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Now showing items 1 - 16 of 230

  • Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains

    Huo, Yong-Biao   Chan, Yuki   Lacap-Bugler, Donnabella C.   Mo, Sisu   Woo, Patrick C. Y.   Leung, W. Keung   Watt, Rory M.  

    More than 75 "species-level" phylotypes of spirochete bacteria belonging to the genus Treponema reside within the human oral cavity. The majority of these oral treponeme phylotypes correspond to as-yet-uncultivated taxa or strains of uncertain standing in taxonomy. Here, we analyze phylogenetic and taxonomic relationships between oral treponeme strains using a multilocus sequence analysis (MLSA) scheme based on the highly conserved 16S rRNA, pyrH, recA, and flaA genes. We utilized this MLSA scheme to analyze genetic data from a curated collection of oral treponeme strains (n =3D 71) of diverse geographical origins. This comprises phylogroup 1 (n =3D 23) and phylogroup 2 (n =3D 48) treponeme strains, including all relevant American Type Culture Collection reference strains. The taxonomy of all strains was confirmed or inferred via the analysis of ca. 1,450-bp 16S rRNA gene sequences using a combination of bioinformatic and phylogenetic approaches. Taxonomic and phylogenetic relationships between the respective treponeme strains were further investigated by analyzing individual and concatenated flaA (1,074-nucleotide [nt]), recA (1,377-nt), and pyrH (696-nt) gene sequence data sets. Our data confirmed the species differentiation between Treponema denticola (n =3D 41) and Treponema putidum (n =3D 7) strains. Notably, our results clearly supported the differentiation of the 23 phylogroup 1 treponeme strains into five distinct "species-level" phylotypes. These respectively corresponded to "Treponema vincentii" (n =3D 11), Treponema medium (n =3D 1), "Treponema sinensis" (Treponema sp. IA; n =3D 4), Treponema sp. IB (n =3D 3), and Treponema sp. IC (n =3D 4). In conclusion, our MLSA-based approach can be used to effectively discriminate oral treponeme taxa, confirm taxonomic assignment, and enable the delineation of species boundaries with high confidence. IMPORTANCE Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.
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  • In-depth snapshot of the equine subgingival microbiome

    Gao, Wenling   Chan, Yuki   You, Meng   Lacap-Bugler, Donnabella C.   Leung, W. Keung   Watt, Rory M.  

    This study explored the range of bacterial taxa present within healthy subgingival (below the gum-line) niches in the horse oral cavity using 16S rRNA gene amplicon pyrosequencing. Pooled subgingival plaque samples were collected from approximately 200 sulcus sites from two horses (EQ1, EQ2) for analysis. A total of 14,260 quality-filtered pyrosequencing reads were obtained, which were assigned to 3875 operational taxonomic units (OTUs; 99% identity cut-off); 1907 OTUs for EQ1 and 2156 OTUs for EQ2. Diverse taxa from 12 phyla were identified, including Actinobacteria (3.17%), Bacteroidetes (25.11%), Chloroflexi (0.04%), Firmicutes (27.57%), Fusobacteria (5.15%), Proteobacteria (37.67%), Spirochaetes (0.15%), Synergistetes (0.22%), Tenericutes (0.16%), GN02 (0.19%), SR1 (0.01%) and TM7 (0.37%). Many OTUs were not closely related to known phylotypes, and may represent 'equine-specific' taxa. Phylotypes corresponding to Gammaproteobacteria were abundant, including Actinobacillus spp. (8.75%), unclassified Pasteurellaceae (9.90%) and Moraxella spp. (9.58%). PCR targeting the Synergistetes and Spirochaetes phyla was performed, and resultant plasmid libraries of 16S rRNA gene amplicons (ca. 1480 bp) were Sanger sequenced. Twenty-six Spirochaetes OTUs, and 16 Synergistetes OTUs were identified (99% identity cut-off). These 'species-level' OTUs were assigned Equine Oral Taxon (EOT) numbers, whose phylogenies and taxonomy were comprehensively investigated, in conjunction with corresponding Synergistetes and Spirochaetes OTUs identified by pyrosequencing. The vast majority of Spirochaetes taxa belonged to the genus Treponema, which corresponded to 7 of the 10 human oral treponeme phylogroups. Other Spirochaetes taxa belonging to the Leptospiraceae family were observed; but many treponemes commonly implicated in animal hoof/foot and non-oral soft tissue infections; e.g. Treponema phagedenis, Treponema pedis, Treponema refringens, Treponema calligyrum; were not identified here. Diverse Synergistetes taxa corresponding to oral clusters A and B were identified, which included Fretibacterium fastidiosum and Pyramidobacter piscolens. Taken together, our data reveals that equine sub gingival plaque microbiota shares many similarities with the human, canine and feline oral microbiomes. (C) 2015 Elsevier Ltd. All rights reserved.
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  • Two-dimensional gel electrophoresis in bacterial proteomics

    Curreem, Shirly O. T.   Watt, Rory M.   Lau, Susanna K. P.   Woo, Patrick C. Y.  

    Two-dimensional gel electrophoresis (2-DE) is a gel-based technique widely used for analyzing the protein composition of biological samples. It is capable of resolving complex mixtures containing more than a thousand protein components into individual protein spots through the coupling of two orthogonal biophysical separation techniques: isoelectric focusing (first dimension) and polyacrylamide gel electrophoresis (second dimension). 2-DE is ideally suited for analyzing the entire expressed protein complement of a bacterial cell: its proteome. Its relative simplicity and good reproducibility have led to 2-DE being widely used for exploring proteomics within a wide range of environmental and medically-relevant bacteria. Here we give a broad overview of the basic principles and historical development of gel-based proteomics, and how this powerful approach can be applied for studying bacterial biology and physiology. We highlight specific 2-DE applications that can be used to analyze when, where and how much proteins are expressed. The links between proteomics, genomics and mass spectrometry are discussed. We explore how proteomics involving tandem mass spectrometry can be used to analyze (post-translational) protein modifications or to identify proteins of unknown origin by de novo peptide sequencing. The use of proteome fractionation techniques and non-gel-based proteomic approaches are also discussed. We highlight how the analysis of proteins secreted by bacterial cells (secretomes or exoproteomes) can be used to study infection processes or the immune response. This review is aimed at non-specialists who wish to gain a concise, comprehensive and contemporary overview of the nature and applications of bacterial proteomics.
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  • Comparative analysis of oral treponemes associated with periodontal health and disease

    You, Meng   Mo, Sisu   Leung, W. Keung   Watt, Rory M.  

    Background: Periodontal diseases, such as periodontitis, are chronic inflammatory infections affecting the gingivae (gums), underlying connective tissues and bone that support the teeth. Oral treponemes (genus Treponema) are widely-considered to play important roles in periodontal disease etiology and pathogenesis; however, precise relationships remain to be fully established. Methods: A 16S rRNA clone library-based approach was used to comprehensively characterize and compare the diversity of treponeme taxa present in subgingival plaque sampled from periodontitis patients (n = 10) versus periodontitis-free controls (n = 10). 16S rRNA gene sequences were assigned to operational taxonomic units (OTUs) using a 99% identity cut-off A variety of taxonomy (OTU) and phylogeny-based statistical approaches were used to compare populations of treponeme OTUs present in both subject groups. Results: A total of 615 plasmid clones containing ca. 1500 bp Treponema 16S rRNA gene sequences were obtained; 365 from periodontitis subjects, 250 from periodontitis-free controls. These were assigned to 110 treponeme OTUs. 93 OTUs were detected in the periodontitis subjects (mean 9.3 +/- 5.2 OTUs per subject; range 9-26), and 43 OTUs were detected in controls (mean 4.3 +/- 5.9 OTUs per subject; range 3-20). OTUs belonging to oral treponeme phylogroups 1-7 were detected in both subject sets. Phylogroup 1 treponemes had the highest levels of OTU richness (diversity) and clonal abundance within both subject groups. Levels of OTU richness and clonal abundance of phylogroup 2 treponemes were significantly higher in the periodontitis subjects (Mann Whitney U-test, p < 0.001). Both OTU-based and phylogeny-based analyses clearly indicated that there were significant differences in the composition of treponeme communities present in periodontitis versus control subjects. The detection frequency of five OTUs showed a statistically-significant correlation with disease status. The OTU (8P47) that corresponded to the type strain of Treponema denticola had the strongest association with periodontitis (p < 0.01). Conclusions: Higher levels of treponeme taxon richness and clonal abundance were associated with periodontitis. However, our results clearly indicated that subjects free from clinical symptoms of periodontal disease also contained highly diverse populations of treponeme bacteria within their subgingival microbiota. Our data supports the hypothesis that specific treponeme taxa are associated with periodontal disease.
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  • The Two PPX-GppA Homologues from Mycobacterium tuberculosis Have Distinct Biochemical Activities

    Choi, Mei Y.   Wang, Ying   Wong, Leo L. Y.   Lu, Bing-tai   Chen, Wen-yang   Huang, Jian-Dong   Tanner, Julian A.   Watt, Rory M.  

    Inorganic polyphosphate (poly-P), guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) are ubiquitous in bacteria. These molecules play a variety of important physiological roles associated with stress resistance, persistence, and virulence. In the bacterial pathogen Mycobacterium tuberculosis, the identities of the proteins responsible for the metabolism of polyphosphate and (p) ppGpp remain to be fully established. M. tuberculosis encodes two PPX-GppA homologues, Rv0496 (MTB-PPX1) and Rv1026, which share significant sequence similarity with bacterial exopolyphosphatase (PPX) and guanosine pentaphosphate 5'-phosphohydrolase (GPP) proteins. Here we delineate the respective biochemical activities of the Rv0496 and Rv1026 proteins and benchmark these against the activities of the PPX and GPP proteins from Escherichia coli. We demonstrate that Rv0496 functions as an exopolyphosphatase, showing a distinct preference for relatively short-chain poly-P substrates. In contrast, Rv1026 has no detectable exopolyphosphatase activities. Analogous to the E. coli PPX and GPP enzymes, the exopolyphosphatase activities of Rv0496 are inhibited by pppGpp and, to a lesser extent, by ppGpp alarmones, which are produced during the bacterial stringent response. However, neither Rv0496 nor Rv1026 have the ability to hydrolyze pppGpp to ppGpp; a reaction catalyzed by E. coli PPX and GPP. Both the Rv0496 and Rv1026 proteins have modest ATPase and to a lesser extent ADPase activities. pppGpp alarmones inhibit the ATPase activities of Rv1026 and, to a lesser extent, the ATPase activities of Rv0496. We conclude that PPX-GppA family proteins may not possess all the catalytic activities implied by their name and may play distinct biochemical roles involved in polyphosphate and (p) ppGpp metabolic pathways.
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  • Intra-oral single-site comparisons of periodontal and peri-implant microbiota in health and disease

    Yu, Xiao-Lin   Chan, Yuki   Zhuang, Longfei   Lai, Hong-Chang   Lang, Niklaus P.   Leung, Wai Keung   Watt, Rory M.  

    Objective Periodontitis and peri-implantitis are oral infectious-inflammatory diseases that share similarities in their pathology and etiology. Our objective was to characterize the single-site subgingival and submucosal microbiomes of implant-rehabilitated, partially dentate Chinese subjects (n =3D 18) presenting with both periodontitis and peri-implantitis. Materials and methods Subgingival/submucosal plaque samples were collected from four clinically distinct sites in each subject: peri-implantitis submucosa (DI), periodontal pocket (DT), clinically healthy (unaffected) peri-implant submucosa (HI), and clinically healthy (unaffected) subgingival sulcus (HT). The bacterial microbiota present was analyzed using Illumina MiSeq sequencing. Results Twenty-six phyla and 5,726 operational taxonomic units (OTUs, 97% sequence similarity cutoff) were identified. Firmicutes, Proteobacteria, Fusobacteria, Bacteroidetes, Actinobacteria, Synergistetes, TM7, and Spirochaetes comprised 99.6% of the total reads detected. Bacterial communities within the DI, DT, HI, and HT sites shared high levels of taxonomic similarity. Thirty-one "core species" were present in >90% sites, with Streptococcus infantis/mitis/oralis (HMT-070/HMT-071/HMT-638/HMT-677) and Fusobacterium sp. HMT-203/HMT-698 being particularly prevalent and abundant. Beta-diversity analyses (PERMANOVA test, weighted UniFrac) revealed the largest variance in the microbiota was at the subject level (46%), followed by periodontal health status (4%). Differing sets of OTUs were associated with periodontitis and peri-implantitis sites, respectively. This included putative "periodontopathogens," such as Prevotella, Porphyromonas, Tannerella, Bacteroidetes [G-5], and Treponema spp. Interaction network analysis identified several putative patterns underlying dysbiosis in periodontitis/peri-implantitis sites. Conclusions Species (OTU) composition of the periodontal and peri-implant microbiota varied widely between subjects. The inter-subject variations in subgingival/submucosal microbiome composition outweighed differences observed between implant vs. tooth sites, or between diseased vs. healthy (unaffected) peri-implant/periodontal sites.
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  • Structural basis for discriminatory recognition of Plasmodium lactate dehydrogenase by a DNA aptamer

    Cheung, Yee-Wai   Kwok, Jane   Law, Alan W. L.   Watt, Rory M.  

    DNA aptamers have significant potential as diagnostic and therapeutic agents, but the paucity of DNA aptamer-target structures limits understanding of their molecular binding mechanisms. Here, we report a distorted hairpin structure of a DNA aptamer in complex with an important diagnostic target for malaria: Plasmodium falciparum lactate dehydrogenase (PfLDH). Aptamers selected from a DNA library were highly specific and discriminatory for Plasmodium as opposed to human lactate dehydrogenase because of a counterselection strategy used during selection. Isothermal titration calorimetry revealed aptamer binding to PfLDH with a dissociation constant of 42 nM and 2: 1 protein: aptamer molar stoichiometry. Dissociation constants derived from electrophoretic mobility shift assays and surface plasmon resonance experiments were consistent. The aptamer: protein complex crystal structure was solved at 2.1-angstrom resolution, revealing two aptamers bind per PfLDH tetramer. The aptamers showed a unique distorted hairpin structure in complex with PfLDH, displaying a Watson-Crick basepaired stem together with two distinct loops each with one base flipped out by specific interactions with PfLDH. Aptamer binding specificity is dictated by extensive interactions of one of the aptamer loops with a PfLDH loop that is absent in human lactate dehydrogenase. We conjugated the aptamer to gold nanoparticles and demonstrated specificity of colorimetric detection of PfLDH over human lactate dehydrogenase. This unique distorted hairpin aptamer complex provides a perspective on aptamer-mediated molecular recognition and may guide rational design of better aptamers for malaria diagnostics.
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  • Mechanism of 8-amino-7-oxononanoate synthase: Spectroscopic, kinetic, and crystallographic studies

    Webster, Scott P.   Alexeev, Dmitriy   Campopiano, Dominic J.   Watt, Rory M.   Alexeeva, Marina   Sawyer, Lindsay   Baxter, Robert L.  

    8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 ANG (R = 18.6%, Rfree = 21.2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 ANG (R = 21.2%, Rfree = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k1 = 2 X 104 M-1 s-1). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k1 = 125 M-1 s-1). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase (apprx30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.
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  • Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    Yang, Wen   Chen, Wen-yang   Wang, Hui   Ho, John W. S.   Huang, Jian-Dong   Woo, Patrick C. Y.   Lau, Susanna K. P.   Yuen, Kwok-Yung   Zhang, Qionglin   Zhou, Weihong   Bartlam, Mark   Watt, Rory M.   Rao, Zihe  

    Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced beta-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 A, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of lambda-exonuclease (Exo) from bacteriophage-lambda. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.
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  • Periodontal and peri-implant microbiota in patients with healthy and inflamed periodontal and peri-implant tissues

    Zhuang, Long-Fei   Watt, Rory M.   Mattheos, Nikos   Si, Mi-Si   Lai, Hong-Chang   Lang, Niklaus P.  

    Objective: To compare the prevalence and levels of six bacterial pathogens within the subgingival/submucosal microbiota at teeth versus implants with various clinical conditions. Material and methods: Twenty-two Chinese were included. Four subgingival/submucosal sites were selected for microbiological sampling within each subject, that is, (1) healthy peri-implant tissues; (2) peri-implantitis [PPD >=3D 5 mm, presence of bleeding on probing (BOP) and confirmed radiographic bone loss]; (3) healthy gingiva; and (4) periodontitis (PPD > 4 mm). Subgingival/ submucosal plaque was sampled using paper points. Quantitative real-time polymerase chain reaction (q-PCR) was used to quantify six pathogens, including Porphyromonas gingivalis (P. g.), Treponema denticola (T. d.), Aggregatibacter actinomycetemcomitans (A. a.), Fusobacterium nucleatum (F. n.), Prevotella intermedia (P. i.), and Staphylococcus aureus (S. a.). Counts were log 10-transformed. Results: The most commonly detected species were S. a. and F. n., while A. a. and. P. i. had the lowest detection frequency. The detection frequencies of diseased tooth or implant sites for each of the six target species were either equal to or higher than the respective frequencies at the corresponding healthy sites. There were no statistically significant differences for any of the species or clinical sites (P > 0.05, Cochran's Q test). No statistically significant differences in the bacterial loads were found among the four clinical sites; with the exception of F. nucleatum. This was more abundant in periodontitis sites (P =3D 0.023, Friedman's 2-way ANOVA). Both periodontal and periimplant sites, irrespective of their health status, were revealed to harbor S. aureus cells. The log10-transformed loads of S. aureus were approximately 3.5 within each of the clinical sites (P =3D 0.232). This was the highest of the six species analyzed. Conclusions: Within the same subjects, putative periodontal pathogens were common to both periodontal and peri-implant sites irrespective of health status. The prevalence and levels of P. gingivalis and F. nucleatum were significantly associated with periodontitis, but not with peri-implantitis. A. actinomycetemcomitans was associated with both disease conditions, periodontitis and peri-implantitis, but not with either gingival or mucosal health.
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  • Purification,crystallization and X-ray crystallographic analysis of a putative exopolyphosphatase from Zymomonas mobilis

    Zhang, Aili   Guo, Erhong   Qian, Lanfang   Tang, Nga-Yeung   Watt, Rory M.   Bartlam, Mark  

    Exopolyphosphatase (PPX) enzymes degrade inorganic polyphosphate (poly-P), which is essential for the survival of microbial cells in response to external stresses. In this study, a putative exopolyphosphatase from Zymomonas mobilis (ZmPPX) was crystallized. Crystals of the wild-type enzyme diffracted to 3.3 angstrom resolution and could not be optimized further. The truncation of 29 amino acids from the N-terminus resulted in crystals that diffracted to 1.8 angstrom resolution. The crystals belonged to space group C2, with unit-cell parameters a =3D 122.0, b =3D 47.1, c =3D 89.5 angstrom, alpha =3D gamma =3D 90, beta =3D 124.5 degrees. An active-site mutant that crystallized in the same space group and with similar unit-cell parameters diffracted to 1.56 angstrom resolution. One molecule was identified per asymmetric unit. Analytical ultracentrifugation confirmed that ZmPPX forms a dimer in solution. It was confirmed that ZmPPX possesses exopolyphosphatase activity against a synthetic poly-P substrate.
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  • Thermodynamic and kinetic aspects of metal binding to the histidine-rich protein, Hpn RID B-2177-2009 RID D-3123-2009

    Ge, Ruiguang   Zhang, Yi   Sun, Xuesong   Watt, Rory M.   He, Qing-Yu   Huang, Jian-Dong   Wilcox, Dean E.   Sun, Hongzhe  

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  • The involvement of replication in single stranded oligonucleotide-mediated gene repair RID E-9862-2010 RID B-2177-2009

    Huen, Michael S. Y.   Li, Xin-tian   Lu, Lin-Yu   Watt, Rory M.   Liu, De-Pei   Huang, Jian-Dong  

    Targeted gene repair mediated by single-stranded oligonucleotides (SSOs) has great potential for use in functional genomic studies and gene therapy. Genetic changes have been created using this approach in a number of prokaryotic and eukaryotic systems, including mouse embryonic stem cells. However, the underlying mechanisms remain to be fully established. In one of the current models, the 'annealing-integration' model, the SSO anneals to its target locus at the replication fork, serving as a primer for subsequent DNA synthesis mediated by the host replication machinery. Using a lambda-Red recombination-based system in the bacterium Escherichia coli, we systematically examined several fundamental premises that form the mechanistic basis of this model. Our results provide direct evidence strongly suggesting that SSO-mediated gene repair is mechanistically linked to the process of DNA replication, and most likely involves a replication intermediate. These findings will help guide future experiments involving SSO-mediated gene repair in mammalian and prokaryotic cells, and suggest several mechanisms by which the efficiencies may be reliably and substantially increased.
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  • Laribacter hongkongensis anaerobic adaptation mediated by arginine metabolism is controlled by the cooperation of FNR and ArgR

    Xiong, Lifeng   Yang, Ying   Ye, Yuan-Nong   Teng, Jade L. L.   Chan, Elaine   Watt, Rory M.   Guo, Feng-Biao   Lau, Susanna K. P.   Woo, Patrick C. Y.  

    Laribacter hongkongensis is a fish-borne pathogen associated with invasive infections and gastroenteritis. Its adaptive mechanisms to oxygen-limiting conditions in various environmental niches remain unclear. In this study, we compared the transcriptional profiles of L. hongkongensis under aerobic and anaerobic conditions using RNA-sequencing. Expression of genes involved in arginine metabolism significantly increased under anoxic conditions. Arginine was exploited as the sole energy source in L. hongkongensis for anaerobic respiration via the arginine catabolism pathway: specifically via the arginine deiminase (ADI) pathway. A transcriptional regulator FNR was identified to coordinate anaerobic metabolism by tightly regulating the expression of arginine metabolism genes. FNR executed its regulatory function by binding to FNR boxes in arc operons promoters. Survival of isogenic fnr mutant in macrophages decreased significantly when compared with wild-type; and expression level of fnr increased 8 h post-infection. Remarkably, FNR directly interacted with ArgR, another regulator that influences the biological fitness and intracellular survival of L. hongkongensis by regulating arginine metabolism genes. Our results demonstrated that FNR and ArgR work in coordination to respond to oxygen changes in both extracellular and intracellular environments, by finely regulating the ADI pathway and arginine anabolism pathways, thereby optimizing bacterial fitness in various environmental niches.
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  • 1.56 m 1 watt single frequency semiconductor disk laser.

    Rantamaki, Antti   Rautiainen, Jussi   Sirbu, Alexei   Mereuta, Alexandru   Kapon, Eli   Okhotnikov, Oleg G  

    A single frequency wafer-fused semiconductor disk laser at 1.56 m with 1 watt of output power and a coherence length over 5 km in fiber is demonstrated. The result represents the highest output power reported for a narrow-line semiconductor disk laser operating at this spectral range. The study shows the promising potential of the wafer fusion technique for power scaling of single frequency vertical-cavity lasers emitting in the 1.3-1.6 m range.
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  • Design of a 40 Watt 1.55 mu m Uplink Transmitter for Lunar Laser Communications

    Caplan, D. O.   Carney, J. J.   Lafon, R. E.   Stevens, M. L.  

    A ground-based 40W 1.55 mu m uplink transmitter for lunar laser communications is described. The transmitter, which generates wavelength multiplexed communication and beacon signals, is implemented using four 10W spatial-diversity channels to reduce far-field atmospheric-turbulence-induced fading and facilitate high-power signal generation via parallel-spatial-combining of commercially-available EDFAs. Each transmitter channel can generate a 1 kHz modulated beacon for spatial acquisition, and a multi-rate 4-PPM communication signal at a 311 MHz slot rate with 16:1 and 32:1 duty cycles to support 38.9 Mbit/s and 19.4 Mbit/s channel rates, respectively. Details on the transmitter design, including the mitigation of optical nonlinear effects are discussed.
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