Identification of a Staphylococcal dipeptidase involved in the production of human body odour.

The production of human body odour is the result of the action of commensal skin bacteria, including Staphylococcus hominis, acting to biotransform odourless apocrine gland secretions into volatile chemicals like thioalcohols such as 3-methyl-3-sulphanylhexan-1-ol (3M3SH). As the secreted odour 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 modelling, which suggested mutations that might accommodate this ligand more favourably. Indeed, a D437A resulted in an almost 6-fold increase in the kcat/KM, while other introduced mutations reduced or abolished function. Together these data identify an enzyme capable of catalysing the missing step in an ancient human-specific biochemical transformation and suggest that the production of 3M3SH neither uses a dedicated transporter nor peptidase for its breakdown, with only the final cleavage step, catalysed by PatB C-S ß-lyase, being a unique enzyme.

Structural dissection of the CMP-pseudaminic acid synthetase, PseF.

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.

Enhancement of growth media for extreme iron limitation in Escherichia coli.

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.

Stereoselective synthesis of an advanced trans-decalin intermediate towards the total synthesis of anthracimycin.

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.

Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria.

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.

Interrogation of RNA-protein interaction dynamics in bacterial growth.

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.

Structure and ligand binding in the putative anti-microbial peptide transporter protein, YejA.

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 Retaining Pse5Ac7Ac Pseudaminyltransferase KpsS1 Defines a Previously Unreported glycosyltransferase family (GT118).

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).

Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP.

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.

Flipping the switch: dynamic modulation of membrane transporter activity in bacteria.

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.

Innovation and elaboration on the avian tree of life.

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.

A phase-separated CO2-fixing pyrenoid proteome determined by TurboID in Chlamydomonas reinhardtii.

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.

Characterization of the l-arabinofuranose-specific GafABCD ABC transporter essential for l-arabinose-dependent growth of the lignocellulose-degrading bacterium Shewanella sp. ANA-3.

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, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) 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.

Using pose estimation to identify regions and points on natural history specimens.

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.

A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer.

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.

The fatty membrane that surrounds cells is an essential feature of all living things. It is a selective barrier, only allowing certain substances to enter and exit the cell, and it contains the proteins and carbohydrates that the cell uses to interact with its environment. In bacteria, the carbohydrates on the outer side of the membrane can become 'tagged' or modified with small chemical entities which often prove useful for the cell. Acyl groups, for example, allow disease-causing bacteria to evade the immune system and contribute to infections persisting in the body. As a rule, activated acyl groups are only found inside the cell, so they need to move across the membrane before they can be attached onto the carbohydrates at the surface. This transfer is performed by a group of proteins that sit within the membrane called the acyltransferase-3 (AT3) family. The structure of these proteins and the mechanism by which they facilitate membrane crossing have remained unclear. Newman, Tindall et al. combined computational and structural modelling techniques with existing experimental data to establish how this family of proteins moves acyl groups across the membrane. They focused on OafB, an AT3 protein from the foodborne bacterial pathogen Salmonella typhimurium. The experimental data used by the team included information about which parts of OafB are necessary for this protein to acylate carbohydrates molecules. In their experiments, Newman, Tindall et al. studied how different parts of OafB move, how they interact with the molecules that carry an acyl group to the membrane, and how the acyl group is then transferred to the carbohydrate acceptor. Their results suggest that AT3 family proteins have a central pore or hole, plugged by a loop. This loop moves and therefore 'unplug' the pore, resulting in the emergence of a channel across the membrane. This channel can accommodate the acyl-donating molecule, presenting the acyl group to the outer surface of the membrane where it can be transferred to the acceptor carbohydrate. The AT3 family of proteins participates in many cellular processes involving the membrane, and a range of bacterial pathogens rely on these proteins to successfully infect human hosts. The results of Newman Tindall et al. could therefore be used across the biological sciences to provide more detailed understanding of the membrane, and to inform the design of drugs to fight bacterial diseases.

Siderophore-Linked Ruthenium Catalysts for Targeted Allyl Ester Prodrug Activation within Bacterial Cells.

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.

Two closely related ureotelic fish species of the genus Alcolapia express different levels of ammonium transporters in gills.

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.