The physiological potential of anammox bacteria as revealed by their core genome structure

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although NO2− reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. Kuenenia stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologues. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least five paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH₂OH by self-production for anammox metabolism.     

Are Nonsynonymous Transversions Generally More Deleterious than Nonsynonymous Transitions?

It has been suggested that, due to the structure of the genetic code, nonsynonymous transitions are less likely than transversions to cause radical changes in amino acid physicochemical properties so are on average less deleterious. This view was supported by some but not all mutagenesis experiments. Because laboratory measures of fitness effects have limited sensitivities and relative frequencies of different mutations in mutagenesis studies may not match those in nature, we here revisit this issue using comparative genomics. We extend the standard codon model of sequence evolution by adding the parameter η that quantifies the ratio of the fixation probability of transitional nonsynonymous mutations to that of transversional nonsynonymous mutations. We then estimate η from the concatenated alignment of all protein-coding DNA sequences of two closely related genomes. Surprisingly, η ranges from 0.13 to 2.0 across 90 species pairs sampled from the tree of life, with 51 incidences of η < 1 and 30 incidences of η >1 that are statistically significant. Hence, whether nonsynonymous transversions are overall more deleterious than nonsynonymous transitions is species-dependent. Because the corresponding groups of amino acid replacements differ between nonsynonymous transitions and transversions, η is influenced by the relative exchangeabilities of amino acid pairs. Indeed, an extensive search reveals that the large variation in η is primarily explainable by the recently reported among-species disparity in amino acid exchangeabilities. These findings demonstrate that genome-wide nucleotide substitution patterns in coding sequences have species-specific features and are more variable among evolutionary lineages than are currently thought.     

Controlling Electron Transfer between the Two Cofactor Chains of Photosystem I by the Redox State of One of Their Components

Two functional electron transfer (ET) chains, related by a pseudo-C₂ symmetry, are present in the reaction center of photosystem I (PSI). Due to slight differences in the environment around the cofactors of the two branches, there are differences in both the kinetics of ET and the proportion of ET that occurs on the two branches. The strongest evidence that this is indeed the case relied on the observation that the oxidation rates of the reduced phylloquinone (PhQ) cofactor differ by an order of magnitude. Site-directed mutagenesis of residues involved in the respective PhQ-binding sites resulted in a specific alteration of the rates of semiquinone oxidation. Here, we show that the PsaA-F689N mutation results in an ∼100-fold decrease in the observed rate of PhQA− oxidation. This is the largest change of PhQA− oxidation kinetics observed so far for a single-point mutation, resulting in a lifetime that exceeds that of the terminal electron donor, P700+. This situation allows a second photochemical charge separation event to be initiated before PhQA− has decayed, thereby mimicking in PSI a situation that occurs in type II reaction centers. The results indicate that the presence of PhQA− does not impact the overall quantum yield and leads to an almost complete redistribution of the fractional utilization of the two functional ET chains, in favor of the one that does not bear the charged species. The evolutionary implications of these results are also briefly discussed.     

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas

Chlamydomonas reinhardtii can grow photosynthetically using CO₂ or in the dark using acetate as the carbon source. In the light in air, the CO₂ concentrating mechanism (CCM) of C. reinhardtii accumulates CO₂, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO₂ concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO₂ (0.04% CO₂ [v/v] in air). These strains were not able to grow at very low CO₂ (<0.02% CO₂ [v/v] in air), and their ability to accumulate inorganic carbon (C_i = CO₂ + HCO3−) was reduced. At low CO₂ concentrations, the CCM is needed to both deliver C_i to Rubisco and to minimize the leak of CO₂ generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO₂ released from respiration and photorespiration as well as the CO₂ leaked from the chloroplast to HCO3- thus “recapturing” this potentially lost CO₂.

Mitochondrial carbonic anhydrases CAH4 and CAH5 in Chlamydomonas reinhardtii are involved in maintaining optimal photosynthesis.     

Response of nitrate leaching to no-tillage is dependent on soil,climate, and management factors: A global meta-analysis

No tillage (NT) has been proposed as a practice to reduce the adverse effects of tillage on contaminant (e.g., sediment and nutrient) losses to waterways. Nonetheless, previous reports on impacts of NT on nitrate (${\mathrm{NO}}_3^{-}$) leaching are inconsistent. A global meta-analysis was conducted to test the hypothesis that the response of ${\mathrm{NO}}_3^{-}$ leaching under NT, relative to tillage, is associated with tillage type (inversion vs non-inversion tillage), soil properties (e.g., soil organic carbon [SOC]), climate factors (i.e., water input), and management practices (e.g., NT duration and nitrogen fertilizer inputs). Overall, compared with all forms of tillage combined, NT had 4% and 14% greater area-scaled and yield-scaled ${\mathrm{NO}}_3^{-}$ leaching losses, respectively. The ${\mathrm{NO}}_3^{-}$ leaching under NT tended to be 7% greater than that of inversion tillage but comparable to non-inversion tillage. Greater ${\mathrm{NO}}_3^{-}$ leaching under NT, compared with inversion tillage, was most evident under short-duration NT (<5 years), where water inputs were low (<2 mm day⁻¹), in medium texture and low SOC (<1%) soils, and at both higher (>200 kg ha⁻¹) and lower (0–100 kg ha⁻¹) rates of nitrogen addition. Of these, SOC was the most important factor affecting the risk of NO₃⁻ leaching under NT compared with inversion tillage. Globally, on average, the greater amount of NO₃⁻ leached under NT, compared with inversion tillage, was mainly attributed to corresponding increases in drainage. The percentage of global cropping land with lower risk of NO₃⁻ leaching under NT, relative to inversion tillage, increased with NT duration from 3 years (31%) to 15 years (54%). This study highlighted that the benefits of NT adoption for mitigating ${\mathrm{NO}}_3^{-}$ leaching are most likely in long-term NT cropping systems on high-SOC soils.     

Importance of the description of light interception in crop growth models

Canopy light interception determines the amount of energy captured by a crop, and is thus critical to modeling crop growth and yield, and may substantially contribute to the prediction uncertainty of crop growth models (CGMs). We thus analyzed the canopy light interception models of the 26 wheat (Triticum aestivum) CGMs used by the Agricultural Model Intercomparison and Improvement Project (AgMIP). Twenty-one CGMs assume that the light extinction coefficient (K) is constant, varying from 0.37 to 0.80 depending on the model. The other models take into account the illumination conditions and assume either that all green surfaces in the canopy have the same inclination angle (θ) or that θ distribution follows a spherical distribution. These assumptions have not yet been evaluated due to a lack of experimental data. Therefore, we conducted a field experiment with five cultivars with contrasting leaf stature sown at normal and double row spacing, and analyzed θ distribution in the canopies from three-dimensional canopy reconstructions. In all the canopies, θ distribution was well represented by an ellipsoidal distribution. We thus carried out an intercomparison between the light interception models of the AgMIP–Wheat CGMs ensemble and a physically based K model with ellipsoidal leaf angle distribution and canopy clumping (KellC). Results showed that the KellC model outperformed current approaches under most illumination conditions and that the uncertainty in simulated wheat growth and final grain yield due to light models could be as high as 45%. Therefore, our results call for an overhaul of light interception models in CGMs.     

The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg²⁺ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate MgA2+, while MgA2+ together with Arg233 stabilize the inorganic phosphate (P_i); (ii) Asp168 and three water molecules coordinate MgB2+, and MgB2+ further stabilizes P_i by forming a hydrogen bond with two oxygens of P_i; (iii) Arg201 directly coordinates the P_i; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of MgA2+, MgB2+ and P_i. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP⁺/NAD⁺) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.     

Effect of halide ions on the fluorescence properties of 3-aminoquinoline in aqueous medium

Fluorescence (FL) quenching of 3-aminoquinoline (3AQ) by halide ions $\left({\mathrm{Cl}}^{-},{\mathrm{Br}}^{-}\text{and}{\mathrm{I}}^{-}\right)$ has been explored in an aqueous acidic medium using the steady-state and time-domain FL measurement techniques. The halide ions showed no significant change in the absorption spectra of 3AQ in an aqueous acidic medium. The FL intensity was strongly quenched by ${\mathrm{I}}^{-}$ ions and the order of FL quenching by halide ions was ${\mathrm{I}}^{-}>{\mathrm{Br}}^{-}>{\mathrm{Cl}}^{-}$. The decrease in FL lifetime along with the reduction in FL intensity of 3AQ suggested the dynamic nature of quenching. The obtained ${\mathrm{K}}_{\mathrm{SV}}$ values were 328 ${\mathrm{M}}^{-1}$ for ${\mathrm{I}}^{-}$ ions and 119 ${\mathrm{M}}^{-1}$ for ${\mathrm{Br}}^{-}$ ions and the ${\mathrm{k}}_{\mathrm{q}}$ values were ~ $1.66\times {10}^{10}{\mathrm{M}}^{-1}{\mathrm{s}}^{-1}$ and $6.02\times {10}^9{\mathrm{M}}^{-1}{\mathrm{s}}^{-1}$, respectively. The observations suggested that the likely governing mechanism for FL quenching may be an electron transfer process and the involvement of the heavy atom effects.     

uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable “Kozak” context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.     

Sexually antagonistic genetic variance for fitness in an ancestral and a novel environment

The intersex genetic correlation for fitness (rwfm), a standardized measure of the degree to which male and female fitness covary genetically, has consequences for important evolutionary processes, but few estimates are available and none have explored how it changes with environment. Using a half-sibling breeding design, we estimated the genetic (co)variance matrix (G) for male and female fitness, and the resulting rwfm, in Drosophila serrata. Our estimates were performed in two environments: the laboratory yeast food to which the population was well adapted and a novel corn food. The major axis of genetic variation for fitness in the two environments, accounting for 51.3 per cent of the total genetic variation, was significant and revealed a strong signal of sexual antagonism, loading negatively in both environments on males but positively on females. Consequently, estimates of rwfm were negative in both environments (−0.34 and −0.73, respectively), indicating that the majority of genetic variance segregating in this population has contrasting effects on male and female fitness. The possible strengthening of the negative rwfm in this novel environment may be a consequence of no history of selection for amelioration of sexual conflict. Additional studies from a diverse range of novel environments will be needed to determine the generality of this finding.     

Correlation of a Decline in Aerobic Capacity with Development of Emphysema in Patients with Chronic Obstructive Pulmonary Disease: A Prospective Observational Study

In patients with COPD, CT assessment of emphysema and airway disease is known to be associated with lung function and 6-minute walk distance. However, it remains to be determined whether low attenuation area (LAA) on CT is associated with aerobic capacity assessed using cardiopulmonary exercise testing (CPET). In this prospective observational study, we repeatedly conducted high-resolution CT and CPET using a treadmill in 81 COPD patients over a median interval of 3.5 years. Two investigators independently scored LAA on images obtained at the aortic arch level, tracheal bifurcation level, and supradiaphragmatic level. Grades for the images of each lung were added to yield the total LAA score. Total LAA score was negatively correlated with peak aerobic capacity (V˙O2) (p<0.001, r = -0.485). LAA scores of the upper (aortic arch level) and the lower (supradiaphragmatic level) lungs were both significantly associated with peak V˙O2. There was a significant correlation between total LAA score and peak CO₂ output (V˙CO2) (p<0.001, r = -0.433). Total LAA score was correlated with oxygen saturation at peak exercise (p<0.001, r = -0.634) and the estimated dead space fraction (p<0.001, r = 0.416). The mean annual change in total LAA score was significantly correlated with those in peak V˙O2 (p<0.001, r = -0.546) and peak V˙CO2 (p<0.001, r = -0.488). The extent of emphysema measured by CT was associated with the results of CPET. The time-dependent changes in CPET data were also correlated with that in total LAA score. CT assessment could be a non-invasive tool to predict aerobic capacity in patients with COPD.     

Towards efficient N cycling in intensive maize: role of cover crops and application methods of digestate liquid fraction

Digestate, a by-product of biogas production, is widely recognized as a promising renewable nitrogen (N) source with high potential to replace synthetic fertilizers. Yet, inefficient digestate use can lead to pollutant N losses as ammonia (NH₃) volatilization, nitrous oxide (N₂O) emissions and nitrate (${\mathrm{NO}}_3^{-}$) leaching. Cover crops (CCs) may reduce some of these losses and recycle the N back into the soil after incorporation, but the effect on the N balance depends on the CC species. In a one-year field study, we tested two application methods (i.e., surface broadcasting, BDC; and shallow injection, INJ) of the liquid fraction of separated co-digested cattle slurry (digestate liquid fraction [DLF]), combined with different winter cover crop (CC) options (i.e., rye, white mustard or bare fallow), as starter fertilizer for maize. Later, side-dressing with urea was required to fulfil maize N-requirements. We tested treatment effects on yield, N-uptake, N-use efficiency parameters, and N-losses in the form of N₂O emissions and ${\mathrm{NO}}_3^{-}$ leaching. CC development and biomass production were strongly affected by their contrasting frost tolerance, with spring-regrowth for rye, while mustard was winter killed. After the CCs, injection of DLF increased N₂O emissions significantly compared with BDC (emission factor of 2.69% vs. 1.66%). Nitrous oxide emissions accounted for a small part (11%–13%) of the overall yield-scaled N losses (0.46–0.97 kg N Mg grain⁻¹). The adoption of CCs reduced fall ${\mathrm{NO}}_3^{-}$ leaching, being 51% and 64% lower for mustard and rye than under bare soil. In addition, rye reduced ${\mathrm{NO}}_3^{-}$ leaching during spring and summer after termination by promoting N immobilization, thus leading to −57% lower annual leaching losses compared with mustard. DLF application method modified N-loss pathways, but not the cumulative yield-scaled N losses. Overall, these insights contribute to inform an evidence-based design of cropping systems in which nutrients are recycled more efficiently.     

Topography Mediates the Influence of Cover Crops on Soil Nitrate Levels in Row Crop Agricultural Systems

Supplying adequate amounts of soil N for plant growth during the growing season and across large agricultural fields is a challenge for conservational agricultural systems with cover crops. Knowledge about cover crop effects on N comes mostly from small, flat research plots and performance of cover crops across topographically diverse agricultural land is poorly understood. Our objective was to assess effects of both leguminous (red clover) and non-leguminous (winter rye) cover crops on potentially mineralizable N (PMN) and NO3--N levels across a topographically diverse landscape. We studied conventional, low-input, and organic managements in corn-soybean-wheat rotation. The rotations of low-input and organic managements included rye and red clover cover crops. The managements were implemented in twenty large undulating fields in Southwest Michigan starting from 2006. The data collection and analysis were conducted during three growing seasons of 2011, 2012 and 2013. Observational micro-plots with and without cover crops were laid within each field on three contrasting topographical positions of depression, slope and summit. Soil samples were collected 4–5 times during each growing season and analyzed for NO3--N and PMN. The results showed that all three managements were similar in their temporal and spatial distributions of NO₃ ^—N. Red clover cover crop increased NO3--N by 35% on depression, 20% on slope and 32% on summit positions. Rye cover crop had a significant 15% negative effect on NO3--N in topographical depressions but not in slope and summit positions. The magnitude of the cover crop effects on soil mineral nitrogen across topographically diverse fields was associated with the amount of cover crop growth and residue production. The results emphasize the potential environmental and economic benefits that can be generated by implementing site-specific topography-driven cover crop management in row-crop agricultural systems.     

Influence of Adjuvant Therapy in Cancer Survivors on Endothelial Function and Skeletal Muscle Deoxygenation

The cardiotoxic effects of adjuvant cancer treatments (i.e., chemotherapy and radiation treatment) have been well documented, but the effects on peripheral cardiovascular function are still unclear. We hypothesized that cancer survivors i) would have decreased resting endothelial function; and ii) altered muscle deoxygenation response during moderate intensity cycling exercise compared to cancer-free controls. A total of 8 cancer survivors (~70 months post-treatment) and 9 healthy controls completed a brachial artery FMD test, an index of endothelial-dependent dilation, followed by an incremental exercise test up to the ventilatory threshold (VT) on a cycle ergometer during which pulmonary V˙O2 and changes in near-infrared spectroscopy (NIRS)-derived microvascular tissue oxygenation (TOI), total hemoglobin concentration ([Hb]_total), and muscle deoxygenation ([HHb] ≈ fractional O₂ extraction) were measured. There were no significant differences in age, height, weight, and resting blood pressure between cancer survivors and control participants. Brachial artery FMD was similar between groups (P = 0.98). During exercise at the VT, TOI was similar between groups, but [Hb]_total and [HHb] were significantly decreased in cancer survivors compared to controls (P < 0.01) The rate of change for TOI (ΔTOIΔ/V˙O2) and [HHb] (Δ[HHb]/ΔV˙O2) relative to ΔV˙O2 were decreased in cancer survivors compared to controls (P = 0.02 and P = 0.03 respectively). In cancer survivors, a decreased skeletal muscle microvascular function was observed during moderate intensity cycling exercise. These data suggest that adjuvant cancer therapies have an effect on the integrated relationship between O₂ extraction, V˙O2 and O₂ delivery during exercise.     

Technical note: Measures of differentiation for quantitative traits

Studies of anthropological genetics and bioarcheology often examine the degree of among-group variation in quantitative traits such as craniometrics and anthropometrics. One comparative index of among-group differentiation is the minimum value of Wright's $F_{\mathrm{ST}}$ as estimated from quantitative traits. This measure has been used in certain population-genetic applications such as comparison with $F_{\mathrm{ST}}$ estimated from genetic data, although some inferences are limited by how well the data and study design fit the underlying population-genetic model. In many cases, all that is needed is a simple measure of among-group variation. One such measure is $R^2$, the proportion of total phenotypic variation accounted for by among-group phenotypic variation, a measure easily obtained from analysis of variance and regression methods. This paper shows that $R^2$ and minimum $F_{\mathrm{ST}}$ are closely related as $\mathrm{Min}{F}_{\mathrm{ST}}\approx R^2/\left(2-R^2\right)$. $R^2$ is computationally easy and may be useful in cases where all we need is a simple measure of relative among-group differentiation.