Soil Resources Influence Spatial Patterns of Denitrifying Communities at Scales Compatible with Land Management

Knowing spatial patterns of functional microbial guilds can increase our understanding of the relationships between microbial community ecology and ecosystem functions. Using geostatistical modeling to map spatial patterns, we explored the distribution of the community structure, size, and activity of one functional group in N cycling, the denitrifiers, in relation to 23 soil parameters over a 44-ha farm divided into one organic and one integrated crop production system. The denitrifiers were targeted by the nirS and nirK genes that encode the two mutually exclusive types of nitrite reductases, the cd₁ heme-type and copper reductases, respectively. The spatial pattern of the denitrification activity genes was reflected by the maps of the abundances of nir genes. For the community structure, only the maps of the nirS community were related to the activity. The activity was correlated with nitrate and dissolved organic nitrogen and carbon, whereas the gene pools for denitrification, in terms of size and composition, were influenced by the soil structure. For the nirS community, pH and soil nutrients were also important in shaping the community. The only unique parameter related to the nirK community was the soil Cu content. However, the spatial pattern of the nirK denitrifiers corresponded to the division of the farm into the two cropping systems. The different community patterns, together with the spatial distribution of the nirS/nirK abundance ratio, suggest habitat selection on the nirS- and nirK-type denitrifiers. Our findings constitute a first step in identifying niches for denitrifiers at scales relevant to land management.Soil microorganisms are abundant and diverse (46), drive key processes in biogeochemical cycles, and, thus, play crucial roles in ecosystem functioning (2). They are not randomly distributed but exhibit spatial patterns at different scales (26). Spatial patterns ranging from the micrometer up to the meter scale have been reported (19, 20, 32, 37), and an understanding of such patterns can give clues to how microbial communities are generated and maintained (17). Spatial patterns of microorganisms at the field and landscape scales warrant special attention, since they could be associated with land use and aid in creating knowledge-based management strategies for agricultural production (5, 42). However, our understanding of key habitat-selective factors is limited, and few studies have specified which factors influence the spatial patterns of soil microbial communities at larger scales. Lauber et al. (30) recently demonstrated that pH could predict the community composition of soil bacteria at the continental scale. The importance of pH as a key edaphic driver of bacterial community structure has also been shown in other studies (11, 47). Another major, but complex, factor pointed out in a few studies is the soil type (4, 5, 14). Most studies have included only a limited number of properties that are easy to measure; most often, carbon and nitrogen pools and soil physical factors have been neglected in microbial community ecology. Since these factors delineate soil oxygen and water content, they may exert a stronger impact on microbial communities than other soil resources.Reports on the field or landscape scale spatial distribution of soil bacteria have had a taxon-centered perspective at either the species or total-community level, but there is emerging interest in the biogeography of functional traits possessed by microorganisms (18). Bacterial species composition is likely important for soil ecosystem functions, but species affiliation rarely predicts in which way. In addition, the fuzzy species concept of bacteria makes it all the more difficult to link species to niches. Analysis of functional guilds, i.e., assemblages of populations sharing certain traits, can bridge this gap, and one guild of global concern that has been suggested and recently used as a model in functional ecology is the denitrifiers (39, 49). Denitrification is an anaerobic respiration pathway during which NO₃⁻ is reduced to N₂ by a wide range of unrelated taxa. The process is an essential route for N loss from agricultural soil and a major source of the greenhouse gas N₂O. It was recently shown that the spatial distribution of the relative abundances of denitrifiers with the genetic capacity to perform the last step in the denitrification pathway, reduction of N₂O to N₂, is linked to areas with high denitrification rates and low N₂O emissions (38). Adding field scale predicted patterns of the denitrifier community structure to the abundance and activity would not only give insight into the mechanisms shaping the community, but also deepen our understanding of the relationships between the ecology of denitrifiers, N loss, and the agroecosystems'' impact on climate change.We hypothesize that spatial autocorrelations of the structures, sizes, and activities of communities of denitrifying bacteria is governed by soil-based resources at a scale compatible with land management. To test this, and to elucidate the effects of crop production systems and the importance of soil physical and chemical factors in the denitrifying community, we explored the spatial distribution of community structure, size, and activity in relation to 23 soil parameters at a 44-ha farm divided into one organic and one integrated crop production system. The denitrifier community was described in terms of the signature genes that encode the two different types of nitrite reductases in the denitrification pathway, the cd₁ heme-type reductase (NirS), encoded by the nirS gene, and the copper oxidoreductase (NirK), encoded by nirK. The spatial patterns were mapped by geostatistical modeling, and correlation structures were explored.     

Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1

Microsomal prostaglandin E synthase-1 (MPGES1) is induced during an inflammatory reaction from low basal levels by pro-inflammatory cytokines and subsequently involved in the production of the important mediator of inflammation, prostaglandin E₂. Nonsteroidal anti-inflammatory drugs prevent prostaglandin E₂ production by inhibiting the upstream enzymes cyclooxygenases 1 and 2. In contrast to these conventional drugs, a new generation of NSAIDs targets the terminal enzyme MPGES1. Some of these compounds potently inhibit human MPGES1 but do not have an effect on the rat orthologue. We investigated this interspecies difference in a rat/human chimeric form of the enzyme as well as in several mutants and identified key residues Thr-131, Leu-135, and Ala-138 in human MPGES1, which play a crucial role as gate keepers for the active site of MPGES1. These residues are situated in transmembrane helix 4, lining the entrance to the cleft between two subunits in the protein trimer, and regulate access of the inhibitor in the rat enzyme. Exchange toward the human residues in rat MPGES1 was accompanied with a gain of inhibitor activity, whereas exchange in human MPGES1 toward the residues found in rat abrogated inhibitor activity. Our data give evidence for the location of the active site at the interface between subunits in the homotrimeric enzyme and suggest a model of how the natural substrate PGH₂, or competitive inhibitors of MPGES1, enter the active site via the phospholipid bilayer of the membrane.     

Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants.

Here the structure of human glyoxalase II has been investigated by studying unfolding at equilibrium and refolding. Human glyoxalase II contains two tryptophan residues situated at the N-terminal (Trp57) and C-terminal (Trp199) regions of the molecule. Trp57 is a non-conserved residue located within a "zinc binding motif" (T/SHXHX57DH) which is strictly conserved in all known glyoxalase II sequences as well as in metal-dependent beta-lactamase and arylsulfatase. Site-directed mutagenesis has been used to construct single-tryptophan mutants in order to characterize better the guanidine-induced unfolding intermediates. The denaturation at equilibrium of wild-type glyoxalase II, as followed by activity, intrinsic fluorescence and CD, is multiphasic, suggesting that different regions of varying structural stability characterize the native structure of glyoxalase II. At intermediate denaturant concentration (1.2 M guanidine) a molten globule state is attained. The reactivation of the denatured wild-type enzyme occurs only in the presence of Zn(II) ions. The results show that Zn(II) is essential for the maintenance of the native structure of glyoxalase II and that its binding to the apoenzyme occurs during an essential step of refolding. The comparison of unfolding fluorescence transitions of single-trypthophan mutants with that of wild-type enzyme indicates that the strictly conserved "zinc binding motif" is located in a flexible region of the active site in which Zn(II) participates in catalysis.     

Evolution of clonality and polyploidy in a weevil system

The increased interest in asexual organisms calls for in-depth studies of asexual complexes that actively give rise to new clones. We present an extensive molecular study of the Otiorhynchus scaber (Coleoptera, Curculionidae) weevil system. Three forms have traditionally been recognized: diploid sexuals, triploid, and tetraploid parthenogens. All forms coexist in a small central area, but only the polyploid parthenogens have colonized marginal areas. Analyzing the phylogenetic relationship, based on three partial mitochondrial genes, of 95 individuals from 19 populations, we find that parthenogenesis and polyploidy have originated at least three times from different diploid lineages. We observe two major mitochondrial lineages, with over 2.5% sequence divergence between the most basal groups within them, and find that current distribution and phylogenetic relationships are weakly correlated. Quite unexpectedly, we also discover diploid clones that coexist with, and are morphologically indistinguishable from, the diploid sexual females. Our results support that these diploid clones are derived directly from the diploid sexuals. We also find that it is mainly an increase in ploidy level and not the benefits of asexual reproduction that confers to polyploid parthenogens the advantage over their diploid sexual relatives.     

Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein

By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages.     

Implications of shoot structure on the rate of photosynthesis at different levels in a coniferous canopy using a model incorporating grouping and penumbra

1. The connection between high leaf area index (LAI) and photosynthetic production with two attributes of coniferous canopy structure: small leaf size and grouping of needles on shoots, was analysed using a simulation model.
2. The small size of conifer needles gives rise to penumbras, which even out the distribution of direct sunlight on the leaf area and thereby act to increase the rate of canopy photosynthesis per unit of LAI.
3. Grouping, by producing a non-uniform distribution of leaf area, causes a decrease in total canopy light interception at any given LAI, but improves the photosynthetic light capture by shoots in the lower canopy.
4. Application of the model on a case study showed that: (a) grouping had a negative effect on the rate of photosynthesis in the upper canopy, but deeper down in the canopy the situation was reversed; (b) in the lower canopy, photosynthetic rates were up to 10 times higher as a result from the combined effect of grouping and penumbra; (c) grouping did not improve the rate of canopy photosynthesis per unit of LAI, however, it can have a positive effect on the total photosynthetic production by allowing a higher productive LAI to be maintained; (d) penumbra, on the other hand, increased the rate of canopy photosynthesis by as much as 40% for moderate values of the LAI.     

Use of recombinant virus to assess human cytomegalovirus early and late promoters in the context of the viral genome.

We have developed a system to study human cytomegalovirus (HCMV) cis-acting promoter elements within the context of the viral genome. A recombinant HCMV (RV134) containing a marker gene (beta-glucuronidase) was used to insert HCMV promoter-chloramphenicol acetyltransferase gene constructs into the viral genome between open reading frames US9 and US10. Using this system, we have studied the promoters for the early DNA polymerase gene (UL54), the early-late lower matrix phosphoprotein gene (pp65, UL83), and the true late 28-kDa structural phosphoprotein gene (pp28, UL99). Transient-expression assays demonstrated that the pp65 and pp28 promoters are activated earlier and to higher levels than typically observed with the endogenous gene. In contrast, insertion of these promoters into the viral genome resulted in kinetics which mimicked that of the endogenous genes. In addition, we have also tested a variant of the pp28 promoter (d24/26CAT) which is deleted from -609 to -41. This promoter behaved similarly to the wild-type pp28 promoter, indicating that sequences from -40 to +106 are sufficient for conferring true late kinetics. Taken together, these data demonstrate that the viral genome affords a level of regulation on HCMV gene expression that has been previously unrealized. Therefore, these experiments provide a model system for the analysis of cis-acting promoter regulatory elements in the context of the viral genome.