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Journal articleTosi T, Hoshiga F, Millership C, et al., 2019,
Inhibition of the Staphylococcus aureus c-di-AMP cyclase DacA by direct interaction with the phosphoglucosamine mutase GlmM
, PLoS Pathogens, Vol: 15, Pages: 1-28, ISSN: 1553-7366c-di-AMP is an important second messenger molecule that plays a pivotal role in regulating fundamental cellular processes, including osmotic and cell wall homeostasis in many Gram-positive organisms. In the opportunistic human pathogen Staphylococcus aureus, c-di-AMP is produced by the membrane-anchored DacA enzyme. Inactivation of this enzyme leads to a growth arrest under standard laboratory growth conditions and a re-sensitization of methicillin-resistant S. aureus (MRSA) strains to ß-lactam antibiotics. The gene coding for DacA is part of the conserved three-gene dacA/ybbR/glmM operon that also encodes the proposed DacA regulator YbbR and the essential phosphoglucosamine mutase GlmM, which is required for the production of glucosamine-1-phosphate, an early intermediate of peptidoglycan synthesis. These three proteins are thought to form a complex in vivo and, in this manner, help to fine-tune the cellular c-di-AMP levels. To further characterize this important regulatory complex, we conducted a comprehensive structural and functional analysis of the S. aureus DacA and GlmM enzymes by determining the structures of the S. aureus GlmM enzyme and the catalytic domain of DacA. Both proteins were found to be dimers in solution as well as in the crystal structures. Further site-directed mutagenesis, structural and enzymatic studies showed that multiple DacA dimers need to interact for enzymatic activity. We also show that DacA and GlmM form a stable complex in vitro and that S. aureus GlmM, but not Escherichia coli or Pseudomonas aeruginosa GlmM, acts as a strong inhibitor of DacA function without the requirement of any additional cellular factor. Based on Small Angle X-ray Scattering (SAXS) data, a model of the complex revealed that GlmM likely inhibits DacA by masking the active site of the cyclase and preventing higher oligomer formation. Together these results provide an important mechanistic insight into how c-di-AMP production can be regulated in the cell.
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Journal articleExley K, Reynolds C, Suckling L, et al., 2019,
Utilising datasheets for the informed automated design and build of a synthetic metabolic pathway
, Journal of Biological Engineering, Vol: 13, ISSN: 1754-1611BackgroundThe automation of modular cloning methodologies permits the assembly of many genetic designs. Utilising characterised biological parts aids in the design and redesign of genetic pathways. The characterisation information held on datasheets can be used to determine whether a biological part meets the design requirements. To manage the design of genetic pathways, researchers have turned to modelling-based computer aided design software tools.ResultAn automated workflow has been developed for the design and build of heterologous metabolic pathways. In addition, to demonstrate the powers of electronic datasheets we have developed software which can transfer part information from a datasheet to the Design of Experiment software JMP. To this end we were able to use Design of Experiment software to rationally design and test randomised samples from the design space of a lycopene pathway in E. coli. This pathway was optimised by individually modulating the promoter strength, RBS strength, and gene order targets.ConclusionThe use of standardised and characterised biological parts will empower a design-oriented synthetic biology for the forward engineering of heterologous expression systems. A Design of Experiment approach streamlines the design-build-test cycle to achieve optimised solutions in biodesign. Developed automated workflows provide effective transfer of information between characterised information (in the form of datasheets) and DoE software.
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Conference paperAppuswamy R, Brigand KL, Barbry P, et al., 2019,
OligoArchive: Using DNA in the DBMS storage hierarchy
, CIDR 2019 -
Journal articleRamlaul K, Palmer CM, Aylett CHS, 2019,
A local agreement filtering algorithm for transmission EM reconstructions
, Journal of Structural Biology, Vol: 205, Pages: 30-40, ISSN: 1047-8477We present LAFTER, an algorithm for de-noising single particle reconstructions from cryo-EM.Single particle analysis entails the reconstruction of high-resolution volumes from tens of thousands of particle images with low individual signal-to-noise. Imperfections in this process result in substantial variations in the local signal-to-noise ratio within the resulting reconstruction, complicating the interpretation of molecular structure. An effective local de-noising filter could therefore improve interpretability and maximise the amount of useful information obtained from cryo-EM maps.LAFTER is a local de-noising algorithm based on a pair of serial real-space filters. It compares independent half-set reconstructions to identify and retain shared features that have power greater than the noise. It is capable of recovering features across a wide range of signal-to-noise ratios, and we demonstrate recovery of the strongest features at Fourier shell correlation (FSC) values as low as 0.144 over a 2563-voxel cube. A fast and computationally efficient implementation of LAFTER is freely available.We also propose a new way to evaluate the effectiveness of real-space filters for noise suppression, based on the correspondence between two FSC curves: 1) the FSC between the filtered and unfiltered volumes, and 2) Cref, the FSC between the unfiltered volume and a hypothetical noiseless volume, which can readily be estimated from the FSC between two half-set reconstructions.
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Conference paperAppuswamy R, Lebrigand K, Barbry P, et al., 2019,
Oligoarchive: Using DNA in the DBMS storage hierarchy
© 2019 Conference on Innovative Data Systems Research (CIDR). All rights reserved. The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.
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Conference paperAppuswamy R, Lebrigand K, Barbry P, et al., 2019,
Oligoarchive: Using DNA in the DBMS storage hierarchy
The demand for data-driven decision making coupled with need to retain data to meet regulatory compliance requirements has resulted in a rapid increase in the amount of archival data stored by enterprises. As data generation rate far outpaces the rate of improvement in storage density of media like HDD and tape, researchers have started investigating new architectures and media types that can store such “cold”, infrequently accessed data at very low cost. Synthetic DNA is one such storage media that has received some attention recently due to its high density and durability. In this paper, we investigate the problem of integrating DNA in the database storage hierarchy. More specifically, we ask the following two questions: (i) how can database knowledge help optimize DNA encoding and decoding? and (ii) how can biochemical mechanisms used for DNA manipulation be used to perform in-vitro, near-data SQL query processing? In answering these questions, we present OligoArchive, an architecture for using DNA-based storage system as the archival tier of a relational database. We demonstrate that OligoArchive can be realized in practice by building archiving and recovery tools (pg_oligo_dump and pg_oligo_restore) for PostgreSQL that perform schema-aware encoding and decoding of relational data on DNA, and using these tools to archive a 12KB TPC-H database to DNA, perform in-vitro computation, and restore it back again.
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Conference paperWebb AJ, Landeryou T, Kelwick R, et al., 2019,
SPECIFIC NUCLEIC ACIDS LIGATION FOR DETECTION OF SCHISTOSOMES: SNAILS
, 68th Annual Meeting of the American-Society-for-Tropical-Medicine-and-Hygiene (ASTMH), Publisher: AMER SOC TROP MED & HYGIENE, Pages: 182-182, ISSN: 0002-9637 -
Journal articleYates LA, Aramayo RJ, Pokhrel N, et al., 2018,
A structural and dynamic model for the assembly of Replication Protein A on single-stranded DNA
, Nature Communications, Vol: 9, ISSN: 2041-1723Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (∼55 kDa) at ∼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.
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Journal articleSilhan J, Zhao Q, Boura E, et al., 2018,
Structural basis for recognition and repair of the 3'-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis
, Nucleic Acids Research, Vol: 46, Pages: 11980-11989, ISSN: 0305-1048NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3'-phosphate and other 3'-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3'-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3'-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3'-phosphatase NExo bound to DNA in the presence and absence of a 3'-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3'-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.
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Conference paperWebb AJ, Allan F, Kelwick R, et al., 2018,
Protease-based bioreporters for the detection of schistosome cercariae
, American Society of Tropical Medicine and Hygiene (ASTMH) 67th Annual Meeting, New Orleans, Louisiana, USA
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