The Molecular Switch of Telomere Phages: High Binding Specificity of the PY54 Cro Lytic Repressor to a Single Operator Site

Temperate bacteriophages possess a molecular switch, which regulates the lytic and lysogenic growth. The genomes of the temperate telomere phages N15, PY54 and ɸKO2 harbor a primary immunity region (immB) comprising genes for the prophage repressor, the lytic repressor and a putative antiterminator. The roles of these products are thought to be similar to those of the lambda proteins CI, Cro and Q, respectively. Moreover, the gene order and the location of several operator sites in the prototype telomere phage N15 and in ɸKO2 are also reminiscent of lambda-like phages. By contrast, in silico analyses revealed the presence of only one operator (O$_{\rm{R}}$3) in PY54. The purified PY54 Cro protein was used for EMSA studies demonstrating that it exclusively binds to a 16-bp palindromic site (O$_{\rm{R}}$3) upstream of the prophage repressor gene. The O$_{\rm{R}}$3 operator sequences of PY54 and ɸKO2/N15 only differ by their peripheral base pairs, which are responsible for Cro specificity. PY54 cI and cro transcription is regulated by highly active promoters initiating the synthesis of a homogenious species of leaderless mRNA. The location of the PY54 Cro binding site and of the identified promoters suggests that the lytic repressor suppresses cI transcription but not its own synthesis. The results indicate an unexpected diversity of the growth regulation mechanisms in lambda-related phages

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail

Spongiibacter marinus gen. nov., sp. nov., a halophilic marine bacterium isolated from the boreal sponge Haliclona sp. 1

Strain HAL40bT was isolated from the marine sponge Haliclona sp. 1 collected at the Sula Ridge off the Norwegian coast and characterized by physiological, biochemical and phylogenetic analyses. The isolate was a small rod with a polar flagellum. It was aerobic, Gram-negative and oxidase- and catalase-positive. Optimal growth was observed at 20–30 °C, pH 7–9 and in 3 % NaCl. Substrate utilization tests were positive for arabinose, Tween 40 and Tween 80. Enzyme tests were positive for alkaline phosphatase, esterase lipase (C8), leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase. The predominant cellular fatty acid was C17 : 1 ω8, followed by C17 : 0 and C18 : 1 ω7. Analysis by matrix-assisted laser desorption/ionization time-of-flight MS was used to characterize the strain, producing a characteristic low-molecular-mass protein pattern that could be used as a fingerprint for identification of members of this species. The DNA G+C content was 69.1 mol%. Phylogenetic analysis supported by 16S rRNA gene sequence comparison classified the strain as a member of the class Gammaproteobacteria. Strain HAL40bT was only distantly related to other marine bacteria including Neptunomonas naphthovorans and Marinobacter daepoensis (type strain sequence similarity >90 %). Based on its phenotypic, physiological and phylogenetic characteristics, it is proposed that the strain should be placed into a new genus as a representative of a novel species, Spongiibacter marinus gen. nov., sp. nov.; the type strain of Spongiibacter marinus is HAL40bT (=DSM 17750T =CCUG 54896T)

Squirrelpox in a red squirrel in Fife

SQUIRRELPOX has been identified as a key factor in red squirrel (Sciurus vulgaris) decline in the UK.1 Grey squirrels (Sciurus carolinensis) are thought to act as reservoir hosts for squirrelpox virus (SQPV), the causative agent of squirrelpox, with a reported asymptomatic seroprevalence of 61 per cent.2SQPV is implicated in the complete replacement of red squirrels by grey squirrels throughout mainland England and Wales,1 and poses a major threat to Scottish red squirrel populations since being first detected in 2007.3 To combat this threat, an mortality surveillance programme has been established at the Royal (Dick) School of Veterinary Studies, University of Edinburgh, using opportunistic sampling of red squirrel carcases. This has been ongoing for several years, including a summary publication covering 262 cases submitted from 2005 to 2009.4As part of this monitoring programme, an adult female red squirrel carcase was submitted by a member of the public, after having been found in Townhall Wood (NT110894), on the outskirts of Dunfermline, Fife, in March 2024. Postmortem examination identified multiple lesions typically associated with squirrelpox, including ulcerative and exudative dermatitis of the periocular and perioral skin (Fig 1). Histopathology of the affected skin identified extensive ulceration alongside remnant areas of epithelium with marked ballooning degeneration and eosinophilic intracytoplasmic inclusion bodies. Transmission electron microscopy of the affected tissue also identified numerous pox virions within the affected tissue, which through their size, shape and available surface morphology (Fig 2), were consistent with those of SQPV. In Britain there have been no additional identified diseases in red squirrels that present with periocular or perioral, ulcerative to exudative dermatitis due to a poxvirus,5indicating this case is highly likely due to SQPV.This finding represents the first identification of squirrelpox north of the central belt and is consistent with the predictions of previous modelling, which identified a high risk of northern SQPV spread from 2023 onwards.6 This modelling also suggests a rapid increase and spread of squirrelpox into more northerly and naive red squirrel populations is likely following establishment north of the central belt in central Scotland.6This case and modelling supports an increased requirement for targeted investigations, ongoing monitoring and grey squirrel interventions both around Dunfermline itself and within adjacent areas to establish the disease burden in this locality and limit further northerly squirrelpox spread.LA Wilson, veterinary pathology lecturer, M Marr, postdoctoral research fellow, C Logie, postmortem room technician, K Beckmann, conservation medicine lecturer, PWW Lurz, squirrel ecologist, R Ogden, director of conservation science, E Milne, professor emerita of veterinary clinical pathology Royal (Dick) School of Veterinary Studies and Roslin Institute, University of Edinburgh, Easter Bush Campus, Roslin, Midlothian EH25 9RG email: [email protected] DJ Everest, pathology scientist APHA Weybridge, Addlestone, Surrey KT15 3NBReferences1 Tompkins DM, White AR, Boots M. Ecological replacement of native red squirrels by invasive greys driven by disease. Ecol Lett2003;6:189–962 Sainsbury AW, Nettleton P, Gilray J, et al. Grey squirrels have high seroprevalence to a parapoxvirus associated with deaths in red squirrels. Anim Conserv2000;3:229–33 3 Mclnnes CJ, Coulter L, Dagleish MP, et al. First cases of squirrelpox in red squirrels (Sciurus vulgaris) in Scotland. Vet Rec2009;164:528–314 LaRose JP, Meredith AL, Everest DJ, et al. Epidemiological and postmortem findings in 262 red squirrels (Sciurus vulgaris) in Scotland, 2005 to 2009. Vet Rec2010;167:297–3025 Everest DJ, Tolhurst-Cherriman DAR, Davies H, et al. Assessing a potential non-invasive method for viral diagnostic purposes in European squirrels. Hystrix 2019;30:44–506 White A, Lurz PWW. A modelling assessment of control strategies to prevent/reduce squirrelpox spread. 2014. Scottish Natural Heritage Commissioned Report No 627. https://bit.ly/441TOgM (accessed 8 April 2024)PROFESSIONHistory of the veterinary professionI WRITE in response to the debate article by Bruce Vivash Jones (VR, 16/23 March 2024, vol 194, p 236). As a PhD researcher on the history of the RCVS and veterinary regulation, and a veterinary nurse, I would like to raise some issues with Vivash Jones’ historical evidence. He states that changes to the council structure would create an oligarchy. From his interpretation of an oligarchy I can assure him and our profession that the first RCVS council did work as This finding represents the first identification of squirrelpox north of the central belt20/27 April 2024 | VET RECORD312Fig 2: Squirrelpox virus virions detected in the affected tissue. Bar = 200 nmFig 1: Macroscopic lesions of ulcerative and exudative dermatitis surrounding the eye in a red squirrel (Sciurus vulgaris)Letters 20 April.indd 312Letters 20 April.indd 31216/04/2024 12:4816/04/2024 12:4

Egocentric bias across mental and non-mental representations in the Sandbox Task

In the Sandbox Task, participants indicate where a protagonist who has a false belief about the location of an object will look for that object in a trough filled with a substrate that conceals the hidden object’s location. Previous findings that participants tend to indicate a location closer to where they themselves know the object to be located have been interpreted as evidence of egocentric bias when attributing mental states to others. We tested the assumption that such biases occur as a result of reasoning about mental states specifically. We found that participants showed more egocentric bias when reasoning from a protagonist’s false belief than from their own memory, but found equivalent levels of bias when they were asked to indicate where a false film would depict the object as when they were asked about a protagonist’s false belief. Our findings suggest that that egocentric biases found in adult false belief tasks are more likely due to a general difficulty with reasoning about false representations than a specialised difficulty with reasoning about false mental states. </jats:p

The even darker side of the eastern gray squirrel (Sciurus carolinensis): a review of global introductions, invasion biology, and pest management strategies

Huynh, H.M., Bertolino, S., Lurz, P.W.W., Koprowski, J.L., Williams, G.R., Thompson, C.W., McAlpine, D.F

In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

The origin recognition complex (ORC) of Saccharomyces cerevisiae binds origin DNA and cooperates with Cdc6 and Cdt1 to load the replicative helicase MCM2–7 onto DNA. Helicase loading involves two MCM2–7 hexamers that assemble into a double hexamer around double-stranded DNA. This reaction requires ORC and Cdc6 ATPase activity, but it is unknown how these proteins control MCM2–7 double hexamer formation. We demonstrate that mutations in Cdc6 sensor-2 and Walker A motifs, which are predicted to affect ATP binding, influence the ORC–Cdc6 interaction and MCM2–7 recruitment. In contrast, a Cdc6 sensor-1 mutant affects MCM2–7 loading and Cdt1 release, similar as a Cdc6 Walker B ATPase mutant. Moreover, we show that Orc1 ATP hydrolysis is not involved in helicase loading or in releasing ORC from loaded MCM2–7. To determine whether Cdc6 regulates MCM2–7 double hexamer formation, we analysed complex assembly. We discovered that inhibition of Cdc6 ATPase restricts MCM2–7 association with origin DNA to a single hexamer, while active Cdc6 ATPase promotes recruitment of two MCM2–7 hexamer to origin DNA. Our findings illustrate how conserved Cdc6 AAA+ motifs modulate MCM2–7 recruitment, show that ATPase activity is required for MCM2–7 hexamer dimerization and demonstrate that MCM2–7 hexamers are recruited to origins in a consecutive process

Vitamin B12 Deficiency Alters the Gut Microbiota in a Murine Model of Colitis

Purpose: Inflammatory bowel disease (IBD) refers to a spectrum of autoimmune diseases, which result in chronic intestinal inflammation. Previous findings suggest a role for diet, nutrition and dysbiosis of the gut microbiota in both the development and progression of the condition. Vitamin B12 is a key cofactor of methionine synthase and is produced solely by microbes. Previous work links increased levels of homocysteine, a substrate of methionine synthase, MetH, to IBD indicating a potential role for vitamin B12 deficiency in intestinal injury and inflammation. This study assessed the role of vitamin B12 in shaping the gut microbiota and determining responses to intestinal injury using a reproducible murine model of colitis. Methods: The effects of vitamin B12 supplementation and deficiency were assessed in vivo; 3-week-old post-weanling C57Bl/6 mice were divided into three dietary treatment groups: (1) sufficient vitamin B12 (50 mg/Kg), (2) deficient vitamin B12 (0 mg/Kg) and (3) supplemented vitamin B12 (200 mg/Kg) for a period of 4 weeks. Intestinal injury was induced with 2% dextran sodium sulphate (DSS) via drinking water for 5 days. The impact of varying levels of dietary vitamin B12 on gut microbiota composition was assessed using 16S rRNA gene sequencing from fecal samples collected at day 0 and day 28 of the dietary intervention, and 7 days following induction of colitis on day 38, when blood and colonic tissues were also collected. Results: No significant alterations were found in the gut microbiota composition of disease-free animals in response to dietary interventions. By contrast, after DSS-induced colitis, >30 genera were significantly altered in vitamin B12 deficient mice. Altered B12 levels produced no significant effect on composite disease-activity scores; however, administration of a B12 deficient diet resulted in reduced DSS-induced epithelial tissue damage. Conclusions: Vitamin B12 supplementation does not alter the gut microbiota composition under healthy conditions, but does contribute to differential microbial responses and intestinal dysbiosis following the induction of experimental colitis