Phylogenetic placement refers to a family of tools and methods to analyze, visualize, and interpret the tsunami of metagenomic sequencing data generated by high-throughput sequencing. Compared to alternative (e.g., similarity-based) methods, it puts metabarcoding sequences into a phylogenetic context using a set of known reference sequences and taking evolutionary history into account. Thereby, one can increase the accuracy of metagenomic surveys and eliminate the requirement for having exact or close matches with existing sequence databases. Phylogenetic placement constitutes a valuable analysis tool per se, but also entails a plethora of downstream tools to interpret its results. A common use case is to analyze species communities obtained from metagenomic sequencing, for example via taxonomic assignment, diversity quantification, sample comparison, and identification of correlations with environmental variables. In this review, we provide an overview over the methods developed during the first 10years. In particular, the goals of this review are 1) to motivate the usage of phylogenetic placement and illustrate some of its use cases, 2) to outline the full workflow, from raw sequences to publishable figures, including best practices, 3) to introduce the most common tools and methods and their capabilities, 4) to point out common placement pitfalls and misconceptions, 5) to showcase typical placement-based analyses, and how they can help to analyze, visualize, and interpret phylogenetic placement data.

Cell wall remodeling is essential for the control of growth and development as well as the regulation of stress responses. However, the underlying cell wall monitoring mechanisms remain poorly understood. Regulation of root hair fate and flower development in Arabidopsis thaliana requires signaling mediated by the atypical receptor kinase STRUBBELIG (SUB). Furthermore, SUB is involved in cell wall integrity signaling and regulates the cellular response to reduced levels of cellulose, a central component of the cell wall. Here, we show that continuous exposure to sub-lethal doses of the cellulose biosynthesis inhibitor isoxaben results in altered root hair patterning and floral morphogenesis. Genetically impairing cellulose biosynthesis also results in root hair patterning defects. We further show that isoxaben exerts its developmental effects through the attenuation of SUB signaling. Our evidence indicates that downregulation of SUB is a multi-step process and involves changes in SUB complex architecture at the plasma membrane, enhanced removal of SUB from the cell surface, and downregulation of SUB transcript levels. The results provide molecular insight into how the cell wall regulates cell fate and tissue morphogenesis.

Signaling pathways rely on the precise control of protein-protein interactions. Therefore, it is essential to be able to investigate such interactions with spatiotemporal resolution and in live cells. Here we describe a microscope-based fluorescence spectrometry technique to investigate homotypic interactions between GFP-labeled fusion proteins in a rapid and reproducible fashion using fluorescence anisotropy. This method is of great value for the study of protein complexes in live tissue with subcellular resolution.

The green alga Chlamydomonas is a valuable model system capable of assimilating different forms of nitrogen (N). Nitrate (NO3-) has a relevant role in plant-like organisms, first as a nitrogen source for growth and second as a signalling molecule. Several modules are necessary for Chlamydomonas to handle nitrate, including transporters, nitrate reductase (NR), nitrite reductase (NiR), GS/GOGAT enzymes for ammonium assimilation, and regulatory protein(s). Transporters provide a first step for influx/efflux, homeostasis, and sensing of nitrate; and NIT2 is the key transcription factor (RWP-RK) for mediating the nitrate-dependent activation of a number of genes. Here, we review how NR participates in the cycle NO3--> NO2--> NO -> NO3-. NR uses the partner protein amidoxime-reducing component/nitric oxide-forming nitrite reductase (ARC/NOFNiR) for the conversion of nitrite (NO2-) into nitric oxide (NO). It also uses the truncated haemoglobin THB1 in the conversion of nitric oxide to nitrate. Nitric oxide is a negative signal for nitrate assimilation; it inhibits the activity and expression of high-affinity nitrate/nitrite transporters and NR. During this cycle, the positive signal of nitrate is transformed into the negative signal of nitric oxide, which can then be converted back into nitrate. Thus, NR is back in the spotlight as a strategic regulator of the nitric oxide cycle and the nitrate assimilation pathway.

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3-) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalgaChlamydomonas reinhardtiias an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms.

Nitrous oxide (N2O) is a powerful greenhouse gas and an ozone-depleting compound whose synthesis and release have traditionally been ascribed to bacteria and fungi. Although plants and microalgae have been proposed as N2O producers in recent decades, the proteins involved in this process have been only recently unveiled. In the green microalga Chlamydomonas reinhardtii, flavodiiron proteins (FLVs) and cytochrome P450 (CYP55) are two nitric oxide (NO) reductases responsible for N2O synthesis in the chloroplast and mitochondria, respectively. However, the molecular mechanisms feeding these NO reductases are unknown. In this work, we use cavity ring-down spectroscopy to monitor N2O and CO2 in cultures of nitrite reductase mutants, which cannot grow on nitrate or nitrite and exhibit enhanced N2O emissions. We show that these mutants constitute a very useful tool to study the rates and kinetics of N2O release under different conditions and the metabolism of this greenhouse gas. Our results indicate that N2O production, which was higher in the light than in the dark, requires nitrate reductase as the major provider of NO as substrate. Finally, we show that the presence of nitrate reductase impacts CO2 emissions in both light and dark conditions, and we discuss the role of NO in the balance between CO2 fixation and release.

Microbial communities are of considerable significance for biogeochemical processes, for the health of both animals and plants, and for biotechnological purposes. A key feature of microbial interactions is the exchange of nutrients between cells. Isotope labelling followed by analysis with secondary ion mass spectrometry (SIMS) can identify nutrient fluxes and heterogeneity of substrate utilisation on a single cell level. Here we present a novel approach that combines SIMS experiments with mechanistic modelling to reveal otherwise inaccessible nutrient kinetics. The method is applied to study the onset of a synthetic mutualistic partnership between a vitamin B-12-dependent mutant of the alga Chlamydomonas reinhardtii and the B-12-producing, heterotrophic bacterium Mesorhizobium japonicum, which is supported by algal photosynthesis. Results suggest that an initial pool of fixed carbon delays the onset of mutualistic cross-feeding; significantly, our approach allows the first quantification of this expected delay. Our method is widely applicable to other microbial systems, and will contribute to furthering a mechanistic understanding of microbial interactions.

Background: Modelling COVID-19 transmission at live events and public gatherings is essential to controlling the probability of subsequent outbreaks and communicating to participants their personalized risk. Yet, despite the fast-growing body of literature on COVID-19 transmission dynamics, current risk models either neglect contextual information including vaccination rates or disease prevalence or do not attempt to quantitatively model transmission. Objective: This paper attempted to bridge this gap by providing informative risk metrics for live public events, along with a measure of their uncertainty. Methods: Building upon existing models, our approach ties together 3 main components: (1) reliable modelling of the number of infectious cases at the time of the event, (2) evaluation of the efficiency of pre-event screening, and (3) modelling of the event's transmission dynamics and their uncertainty using Monte Carlo simulations. Results: We illustrated the application of our pipeline for a concert at the Royal Albert Hall and highlighted the risk's dependency on factors such as prevalence, mask wearing, and event duration. We demonstrate how this event held on 3 different dates (August 20, 2020; January 20, 2021; and March 20, 2021) would likely lead to transmission events that are similar to community transmission rates (0.06 vs 0.07, 2.38 vs 2.39, and 0.67 vs 0.60, respectively). However, differences between event and background transmissions substantially widened in the upper tails of the distribution of the number of infections (as denoted by their respective 99th quantiles: 1 vs 1, 19 vs 8, and 6 vs 3, respectively, for our 3 dates), further demonstrating that sole reliance on vaccination and antigen testing to gain entry would likely significantly underestimate the tail risk of the event. Conclusions: Despite the unknowns surrounding COVID-19 transmission, our estimation pipeline opens the discussion on contextualized risk assessment by combining the best tools at hand to assess the order of magnitude of the risk. Our model can be applied to any future event and is presented in a user-friendly RShiny interface. Finally, we discussed our model's limitations as well as avenues for model evaluation and improvement.

Cobalamin (vitamin B-12) is a cofactor for essential metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonize the algal phycosphere, forming stable communities that gain preferential access to photosynthate and in return provide compounds such as B-12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B-12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B-12 producers, its growth in co-culture was slower than the B-12-independent wild-type, suggesting that high bacterial B-12 provision may be necessary to favour B-12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B-12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B-12 release provided an advantage in co-cultures. We hypothesize that, given the right conditions, bacteria that release more B-12 may be selected for, particularly if they form close interactions with B-12-dependent algae.

Using argon as the pressure medium, the structural and elastic properties of NiO have been investigated up to 67 GPa by the in situ synchrotron x-ray diffraction in a diamond anvil cell. Up to 67 GPa, NiO remains in the rhombohedral distorted rocksalt structure without phase transition. The lattice parameters of a and c, indexed in the hexagonal lattice, were found to decrease monotonically with increasing pressure, while the c/a ratio remains almost constant. The elastic properties of NiO were studied by analyzing the linewidth of various diffraction perks, which indicates that the factor S = (S-11-S-12-S-44/2) is negative although the single-crystal elastic compliances S11 is positive, respectively, in the investigated pressure range. (c) 2008 American Institute of Physics. [DOI: 10.1063/1.3031697]