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Plant–microbe symbioses span a continuum from pathogenic to mutualistic, with functional consequences for both organisms in the symbiosis. In order to increase sustainable food and fuel production in the future, it is imperative that we harness these symbioses. The tree genus Populus is an excellent model system for studies examining plant–microbe interactions due to the wealth of genomic information available and the molecular tools that have been developed to manipulate Populus–microbe symbioses. In this review, we highlight how Populus can serve as a model system to explore plant–microbe interactions. Specifically, we highlight research linking Populus–microbe interactions from the gene to the ecosystem level. We explore why Populus is an excellent model for perennial plant systems, the molecular underpinnings of Populus–microbe interactions, how host genetics influence microbial community composition, and how microbial communities vary at fine spatial scales and between Populus spp. Furthermore, we explore how changes in the microbiome may affect ecosystem-level functions in managed and natural ecosystems. Understanding and manipulating these interactions in Populus has the potential to improve plant health and affect ecosystem sustainability and processes because Populus trees function as foundational species in many natural ecosystems and are also deployed in managed ecosystems for various agroforestry applications. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The use of enterococci as a fecal indicator bacterial group for public health risk assessment has been brought into question by recent studies showing that “naturalized” populations of Enterococcus faecalis exist in the extraenteric environment. The extent to which these naturalized E. faecalis organisms can confound water quality monitoring is unclear. To determine if strains isolated from different habitats display different survival strategies and responses, we compared the decay patterns of three E. faecalis isolates from the natural environment (environmental strains) against three human gut isolates (enteric strains) in laboratory mesocosms that simulate an oligotrophic, aerobic freshwater environment. Our results showed similar overall decay rates between enteric and environmental isolates based on viable plate and quantitative PCR (qPCR) counts. However, the enteric isolates exhibited a spike in copy number ratios of 16S rRNA gene transcripts to 16S rRNA gene DNA copies (rRNA:rDNA ratios) between days 1 and 3 of the mesocosm incubations that was not observed in environmental isolates, which could indicate a different stress response. Nevertheless, there was no strong evidence of differential gene expression between environmental and enteric isolates related to habitat adaptation in the accompanying mesocosm metatranscriptomes. Overall, our results provide novel information on how rRNA levels may vary over different growth conditions (e.g., standard lab versus oligotrophic) for this important indicator bacteria. We also observed some evidence for habitat adaptation in E. faecalis; however, this adaptation may not be substantial or consistent enough for integration in water quality monitoring. IMPORTANCE Enterococci are commonly used worldwide to monitor environmental fecal contamination and public health risk for waterborne diseases. However, closely related enterococci strains adapted to living in the extraenteric environment may represent a lower public health risk and confound water quality estimates. We developed an rRNA:rDNA viability assay for E. faecalis (a predominant species within this fecal group) and tested it against both enteric and environmental isolates in freshwater mesocosms to assess whether this approach can serve as a more sensitive water quality monitoring tool. We were unable to reliably distinguish the different isolate types using this assay under the conditions tested; thus, environmental strains should continue to be counted during routine water monitoring. However, this assay could be useful for distinguishing more recent (i.e., higher-risk) fecal pollution because rRNA levels significantly decreased after 1 week in all isolates.

Fecal material in the environment is a primary source of pathogens that cause waterborne diseases and affect over a billion people worldwide. Microbial source tracking (MST) assays based on single genes (e.g., 16S rRNA) do not always provide the resolution needed to attribute fecal contamination sources. In this work, we used dialysis bag mesocosms simulating a freshwater habitat that were spiked separately with cow, pig, or human feces to monitor the decay of host-specific fecal signals over time with metagenomics, traditional qPCR, and culture-based methods. Sequencing of the host fecal communities used as inocula recovered 79 non-redundant metagenome-assembled genomes (MAGs) whose abundance patterns showed that the majority of the fecal community signal was not detectable in the mesocosm metagenomes after four days. Several MAGs showed high host specificity, and thus are promising candidates for biomarkers for their respective host type. Traditional qPCR methods varied in their correlation with MAG decay kinetics. Notably, the human-specific Bacteroides assay, HF183/BFDRev, consistently under-estimated fecal pollution due to not being present in all hosts and/or primer mismatches. This work provides new insights on the persistence and decay kinetics of host-specific gut microbes in the environment and identifies several MAGs as putative biomarkers for improved MST.SYNOPSISWe track cow, pig, and human fecal pollution in lake water over time with metagenomics and benchmark these novel protocols against standard culture-based and qPCR tests for water quality monitoring.

In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO(2), CH(4), and CO(2):CH(4) porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO(2) and CH(4) dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate–peatland feedbacks.

Northern-latitude tundra soils harbor substantial carbon (C) stocks that are highly susceptible to microbial degradation with rising global temperatures. Understanding the magnitude and direction (e.g., C release or sequestration) of the microbial responses to warming is necessary to accurately model climate change. In this study, Alaskan tundra soils were subjected to experimental in situ warming by ∼1.1 °C above ambient temperature, and the microbial communities were evaluated using metagenomics after 4.5 years, at 2 depths: 15 to 25 cm (active layer at outset of the experiment) and 45 to 55 cm (transition zone at the permafrost/active layer boundary at the outset of the experiment). In contrast to small or insignificant shifts after 1.5 years of warming, 4.5 years of warming resulted in significant changes to the abundances of functional traits and the corresponding taxa relative to control plots (no warming), and microbial shifts differed qualitatively between the two soil depths. At 15 to 25 cm, increased abundances of carbohydrate utilization genes were observed that correlated with (increased) measured ecosystem carbon respiration. At the 45- to 55-cm layer, increased methanogenesis potential was observed, which corresponded with a 3-fold increase in abundance of a single archaeal clade of the Methanosarcinales order, increased annual thaw duration (45.3 vs. 79.3 days), and increased CH 4 emissions. Collectively, these data demonstrate that the microbial responses to warming in tundra soil are rapid and markedly different between the 2 critical soil layers evaluated, and identify potential biomarkers for the corresponding microbial processes that could be important in modeling.

Tropical ecosystems are an important sink for atmospheric CO2; however, plant growth is restricted by phosphorus (P) availability. Although soil microbiota facilitate organic P turnover and inorganic P mobilization, their role in carbon-phosphorus coupled processes remains poorly understood. To advance this topic, soils collected from four sites representing highly weathered tropical soils in the El Yunque National Forest, Puerto Rico were incubated with exogenous PO43− under controlled laboratory conditions. P amendment increased CO2 respiration by 14–23% relative to control incubations for soils sampled from all but the site with the greatest total and bioavailable soil P. Metatranscriptomics revealed an increase in the relative transcription of genes involved in cell growth and uptake of other nutrients in response to P amendment. A new methodology to normalize gene expression by population-level relative (DNA) abundance revealed that the pattern of increased transcription of cell growth and division genes with P amendment was community-wide. Soil communities responsive to P amendment possessed a greater relative abundance of α-glucosyl polysaccharide biosynthesis genes, suggestive of enhanced C storage under P-limiting conditions. Phosphorylase genes governing the degradation of α-glucosyl polysaccharides were also more abundant and increased in relative transcription with P amendment, indicating a shift from energy storage towards growth. Conversely, microbial communities in soils nonresponsive to P amendment were found to have metabolisms tuned for the phosphorolysis of labile plant-derived substrates, such as β-glucosyl polysaccharides. Collectively, our results provided quantitative estimates of increased soil respiration upon alleviation of P constraints and elucidated several underlying ecological and molecular mechanisms involved in this response.

The use of enterococci as a fecal indicator bacterial group for public health risk assessment has been brought into question by recent studies showing that “naturalized” populations of E. faecalis exist in the extraenteric environment in a viable but not culturable (VBNC) state. The extent to which these naturalized or VBNC E. faecalis can confound water quality monitoring is unclear. To determine if strains isolated from different habitats display different survival strategies and responses, we compared the decay patterns of three E. faecalis isolates from the natural environment (environmental strains) against three human gut isolates (enteric strains) in laboratory mesocosms that simulate an oligotrophic, aerobic freshwater environment. Our results showed similar overall decay rates between enteric and environmental isolates based on viable plate and qPCR counts. However, the enteric isolates exhibited a spike in rRNA:rDNA ratios between days 1 and 3 of the mesocosm incubations that was not observed in environmental isolates, which could indicate a different stress response. Nevertheless, there was no strong evidence of differential expression of genes thought to be related to habitat adaptation in the accompanying mesocosm metatranscriptomes when compared between environmental and enteric isolates. Overall, our results provide novel information on how rRNA levels may vary over different metabolic states (i.e., alive vs. VBNC) for this important indicator bacteria. We also observed some evidence for habitat adaptation in E. faecalis; however, this adaptation may not be substantial or consistent enough for integration in water quality monitoring.IMPORTANCEEnterococci are commonly used worldwide to monitor environmental fecal contamination and public health risk for waterborne diseases. However, some species within this group can enter an inactive, viable but not culturable (VBNC) state that make it difficult to accurately quantify during routine monitoring. Furthermore, lower-risk, environmental enterococci strains may also confound water quality estimates. We developed an rRNA:rDNA viability assay for E. faecalis (a predominant species within this fecal group) and tested it against both enteric and environmental isolates in freshwater mesocosms to assess whether this approach can serve as a more sensitive water quality monitoring tool. We were unable to reliably distinguish the different isolate types using this assay under the conditions tested here; thus, environmental strains should continue to be counted during routine water monitoring. However, this assay could be useful for distinguishing more recent (i.e., higher risk) fecal pollution because rRNA levels significantly decreased after one week in all isolates.

The phylogenetic and functional diversities of microbial communities in tropical rainforests and how these differ from those of temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N(2)O) from the tropics. Toward closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing distinct life zones and an elevation gradient from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity compared to those of temperate grasslands or agricultural soils. In contrast to the overall distinct community composition, the relative abundances and nucleotide sequences of N(2)O reductases (nosZ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N(2)O emissions. Nitrogen fixation (nifH) also showed higher relative abundance in rainforest than in temperate soils, i.e., 20% versus 0.1 to 0.3% of bacterial genomes in each soil type harbored the gene, respectively. Finally, unlike temperate soils, LEF soils showed little stratification with depth in the first 0 to 30 cm, with ∼45% of community composition differences explained solely by location. Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities and should facilitate future ecological studies of these ecosystems. IMPORTANCE Tropical rainforests are the largest terrestrial sinks of atmospheric CO(2) and the largest natural source of N(2)O emissions, two greenhouse gases that are critical for the climate. The microbial communities of rainforest soils that directly or indirectly, through affecting plant growth, contribute to these fluxes remain poorly described by cultured-independent methods. To close this knowledge gap, the present study applied shotgun metagenomics to samples selected from three distinct life zones within the Puerto Rico rainforest. The results advance our understanding of microbial community diversity in rainforest soils and should facilitate future studies of natural or manipulated perturbations of these critical ecosystems.

Iron reduction and sulfate reduction are two of the major biogeochemical processes that occur in anoxic sediments. Microbes that catalyze these reactions are therefore some of the most abundant organisms in the subsurface, and some of the most important. Due to the variety of mechanisms that microbes employ to derive energy from these reactions, including the use of soluble electron shuttles, the dynamics between iron- and sulfate-reducing populations under changing biogeochemical conditions still elude complete characterization. Here, we amended experimental bioreactors comprised of freshwater aquifer sediment with ferric iron, sulfate, acetate, and the model electron shuttle AQDS (9,10-anthraquinone-2,6-disulfonate) and monitored both the changing redox conditions as well as changes in the microbial community over time. The addition of the electron shuttle AQDS did increase the initial rate of FeIIIreduction; however, it had little effect on the composition of the microbial community. Our results show that in both AQDS- and AQDS+ systems there was an initial dominance of organisms classified asGeobacter(a genus of dissimilatory FeIII-reducing bacteria), after which sequences classified asDesulfosporosinus(a genus of dissimilatory sulfate-reducing bacteria) came to dominate both experimental systems. Furthermore, most of the ferric iron reduction occurred under this later, ostensibly “sulfate-reducing” phase of the experiment. This calls into question the usefulness of classifying subsurface sediments by the dominant microbial process alone because of their interrelated biogeochemical consequences. To better inform models of microbially-catalyzed subsurface processes, such interactions must be more thoroughly understood under a broad range of conditions.

Background Peatlands are expected to experience sustained yet fluctuating higher temperatures due to climate change, leading to increased microbial activity and greenhouse gas emissions. Despite mounting evidence for viral contributions to these processes in peatlands underlain with permafrost, little is known about viruses in other peatlands. More generally, soil viral biogeography and its potential drivers are poorly understood at both local and global scales. Here, 87 metagenomes and five viral size-fraction metagenomes (viromes) from a boreal peatland in northern Minnesota (the SPRUCE whole-ecosystem warming experiment and surrounding bog) were analyzed for dsDNA viral community ecological patterns, and the recovered viral populations (vOTUs) were compared with our curated PIGEON database of 266,125 vOTUs from diverse ecosystems. Results Within the SPRUCE experiment, viral community composition was significantly correlated with peat depth, water content, and carbon chemistry, including CH4 and CO2 concentrations, but not with temperature during the first 2 years of warming treatments. Peat vOTUs with aquatic-like signatures (shared predicted protein content with marine and/or freshwater vOTUs) were significantly enriched in more waterlogged surface peat depths. Predicted host ranges for SPRUCE vOTUs were relatively narrow, generally within a single bacterial genus. Of the 4326 SPRUCE vOTUs, 164 were previously detected in other soils, mostly peatlands. None of the previously identified 202,371 marine and freshwater vOTUs in our PIGEON database were detected in SPRUCE peat, but 0.4% of 80,714 viral clusters (VCs, grouped by predicted protein content) were shared between soil and aquatic environments. On a per-sample basis, vOTU recovery was 32 times higher from viromes compared with total metagenomes. Conclusions Results suggest strong viral “species” boundaries between terrestrial and aquatic ecosystems and to some extent between peat and other soils, with differences less pronounced at higher taxonomic levels. The significant enrichment of aquatic-like vOTUs in more waterlogged peat suggests that viruses may also exhibit niche partitioning on more local scales. These patterns are presumably driven in part by host ecology, consistent with the predicted narrow host ranges. Although more samples and increased sequencing depth improved vOTU recovery from total metagenomes, the substantially higher per-sample vOTU recovery after viral particle enrichment highlights the utility of soil viromics.

The phylogenetic and functional diversity of microbial communities in tropical rainforests, and how these differ from temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N2O) from the tropics. Towards closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing distinct life zones from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity when compared to temperate grasslands or agricultural soils. Unlike temperate soils, LEF soils showed little stratification with depth in the first 0-30cm, with ~45% of community composition differences explained solely by location. The relative abundances and nucleotide sequences of N2O reductases (nosZ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N2O emissions. Nitrogen fixation (nifH) also showed higher relative abundance in rainforest compared to temperate soils (20% vs. 0.1-0.3% of bacterial genomes in each soil type harbored the gene, respectively). Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities, and should facilitate future ecological modeling efforts.ImportanceTropical rainforests are the largest terrestrial sinks of atmospheric CO2 and the largest natural source of N2O emissions, two critical greenhouse gases for the climate. The microbial communities of rainforest soils that directly or indirectly, through affecting plant growth, contribute to these fluxes remain poorly described by cultured-independent methods. To close this knowledge gap, the present study applied shotgun metagenomics to samples selected from 3 distinct life zones within the Puerto Rico rainforest. The results advance our understanding of microbial community diversity in rainforest soils and should facilitate future studies of natural or manipulated perturbations of these critical ecosystems.

Background In a warmer world, microbial decomposition of previously frozen organic carbon (C) is one of the most likely positive climate feedbacks of permafrost regions to the atmosphere. However, mechanistic understanding of microbial mediation on chemically recalcitrant C instability is limited; thus, it is crucial to identify and evaluate active decomposers of chemically recalcitrant C, which is essential for predicting C-cycle feedbacks and their relative strength of influence on climate change. Using stable isotope probing of the active layer of Arctic tundra soils after depleting soil labile C through a 975-day laboratory incubation, the identity of microbial decomposers of lignin and, their responses to warming were revealed. Results The β-Proteobacteria genus Burkholderia accounted for 95.1% of total abundance of potential lignin decomposers. Consistently, Burkholderia isolated from our tundra soils could grow with lignin as the sole C source. A 2.2 °C increase of warming considerably increased total abundance and functional capacities of all potential lignin decomposers. In addition to Burkholderia, α-Proteobacteria capable of lignin decomposition (e.g. Bradyrhizobium and Methylobacterium genera) were stimulated by warming by 82-fold. Those community changes collectively doubled the priming effect, i.e., decomposition of existing C after fresh C input to soil. Consequently, warming aggravates soil C instability, as verified by microbially enabled climate-C modeling. Conclusions Our findings are alarming, which demonstrate that accelerated C decomposition under warming conditions will make tundra soils a larger biospheric C source than anticipated.

The magnetization differential equations of Bloch are integrated using a matrix diagonalization method. The solution describes several limiting cases and leads to compact expressions of wide validity for a spin ensemble initially at equilibrium.

Different environmental changes may have some consistent effects on soil microbial community, which remains largely unexplored. Here, we report a novel and general pattern that different environmental changes consistently selected for the sub-dominant soil bacterial phyla. We mimicked 16 types of environmental changes relevant to the Eurasian steppe ecosystem, including removing different number of plant functional groups, mowing, nitrogen adding, phosphorus adding, watering, warming, and some of their combinations. While the absolute abundance of each of the four dominant bacteria phyla (Acidobacteria, Actinobacteria, Chloroflexi, and Proteobacteria) did not show a consistent response to these treatments, that of the five sub-dominant phyla (Bacteroidetes, Firmicutes, Gemmatimonadetes, Nitrospirae, and Planctomycetes) showed a consistent increase. The dominant phyla had high abundances of genes responsible for energy production/conversion and transcription, exhibiting oligotrophic traits; in contrast, the sub-dominant phyla had high abundance of 16S rDNA sequences, which could be due to greater rRNA copy number or smaller genomes, showing large growth/propagation potential and copiotrophic trait. Stochastic processes (e.g., random birth, death, colonization) played more important roles in structuring the sub-dominant than dominant phyla, and the treatments promoted the stochastic processes. The copiotrophy and stochasticity of sub-dominant phyla led to their positive responses to the treatments.

Many anthropogenic environmental changes are leading to a rapid decline in soil microbial functional diversity. However, ecological mechanisms that can serve to counteract/resist the diversity loss remain largely underexplored. In particular, although intermediate disturbance and increased amount of effective resources can promote the diversity of higher organisms, the potential role of these factors, and their combination, in maintaining microbial functional diversity is poorly studied. We conducted a 5-year experiment in a Eurasian steppe, manipulating mowing, nitrogen addition, phosphorus addition and their combinations. Nitrogen addition decreased soil pH by ~0.6 and bacterial abundance by ~19.5%, causing a disturbance effect. Phosphorus addition significantly decreased the effective amount of soil carbon-, nitrogen-, phosphorus- and water-relevant resources. Across all nitrogen-addition treatments subject to intermediate disturbance, there was a significant positive correlation between soil effective resource amount and microbial gene richness (r > 0.6, p < 0.01), which was elevated, in part, due to the increased fungal abundance. In contrast, significant correlations between gene richness and resource amount were not found under low-disturbance conditions. Overall, gene richness was greatest under conditions of both intermediate disturbance and ample effective resources, suggesting that the two factors could be manipulated in combination for the maintenance of microbial functional diversity.

Antarctic soils generally have low temperatures and limited availability of liquid water and nutrients. However, animals can increase the nutrient availability of ice-free areas by transferring nutrients from marine to terrestrial ecosystems, mainly through their excreta. In this study, we employed shotgun metagenomics and population genome binning techniques to study the diversity of microbial communities in Antarctic soils impacted by marine pinnipeds and birds relative to soils with no evident animal presence. We obtained ~285,000 16S rRNA gene-carrying metagenomic reads representing ~60 phyla and 100 metagenome-assembled genomes (MAGs) representing eight phyla. Only nine of these 100 MAGs represented previously described species, revealing that these soils harbor extensive novel diversity. Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant phyla in all samples, with Rhodanobacter being one of the most abundant genera in the bird-impacted soils. Further, the relative abundance of genes related to denitrification was at least double in soils impacted by birds than soils without animal influence. These results advance our understanding of the microbial populations and their genes involved in nitrous oxide emissions in ice-free coastal Antarctic soils impacted by marine animals and reveal novel microbial diversity associated with these ecosystems.

Anthropogenic environmental changes are accelerating the rate of biodiversity loss on Earth. Plant diversity loss is predicted to reduce soil microbial diversity primarily due to the decreased variety of carbon/energy resources. However, this intuitive hypothesis is supported by sparse empirical evidence, and most underlying mechanisms remain underexplored or obscure altogether. We constructed four diversity gradients (0-3) in a five-year plant functional group removal experiment in a steppe ecosystem in Inner Mongolia, China, and quantified microbial taxonomic and functional diversity with shotgun metagenome sequencing. The treatments had little effect on microbial taxonomic diversity, but were found to decrease functional gene diversity. However, the observed decrease in functional gene diversity was more attributable to a loss in plant productivity, rather than to the loss of any individual plant functional group per se. Reduced productivity limited fresh plant resources supplied to microorganisms, and thus, intensified the pressure of ecological filtering, favoring genes responsible for energy production/conversion, material transport/metabolism and amino acid recycling, and accordingly disfavored many genes with other functions. Furthermore, microbial respiration was correlated with the variation in functional composition but not taxonomic composition. Overall, the amount of carbon/energy resources driving microbial gene diversity was identified to be the critical linkage between above- and belowground communities, contrary to the traditional framework of linking plant clade/taxonomic diversity to microbial taxonomic diversity.

Our results demonstrated increased ecosystem nitrogen and water content as the primary drivers of the core and accessory components of soil microbial community functional diversity, respectively. Our findings suggested that more attention should be paid to certain components of community functional diversity under specific global change conditions. Our findings also indicated that microbial communities have adapted to nitrogen addition by strengthening the function of ammonia oxidization to deplete the excess nitrogen, thus maintaining ecosystem homeostasis. Because community gene richness is primarily determined by the presence/absence of accessory community genes, our findings further implied that strategies such as maintaining the amount of soil organic matter could be adopted to effectively improve the functional gene diversity of soil microbial communities subject to global change factors., It is a central ecological goal to explore the effects of global change factors on soil microbial communities. The vast functional gene repertoire of soil microbial communities is composed of both core and accessory genes, which may be governed by distinct drivers. This intuitive hypothesis, however, remains largely unexplored. We conducted a 5-year nitrogen and water addition experiment in the Eurasian steppe and quantified microbial gene diversity via shotgun metagenomics. Nitrogen addition led to an 11-fold increase in the abundance (based on quantitative PCR [qPCR]) of ammonia-oxidizing bacteria, which have mainly core community genes and few accessory community genes. Thus, nitrogen addition substantially increased the relative abundance of many core genes at the whole-community level. Water addition stimulated both plant diversity and microbial respiration; however, increased carbon/energy resources from plants did not counteract increased respiration, so soil carbon/energy resources became more limited. Thus, water addition selected for microorganisms with genes responsible for degrading recalcitrant soil organic matter. Accordingly, many other microorganisms without these genes (but likely with other accessory community genes due to relatively stable average microbial genome size) were selected against, leading to the decrease in the diversity of accessory community genes. In summary, nitrogen addition primarily affected core community genes through nitrogen-cycling processes, and water addition primarily regulated accessory community genes through carbon-cycling processes. Although both gene components may significantly respond as the intensity of nitrogen/water addition increases, our results demonstrated how these common global change factors distinctly impact each component. IMPORTANCE Our results demonstrated increased ecosystem nitrogen and water content as the primary drivers of the core and accessory components of soil microbial community functional diversity, respectively. Our findings suggested that more attention should be paid to certain components of community functional diversity under specific global change conditions. Our findings also indicated that microbial communities have adapted to nitrogen addition by strengthening the function of ammonia oxidization to deplete the excess nitrogen, thus maintaining ecosystem homeostasis. Because community gene richness is primarily determined by the presence/absence of accessory community genes, our findings further implied that strategies such as maintaining the amount of soil organic matter could be adopted to effectively improve the functional gene diversity of soil microbial communities subject to global change factors.

Peatlands play outsized roles in the global carbon cycle. Despite occupying a rather small fraction of the terrestrial biosphere (∼3%), these ecosystems account for roughly one third of the global soil carbon pool. This carbon is largely comprised of undecomposed deposits of plant material (peat) that may be meters thick. The fate of this deep carbon stockpile with ongoing and future climate change is thus of great interest and has large potential to induce positive feedback to climate warming. Recentin situwarming of an ombrotrophic peatland indicated that the deep peat microbial communities and decomposition rates were resistant to elevated temperatures. In this experiment, we sought to understand how nutrient and pH limitations may interact with temperature to limit microbial activity and community composition. Anaerobic microcosms of peat collected from 1.5 to 2 meters in depth were incubated at 6°C and 15°C with elevated pH, nitrogen (NH4Cl), and/or phosphorus (KH2PO4) in a full factorial design. The production of CO2and CH4was significantly greater in microcosms incubated at 15°C, although the structure of the microbial community did not differ between the two temperatures. Increasing the pH from ∼3.5 to ∼5.5 altered microbial community structure, however increases in CH4production were non-significant. Contrary to expectations, N and P additions did not increase CO2and CH4production, indicating that nutrient availability was not a primary constraint in microbial decomposition of deep peat. Our findings indicate that temperature is a key factor limiting the decomposition of deep peat, however other factors such as the availability of O2or alternative electron donors and high concentrations of phenolic compounds, may also exert constraints. Continued experimental peat warming studies will be necessary to assess if the deep peat carbon bank is susceptible to increased temperatures over the longer time scales.

Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens originating from agricultural activities such as Shiga Toxin-producingE. coli(STEC) in such sediments remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley was sampled over a nine-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and comparable to the functional and taxonomic diversity observed in soils. With our sequencing effort (~4 Gbp per library), we were unable to detect any pathogenicEscherichia coliin the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low pathogen abundance. Further, no significant differences were detected in the abundance of human- or cow-specific gut microbiome sequences compared to upstream, more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, a high baseline level of metagenomic reads encoding antibiotic resistance genes (ARGs) was found in all samples and was significantly higher compared to ARG reads in metagenomes from other environments, suggesting that these communities may be natural reservoirs of ARGs. Overall, our metagenomic results revealed that creek sediments are not a major sink for anthropogenic runoff and the public health risk associated with these sediment microbial communities may be low.IMPORTANCECurrent agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food and water-borne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health is assessed by culture-based tests for the intestinal bacterium,E. coli. However, the accuracy of these traditional methods (e.g., low quantification, and false positive signal when PCR-based) and their suitability for sediments remains unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the U.S. in order to assess how agricultural runoff affects the native microbial communities and if the presence of STEC in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

Se piensa que las comunidades microbianas del suelo son responsables del efecto de preparación de la rizósfera (RPE). Sin embargo, desde que las comunidades microbiales están compuestas de diversos componentes, se conoce muy poco acerca de cuál es el componente que tiene el rol principal en dicho efecto. En este estudio, se hicieron crecer soja y algodón en dos lugares a diferentes latitudes con diferentes condiciones de luz y temperatura in situ. Se cuantificó RPE usando un método natural de δC13 y se midió la abundancia, riqueza y composición de las comunidades de hongos y bacterias con métodos moleculares basados en el ADN. Entre todas las variables potenciales, incluyendo los tres índices de comunidades de hongos y bacterias anteriormente mencionados, e índices vegetales y físico-químicos del suelo, se mostró que la abundancia de bacterias explicó una gran proporción de la variación en RPE. Nuestro estudio identificó el mecanismo biológico que subyace este importante proceso ecológico. Soil microbial communities are thougth to be responsible for the rhizosphere priming effect (RPE). However, because soil microbial communities are comprised of diverse components, very little is known about which component plays the critical role. Here, soybean and cottonwood were grown at two latitudinal locations with different temperature and light conditions in-situ. We quantified RPE using a natural 13C method, and measured the abundance, richness and composition of bacteria and fungi communities with DNA-based molecular methods. Among all potential variables, including the three aforementioned indexes of bacteria and fungi communities and soil physiochemical and plant indexes, bacterial abundance was found to explain a large proportion of variation in RPE. Our study identified the biological mechanism underlying this important ecological process. Fil: Ma, Y.P.. Chinese Academy of Sciences. Institute of Applied Ecology; China. Chinese Academy of Agricultural Sciences. Institute of Environment and Sustainable Development in Agriculture. Key Laboratory of Dryland Agriculture;; China. University of Chinese Academy of Science; China Fil: Zhang, Z.J.. Chinese Academy of Sciences. Institute of Applied Ecology; China. Chinese Academy of Agricultural Sciences. Institute of Environment and Sustainable Development in Agriculture. Key Laboratory of Dryland Agriculture;; China Fil: Su, T.Q.. Chinese Academy of Sciences. Institute of Applied Ecology; China Fil: Busso, Carlos Alberto. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Johnston, E.R.. Georgia Institute of Technology. School of Civil and Environmental Engineering; Estados Unidos Fil: Han, X.G.. Chinese Academy of Sciences. Institute of Applied Ecology; China. Chinese Academy of Sciences. Institute of Botany. State Key Laboratory of Vegetation and Environmental Change; China Fil: Zhang, X.M.. Chinese Academy of Agricultural Sciences. Institute of Environment and Sustainable Development in Agriculture. Key Laboratory of Dryland Agriculture;; China

We investigated patterns in denitrifier and NH3-oxidizer community structure and function along environmental gradients in midwestern USA wetlands. Denitrifier and NH3-oxidizer functional groups have contrasting sensitivities to O2 levels and other soil factors. Thus, variable environmental tolerance among and within functional groups can result in a range of microbial responses to the same environmental gradient. We compared microbial structure–function relationships in 2 restored wetlands in Illinois and 2 natural wetlands in Michigan. At each site, we established 4 transects perpendicular to the shore and placed 4 plots along each transect. We analyzed denitrifier and NH3-oxidizer community structure (assessed by molecular analysis of functional genes nosZ and amoA) and potential denitrification and nitrification activities (function) across the soil hydrologic gradient. Soil moisture and physicochemical measurements, including organic-matter content, pH, inorganic N levels, and texture, varied...

BackgroundPrecisely engineered mesoporous silica has been shown to induce weight loss in mice, but whether it is safe to use in humans have not investigated.ObjectiveThe aim was to determine whether oral dosing, up to 9 grams/day, of precisely engineered mesoporous silica as a food additive can be used safely in male humans.DesignThis single blinded safety study consisted of two study arms including 10 males each (18-35 years). One arm consisted of participants with normal weight and one with obesity. After a placebo run-in period, all subjects were given porous silica three times daily, with increasing dose up to 9 grams/day (Phase 1). Subjects with obesity continued the study with highest dose for additional 10 weeks (Phase 2).ResultsAll participants completed Phase 1 and 90% completed Phase 2, with approximately 1% missed doses. Participants reported no abdominal discomfort, and changes in bowel habits were minor and inconsistent. The side effects observed were mild and tolerable, biomarkers did not give any safety concern, and no severe adverse events occurred.ConclusionMesoporous silica intake of up to 9 grams/day can be consumed by males without any major adverse events or safety concerns.

Many anthropogenic environmental changes are leading to a rapid decline in soil microbial functional diversity. However, ecological mechanisms that can serve to counteract/resist the diversity loss remain largely underexplored. In particular, although intermediate disturbance and increased amount of effective resources can promote the diversity of higher organisms, the potential role of these factors, and their combination, in maintaining microbial functional diversity is poorly studied. We conducted a 5-year experiment in a Eurasian steppe, manipulating mowing, nitrogen addition, phosphorus addition and their combinations. Nitrogen addition decreased soil pH by ~0.6 and bacterial abundance by ~19.5%, causing a disturbance effect. Phosphorus addition significantly decreased the effective amount of soil carbon-, nitrogen-, phosphorus- and water-relevant resources. Across all nitrogen-addition treatments subject to intermediate disturbance, there was a significant positive correlation between soil effective resource amount and microbial gene richness (r0.6, p0.01), which was elevated, in part, due to the increased fungal abundance. In contrast, significant correlations between gene richness and resource amount were not found under low-disturbance conditions. Overall, gene richness was greatest under conditions of both intermediate disturbance and ample effective resources, suggesting that the two factors could be manipulated in combination for the maintenance of microbial functional diversity.

Although dissimilatory iron and sulfate reduction (DIR and DSR) profoundly affect the biogeochemical cycling of C, Fe, and S in subsurface systems, the dynamics of DIR and DSR in the presence of both FeIII (hydr)oxides and sulfate have not been well-studied with mixed microbial populations. This study examined the response of native microbial communities in subsurface sediment from the U.S. Department of Energy’s Integrated Field Research Challenge site in Rifle, CO to the availability of sulfate and specific FeIII (hydr)oxide minerals in experimental systems containing lactate as the electron donor, with ferrihydrite, goethite, or lepidocrocite and high (10.2 mM) or low (0.2 mM) sulfate as electron acceptors. We observed rapid fermentation of lactate to acetate and propionate. FeIII reduction was slow and limited in the presence of low-sulfate, but the extent of FeIII reduction increased more than 10 times with high-sulfate amendments. Furthermore, the extent of FeIII reduction was higher in ferrihydrite or lepidocrocite incubations than in goethite incubations. Propionate produced during fermentation of lactate was used as the electron donor for DSR. The concurrence of sulfate reduction and FeII production suggests that FeII production was driven primarily by reduction of FeIII by biogenic sulfide. X-ray absorption fine-structure analysis confirmed the formation of ferrous sulfide and the presence of O-coordinated ferrous species. 16S rRNA-based microbial community analysis revealed the development of distinct communities with different FeIII (hydr)oxides. These results highlight the highly coupled nature of C, Fe, and S biogeochemical cycles during DIR and DSR and provide new insight into the effects of electron donor utilization, sulfate concentration, and the presence of specific FeIII (hydr)oxide phases on microbial community development.

Cognitive control is defined by a set of neural processes that allow us to interact with our complex environment in a goal-directed manner. Humans regularly challenge these control processes when attempting to simultaneously accomplish multiple goals (multitasking), generating interference as the result of fundamental information processing limitations. It is clear that multitasking behaviour has become ubiquitous in today's technologically dense world, and substantial evidence has accrued regarding multitasking difficulties and cognitive control deficits in our ageing population. Here we show that multitasking performance, as assessed with a custom-designed three-dimensional video game (NeuroRacer), exhibits a linear age-related decline from 20 to 79 years of age. By playing an adaptive version of NeuroRacer in multitasking training mode, older adults (60 to 85 years old) reduced multitasking costs compared to both an active control group and a no-contact control group, attaining levels beyond those achieved by untrained 20-year-old participants, with gains persisting for 6 months. Furthermore, age-related deficits in neural signatures of cognitive control, as measured with electroencephalography, were remediated by multitasking training (enhanced midline frontal theta power and frontal-posterior theta coherence). Critically, this training resulted in performance benefits that extended to untrained cognitive control abilities (enhanced sustained attention and working memory), with an increase in midline frontal theta power predicting the training-induced boost in sustained attention and preservation of multitasking improvement 6 months later. These findings highlight the robust plasticity of the prefrontal cognitive control system in the ageing brain, and provide the first evidence, to our knowledge, of how a custom-designed video game can be used to assess cognitive abilities across the lifespan, evaluate underlying neural mechanisms, and serve as a powerful tool for cognitive enhancement.

© 2016 Johnston, Rodriguez-R, Luo, Yuan, Wu, He, Schuur, Luo, Tiedje, Zhou and Konstantinidis. How soil microbial communities contrast with respect to taxonomic and functional composition within and between ecosystems remains an unresolved question that is central to predicting how global anthropogenic change will affect soil functioning and services. In particular, it remains unclear how small-scale observations of soil communities based on the typical volume sampled (1-2 g) are generalizable to ecosystem-scale responses and processes. This is especially relevant for remote, northern latitude soils, which are challenging to sample and are also thought to be more vulnerable to climate change compared to temperate soils. Here, we employed well-replicated shotgun metagenome and 16S rRNA gene amplicon sequencing to characterize community composition and metabolic potential in Alaskan tundra soils, combining our own datasets with those publically available from distant tundra and temperate grassland and agriculture habitats. We found that the abundance of many taxa and metabolic functions differed substantially between tundra soil metagenomes relative to those from temperate soils, and that a high degree of OTU-sharing exists between tundra locations. Tundra soils were an order of magnitude less complex than their temperate counterparts, allowing for near-complete coverage of microbial community richness (~92% breadth) by sequencing, and the recovery of 27 high-quality, almost complete (>80% completeness) population bins. These population bins, collectively, made up to ~10% of the metagenomic datasets, and represented diverse taxonomic groups and metabolic lifestyles tuned toward sulfur cycling, hydrogen metabolism, methanotrophy, and organic matter oxidation. Several population bins, including members of Acidobacteria, Actinobacteria, and Proteobacteria, were also present in geographically distant (~100-530 km apart) tundra habitats (full genome representation and up to 99.6% genome-derived average nucleotide identity). Collectively, our results revealed that Alaska tundra microbial communities are less diverse and more homogenous across spatial scales than previously anticipated, and provided DNA sequences of abundant populations and genes that would be relevant for future studies of the effects of environmental change on tundra ecosystems.

Identifying soil microbial feedbacks to increasing temperatures and moisture alterations is critical for predicting how terrestrial ecosystems will respond to climate change. We performed a 5-year field experiment manipulating warming, watering and their combination in a semiarid temperate steppe in northern China. Warming stimulated the abundance of genes responsible for degrading recalcitrant soil organic matter (SOM) and reduced SOM content by 13%. Watering, and warming plus watering also increased the abundance of recalcitrant SOM catabolism pathways, but concurrently promoted plant growth and increased labile SOM content, which somewhat offset SOM loss. The treatments also increased microbial biomass, community complexity and metabolic potential for nitrogen and sulfur assimilation. Both microbial and plant community composition shifted with the treatment conditions, and the sample-to-sample compositional variations of the two communities (pairwise β-diversity distances) were significantly correlated. In particular, microbial community composition was substantially correlated with the dominant plant species (~0.54 Spearman correlation coefficient), much more than with measured soil indices, affirming a tight coupling between both biological communities. Collectively, our study revealed the direction and underlying mechanisms of microbial feedbacks to warming and suggested that semiarid regions of northern steppes could act as a net carbon source under increased temperatures, unless precipitation increases concurrently.

Although iron- and sulfate-reducing bacteria in subsurface environments have crucial roles in biogeochemical cycling of C, Fe, and S, how specific electron donors impact the compositional structure and activity of native iron- and/or sulfate-reducing communities is largely unknown. To understand this better, we created bicarbonate-buffered batch systems in duplicate with three different electron donors (acetate, lactate, or glucose) paired with ferrihydrite and sulfate as the electron acceptors and inoculated them with subsurface sediment as the microbial inoculum. Sulfate and ferrihydrite reduction occurred simultaneously and were faster with lactate than with acetate. 16S rRNA-based sequence analysis of the communities over time revealed that Desulfotomaculum was the major driver for sulfate reduction coupled with propionate oxidation in lactate-amended incubations. The reduction of sulfate resulted in sulfide production and subsequent abiotic reduction of ferrihydrite. In contrast, glucose promoted faster reduction of ferrihydrite, but without reduction of sulfate. Interestingly, the glucose-amended incubations led to two different biogeochemical trajectories among replicate bottles that resulted in distinct coloration (white and brown). The two outcomes in geochemical evolution might be due to the stochastic evolution of the microbial communities or subtle differences in the initial composition of the fermenting microbial community and its development via the use of different glucose fermentation pathways available within the community. Synchrotron-based x-ray analysis indicated that siderite and amorphous Fe(II) were formed in the replicate bottles with glucose, while ferrous sulfide and vivianite were formed with lactate or acetate. These data sets reveal that use of different C utilization pathways projects significant changes in microbial community composition over time that uniquely impact both the geochemistry and mineralogy of subsurface environments.

Soil microbial communities are extremely complex, being composed of thousands of low-abundance species (2 production (∼10%), and to nitrogen cycling, including denitrification (∼12%), were enriched under warming, which was consistent with independent physicochemical measurements. These community shifts were interlinked, in part, with higher primary productivity of the aboveground plant communities stimulated by warming, revealing that most of the additional, plant-derived soil carbon was likely respired by microbial activity. Warming also enriched for a higher abundance of sporulation genes and genomes with higher G+C content. Collectively, our results indicate that microbial communities of temperate grassland soils play important roles in mediating feedback responses to climate change and advance the understanding of the molecular mechanisms of community adaptation to environmental perturbations.

Dynamic two-dimensional 1H and 13C NMR studies of the conformationally restricted amide N-(1-naphthoyl)-cis-2,6-dimethylpiperidine reveal correlated rotation about the aryl–CO and amide bonds. The naphthalene and piperidine rings are tightly meshed and their rotation is virtually entirely (>99%) concerted, as evidenced by separate NMR measurement of the much slower rates of rotation about the individual aryl–CO and amide bonds. A disrotatory cogwheeling mechanism for the concerted rotation is proposed. Copyright © 2000 John Wiley & Sons, Ltd.

The high barrier to reversal of the bent trisulfide bridge in 3-n-butyl-[3]-trithiaferroceneophane results in syn and anti stereoisomers which are distinguishable by NMR at 300 K. All seven ring hydrogen atoms and their directly attached carbon atoms in each stereoisomer are nonequivalent and have been uniquely assigned using a suite of one- and two-dimensional experiments. Row slices of the total correlation (TOCSY) spectrum provide the individual ring 1H NMR subspectra. The two ring proton sets of each stereoisomer can be correlated via inter-ring nuclear Overhauser effects across the iron atom of the sandwich. The syn and anti isomers are in a slow dynamic equilibrium brought about by reversal of the trisulfide bridge, which interconverts the hydrogen-atom pairs in the two stereoisomers. The rate of this process has been measured with high accuracy by 2D exchange spectroscopy. The rate constant k = 0.07 s-1 at 300 K corresponds to an activation barrier of 19.1 kcal/mol, virtually identical with that of...

The effect of deuterium substitution on the positional equilibrium and rate of the Cope rearrangement in barbaralone-d1 has been investigated by 1H and 13C dynamic NMR spectroscopy. Deuterium favors the allylic position over the cyclopropyl position. Skewing of the equilibrium requires that the Cope rearrangement be considered as a nonmutual exchange process that transports 13C−1H spin pairs between unequally populated molecular configurations. The NMR fast-exchange results are critically interpreted in light of a recently proposed alternative averaging theory (Jones, D. H.; Kurur, N. D.; Weitekamp, D. P. Bull. Magn. Reson. 1992, 14, 214). Our experimental results are consistent with the traditional theory but do not agree with the prediction of the alternative theory. The use of 1H−13C inverse detection is demonstrated for the observation of very broad 13C resonances in the coalescence region via their directly attached exchange-narrowed 1H signals, and the contribution of rapid chemical exchange to the ...

The slow inversion at pyramidal nitrogen in 2,2-dimethylaziridine was investigated by NMR spectroscopy at 304 K using one-dimensional spin locking methods and two-dimensional exchange spectroscopy. The activation free energy was found to be 17.5 kcal mol−1 (73.2 kJ mol−1) at this temperature. This value represents a lower limit for the unimolecular inversion barrier height as autocatalytic and water-catalyzed inversion pathways can in principle also contribute to the observed rate. The spin locking experiments described employ non-selective pulses and are particularly useful for the quantitative study of slow chemical exchange between nuclei of similar resonance frequency where selective magnetization transfer experiments are difficult or impossible.

NMR is a powerful tool for the study of chemical rate processes and the mechanisms by which they occur. Dynamic processes give rise to characteristic changes in transverse and longitudinal relaxation, which manifest themselves in the NMR lineshape and in magnetization transfer phenomena, respectively. Analysis of these effects permits the extraction of kinetic data. The mathematical theory central to this analysis is illustrated using the formalism of the density matrix for a two-spin system undergoing mutual intramolecular exchange in isotropic solution. The spin Hamiltonian and exchange operators are introduced, and the quantum mechanical equation of motion of the density matrix is presented, emphasizing those density matrix elements that correspond classically to longitudinal and transverse magnetization. The necessary Liouville and exchange superoperators are explicitly constructed from the corresponding Hamiltonian and exchange operators using tensor algebra. The equation of motion is then solved for the populations and single-quantum coherences to illustrate the origin of magnetization transfer and dynamic lineshape effects. The extension of the theory to arbitrarily large exchanging spin systems is addressed and illustrated for two mutually exchanging spins scalar coupled to a third spin. Finally, the two-spin lineshape calculation is fully worked out using a steady-state method that reduces the problem to the solution of a set of simultaneous algebraic equations.

Ac-(D)Phe-Pro-boroArg-OH is a potent, competitive inhibitor of thrombin (Ki = 40 pM). 1H-NMR studies have shown that the peptide portion, -(D)Phe-Pro-, has secondary structure in aqueous solutions. This structure corresponds fairly closely to the structure of H-(D)Phe-Pro-ArgCH2Cl complexed to thrombin in the protein crystal structure (Bode, W.; et al. EMBO J. 1989, 193, 3467-3475.). These results indicate that, in addition to enthalpic interactions in the active site of the enzyme, there are significant entropic advantages in binding this molecule not previously recognized. We estimate that they contribute approximately 10-fold to binding. The structure we have observed can be explained by pi-pi interactions between the phenyl side chain of (D)Phe and the (D)Phe-Pro peptide bond. Assignment of structure is based first on the 0.8-1.2 ppm difference between the two Pro C delta protons. The magnitude of these chemical shifts are consistent with aromatic ring current-induced effects expected for distances in our structure. The structure was further defined by interproton distances and correlation times calculated by backtransformation and correction of the NOESY and ROESY data to the longitudinal and transverse cross relaxation rates. Analysis of the vicinal coupling constants show that Phe chi 1 is not fixed. Correlation times for the peptide side chains and backbone indicate that the phenyl ring and boroArg side chain possess various degrees of internal motion, and that the rest of the peptide has a fairly rigid conformation.