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1.  A Comparison between Ultraviolet Disinfection and Copper Alginate Beads within a Vortex Bioreactor for the Deactivation of Bacteria in Simulated Waste Streams with High Levels of Colour, Humic Acid and Suspended Solids 
PLoS ONE  2014;9(12):e115688.
We show in this study that the combination of a swirl flow reactor and an antimicrobial agent (in this case copper alginate beads) is a promising technique for the remediation of contaminated water in waste streams recalcitrant to UV-C treatment. This is demonstrated by comparing the viability of both common and UV-C resistant organisms in operating conditions where UV-C proves ineffective - notably high levels of solids and compounds which deflect UV-C. The swirl flow reactor is easy to construct from commonly available plumbing parts and may prove a versatile and powerful tool in waste water treatment in developing countries.
PMCID: PMC4277313  PMID: 25541706
2.  Exploring nicotinamide cofactor promiscuity in NAD(P)H-dependent flavin containing monooxygenases (FMOs) using natural variation within the phosphate binding loop. Structure and activity of FMOs from Cellvibrio sp. BR and Pseudomonas stutzeri NF13 
Graphical abstract
•Genes encoding ‘Type II FMOs’ CFMO and PSFMO were selected for diversity in the NADPH binding loop.•CFMO and PSFMO were shown to both accept either NADH or NADPH as cofactor in the reduction of FAD.•The activities with both NADPH and NADH have been evaluated in the oxidation of sulfide substrates.•Structures of CFMO and PSFMO were solved, revealing the nature of the NADPH phosphate binding loop.
Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg–Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln–His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872–878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr–Ser and Gln–Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix–loop–helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.
PMCID: PMC4220118  PMID: 25383040
Flavoprotein; Monooxygenase; FAD; Biotransformations; Sulfide
3.  Draft Genome Sequence of Four Coccolithoviruses: Emiliania huxleyi Virus EhV-88, EhV-201, EhV-207, and EhV-208 
Journal of Virology  2012;86(5):2896-2897.
The Coccolithoviridae are a group of viruses which infect the marine coccolithophorid microalga Emiliania huxleyi. The Emiliania huxleyi viruses (known as EhVs) described herein have 160- to 180-nm diameter icosahedral structures, have genomes of approximately 400 kbp, and consist of more than 450 predicted coding sequences (CDSs). Here, we describe the genomic features of four newly sequenced coccolithoviruses (EhV-88, EhV-201, EhV-207, and EhV-208) together with their draft genome sequences and their annotations, highlighting the homology and heterogeneity of these genomes to the EhV-86 model reference genome.
PMCID: PMC3302265  PMID: 22328700
4.  Draft Genome Sequence of the Coccolithovirus Emiliania huxleyi Virus 202 
Journal of Virology  2012;86(4):2380-2381.
Emiliania huxleyi virus 202 (EhV-202) is a member of the Coccolithoviridae, a group of viruses that infect the marine coccolithophorid Emiliania huxleyi. EhV-202 has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 407 kbp, consisting of 485 coding sequences (CDSs). Here we describe the genomic features of EhV-202, together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.
PMCID: PMC3302404  PMID: 22282334
5.  The Bactericidal Effect of Dendritic Copper Microparticles, Contained in an Alginate Matrix, on Escherichia coli 
PLoS ONE  2014;9(5):e96225.
Although the bactericidal effect of copper has been known for centuries, there is a current resurgence of interest in the use of this element as an antimicrobial agent. During this study the use of dendritic copper microparticles embedded in an alginate matrix as a rapid method for the deactivation of Escherichia coli ATCC 11775 was investigated. The copper/alginate produced a decrease in the minimum inhibitory concentration from free copper powder dispersed in the media from 0.25 to 0.065 mg/ml. Beads loaded with 4% Cu deactivated 99.97% of bacteria after 90 minutes, compared to a 44.2% reduction in viability in the equivalent free copper powder treatment. There was no observed loss in the efficacy of this method with increasing bacterial loading up to 106 cells/ml, however only 88.2% of E. coli were deactivated after 90 minutes at a loading of 108 cells/ml. The efficacy of this method was highly dependent on the oxygen content of the media, with a 4.01% increase in viable bacteria observed under anoxic conditions compared to a >99% reduction in bacterial viability in oxygen tensions above 50% of saturation. Scanning electron micrographs (SEM) of the beads indicated that the dendritic copper particles sit as discrete clusters within a layered alginate matrix, and that the external surface of the beads has a scale-like appearance with dendritic copper particles extruding. E. coli cells visualised using SEM indicated a loss of cellular integrity upon Cu bead treatment with obvious visible blebbing. This study indicates the use of microscale dendritic particles of Cu embedded in an alginate matrix to effectively deactivate E. coli cells and opens the possibility of their application within effective water treatment processes, especially in high particulate waste streams where conventional methods, such as UV treatment or chlorination, are ineffective or inappropriate.
PMCID: PMC4022509  PMID: 24831035
6.  Draft genome sequence of the coccolithovirus EhV-84 
The Coccolithoviridae is a recently discovered group of viruses that infect the marine coccolithophorid Emiliania huxleyi. Emiliania huxleyi virus 84 (EhV-84) has a 160 -180 nm diameter icosahedral structure and a genome of approximately 400 kbp. Here we describe the structural and genomic features of this virus, together with a near complete draft genome sequence (~99%) and its annotation. This is the fourth genome sequence of a member of the coccolithovirus family.
PMCID: PMC3236045  PMID: 22180805
coccolithovirus; marine; phycodnavirus; algae; virus
7.  Mutations of an NAD(P)H-dependent flavoprotein monooxygenase that influence cofactor promiscuity and enantioselectivity☆ 
FEBS Open Bio  2013;3:473-478.
The flavoprotein monooxygenase (FPMO) from Stenotrophomonas maltophilia (SMFMO, Uniprot: B2FLR2) catalyses the asymmetric oxidation of thioethers and is unusual amongst FPMOs in its ability to use the non-phosphorylated cofactor NADH, as well as NADPH, for the reduction of the FAD coenzyme. In order to explore the basis for cofactor promiscuity, structure-guided mutation of two residues in the cofactor binding site, Gln193 and His194, in SMFMO were performed in an attempt to imitate the cofactor binding site of the NADPH-dependent FMO from Methylophaga aminisulfidivorans sp. SK1 (mFMO), in which structurally homologous residues Arg234 and Thr235 bind the NADPH 2′-ribose phosphate. Mutation of His194 to threonine proved most significant, with a switch in specificity from NADH to NADPH [(kcat/Km NADH)/kcat/Km NADPH) from 1.5:1 to 1:3.5, mostly as a result of a reduced Km for NADPH of approximately sevenfold in the His194Thr mutant. The structure of the Gln193Arg/His194Thr mutant revealed no substantial changes in the backbone of the enzyme or orientation of side chains resulting from mutation. Mutation of Phe52, in the vicinity of FAD, and which in mFMO is an asparagine thought to be responsible for flavin hydroperoxide stabilisation, is, in SMFMO, a determinant of enantioselectivity in sulfoxidation. Mutation of Phe52 to valine resulted in a mutant that transformed para-tolyl methyl sulfide into the (S)-sulfoxide with 32% e.e., compared to 25% (R)- for the wild type. These results shed further light both on the cofactor specificity of FPMOs, and their determinants of enantioselectivity, with a view to informing engineering studies of FPMOs in the future.
•SMFMO was mutated to investigate cofactor specificity and enantioselectivity.•The Gln193Arg/His194Thr mutant displayed a preference for NADPH, rather than NADH.•The structure of the Gln193Arg/His194Thr mutant was determined.•Active site mutants were assessed for enantioselectivity in sulfoxidation reactions.•The Phe52Val mutant displayed inverted enantioselectivity.
PMCID: PMC3829993  PMID: 24251114
Flavoprotein monoxygenase; Baeyer–Villiger monoxygenase; Sulfoxide; Biocatalyst; NADPH; SMFMO, Stenotrophomonas maltophilia flavin monoxygenase; mFMO, flavin monooxygenase from Methylophaga aminisulfidivorans; PAMO, phenylacetone monooxygenase; CHMO, cyclohexanone monooxygenase
8.  Mutant SOD1 Forms Ion Channel: Implications for ALS Pathophysiology 
Neurobiology of Disease  2011;45(3):831-838.
Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1. AFM of A4VSOD1 reconstituted in lipid membrane shows discrete tetrameric pore-like structure with outer and inner diameters 12.2 and 3.0 nm respectively. Electrophysiological recordings show distinct ionic conductances across bilayer for A4VSOD1 and none for wild-type SOD1. Mouse neuroblastoma cells exposed to A4VSOD1 undergo membrane depolarization and increases in intracellular calcium. These results provide compelling new evidence that a mutant SOD1 is capable of disrupting cellular homeostasis via an unregulated ion channel mechanism. Such a “toxic channel” mechanism presents a new therapeutic direction for ALS research.
PMCID: PMC3384721  PMID: 21930207
amyotrophic lateral sclerosis; superoxide dismutase; A4V; protein misfolding; atomic force microscopy; Fluo-4 calcium assay; DiBAC4(3) membrane potential measurement
9.  Genome Sequence of Stenotrophomonas maltophilia PML168, Which Displays Baeyer-Villiger Monooxygenase Activity 
Journal of Bacteriology  2012;194(17):4753-4754.
Stenotrophomonas maltophilia PML168 was isolated from Wembury Beach on the English Coast from a rock pool following growth and selection on agar plates. Here we present the permanent draft genome sequence, which has allowed prediction of function for several genes encoding enzymes relevant to industrial biotechnology, including a novel flavoprotein monooxygenase.
PMCID: PMC3415523  PMID: 22887661
10.  Coxibs and cardiovascular risk 
PMCID: PMC1247685  PMID: 16217098
11.  Draft Genome Sequence of the Coccolithovirus Emiliania huxleyi Virus 203 
Journal of Virology  2011;85(24):13468-13469.
The Coccolithoviridae are a recently discovered group of viruses that infect the marine coccolithophorid Emiliania huxleyi. Emiliania huxleyi virus 203 (EhV-203) has a 160- to 180-nm-diameter icosahedral structure and a genome of approximately 400 kbp, consisting of 464 coding sequences (CDSs). Here we describe the genomic features of EhV-203 together with a draft genome sequence and its annotation, highlighting the homology and heterogeneity of this genome in comparison with the EhV-86 reference genome.
PMCID: PMC3233127  PMID: 22106382
12.  Genome Sequence of Ostreococcus tauri Virus OtV-2 Throws Light on the Role of Picoeukaryote Niche Separation in the Ocean▿ 
Journal of Virology  2011;85(9):4520-4529.
Ostreococcus tauri, a unicellular marine green alga, is the smallest known free-living eukaryote and is ubiquitous in the surface oceans. The ecological success of this organism has been attributed to distinct low- and high-light-adapted ecotypes existing in different niches at a range of depths in the ocean. Viruses have already been characterized that infect the high-light-adapted strains. Ostreococcus tauri virus (OtV) isolate OtV-2 is a large double-stranded DNA algal virus that infects a low-light-adapted strain of O. tauri and was assigned to the algal virus family Phycodnaviridae, genus Prasinovirus. Our working hypothesis for this study was that different viruses infecting high- versus low-light-adapted O. tauri strains would provide clues to propagation strategies that would give them selective advantages within their particular light niche. Sequence analysis of the 184,409-bp linear OtV-2 genome revealed a range of core functional genes exclusive to this low-light genotype and included a variety of unexpected genes, such as those encoding an RNA polymerase sigma factor, at least four DNA methyltransferases, a cytochrome b5, and a high-affinity phosphate transporter. It is clear that OtV-2 has acquired a range of potentially functional genes from its host, other eukaryotes, and even bacteria over evolutionary time. Such piecemeal accretion of genes is a trademark of large double-stranded DNA viruses that has allowed them to adapt their propagation strategies to keep up with host niche separation in the sunlit layers of the oceanic environment.
PMCID: PMC3126241  PMID: 21289127
13.  Coccolithophores: Functional Biodiversity, Enzymes and Bioprospecting 
Marine Drugs  2011;9(4):586-602.
Emiliania huxleyi is a single celled, marine phytoplankton with global distribution. As a key species for global biogeochemical cycling, a variety of strains have been amassed in various culture collections. Using a library consisting of 52 strains of E. huxleyi and an ‘in house’ enzyme screening program, we have assessed the functional biodiversity within this species of fundamental importance to global biogeochemical cycling, whilst at the same time determining their potential for exploitation in biocatalytic applications. Here, we describe the screening of E. huxleyi strains, as well as a coccolithovirus infected strain, for commercially relevant biocatalytic enzymes such as acid/alkali phosphodiesterase, acid/alkali phosphomonoesterase, EC1.1.1-type dehydrogenase, EC1.3.1-type dehydrogenase and carboxylesterase.
PMCID: PMC3124974  PMID: 21731551
functional biodiversity; bioprospecting; biocatalysis; coccolithophore
14.  The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription 
The Journal of Experimental Medicine  2009;206(5):1057-1071.
The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes.
PMCID: PMC2715043  PMID: 19380635
15.  Interaction between clopidogrel and proton pump inhibitors 
PMCID: PMC2691442  PMID: 19506284
16.  The minimum information about a genome sequence (MIGS) specification 
Nature biotechnology  2008;26(5):541-547.
With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the ‘transparency’ of the information contained in existing genomic databases.
PMCID: PMC2409278  PMID: 18464787
17.  Proteomic analysis of the EhV-86 virion 
Proteome Science  2008;6:11.
Emiliania huxleyi virus 86 (EhV-86) is the type species of the genus Coccolithovirus within the family Phycodnaviridae. The fully sequenced 407,339 bp genome is predicted to encode 473 protein coding sequences (CDSs) and is the largest Phycodnaviridae sequenced to date. The majority of EhV-86 CDSs exhibit no similarity to proteins in the public databases.
Proteomic analysis by 1-DE and then LC-MS/MS determined that the virion of EhV-86 is composed of at least 28 proteins, 23 of which are predicted to be membrane proteins. Besides the major capsid protein, putative function can be assigned to 4 other components of the virion: two lectin proteins, a thioredoxin and a serine/threonine protein kinase.
This study represents the first steps toward the identification of the protein components that make up the EhV-86 virion. Aside from the major capsid protein, whose function in the virion is well known and defined, the nature of the other proteins suggest roles involved with viral budding, caspase activation, signalling, anti-oxidation, virus adsorption and host range determination.
PMCID: PMC2322966  PMID: 18346272
18.  Locus-Specific Gene Expression Pattern Suggests a Unique Propagation Strategy for a Giant Algal Virus†  
Journal of Virology  2006;80(15):7699-7705.
Emiliania huxleyi virus strain 86 is the largest algal virus sequenced to date and is unique among the Phycodnaviridae since its genome is predicted to contain six RNA polymerase subunit genes. We have used a virus microarray to profile the temporal transcription strategy of this unusual virus during infection. There are two distinct transcription phases to the infection process. The primary phase is dominated by a group of coding sequences (CDSs) expressed by 1 h postinfection that are localized to a subregion of the genome. The CDS of the primary group have no database homologues, and each is associated with a unique promoter element. The remainder of the CDSs are expressed in a secondary phase between 2 and 4 hours postinfection. Compartmentalized transcription of the two distinctive phases is discussed. We hypothesize that immediately after infection the nucleic acid of the virus targets the host nucleus, where primary-phase genes are transcribed by host RNA polymerase which recognizes the viral promoter. Secondary-phase transcription may then be conducted in the cytoplasm.
PMCID: PMC1563701  PMID: 16840348
19.  Genome comparison of two Coccolithoviruses 
Virology Journal  2006;3:15.
The Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163.
We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes.
The sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163.
PMCID: PMC1440845  PMID: 16553948

Results 1-19 (19)