CONSTRUCTION OF A RECOMBINATION-BASED REPORTER SYSTEM TO IDENTIFY GENE EXPRESSION IN STAPHYLOCOCCUS AUREUS

Staphylococcus aureus harbours several iron acquisition systems, allowing for survival within the iron-restricted host environment. Five such systems have been identified in S. aureus; it is unknown why S. aureus maintains so many of these systems. Studies have shown that some iron transport proteins might be differentially expressed in different organs in vivo. The purpose of this study was to construct an in vivo expression technology (IVET) system to identify temporal expression patterns of S. aureus iron transport systems. An IVET utilizing the CrelloxP recombination system was constructed for use in S. aureus. Using genome-integrated and multi-copy ere constructs driven by the S. aureus fhuCBG promoter, resolution of an in vitro reporter cassette was determined. Although fhuCBG driven ere expression was confirmed using the multi-copy ere constructs, no detectable Cre was expressed by genome-integrated cre constructs

Zero-Transmission Law for Multiport Beam Splitters

The Hong-Ou-Mandel effect is generalized to a configuration of n bosons prepared in the n input ports of a Bell multiport beam splitter. We derive a strict suppression law for most possible output events, consistent with a generic bosonic behavior after suitable coarse graining.Comment: Version accepted by PR

Mechanisms controlling global mean sea surface temperature determined from a state estimate

Author Posting. © American Geophysical Union, 2018. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Geophysical Research Letters 45 (2018): 3221-3227, doi:10.1002/2017GL076821.Global mean sea surface temperature ((T) over bar) is a variable of primary interest in studies of climate variability and change. The temporal evolution of (T) over bar) can be influenced by surface heat fluxes ((F) over bar)) and by diffusion ((D) over bar)) and advection ((A) over bar)) processes internal to the ocean, but quantifying the contribution of these different factors from data alone is prone to substantial uncertainties. Here we derive a closed (T) over bar) budget for the period 1993-2015 based on a global ocean state estimate, which is an exact solution of a general circulation model constrained to most extant ocean observations through advanced optimization methods. The estimated average temperature of the top (10-m thick) level in the model, taken to represent (T) over bar), shows relatively small variability at most time scales compared to (F) over bar), (D) over bar), or (A) over bar), reflecting the tendency for largely balancing effects from all the latter terms. The seasonal cycle in (T) over bar) is mostly determined by small imbalances between (F) over bar) and (D) over bar), with negligible contributions from (A) over bar). While (D) over bar) seems to simply damp (F) over bar) at the annual period, a different dynamical role for (D) over bar) at semiannual period is suggested by it being larger than (F) over bar). At periods longer than annual, (A) over bar) contributes importantly to (T) over bar) variability, pointing to the direct influence of the variable ocean circulation on (T) over bar) and mean surface climate. Plain Language Summary Global mean sea surface temperature (T) over bar) is a key metric when defining the Earth's climate. Determining what controls the evolution of (T) over bar )T is thus vital for understanding past climate variability and predicting its future evolution. Processes that control (T) over bar) involve forcing surface heat fluxes, as well as advection and diffusion of heat internal to the ocean, but their relative contributions are poorly known and difficult to assess from observations alone. Here we use advanced methods to combine models and data and derive a closed budget for (T) over bar) variability in terms of the forcing, advection, and diffusion processes. The estimated (T) over bar) shows relatively small variability compared to surface forcing, advection, or diffusion, reflecting the tendency for largely balancing effects from all the latter terms. The seasonal cycle in (T) over bar) is mostly determined by small imbalances between forcing and diffusion, with negligible contributions from advection. Diffusion does not always act as a simple damping of forcing surface fluxes, however. In addition, at periods longer than annual, advection contributes importantly to (T) over bar) variability. The results point to the direct influence of the variable ocean circulation on (T) over bar) and the Earth's surface climate.NSF Grant Number: PLR-15133962018-09-2

Active site remodelling of a cyclodipeptide synthase redefines substrate scope

Funding: Wellcome Trust (210486/Z/18/Z), Cunningham Trust (PhD-CT-18-41).Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.Publisher PDFPeer reviewe

Numerical Modelling of Melt Behaviour in the Lower Vessel Head of a Nuclear Reactor

Acknowledgements The authors would like to thank the EPSRC MEMPHIS multi-phase programme grant, the EPSRC Computational modelling for advanced nuclear power plants project and the EU FP7 projects THINS and GoFastR for helping to fund this work.Peer reviewedPublisher PD

An anti-biofilm cyclic peptide targets a secreted aminopeptidase from P. aeruginosa

Funding: CMC and CJH are funded by the Wellcome Trust (210486/Z/18/Z), additional funding was provided by the University of St Andrews Impact Innovation Fund. MB (Bergkessel) is funded by the University of Dundee/Wellcome Trust Institutional Strategic Support Fund [204816/Z/16/Z] and UKRI Future Leaders Fellowship (funded by the Medical Research Council) [MR/T041811/1].Pseudomonas aeruginosa is an opportunistic pathogen that causes serious illness, especially in immunocompromised individuals. P. aeruginosa forms biofilms that contribute to growth and persistence in a wide range of environments. Here we investigated the aminopeptidase, P. aeruginosa aminopeptidase (PaAP) from P. aeruginosa, which is highly abundant in the biofilm matrix. PaAP is associated with biofilm development and contributes to nutrient recycling. We confirmed that post-translational processing was required for activation and PaAP is a promiscuous aminopeptidase acting on unstructured regions of peptides and proteins. Crystal structures of wild-type enzymes and variants revealed the mechanism of autoinhibition, whereby the C-terminal propeptide locks the protease-associated domain and the catalytic peptidase domain into a self-inhibited conformation. Inspired by this, we designed a highly potent small cyclic-peptide inhibitor that recapitulates the deleterious phenotype observed with a PaAP deletion variant in biofilm assays and present a path toward targeting secreted proteins in a biofilm context.Publisher PDFPeer reviewe