Systemic analysis of lipid metabolism from individuals to multi-organism systems.

Lipid metabolism is recognised as being central to growth, disease and health. Lipids, therefore, have an important place in current research on globally significant topics such as food security and biodiversity loss. However, answering questions in these important fields of research requires not only identification and measurement of lipids in a wider variety of sample types than ever before, but also hypothesis-driven analysis of the resulting 'big data'. We present a novel pipeline that can collect data from a wide range of biological sample types, taking 1 000 000 lipid measurements per 384 well plate, and analyse the data systemically. We provide evidence of the power of the tool through proof-of-principle studies using edible fish (mackerel, bream, seabass) and colonies of Bombus terrestris. Bee colonies were found to be more like mini-ecosystems and there was evidence for considerable changes in lipid metabolism in bees through key developmental stages. This is the first report of either high throughput LCMS lipidomics or systemic analysis in individuals, colonies and ecosystems. This novel approach provides new opportunities to analyse metabolic systems at different scales at a level of detail not previously feasible, to answer research questions about societally important topics.

Synthesis and biochemical evaluation of new 3-amido-4-substituted monocyclic ß-lactams as inhibitors of penicillin-binding protein(s).

In the final phases of bacterial cell wall synthesis, penicillin-binding proteins (PBPs) catalyze the cross-linking of peptidoglycan. For many decades, effective and non-toxic ß-lactam antibiotics have been successfully used as mimetics of the d-Ala-d-Ala moiety of the natural substrate and employed as irreversible inhibitors of PBPs. In the years following their discovery, the emergence of resistant bacteria led to a decline in their clinical efficacy. Using Staudinger cycloaddition, we synthesized a focused library of novel monocyclic ß-lactams in which different substituents were introduced at the C4 position of the ß-lactam ring, at the C3 amino position, and at the N1 lactam nitrogen. In biochemical assays, the compounds were evaluated for their inhibitory effect on the model enzyme PBP1b from Streptococcus pneumoniae. Upon investigation of the antibacterial activity of the newly prepared compounds against ESKAPE pathogens, some compounds showed moderate inhibition. We also examined their reactivity and selectivity in a biochemical assay with other enzymes that have a catalytic serine in the active site, such as human cholinesterases, where they also showed no inhibitory activity, highlighting their specificity for bacterial targets. These compounds form the basis for further work on new monocyclic ß-lactams with improved antibacterial activity.

Differential impacts of land-use change on multiple components of common milkweed (Asclepias syriaca) pollination success.

Land-use change is one the greatest threats to biodiversity and is projected to increase in magnitude in the coming years, stressing the importance of better understanding how land-use change may affect vital ecosystem services, such as pollination. Past studies on the impact of land-use change have largely focused on only one aspect of the pollination process (e.g., pollinator composition, pollinator visitation, and pollen transfer), potentially misrepresenting the full complexity of land-use effects on pollination services. Evaluating the impacts across multiple components of the pollination process can also help pinpoint the underlying mechanisms driving land-use change effects. This study evaluates how land-use change affects multiple aspects of the pollination process in common milkweed populations, including pollinator community composition, pollinator visitation rate, pollen removal, and pollen deposition. Overall, land-use change altered floral visitor composition, with small bees having a larger presence in developed areas. Insect visitation rate and pollen removal were also higher in more developed areas, perhaps suggesting a positive impact of land-use change. However, pollen deposition did not differ between developed and undeveloped sites. Our findings highlight the complexity evaluating land-use change effects on pollination, as these likely depend on the specific aspect of pollination evaluated and on the of the intensity of disturbance. Our study stresses the importance of evaluating multiple components of the pollination process in order to fully understand overall effects and mechanisms underlying land-use change effects on this vital ecosystem service.

Lateralized Movements during the Mating Behavior, Which Are Associated with Sex and Sexual Experience, Increase the Mating Success in Alphitobius diaperinus (Coleoptera: Tenebrionidae).

In the present study, we explored the effects of displacement directionality in mating behavior (i.e., lateralized and non-lateralized movements) on mating success (i.e., copulation occurs) and efficiency (i.e., time length at which copulation is achieved), and its association with sex and sexual experience in A. diaperinus. To do so, we carried out mating experiments and recorded the behavior of the mating pair during the whole mating sequence (i.e., precopulatory and copulatory phases). During the precopulatory phase, independently of sex and sexual experience, all beetles performed non-lateralized (i.e., backside or frontside) approaches; however, only sexually experienced beetles showed lateralized approaches (i.e., right-side and left-side). Notably, experienced males exhibited greater mating success than virgin males. After the approach, both virgin and experienced males displayed lateralized and non-lateralized mounts on the females with distinct mating success. Regardless of their sexual experience, 100% of successful mating attempts were achieved when males mounted from the females' right side. Furthermore, the development of lateralized approaches and mounts reduces the time of mating sequence span compared with non-lateralized behaviors. We highlight the importance of lateralization in mating behavior and sexual experience to achieve higher mating success, addressing a potential learning ability of beetles based on experience.

Sterol composition in plants is specific to pollen, leaf, pollination and pollinator.

Sterols have several roles in planta, including as membrane components. Sterols are also essential nutrients for insects. Based on this, and the different functions of leaves and pollen, we tested the hypotheses that (a) the sterolome is different in leaves and pollen from the same plant, (b) pollens from wind- and insect pollinated plants comprise different sterols, and (c) sterol provision in pollen-rewarding angiosperms differs from nectar-rewarding species. A novel approach to sterolomics was developed, using LCMS to determine the sterol profile of leaf and pollen from a taxonomically diverse range of 36 plant species. Twenty-one sterols were identified unambiguously, with several more identified in trace amounts. C29 sterols dominated the sterolome in most plants. The sterol composition was significantly different in leaf and pollen and their main sterols evolved in different ways. The sterolome of pollen from animal- and wind-pollinated was also significantly different, but not between nectar- and pollen-rewarding species. Our results suggest that the sterol composition in different plant tissues is linked to their biological functions. Sterol composition in pollen might be driven by physical role rather than the nutrient needs of pollinating insects.

Architecture of a PKS-NRPS hybrid megaenzyme involved in the biosynthesis of the genotoxin colibactin.

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications.

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with ß-Lactones.

ß-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to ß-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of ß-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four ß-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the ß-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

Chemical Signals Associated With Gender and Sexual Experience Affect Mating and the Attractiveness of the Poultry Pest, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

Alphitobius diaperinus is one of the most significant pests in the poultry industry. Identifying the role of self-produced chemical signals can help control it. Here, we exposed adults to the olfactory signals of other adults of similar and different genders (either males or females) and sexual experiences (i.e., virgin and experienced) to assess their long-range attractiveness and, at short-range, their mating behavior responses (i.e., touching, mounting, and copulation). In olfactometric experiments, our results indicate that adults are attracted to the olfactory signals of other male adults, independently of gender, or sexual condition, indicating the presence of generalized long-range attractive signals, in contrast to female signals, can be both factor-dependent. However, in mating experiments, virgin males developed more robust mating responses (i.e., they mount and copulate longer with females) compared to sexually experienced males, even though they both have similar precopulatory behavioral responses (i.e., time of antennal and leg touching). These results address the importance of short-range chemical signals in eliciting copulation. Furthermore, when virgins of both genders were tested, their mating responses were significantly longer than any other pair combination, indicating that sexual experience also affects mating behavior. Chemical analyses of adult extracts showed that sexual experience, but not gender, is linked to differences in chemical profiles of adults, primarily involved in short-range signaling. These findings provide new insights into the attractiveness and mating responses of A. diaperinus and the role of sexual experience in shaping the behavior and chemical profile of insects that mate multiple times during their lifetime.

Structural Plastome Evolution in Holoparasitic Hydnoraceae with Special Focus on Inverted and Direct Repeats.

Plastome condensation during adaptation to a heterotrophic lifestyle is generally well understood and lineage-independent models have been derived. However, understanding the evolutionary trajectories of comparatively old heterotrophic lineages that are on the cusp of a minimal plastome, is essential to complement and expand current knowledge. We study Hydnoraceae, one of the oldest and least investigated parasitic angiosperm lineages. Plastome comparative genomics, using seven out of eight known species of the genus Hydnora and three species of Prosopanche, reveal a high degree of structural similarity and shared gene content; contrasted by striking dissimilarities with respect to repeat content [inverted and direct repeats (DRs)]. We identified varying inverted repeat contents and positions, likely resulting from multiple, independent evolutionary events, and a DR gain in Prosopanche. Considering different evolutionary trajectories and based on a fully resolved and supported species-level phylogenetic hypothesis, we describe three possible, distinct models to explain the Hydnoraceae plastome states. For comparative purposes, we also report the first plastid genomes for the closely related autotrophic genera Lactoris (Lactoridaceae) and Thottea (Aristolochiaceae).

Structural insights into ring-building motif domains involved in bacterial sporulation.

Components of specialized secretion systems, which span the inner and outer membranes in Gram-negative bacteria, include ring-forming proteins whose oligomerization was proposed to be promoted by domains called RBM for "Ring-Building Motifs". During spore formation in Gram-positive bacteria, a transport system called the SpoIIIA-SpoIIQ complex also assembles in the double membrane that surrounds the forespore following its endocytosis by the mother cell. The presence of RBM domains in some of the SpoIIIA proteins led to the hypothesis that they would assemble into rings connecting the two membranes and form a conduit between the mother cell and forespore. Among them, SpoIIIAG forms homo-oligomeric rings in vitro but the oligomerization of other RBM-containing SpoIIIA proteins, including SpoIIIAH, remains to be demonstrated. In this work, we identified RBM domains in the YhcN/YlaJ family of proteins that are not related to the SpoIIIA-SpoIIQ complex. We solved the crystal structure of YhcN from Bacillus subtilis, which confirmed the presence of a RBM fold, flanked by additional secondary structures. As the protein did not show any oligomerization ability in vitro, we investigated the structural determinants of ring formation in SpoIIIAG, SpoIIIAH and YhcN. We showed that in vitro, the conserved core of RBM domains alone is not sufficient for oligomerization while the ß-barrel forming region in SpoIIIAG forms rings on its own. This work suggests that some RBMs might indeed participate in the assembly of homomeric rings but others might have evolved toward other functions.

Spatial variation in bidirectional pollinator-mediated interactions between two co-flowering species in serpentine plant communities.

Pollinator-mediated competition and facilitation are two important mechanisms mediating co-flowering community assembly. Experimental studies, however, have mostly focused on evaluating outcomes for a single interacting partner at a single location. Studies that evaluate spatial variation in the bidirectional effects between co-flowering species are necessary if we aim to advance our understanding of the processes that mediate species coexistence in diverse co-flowering communities. Here, we examine geographic variation (i.e. at landscape level) in bidirectional pollinator-mediated effects between co-flowering Mimulus guttatus and Delphinium uliginosum. We evaluated effects on pollen transfer dynamics (conspecific and heterospecific pollen deposition) and plant reproductive success. We found evidence of asymmetrical effects (one species is disrupted and the other one is facilitated) but the effects were highly dependent on geographical location. Furthermore, effects on pollen transfer dynamics did not always translate to effects on overall plant reproductive success (i.e. pollen tube growth) highlighting the importance of evaluating effects at multiple stages of the pollination process. Overall, our results provide evidence of a spatial mosaic of pollinator-mediated interactions between co-flowering species and suggest that community assembly processes could result from competition and facilitation acting simultaneously. Our study highlights the importance of experimental studies that evaluate the prevalence of competitive and facilitative interactions in the field, and that expand across a wide geographical context, in order to more fully understand the mechanisms that shape plant communities in nature.

MagC is a NplC/P60-like member of the α-2-macroglobulin Mag complex of Pseudomonas aeruginosa that interacts with peptidoglycan.

Bacterial α-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In Pseudomonas aeruginosa, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

Self-association of MreC as a regulatory signal in bacterial cell wall elongation.

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.

Mass Spectrometry: A Rosetta Stone to Learn How Fungi Interact and Talk.

Fungi are a highly diverse group of heterotrophic organisms that play an important role in diverse ecological interactions, many of which are chemically mediated. Fungi have a very versatile metabolism, which allows them to synthesize a large number of still little-known chemical compounds, such as soluble compounds that are secreted into the medium and volatile compounds that are chemical mediators over short and long distances. Mass spectrometry (MS) is currently playing a dominant role in mycological studies, mainly due to its inherent sensitivity and rapid identification capabilities of different metabolites. Furthermore, MS has also been used as a reliable and accurate tool for fungi identification (i.e., biotyping). Here, we introduce the readers about fungal specialized metabolites, their role in ecological interactions and provide an overview on the MS-based techniques used in fungal studies. We particularly present the importance of sampling techniques, strategies to reduce false-positive identification and new MS-based analytical strategies that can be used in mycological studies, further expanding the use of MS in broader applications. Therefore, we foresee a bright future for mass spectrometry-based research in the field of mycology.

Análisis de la categorización del estado de conservación de las orquídeas en el Perú: el caso del género Telipogon / Analysis of the conservation status of Peruvian orchids: The case of the genus Telipogon

Resumen Aunque la familia de las orquídeas es uno de los grupos de plantas mejor representados en los listados de conservación a nivel mundial, aun este número de representantes es pequeño considerando su alta diversidad y vulnerabilidad. Esto es particularmente notorio en los listados de la flora amenazada del Perú. En el presente comentario se analiza la representatividad de las orquídeas, con foco en las especies del género Telipogon incluidas en los listados de categorización de conservación en el Perú, enfatizando la importancia de realizar correctos listados y categorizaciones coherentes con las metas Aichi del Plan Estratégico para la Diversidad Biológica 2011-2020.

Abstract The orchid family is one of the plant groups with the highest number of species included in conservation lists worldwide. However, this number is still small considering the high orchid diversity and vulnerability. This is particularly manifest in Peruvian lists of threatened flora. In this comment, I analyse their representativeness in conservation lists in Peru, with an emphasis on species of the genus Telipogon. My analysis highlights the importance of elaborating accurate lists, consistent with the Aichi Biodiversity Targets of the Strategic Plan for Biological Diversity 2011-2020.

Bacterial secretins: Mechanisms of assembly and membrane targeting.

Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS secretins, highlighting recent near-atomic resolution cryo-EM secretin complex structures and underlining the importance of these interactions for secretin functionality.

The chemical and visual bases of the pollination of the Neotropical sexually deceptive orchid Telipogon peruvianus.

Deception of floral visitors in pollination systems is widely distributed among flowering plants. In deceptive systems, the flower (or part of it) or inflorescence mimics either a specific or an unspecific model to attract pollinators. A previous study showed that Telipogon peruvianus flowers developed sexual deception for pollination. However, it was unknown which stimuli were playing a role in pollination. Therefore, we aim to throw some light onto these questions using colour and chemical analysis and biotests. Interestingly, using spectral reflectance, we show here that the flowers present high contrast similar to that produced by a female tachinid fly sitting on a daisy inflorescence, which is used as food resource. We also tested the role of chemical signals in pollinator attraction by collecting floral and female extracts for chemical and electrophysiological analyses, and carried out behavioural tests. For biotests, various treatments, including synthetic mixtures of the electrophysiologically active compounds found in common in females and flowers, have demonstrated that T. peruvianus flowers mimic the sexual pheromone of their pollinator's females. Thus, we give evidence that T. peruvianus flowers mimic a model composed of two organisms. Our study contributes to the understanding of the evolution of deceptive pollination.

Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.