponding phage clones, could inhibit the binding of the two clones to their respective mAb. Interestingly, the peptide insert in PhaM8C1 was identical to that in PhaB3C4, a previously reported mimotope of alpha(2-->8) polysialic acid, Neisseria meningitidis group B PS. Indeed, PhaM8C1 bound to HmenB3 (a meningococcal Ab), and their association could be inhibited with alpha(2-8) polysialic acid, but not with 6B PS. Conversely, alpha(2-8) polysialic acid could not inhibit the binding of PhaM8C1 to Hyp6BM8. The two-dimensional nuclear magnetic resonance studies indicate that PepM8C1 peptide can assume several conformations in solution. The ability of this peptide to assume multiple conformations might account for its ability to mimic more than one Ag type.