Biomedical subjects
Publications and source records attributed to D J Richardson.
We report compression of low-power femtosecond pulses at 1.06 microm in a dispersion-decreasing holey fiber. Near-adiabatic compression of 130 fs pulses down to 60 fs has been observed. Measured spectra and pulse shapes agree well with numerical simulations. Compression factors of ten are possible in optimized fibers.
We report the results of detailed measurements on the Brillouin frequency shift (BFS), gain bandwidth, and gain coefficients of several small-core holey optical fibers (HFs) of both uniform and axially varying structural characteristics and compare these with measurements on more conventional fibers. Our measurements show that the BFS of HFs is first-order proportional to the modal index for light propagating along the fiber and is thus extremely sensitive to its precise structural parameters. Our results highlight the possibility of using distributed Brillouin scattering measurements to perform nondestructive structural characterization of HFs, and the possibility of producing Brillouin-suppressed HFs using controlled structural variation along the fiber length.
We demonstrate a novel frequency-resolved optical gating (FROG) configuration based on cascaded second-order nonlinear interactions. Its implementation in a 2.6 cm long quasi-phase-matched LiNbO3 waveguide allowed high-quality retrieval of 2 ps to 80 fJ pulses at 1.56 microm.
Haloferax mediterranei cells are able to use high nitrate or nitrite concentrations as electron acceptors under anoxic conditions. The nar operon, which has eight open reading frames, has been sequenced and its regulation has been characterized at the transcriptional level. The narG and narH genes encode the Nar (respiratory nitrate reductase) catalytic subunit (NarG) and the electron transfer Nar subunit (NarH) respectively. Nar has been purified and characterized in vitro. This characterization has included protein-film voltammetry and preliminary EPR studies.
The AF0174-AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31-32%) and similarity (41-42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO(3)(-)) rather than tetrahedral (SeO(4)(2-) and ClO(4)(-)) substrates.
Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.
Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.
The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer-monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.
c-Type cytochromes are a widespread class of proteins that play a vital role in the energy-conserving metabolism of prokaryotic and eukaryotic organisms. The key event in cytochrome c biogenesis is the covalent attachment of the haem cofactor to two (or rarely one) cysteine residues arranged in a haem c-binding motif such as CX(2-4)CH, CXXCK or X(3)CH. This reaction is catalysed by the membrane-bound enzyme CCHL (cytochrome c haem lyase). Different CCHLs have been described and some of them are dedicated to distinct haem c-binding motifs of cytochromes that are encoded in the vicinity of the respective CCHL gene. Various bacterial genomes contain multiple copies of CCHL-encoding genes, suggesting the presence of non-conventional or even as yet unrecognized haem c-binding motifs. This assumption is exemplified in the present study using the proteobacterium Wolinella succinogenes as a model organism whose genome encodes three CCHL isoenzymes. The discovery of a novel conserved multihaem cytochrome c (MccA) is described.
The identification of nitric oxide-bound leghaemoglobin within soya bean nodules has led to the question of how Bradyrhizobium japonicum bacteroids overcome the toxicity of this nitric oxide. It has previously been shown that one candidate for nitric oxide detoxification, the respiratory nitric oxide reductase, is expressed in soya bean nodules from plants supplied with nitrate. In this paper, the role of this enzyme in nitric oxide detoxification is assessed and discussion is provided on other possible B. japonicum nitric oxide detoxification systems.
Expression of the nap operon, encoding the periplasmic nitrate reductase in Paracoccus pantotrophus, is maximal when cells are grown aerobically, but not anaerobically, with butyrate. Two promoters, termed P1 and P2, control operon expression and the operon-proximal P2 promoter is primarily responsible for increased nap expression in the presence of butyrate. A near-perfect palindromic sequence is centred at +7, relative to the P2 transcription start site. Mutation of this palindrome demonstrated that it is important for regulation of nap operon expression in response to both the redox and the oxidation state of the carbon substrate. A 5' deletion analysis of the nap promoter fused to lacZ revealed that full redox control of expression was retained when the DNA sequence up to position -49 bp, relative to the operon-distal P1 transcription start site, was removed. Encroaching beyond this position resulted in an approximately 4-fold reduction in expression when cells were grown aerobically with butyrate. Additionally, point mutations at position -38 and -45 relative to P1 also resulted in a reduction in expression during aerobic growth with butyrate. A GC-rich region of nap promoter DNA, centred on position -41 relative to the P1 transcription start site is thus proposed as a second DNA motif that is important for efficient expression of the nap operon.
Over the last 10 years, during the lifetime of the nitrogen cycle meetings, structural biology, coupled with spectroscopy, has had a major impact of our understanding enzymology of the nitrogen cycle. The three-dimensional structures for many of the key enzymes have now been resolved and have provided a wealth of information regarding the architecture of redox active metal sites, as well as revealing novel structural folds. Coupled with structure-based spectroscopic analysis, this has led to new insight into the reaction mechanisms of the diverse chemical transformations that together cycle nitrogen in the biosphere. An overview of the some of the key developments in field over the last decade is presented.
Controlled targeting and transport of redox enzymes to and across the bacterial cytoplasmic membrane is essential for bacterial respiration. A subset of bacterial redox enzymes is exported as folded proteins on the Tat (twin-arginine transport) pathway. Protein export is the point-of-no-return for passenger proteins on the Tat pathway and it is crucial that complex, cofactor-containing enzymes are fully assembled before export is attempted. Using the Escherichia coli trimethylamine N-oxide reductase system as a model, we discuss here the molecular processes governing assembly and export of Tat-dependent enzymes.
The recent structural characterization of the NrfA from Escherichia coli provides a framework to rationalize the spectroscopic and functional properties of this enzyme. Analyses by EPR and magnetic CD spectroscopies have been complemented by protein-film voltammetry and these are discussed in relation to the essential structural features of the enzyme.
A number of biochemically distinct systems have been characterized for the microbial reduction of the oxyanions, selenate (SeO(4)(2-)) and nitrate (NO(3)(-)). Two classes of molybdenum-dependent nitrate reductase catalyse the respiratory-linked reduction of nitrate (NO(3)(-)) to nitrite (NO(2)(-)). The main respiratory nitrate reductase (NAR) is membrane-anchored, with its active site facing the cytoplasmic compartment. The other enzyme (NAP) is water-soluble and located in the periplasm. In recent years, our understanding of each of these enzyme systems has increased significantly. The crystal structures of both NAR and NAP have now been solved and they provide new insight into the structure, function and evolution of these respiratory complexes. In contrast, our understanding of microbial selenate (SeO(4)(2-)) reduction and respiration is at an early stage; however, similarities to the nitrate reductase systems are emerging. This review will consider some of the common themes and variations between the different classes of nitrate and selenate reductases.
Salmonella possesses multiple enzymes that utilize NO as a substrate, and could therefore contribute to the organism's ability to resist nitrosative killing by macrophages. Flavorubredoxin is an oxygen-sensitive enzyme that reduces NO to nitrous oxide. The Salmonella enterica serovar Typhimurium norV gene encoding flavorubredoxin was disrupted and the NO sensitivity of the mutant was determined. The norV mutant showed a greater sensitivity to NO than wild-type S. Typhimurium, but did recover growth after a transient inhibition. The mutant phenotype suggests that multiple enzymes are employed by S. Typhimurium to detoxify NO under anaerobic conditions, one of which is flavorubredoxin.
We demonstrate that nonlinear fiber compression is possible at unprecedented average power levels by use of a large-mode-area holey (microstructured) fiber and a passively mode-locked thin disk Yb:YAG laser operating at 1030 nm. We broaden the optical spectrum of the 810-fs pump pulses by nonlinear propagation in the fiber and remove the resultant chirp with a dispersive prism pair to achieve 18 W of average power in 33-fs pulses with a peak power of 12 MW and a repetition rate of 34 MHz. The output beam is nearly diffraction limited and is linearly polarized.
Rhodobacter capsulatus N22DNAR(+) possesses a periplasmic nitrate reductase and is capable of reducing nitrate to nitrite under anaerobic conditions. In the absence of light this ability cannot support chemoheterotrophic growth in batch cultures. This study investigated the effect of nitrate reduction on the growth of R. capsulatus N22DNAR(+) during multiple light-dark cycles of anaerobic photoheterotrophic/dark chemoheterotrophic growth conditions in carbon-limited continuous cultures. The reduction of nitrate did not affect the photoheterotrophic growth yield of R. capsulatus N22DNAR(+). After a transition from photoheterotrophic to dark chemoheterotrophic growth conditions, the reduction of nitrate slowed the initial washout of a R. capsulatus N22DNAR(+) culture. Towards the end of a period of darkness nitrate-reducing cultures maintained higher viable cell counts than non-nitrate-reducing cultures. During light-dark cycling of a mixed culture, the strain able to reduce nitrate (N22DNAR(+)) outcompeted the strain which was unable to reduce nitrate (N22). The evidence indicates that the periplasmic nitrate reductase activity supports slow growth that retards the washout of a culture during anaerobic chemoheterotrophic conditions, and provides a protonmotive force for cell maintenance during the dark period before reillumination. This translates into a selective advantage during repeated light-dark cycles, such that in mixed culture N22DNAR(+) outcompetes N22. Exposure to light-dark cycles will be a common feature for R. capsulatus in its natural habitats, and this study shows that nitrate respiration may provide a selective advantage under such conditions.