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Sexual size dimorphism (SSD) is highly prevalent in nature. Several hypotheses aim to explain its evolution including sexual selection, differential equilibrium and ecological niche divergence. Disentangling the causal mechanism behind the evolution of SSD is challenging, as selection arising from multiple pressures on fitness may act simultaneously to generate observed patterns. Here, we use phylogenetic comparative methods to study the evolution of SSD across tetrapods globally. We estimate directional changes in body size evolution, and compare the number, phylogenetic position and magnitude of size changes between sexes. We find evidence that directional changes in size associated with SSD are typically more common in males-even in lineages where females are larger. However, underlying mechanisms differ among lineages-whereas SSD in amphibians becomes more male-biased with greater increases in male size and mammalian SSD becomes more female-biased with greater decreases in male size. Thus, differing mechanisms of directional body size evolution across sexes are essential to explain observed SSD patterns., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Pseudaminic acid is a non-mammalian sugar found in the surface glycoconjugates of many bacteria, including several human pathogens, and is a virulence factor thought to facilitate immune evasion. The final step in the biosynthesis of the nucleotide activated form of the sugar, CMP-Pse5Ac7Ac is performed by a CMP-Pse5Ac7Ac synthetase (PseF). Here we present the biochemical and structural characterization of PseF from Aeromonas caviae (AcPseF), with AcPseF displaying metal-dependent activity over a broad pH and temperature range. Upon binding to CMP-Pse5Ac7Ac, AcPseF undergoes dynamic movements akin to other CMP-ulosonic acid synthetases. The enzyme clearly discriminates Pse5Ac7Ac from other ulosonic acids, through active site interactions with side-chain functional groups and by positioning the molecule in a hydrophobic pocket. Finally, we show that AcPseF binds the CMP-Pse5Ac7Ac side chain in the lowest energy conformation, a trend that we observed in the structures of other enzymes of this class., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in prokaryotes and are responsible for the transport of a variety of different ligands, primarily organic acids. TRAP transporters can be divided into two subclasses; DctP-type and TAXI type, which share the same overall architecture and substrate-binding protein requirement. DctP-type transporters are very well studied and have been shown to transport a range of compounds including dicarboxylates, keto acids, and sugar acids. However, TAXI-type transporters are relatively poorly understood. To address this gap in our understanding, we have structurally and biochemically characterized VC0430 from Vibrio cholerae. We show it is a monomeric, high affinity glutamate-binding protein, which we thus rename VcGluP. VcGluP is stereoselective, binding the L-isomer preferentially, and can also bind L-glutamine and L-pyroglutamate with lower affinity. Structural characterization of ligand-bound VcGluP revealed details of its binding site and biophysical characterization of binding site mutants revealed the substrate binding determinants, which differ substantially from those of DctP-type TRAPs. Finally, we have analyzed the interaction between VcGluP and its cognate membrane component, VcGluQM (formerly VC0429) in silico, revealing an architecture hitherto unseen. To our knowledge, this is the first transporter in V. cholerae to be identified as specific to glutamate, which plays a key role in the osmoadaptation of V. cholerae, making this transporter a potential therapeutic target., (© 2024 Davies et al.)

The production of human body odor is the result of the action of commensal skin bacteria, including Staphylococcus hominis, acting to biotransform odorless apocrine gland secretions into volatile chemicals like thioalcohols such as 3-methyl-3-sulphanylhexan-1-ol (3M3SH). As the secreted odor precursor Cys-Gly-3M3SH contains a dipeptide, yet the final enzyme in the biotransformation pathway only functions on Cys-3M3SH, we sought to identify the remaining step in this human-adapted biochemical pathway using a novel coupled enzyme assay. Purification of this activity from S. hominis extracts led to the identification of the M20A-family PepV peptidase (ShPepV) as the primary Cys-Gly-3M3SH dipeptidase. To establish whether this was a primary substrate for PepV, the recombinant protein was purified and demonstrated broad activity against diverse dipeptides. The binding site for Cys-Gly-3M3SH was predicted using modeling, which suggested mutations that might accommodate this ligand more favorably. Indeed, a D437A resulted in an almost sixfold increase in the k cat /K m , whereas other introduced mutations reduced or abolished function. Together, these data identify an enzyme capable of catalyzing the missing step in an ancient human-specific biochemical transformation and suggest that the production of 3M3SH uses neither a dedicated transporter nor a peptidase for its breakdown, with only the final cleavage step, catalyzed by PatB cysteine-S-conjugate β-lyase, being a unique enzyme., Competing Interests: Conflict of interest The authors declare that B. M. is a current employee of Unilever, whereas A. G. J. was an employee of Unilever when this study was carried out. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The tripartite ATP-independent periplasmic (TRAP) transporters enable Vibrio cholerae and Haemophilus influenzae to acquire sialic acid, aiding their colonization of human hosts. This process depends on SiaP, a substrate-binding protein (SBP) that captures and delivers sialic acid to the transporter. We identified 11 nanobodies that bind specifically to the SiaP proteins from H. influenzae (HiSiaP) and V. cholerae (VcSiaP). Two nanobodies inhibited sialic acid binding. Detailed structural and biophysical studies of one nanobody-SBP complex revealed an allosteric inhibition mechanism, preventing ligand binding and releasing pre-bound sialic acid. A hydrophobic surface pocket of the SBP is crucial for the allosteric mechanism and for the conformational rearrangement that occurs upon binding of sialic acid to the SBP. Our findings provide new clues regarding the mechanism of TRAP transporters, as well as potential starting points for novel drug design approaches to starve these human pathogens of important host-derived molecules., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Iron is an essential nutrient for microbial growth and bacteria have evolved numerous routes to solubilize and scavenge this biometal, which is often present at very low concentrations in host tissue. We recently used a MOPS-based medium to induce iron limitation in Escherichia coli K-12 during the characterization of novel siderophore-conjugated antibiotics. In this study we confirm that growth media derived from commercially available M9 salts are unsuitable for studies of iron-limited growth, probably through the contamination of the sodium phosphate buffer components with over 100 µM iron. In contrast, MOPS-based media that are treated with metal-binding Chelex resin allow the free iron concentration to be reduced to growth-limiting levels. Despite these measures a small amount of E. coli growth is still observed in these iron-depleted media. By growing E. coli in conditions that theoretically increase the demand for iron-dependent enzymes, namely by replacing the glucose carbon source for acetate and by switching to a microaerobic atmosphere, we can reduce background growth even further. Finally, we demonstrate that by adding an exogeneous siderophore to the growth media which is poorly used by E. coli , we can completely prevent growth, perhaps mimicking the situation in host tissue. In conclusion, this short study provides practical experimental insight into low iron media and how to augment the growth conditions of E. coli for extreme iron-limited growth., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 The Authors.)

Progress towards the total synthesis of the macrolide natural product anthracimycin is described. This new approach utilises an intermolecular Diels-Alder strategy followed by epimeirsation to form the key trans -decalin framework. The route culminates in the stereoselective synthesis of an advanced tricyclic lactone intermediate, containing five contiguous sterogenic centres with the correct relative and absolute stereochemistry required for the anthracimycin core motif.

Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them are orthologs of human mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. For the first time, the dynamics of the bacterial RBPome have been interrogated, showcasing how this approach can reveal the function of uncharacterised proteins and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies., (© 2024. The Author(s).)

Cell surface sugar 5,7-diacetyl pseudaminic acid (Pse5Ac7Ac) is a bacterial analogue of the ubiquitous sialic acid, Neu5Ac, and contributes to the virulence of a number of multidrug resistant bacteria, including ESKAPE pathogens Pseudomonas aeruginosa, and Acinetobacter baumannii. Despite its discovery in the surface glycans of bacteria over thirty years ago, to date no glycosyltransferase enzymes (GTs) dedicated to the synthesis of a pseudaminic acid glycosidic linkage have been unequivocally characterised in vitro. Herein we demonstrate that A. baumannii KpsS1 is a dedicated pseudaminyltransferase enzyme (PseT) which constructs a Pse5Ac7Ac-α(2,6)-Glcp linkage, and proceeds with retention of anomeric configuration. We utilise this PseT activity in tandem with the biosynthetic enzymes required for CMP-Pse5Ac7Ac assembly, in a two-pot, seven enzyme synthesis of an α-linked Pse5Ac7Ac glycoside. Due to its unique activity and protein sequence, we also assign KpsS1 as the prototypical member of a previously unreported GT family (GT118)., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

YejABEF is an ATP-binding cassette transporter that is implicated in the sensitivity of Escherichia coli to anti-microbial peptides, the best-characterized example being microcin C, a peptide-nucleotide antibiotic that targets aspartyl-tRNA synthetase. Here the structure of the extracellular solute binding protein, YejA, has been determined, revealing an oligopeptide-binding protein fold enclosing a ligand-binding pocket larger than those of other peptide-binding proteins of known structure. Prominent electron density in this cavity defines an undecapeptide sequence LGEPRYAFNFN, an observation that is confirmed by mass spectrometry. In the structure, the peptide interactions with the protein are mediated by main chain hydrogen bonds with the exception of Arg5 whose guanidinium side chain makes a set of defining polar interactions with four YejA residues. More detailed characterization of purified recombinant YejA, by a combination of ESI and MALDI-mass spectrometry as well as thermal shift assays, reveals a set of YejA complexes containing overlapping peptides 10-19 residues in length. All contain the sequence LGEPRYAFN. Curiously, these peptides correspond to residues 8-26 of the mature YejA protein, which belong to a unique N-terminal extension that distinguishes YejA from other cluster C oligopeptide binding proteins of known structure. This 35-residue extension is well-ordered and packs across the surface of the protein. The undecapeptide ligand occupies only a fraction of the enclosed pocket volume suggesting the possibility that much larger peptides or peptide conjugates could be accommodated, though thermal shift assays of YejA binding to antimicrobial peptides and peptides unrelated to LGEPRYAFNFN have not provided evidence of binding. While the physiological significance of this 'auto-binding' is not clear, the experimental data suggest that it is not an artefact of the crystallization process and that it may have a function in the sensing of periplasmic or membrane stress.

The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa., (© 2024. The Author(s).)

The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While much is known about the transcriptional regulation of genes encoding transporters, less is understood about how transporter activity is modulated once the protein is functional in the membrane, a potentially more rapid and dynamic level of control. In this review, we bring together literature from the bacterial transport community exemplifying the extensive and diverse mechanisms that have evolved to rapidly modulate transporter function, predominantly by switching activity off. This includes small molecule feedback, inhibition by interaction with small peptides, regulation through binding larger signal transduction proteins and, finally, the emerging area of controlled proteolysis. Many of these examples have been discovered in the context of metal transport, which has to finely balance active accumulation of elements that are essential for growth but can also quickly become toxic if intracellular homeostasis is not tightly controlled. Consistent with this, these transporters appear to be regulated at multiple levels. Finally, we find common regulatory themes, most often through the fusion of additional regulatory domains to transporters, which suggest the potential for even more widespread regulation of transporter activity in biology.

Widely documented, megaevolutionary jumps in phenotypic diversity continue to perplex researchers because it remains unclear whether these marked changes can emerge from microevolutionary processes. Here, we tackle this question using new approaches for modeling multivariate traits to evaluate the magnitude and distribution of elaboration and innovation in the evolution of bird beaks. We find that elaboration, evolution along the major axis of phenotypic change, is common at both macro- and megaevolutionary scales, whereas innovation, evolution away from the major axis of phenotypic change, is more prominent at megaevolutionary scales. The major axis of phenotypic change among species beak shapes at megaevolutionary scales is an emergent property of innovation across clades. Our analyses suggest that the reorientation of phenotypes via innovation is a ubiquitous route for divergence that can arise through gradual change alone, opening up further avenues for evolution to explore.

Phase separation underpins many biologically important cellular events such as RNA metabolism, signaling, and CO2 fixation. However, determining the composition of a phase-separated organelle is often challenging due to its sensitivity to environmental conditions, which limits the application of traditional proteomic techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase-separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID-based proximity labeling technique in which proximal proteins in Chlamydomonas chloroplasts are labeled by biotin radicals generated from the TurboID-tagged protein. By fusing 2 core pyrenoid components with the TurboID tag, we generated a high-confidence pyrenoid proxiome that contains most known pyrenoid proteins, in addition to new pyrenoid candidates. Fluorescence protein tagging of 7 previously uncharacterized TurboID-identified proteins showed that 6 localized to a range of subpyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox-sensitive iron-sulfur cluster metabolism. This developed pipeline can be used to investigate a broad range of biological processes in Chlamydomonas, especially at a temporally resolved suborganellar resolution., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafA Sw , binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafA Sw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafA Ec ) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology.

A key challenge in mobilising growing numbers of digitised biological specimens for scientific research is finding high-throughput methods to extract phenotypic measurements on these datasets. In this paper, we test a pose estimation approach based on Deep Learning capable of accurately placing point labels to identify key locations on specimen images. We then apply the approach to two distinct challenges that each requires identification of key features in a 2D image: (i) identifying body region-specific plumage colouration on avian specimens and (ii) measuring morphometric shape variation in Littorina snail shells. For the avian dataset, 95% of images are correctly labelled and colour measurements derived from these predicted points are highly correlated with human-based measurements. For the Littorina dataset, more than 95% of landmarks were accurately placed relative to expert-labelled landmarks and predicted landmarks reliably captured shape variation between two distinct shell ecotypes ('crab' vs 'wave'). Overall, our study shows that pose estimation based on Deep Learning can generate high-quality and high-throughput point-based measurements for digitised image-based biodiversity datasets and could mark a step change in the mobilisation of such data. We also provide general guidelines for using pose estimation methods on large-scale biological datasets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 He et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small-molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Acylation of diverse carbohydrates occurs across all domains of life and can be catalysed by proteins with a membrane bound acyltransferase-3 (AT3) domain (PF01757). In bacteria, these proteins are essential in processes including symbiosis, resistance to viruses and antimicrobials, and biosynthesis of antibiotics, yet their structure and mechanism are largely unknown. In this study, evolutionary co-variance analysis was used to build a computational model of the structure of a bacterial O-antigen modifying acetyltransferase, OafB. The resulting structure exhibited a novel fold for the AT3 domain, which molecular dynamics simulations demonstrated is stable in the membrane. The AT3 domain contains 10 transmembrane helices arranged to form a large cytoplasmic cavity lined by residues known to be essential for function. Further molecular dynamics simulations support a model where the acyl-coA donor spans the membrane through accessing a pore created by movement of an important loop capping the inner cavity, enabling OafB to present the acetyl group close to the likely catalytic resides on the extracytoplasmic surface. Limited but important interactions with the fused SGNH domain in OafB are identified, and modelling suggests this domain is mobile and can both accept acyl-groups from the AT3 and then reach beyond the membrane to reach acceptor substrates. Together this new general model of AT3 function provides a framework for the development of inhibitors that could abrogate critical functions of bacterial pathogens., Competing Interests: KN, ST, SM, SK, GT, MV No competing interests declared, (© 2023, Newman, Tindall et al.)

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake - the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with K d values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu- meso -DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli , suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp , which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes .

Most fish excrete their nitrogenous waste across the gills as ammonia through the activity of the Rhesus glycoprotein ammonium transporters. In contrast, fish of the subgenus Alcolapia (Oreochromis) are the only vertebrates that survive the extreme conditions of the soda lakes of Natron and Magadi in East Africa and have evolved adaptations to the highly alkaline waters including the ability to excrete their nitrogenous waste as urea. Nevertheless, Alcolapia retain the Rhesus glycoprotein genes in their genomes and using two heterologous expression systems, we demonstrate that Alcolapia Rhbg is capable of moving ammonia. Comparing ammonia and urea excretion from two closely related Alcolapia species from the same aquarium, we found that while Alcolapia grahami remains fully ureotelic after many generations in lab conditions, Alcolapia alcalica excretes some of its nitrogenous waste as ammonia. Using in situ hybridisation, we demonstrate robust, localised gene expression of Rhbg, rhcg1 and rhcg2 in the gill tissue in both A. alcalica embryos and adults, similar to that in other ammoniotelic fish. In contrast, the expression of these genes in A. grahami gills is much lower than in A. alcalica, suggesting the rapid evolution of a molecular mechanism underlying the complete ureotelism of A. grahami., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Biodiversity is facing a global extinction crisis that will reduce ecological trait diversity, evolutionary history, and ultimately ecosystem functioning and services. 1-4 A key challenge is understanding how species losses will impact morphological and phylogenetic diversity at global scales. 5 , 6 Here, we test whether the loss of species threatened with extinction according to the International Union for Conservation of Nature (IUCN) leads to morphological and phylogenetic homogenization 7 , 8 across both the whole avian class and within each biome and ecoregion globally. We use a comprehensive set of continuous morphological traits extracted from museum collections of 8,455 bird species, including geometric morphometric beak shape data, 9 and sequentially remove species from those at most to least threat of extinction. We find evidence of morphological, but not phylogenetic, homogenization across the avian class, with species becoming more alike in terms of their morphology. We find that most biome and ecoregions are expected to lose morphological diversity at a greater rate than predicted by species loss alone, with the most imperiled regions found in East Asia and the Himalayan uplands and foothills. Only a small proportion of assemblages are threatened with phylogenetic homogenization, in particular parts of Indochina. Species extinctions will lead to a major loss of avian ecological strategies, but not a comparable loss of phylogenetic diversity. As the decline of species with unique traits and their replacement with more widespread generalist species continues, the protection of assemblages at most risk of morphological and phylogenetic homogenization should be a key conservation priority., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

A novel Trojan Horse conjugate consisting of an SO 2 -releasing 2,4-dinitrobenzenesulfonamide group attached to the monocatecholate siderophore aminochelin was synthesized to examine whether a bidentate catecholate siderophore unit could help potentiate the antimicrobial activity of SO 2 -releasing prodrugs. The conjugate obtained displays rapid SO 2 release on reaction with glutathione, and proved more active against Staphylococcus aureus than a comparable SO 2 -releasing prodrug lacking the siderophore unit, although activity required micromolar concentrations. The conjugate was inactive against wild-type Escherichia coli, but activity was observed against an entA mutant strain that is unable to produce its major siderophores. Hence, the poor activity of the conjugate in wild-type E. coli may be due to the production of native siderophores that can compete with the conjugate for iron binding and uptake., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ultraviolet colouration is thought to be an important form of signalling in many bird species, yet broad insights regarding the prevalence of ultraviolet plumage colouration and the factors promoting its evolution are currently lacking. In this paper, we develop a image segmentation pipeline based on deep learning that considerably outperforms classical (i.e. non deep learning) segmentation methods, and use this to extract accurate information on whole-body plumage colouration from photographs of >24,000 museum specimens covering >4500 species of passerine birds. Our results demonstrate that ultraviolet reflectance, particularly as a component of other colours, is widespread across the passerine radiation but is strongly phylogenetically conserved. We also find clear evidence in support of the role of light environment in promoting the evolution of ultraviolet plumage colouration, and a weak trend towards higher ultraviolet plumage reflectance among bird species with ultraviolet rather than violet-sensitive visual systems. Overall, our study provides important broad-scale insight into an enigmatic component of avian colouration, as well as demonstrating that deep learning has considerable promise for allowing new data to be brought to bear on long-standing questions in ecology and evolution., (© 2022. The Author(s).)

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo., (© 2022. The Author(s).)

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology., (© 2022. The Author(s).)

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxP o ) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxP o , produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

It has long been suggested that tropical species are generally more colourful than temperate species, but whether latitudinal gradients in organismal colourfulness exist remains controversial. Here we quantify global latitudinal trends in colourfulness (within-individual colour diversity) by collating and analysing a photographic dataset of whole-body plumage reflectance information for >4,500 species of passerine birds. We show that male and female birds of tropical passerine species are generally more colourful than their temperate counterparts, both on average and in the extreme. We also show that these geographic gradients can be explained in part by the effects of several latitude-related factors related to classic hypotheses for climatic and ecological determinants of organismal colourfulness. Taken together, our results reveal that species' colourfulness peaks in the tropics for passerine birds, confirming the existence of a long-suspected yet hitherto elusive trend in the distribution of global biodiversity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Streptomyces rimosus ATCC 10970 is the parental strain of industrial strains used for the commercial production of the important antibiotic oxytetracycline. As an actinobacterium with a large linear chromosome containing numerous long repeat regions, high GC content, and a single giant linear plasmid (GLP), these genomes are challenging to assemble. Here, we apply a hybrid sequencing approach relying on the combination of short- and long-read next-generation sequencing platforms and whole-genome restriction analysis by using pulsed-field gel electrophoresis (PFGE) to produce a high-quality reference genome for this biotechnologically important bacterium. By using PFGE to separate and isolate plasmid DNA from chromosomal DNA, we successfully sequenced the GLP using Nanopore data alone. Using this approach, we compared the sequence of GLP in the parent strain ATCC 10970 with those found in two semi-industrial progenitor strains, R6-500 and M4018. Sequencing of the GLP of these three S. rimosus strains shed light on several rearrangements accompanied by transposase genes, suggesting that transposases play an important role in plasmid and genome plasticity in S. rimosus . The polished annotation of secondary metabolite biosynthetic pathways compared to metabolite analysis in the ATCC 10970 strain also refined our knowledge of the secondary metabolite arsenal of these strains. The proposed methodology is highly applicable to a variety of sequencing projects, as evidenced by the reliable assemblies obtained. IMPORTANCE The genomes of Streptomyces species are difficult to assemble due to long repeats, extrachromosomal elements (giant linear plasmids [GLPs]), rearrangements, and high GC content. To improve the quality of the S. rimosus ATCC 10970 genome, producer of oxytetracycline, we validated the assembly of GLPs by applying a new approach to combine pulsed-field gel electrophoresis separation and GLP isolation and sequenced the isolated GLP with Oxford Nanopore technology. By examining the sequenced plasmids of ATCC 10970 and two industrial progenitor strains, R6-500 and M4018, we identified large GLP rearrangements. Analysis of the assembled plasmid sequences shed light on the role of transposases in genome plasticity of this species. The new methodological approach developed for Nanopore sequencing is highly applicable to a variety of sequencing projects. In addition, we present the annotated reference genome sequence of ATCC 10970 with a detailed analysis of the biosynthetic gene clusters.

The acylation of sugars, most commonly via acetylation, is a widely used mechanism in bacteria that uses a simple chemical modification to confer useful traits. For structures like lipopolysaccharide, capsule and peptidoglycan, that function outside of the cytoplasm, their acylation during export or post-synthesis requires transport of an activated acyl group across the membrane. In bacteria this function is most commonly linked to a family of integral membrane proteins - acyltransferase-3 (AT3). Numerous studies examining production of diverse extracytoplasmic sugar-containing structures have identified roles for these proteins in O -acylation. Many of the phenotypes conferred by the action of AT3 proteins influence host colonisation and environmental survival, as well as controlling the properties of biotechnologically important polysaccharides and the modification of antibiotics and antitumour drugs by Actinobacteria. Herein we present the first systematic review, to our knowledge, of the functions of bacterial AT3 proteins, revealing an important protein family involved in a plethora of systems of importance to bacterial function that is still relatively poorly understood at the mechanistic level. By defining and comparing this set of functions we draw out common themes in the structure and mechanism of this fascinating family of membrane-bound enzymes, which, due to their role in host colonisation in many pathogens, could offer novel targets for the development of antimicrobials.

Trapliners are pollinators that visit widely dispersed flowers along circuitous foraging routes. The evolution of traplining in hummingbirds is thought to entail morphological specialization through the reciprocal coevolution of longer bills with the long-tubed flowers of widely dispersed plant species. Specialization, such as that exhibited by traplining hummingbirds, is often viewed as both irreversible and an evolutionary dead end. We tested these predictions in a macroevolutionary framework. Specifically, we assessed the relationship between beak morphology and foraging and tested whether transitions to traplining are irreversible and lead to lower rates of diversification as predicted by the hypothesis that specialization is an evolutionary dead end. We find that there have been multiple independent transitions to traplining across the hummingbird phylogeny, but reversals have been rare or incomplete at best. Multiple independent lineages of trapliners have become morphologically specialized, convergently evolving relatively large bills for their body size. Traplining is not an evolutionary dead end however, since trapliners continue to give rise to new traplining species at a rate comparable to non-trapliners.

The organic polymer lignin is a component of plant cell walls, which like (hemi)-cellulose is highly abundant in nature and relatively resistant to degradation. However, extracellular enzymes released by natural microbial consortia can cleave the β-aryl ether linkages in lignin, releasing monoaromatic phenylpropanoids that can be further catabolised by diverse species of bacteria. Biodegradation of lignin is therefore important in global carbon cycling, and its natural abundance also makes it an attractive biotechnological feedstock for the industrial production of commodity chemicals. Whilst the pathways for degradation of lignin-derived aromatics have been extensively characterised, much less is understood about how they are recognised and taken up from the environment. The purple phototrophic bacterium Rhodopseudomonas palustris can grow on a range of phenylpropanoid monomers and is a model organism for studying their uptake and breakdown. R. palustris encodes a tripartite ATP-independent periplasmic (TRAP) transporter (TarPQM) linked to genes encoding phenylpropanoid-degrading enzymes. The periplasmic solute-binding protein component of this transporter, TarP, has previously been shown to bind aromatic substrates. Here, we determine the high-resolution crystal structure of TarP from R. palustris as well as the structures of homologous proteins from the salt marsh bacterium Sagittula stellata and the halophile Chromohalobacter salexigens, which also grow on lignin-derived aromatics. In combination with tryptophan fluorescence ligand-binding assays, our ligand-bound co-crystal structures reveal the molecular basis for high-affinity recognition of phenylpropanoids by these TRAP transporters, which have potential for improving uptake of these compounds for biotechnological transformations of lignin., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Evolution can involve periods of rapid divergent adaptation and expansion in the range of diversity, but evolution can also be relatively conservative over certain timescales due to functional, genetic-developmental, and ecological constraints. One way in which evolution may be conservative is in terms of allometry, the scaling relationship between the traits of organisms and body size. Here, we investigate patterns of allometric conservatism in the evolution of bird beaks with beak size and body size data for a representative sample of over 5000 extant bird species within a phylogenetic framework. We identify clades in which the allometric relationship between beak size and body size has remained relatively conserved across species over millions to tens of millions of years. We find that allometric conservatism is nonetheless punctuated by occasional shifts in the slopes and intercepts of allometric relationships. A steady accumulation of such shifts through time has given rise to the tremendous diversity of beak size relative to body size across birds today. Our findings are consistent with the Simpsonian vision of macroevolution, with evolutionary conservatism being the rule but with occasional shifts to new adaptive zones., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).)

Antimicrobial resistance (AMR) continues to threaten the effective treatment and prevention of bacterial infections. The spread of resistant infections is accelerated by the lack of fast and cost-effective tests for the detection of AMR at the point-of-care. We aimed to address this challenge by developing a diagnostic tool to detect one of the major forms of AMR, the β-lactamase enzymes. Antibiotic-functionalized gold nanoparticles (AuNPs) have been successfully developed for the detection of β-lactamases in challenging biological media, namely undiluted urine. Furthermore, these tools are compatible with samples containing a urine sample preservative (boric acid) or hematuria (blood). The functionalized AuNPs interact with the active β-lactamases, resulting in the hydrolysis of the surface-bound antibiotics, which then inhibits binding of the AuNPs to a capture protein (a penicillin-binding protein) to indicate the presence of active β-lactamases. We successfully integrated the antibiotic-functionalized AuNPs into a new lateral flow assay (LFA), which can be used to detect active β-lactamases down to the detection limit of 11 nM. While we demonstrate the use of antibiotic-functionalized AuNPs in an LFA format to provide a novel method of detecting active β-lactamases, these functionalized AuNPs are amenable to a range of alternative diagnostic technologies and could lead to vital point-of-care diagnostics for the early detection of multi-drug resistant infections., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper., (This journal is © The Royal Society of Chemistry.)