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Fatima Ulhuq, Amy K. Tooke, Chriselle Mendonca​, Guillermina Casabona​, Johann Habersetzer​, Yaping Yang​, Margarida C. Gomes, Felicity Alcock​, Serge Mostowy​, Tracy Palmer

The Staphylococcus aureus type VIIb secretion system (T7SSb) is a multiprotein secretion system that secretes toxins with antibacterial activity, but which is also required for full virulence in animal models of infection. S. aureus strains carry one of four T7SSb locus types, named essC1 to essC4, each of which encodes a characteristic LXG-family substrate at the T7SS locus. In essC2 strains, this LXG-domain protein is EsxX, which has a glycine zipper sequence in its C-terminus and has potent antibacterial, membrane-depolarizing activity. In this work, we recognize conserved features of the essC2 and essC3 systems, identifying the LXG protein SAR0287 as structurally and functionally similar to EsxX. Using a zebrafish larval hindbrain ventricle infection model, we demonstrate that the T7SSb of essC2 and essC3 representative strains contributes to bacterial replication and zebrafish mortality. However, there is no significant loss of virulence in the model system if EsxX or SAR0287 is absent. These findings indicate that there is no discernible role for either toxin in this virulence model.

Annabelle R de Vries , Lochlan Chadwick , Mark Carine , Robin G Allaby

Adhesives have been integral to the production of herbaria for paper making, securing plant material to paper, and, in the case of bound volumes, for bookbinding. The adhesives used may be of plant, animal, or synthetic origin. Here we investigated herbarium specimens from the Natural History Museum London (NHM), collected between 1698 and 1970, to determine whether information on the adhesives used in the preparation of herbarium specimens can be established using ancient DNA analysis of the mounted plant material. Ancient DNA was obtained from leaf tissue of 14 herbarium specimens of Trochetiopsis and sequenced using Illumina MiSeq. Non-Trochetiopsis DNA was identified using metagenome analysis software (MEGAN). Reads identified as animal were further analysed using the metagenomics pipeline Phylogenetic Intersection Analysis (PIA). Two specimens showed distinct animal reads. One specimen from 1698, which had glue residue observable on the leaf material, showed evidence for Pecora and Bovidae, specifically Bos, and with lower read counts also for both Leporidae and Ovis. The bones of cattle, rabbits, and sheep are all likely to have been used in the preparation of glue in this period, and consequently the animal DNA retrieved is probably from the glue used for mounting. The second sample was from 1970 and showed reads of Pecora, Bovidae, and Bos. Latex adhesives were used at the NHM during the 1970s with synthetic adhesives used thereafter. We infer that the animal DNA retrieved is probably from gelatine used for paper sizing. The results of this study demonstrate that the genetic analysis of plant material can also provide insights into the process of making herbaria.

Bertil B. Fredholm, Lauren May, Christa E. Müller, Joel Linden, Karl-Norbert Klotz, Kenneth A. Jacobson, Adriaan P. IJzerman, Rebecca Hills, Bruno G. Frenguelli, Gary L. Stiles

Adenosine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Adenosine Receptors []) are activated by the endogenous ligand (potentially also at A₃ receptors). Crystal and cryo-EM structures for all four adenosine receptors have been solved, occupied by either agonists (sometimes in the presence of an allosteric modulator) or antagonists. Many of these structures were incorporated in a recent review []. More recently, structures for the A_2B receptor [, ] and the A₃ receptor [, ] were elucidated. The A_2A receptor is used as a workhorse in GPCR structure elucidation: almost 100 structures are available in the Protein Data Bank (www.rcsb.org). , a selective A_2A receptor antagonist, is on the market for the treatment of Parkinson's disease, while caffeine's mechanism of action is largely due to its antagonism of at least three of the four adenosine receptor subtypes. Allosteric modulators, particular PAMs of A₁ and A₃ receptors, have been explored chemically and structurally.

Ning Wang, Linda M. Westermann, Mingyu Li, Chun-Yang Li, Andrew R. J. Murphy, Zengtian Gu, Eleonora Silvano, Claudia A. Blindauer, Ian D. E. A., Yu-Zhong Zhang, David J. Scanlan, Yin Chen

All cells use lipid membranes to maintain cellular integrity and function, though Archaea utilize lipids composed of glycerol-1-phosphate (G1P), while Bacteria and Eukaryotes use glycerol-3-phosphate (G3P). Given that Archaea contribute significantly to global marine biomass, accounting for 0.3 gigatonnes (Gt) of carbon in the oceans, we aimed to uncover how archaeal G1P is recycled by marine microorganisms. Through a multidisciplinary approach combining microbiology, biochemistry, and structural biology, we identified a G1P transporter in marine bacteria, which we named GpxB. Phylogenetic analysis revealed that GpxB belongs to the organic phosphonate transporter (PhnT) family and is widely distributed in the marine microbiome, found in approximately 5 to 10% of microbial cells in surface marine waters. Strikingly, we also identified a second G1P transporter, UgpB, that is known to transport G3P and belongs to the carbohydrate uptake transporter-1 (CUT1) family, in the model bacterium Phaeobacter sp. MED193. To explore the evolutionary pathways that led to the formation of G1P binding sites in both the PhnT and CUT1 families, we determined the structures of GpxB and UgpB bound to G1P and G3P. Using structure-guided mutagenesis and a comparative analysis of the binding pockets within the PhnT and CUT1 families, we traced their evolutionary trajectories, highlighting the distinct strategies through which G1P-binding sites developed in these two protein families.

Nicola Gorringe , Stephanie Topp , Robin Burns , Sota Yamaguchi , Fernando ARabanal , Joiselle B Fernandes , Detlef Weigel , Tetsuji Kakutani , Matthew Naish , Ian R Henderson

Eukaryotic centromeres mediate chromosome segregation during cell division. Plant centromeres are loaded with CENH3-variant nucleosomes, which direct kinetochore formation and spindle-microtubule interaction. Centromeres are frequently composed of megabase-scale satellite repeat arrays, or retrotransposon nests. In monocentric genomes, such as the model plant Arabidopsis thaliana, pericentromeric heterochromatin surrounds the CENH3-occupied satellite arrays. A zone of suppressed meiotic crossover recombination contains the centromere and extends into the pericentromeres. Here, we explore how natural variation in Arabidopsis influences centromere-proximal crossover frequency and segregation distortion when centromeres are heterozygous. We used fluorescent crossover reporters to quantify the effect of genetic variation on centromere-proximal recombination in 12 F₁ hybrids between the reference strain Col-0 and nonreference accessions that captured Eurasian and relict diversity, and in total, we measured 3,037,802 meioses. The majority of the F₁ hybrids (49 of 60) had significantly higher or lower centromere-proximal crossover frequency than inbreds. We relate hybrid crossover frequencies to patterns of nucleotide diversity and centromeric structural variation, and in a subset of 7 accessions, to epigenetic patterns of CENH3 enrichment and DNA methylation. Using linear modeling, we observed that chromosome and accession, and their interaction, together explained 85% of variation in crossover frequency, consistent with cis- and trans-acting modifying effects. The fluorescent reporters also allow segregation distortion through meiosis to be quantified between hybrids and inbreds. We observed a minority of hybrids (18 of 60) with distorted segregation through meiosis compared to inbreds, which occurred with or without a simultaneous change to centromere-proximal crossover frequency. Linear modeling revealed that 56% of variation in segregation distortion is explained by chromosome and accession, but with a stronger effect of accession compared to crossover frequency. We discuss how Arabidopsis centromeric structural heterozygosity may modify recombination and cause segregation distortion through meiosis.

Yayoi Sugita, Amanda J. Dowson, Ichiro Kajisa, Katsuaki Takechi, Yilan E, Jingzhi Zhao, Jiaqi Wang, Xiaofei Lin, Laura Diaz-Saez, Adrian J. Lloyd, Christopher G. Dowson, Hiroyoshi Takano

It is understood that a cyanobacterium was the progenitor of plastids and that the biosynthesis of cell wall peptidoglycan was lost during chloroplast evolution. However, accumulated data, especially from the moss Physcomitrium patens, suggest that peptidoglycan remains essential for plastid division in some land plants. A fundamental set of peptidoglycan biosynthesis (Mur) genes has been identified in the genomes of these land plants, while many angiosperms no longer encode some core Mur genes, including a bifunctional penicillin-binding protein (PBP). Ten incomplete Mur genes were previously identified in the genome of the gymnosperm Picea abies but these could be pseudogenes or encode proteins that have been repurposed. For instance, mutant albino maize and Arabidopsis seedlings possess a defective UDP-N-acetylmuramoyl-l-alanyl-d-glutamate--2,6-diaminopimelate ligase (MurE), an intact MurE ligase being essential for peptidoglycan synthesis. In this study, we isolated a full set of cDNAs for peptidoglycan biosynthesis from P. abies. GFP fusion proteins with either P. abies (Pa)MurE or PaPBP were detected in chloroplasts. Cross-species complementation assays with PaMurE in Arabidopsis albino MurE mutants and Physcomitrium MurE chloroplast division mutants showed that the gymnosperm MurE completely rescued both mutant phenotypes. Enzymatic assay of recombinant PaMurE proteins revealed they catalyze the same reaction performed by their bacterial MurE homologs. Moreover, the expression of the PaPbp cDNA partially rescued the giant chloroplast phenotype in the moss Pbp knockout line. These results are consistent with the operation of a functional Mur gene set in the Norway spruce genome.