Response of Soil Bacterial Community Diversity and Composition to Time, Fertilization, and Plant Species in a Sub-Boreal Climate

Pastures are an important part of crop and food systems in cold climates. Understanding how fertilization and plant species affect soil bacterial community diversity and composition is the key for understanding the role of soil bacteria in sustainable agriculture. To study the response of soil bacteria to different fertilization and cropping managements, a 3-year (2013-2015) field study was established. In the split-plot design, fertilizer treatment (unfertilized control, organic fertilizer, and synthetic fertilizer) was the main plot factor, and plant treatment [clear fallow, red clover (Trifolium pratense), timothy (Phleum pratense), and a mixture of red clover and timothy] was the sub-plot factor. Soil bacterial community diversity and composition, soil properties, and crop growth were investigated through two growing seasons in 2014 and 2015, with different nitrogen input levels. The community diversity measures (richness, Shannon diversity, and Shannon evenness) and composition changed over time (P<0.05) and at different time scales. The community diversity was lower in 2014 than in 2015. The temporal differences were greater than the differences between treatments. The overall correlations of Shannon diversity to soil pH, NO3-, NH4+, and surplus nitrogen were positive and that of bacterial richness to crop dry matter yield was negative (P<0.05). The major differences in diversity and community composition were found between fallow and planted treatments and between organic and synthetic fertilizer treatments. The differences between the planted plots were restricted to individual operational taxonomic units (OTUs). Soil moisture, total carbon content, and total nitrogen content correlated consistently with the community composition (P<0.05). Compared to the unfertilized control, the nitrogen fertilizer loading enhanced the temporal change of community composition in pure timothy and in the mixture more than that in red clover, which further emphasizes the complexity of interactions between fertilization and cropping treatments on soil bacteria.Peer reviewe

Stable nitrogen-cycling capacity in relation to fertilization and intercropping in a sub-boreal grassland

Grasslands are important in sub-boreal climate agricultural systems and are managed with various combinations of N fertilization and plant species. Ammonia-oxidizing and denitrifying microorganisms are key players in determining the fate of nitrogen (N) and thereby also the yield in grassland systems and their impact on gaseous N losses and leaching. We established a three-year field study in southern Finland with fertilizer treatment as a main-plot factor, including organic and synthetic fertilizers and plant species and mixtures thereof as the sub-plot factor. We quantified six genes encoding key N-cycling enzymes by quantitative PCR to determine the abundance of the communities involved in N-transformation processes and also included previously published data on crop yield, soil properties and the overall bacterial community composition. With the exception of ammonia oxidizing bacteria (AOB), which were primarily affected by fertilization, the abundances of all other N-cycling communities changed over time with either an increase or decrease from summer to autumn. Differences in gene abundances between plant species treatments and in fertilizer by plant species interactions were detected mainly in the beginning of the cropping season during the first year. The nirS-type denitrifiers and nosZII nitrous oxide reducers responded more to changes in soil properties than their functional counterpart nirK and nosZI communities. Using structural equation modeling, we show that the overall microbial community composition and diversity played an important role in mediating the management effects on crop yield, genetic potential for N retention and N2O sink capacity. However, a trade-off between the genetic potential for N retention and N2O sink capacity was detected, indicating the challenges in managing grasslands in a sustainable way.Peer reviewe

Stable nitrogen-cycling capacity in relation to fertilization and intercropping in a sub-boreal grassland

Grasslands are important in sub-boreal climate agricultural systems and are managed with various combinations of N fertilization and plant species. Ammonia-oxidizing and denitrifying microorganisms are key players in determining the fate of nitrogen (N) and thereby also the yield in grassland systems and their impact on gaseous N losses and leaching. We established a three-year field study in southern Finland with fertilizer treatment as a main-plot factor, including organic and synthetic fertilizers and plant species and mixtures thereof as the sub-plot factor. We quantified six genes encoding key N-cycling enzymes by quantitative PCR to determine the abundance of the communities involved in N-transformation processes and also included previously published data on crop yield, soil properties and the overall bacterial community composition. With the exception of ammonia oxidizing bacteria (AOB), which were primarily affected by fertilization, the abundances of all other N-cycling communities changed over time with either an increase or decrease from summer to autumn. Differences in gene abundances between plant species treatments and in fertilizer by plant species interactions were detected mainly in the beginning of the cropping season during the first year. The nirS-type denitrifiers and nosZII nitrous oxide reducers responded more to changes in soil properties than their functional counterpart nirK and nosZI communities. Using structural equation modeling, we show that the overall microbial community composition and diversity played an important role in mediating the management effects on crop yield, genetic potential for N retention and N2O sink capacity. However, a trade-off between the genetic potential for N retention and N2O sink capacity was detected, indicating the challenges in managing grasslands in a sustainable way.Peer reviewe

Supercooled Lennard-Jones Liquids and Glasses: a Kinetic Monte Carlo Approach

A kinetic Monte Carlo (KMC) method is used to study the structural properties and dynamics of a supercooled binary Lennard-Jones liquid around the glass transition temperature. This technique permits us to explore the potential energy surface and barrier distributions without suffering the exponential slowing down at low temperature that affects molecular dynamics simulations. In agreement with previous studies we observe a distinct change in behaviour around $T=0.45$, close to the dynamical transition temperature $T_c$ of mode coupling theory (MCT). Below this temperature the number of different local minima visited by the system for the same number of KMC steps decreases by more than an order of magnitude. The mean number of atoms involved in each jump between local minima and the average distance they move also decreases significantly, and new features appear in the partial structure factor. Above $T~0.45$ the probability distribution for the magnitude of the atomic displacement per KMC step exhibits an exponential decay, which is only weakly temperature dependent.Comment: Accepted for J. Non-Cryst. Solid

Comprehensive homing endonuclease target site specificity profiling reveals evolutionary constraints and enables genome engineering applications

Homing endonucleases (HEs) promote the evolutionary persistence of selfish DNA elements by catalyzing element lateral transfer into new host organisms. The high site specificity of this lateral transfer reaction, termed homing, reflects both the length (14–40 bp) and the limited tolerance of target or homing sites for base pair changes. In order to better understand molecular determinants of homing, we systematically determined the binding and cleavage properties of all single base pair variant target sites of the canonical LAGLIDADG homing endonucleases I-CreI and I-MsoI. These Chlorophyta algal HEs have very similar three-dimensional folds and recognize nearly identical 22 bp target sites, but use substantially different sets of DNA-protein contacts to mediate site-specific recognition and cleavage. The site specificity differences between I-CreI and I-MsoI suggest different evolutionary strategies for HE persistence. These differences also provide practical guidance in target site finding, and in the generation of HE variants with high site specificity and cleavage activity, to enable genome engineering applications

Mathematical Modelling of Mosquito Dispersal in a Heterogeneous Environment.

Mosquito dispersal is a key behavioural factor that affects the persistence and resurgence of several vector-borne diseases. Spatial heterogeneity of mosquito resources, such as hosts and breeding sites, affects mosquito dispersal behaviour and consequently affects mosquito population structures, human exposure to vectors, and the ability to control disease transmission. In this paper, we develop and simulate a discrete-space continuous-time mathematical model to investigate the impact of dispersal and heterogeneous distribution of resources on the distribution and dynamics of mosquito populations. We build an ordinary differential equation model of the mosquito life cycle and replicate it across a hexagonal grid (multi-patch system) that represents two-dimensional space. We use the model to estimate mosquito dispersal distances and to evaluate the effect of spatial repellents as a vector control strategy. We find evidence of association between heterogeneity, dispersal, spatial distribution of resources, and mosquito population dynamics. Random distribution of repellents reduces the distance moved by mosquitoes, offering a promising strategy for disease control

Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs

Site-specific homing endonucleases are capable of inducing gene conversion via homologous recombination. Reprogramming their cleavage specificities allows the targeting of specific biological sites for gene correction or conversion. We used computational protein design to alter the cleavage specificity of I-MsoI for three contiguous base pair substitutions, resulting in an endonuclease whose activity and specificity for its new site rival that of wild-type I-MsoI for the original site. Concerted design for all simultaneous substitutions was more successful than a modular approach against individual substitutions, highlighting the importance of context-dependent redesign and optimization of protein–DNA interactions. We then used computational design based on the crystal structure of the designed complex, which revealed significant unanticipated shifts in DNA conformation, to create an endonuclease that specifically cleaves a site with four contiguous base pair substitutions. Our results demonstrate that specificity switches for multiple concerted base pair substitutions can be computationally designed, and that iteration between design and structure determination provides a route to large scale reprogramming of specificity

Comprehensive computational design of mCreI homing endonuclease cleavage specificity for genome engineering

Homing endonucleases (HEs) cleave long (∼20 bp) DNA target sites with high site specificity to catalyze the lateral transfer of parasitic DNA elements. In order to determine whether comprehensive computational design could be used as a general strategy to engineer new HE target site specificities, we used RosettaDesign (RD) to generate 3200 different variants of the mCreI LAGLIDADG HE towards 16 different base pair positions in the 22 bp mCreI target site. Experimental verification of a range of these designs demonstrated that over 2/3 (24 of 35 designs, 69%) had the intended new site specificity, and that 14 of the 15 attempted specificity shifts (93%) were achieved. These results demonstrate the feasibility of using structure-based computational design to engineer HE variants with novel target site specificities to facilitate genome engineering

Computational identification of antibody-binding epitopes from mimotope datasets

Introduction: A fundamental challenge in computational vaccinology is that most B-cell epitopes are conformational and therefore hard to predict from sequence alone. Another significant challenge is that a great deal of the amino acid sequence of a viral surface protein might not in fact be antigenic. Thus, identifying the regions of a protein that are most promising for vaccine design based on the degree of surface exposure may not lead to a clinically relevant immune response.Methods: Linear peptides selected by phage display experiments that have high affinity to the monoclonal antibody of interest (“mimotopes”) usually have similar physicochemical properties to the antigen epitope corresponding to that antibody. The sequences of these linear peptides can be used to find possible epitopes on the surface of the antigen structure or a homology model of the antigen in the absence of an antigen-antibody complex structure.Results and Discussion: Herein we describe two novel methods for mapping mimotopes to epitopes. The first is a novel algorithm named MimoTree that allows for gaps in the mimotopes and epitopes on the antigen. More specifically, a mimotope may have a gap that does not match to the epitope to allow it to adopt a conformation relevant for binding to an antibody, and residues may similarly be discontinuous in conformational epitopes. MimoTree is a fully automated epitope detection algorithm suitable for the identification of conformational as well as linear epitopes. The second is an ensemble approach, which combines the prediction results from MimoTree and two existing methods

Generation of single-chain LAGLIDADG homing endonucleases from native homodimeric precursor proteins

Homing endonucleases (HEs) cut long DNA target sites with high specificity to initiate and target the lateral transfer of mobile introns or inteins. This high site specificity of HEs makes them attractive reagents for gene targeting to promote DNA modification or repair. We have generated several hundred catalytically active, monomerized versions of the well-characterized homodimeric I-CreI and I-MsoI LAGLIDADG family homing endonuclease (LHE) proteins. Representative monomerized I-CreI and I-MsoI proteins (collectively termed mCreIs or mMsoIs) were characterized in detail by using a combination of biochemical, biophysical and structural approaches. We also demonstrated that both mCreI and mMsoI proteins can promote cleavage-dependent recombination in human cells. The use of single chain LHEs should simplify gene modification and targeting by requiring the expression of a single small protein in cells, rather than the coordinate expression of two separate protein coding genes as is required when using engineered heterodimeric zinc finger or homing endonuclease proteins