Supplementary Information Table S1. Up-Regulated Known Orfs Induced
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Recruitment of Genes and Enzymes Conferring Resistance to the Nonnatural Toxin Bromoacetate
Recruitment of genes and enzymes conferring resistance to the nonnatural toxin bromoacetate Kevin K. Desai and Brian G. Miller1 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306-4390 Edited* by Richard Wolfenden, University of North Carolina, Chapel Hill, NC, and approved August 24, 2010 (received for review May 28, 2010) Microbial niches contain toxic chemicals capable of forcing organ- tance of a naïve bacterial population can play a role in combating isms into periods of intense natural selection to afford survival. the toxicity of a nonnatural small-molecule. Revealing the reser- Elucidating the mechanisms by which microbes evade environmen- voir of intrinsic resistance genes that are subject to evolutionary tal threats has direct relevance for understanding and combating recruitment promises to aid our understanding of the processes the rise of antibiotic resistance. In this study we used a toxic small- leading to the emergence of antibiotic resistant pathogens. molecule, bromoacetate, to model the selective pressures imposed We sought to identify the full spectrum of bromoacetate resis- by antibiotics and anthropogenic toxins. We report the results tance mechanisms available to the model bacterium, Escherichia of genetic selection experiments that identify nine genes from coli. The reactivity of bromoacetate is likely to mimic that of elec- Escherichia coli whose overexpression affords survival in the trophilic natural products as well as anthropogenic environmen- presence of a normally lethal concentration of bromoacetate. Eight tal contaminants that microbes may encounter. The clinically of these genes encode putative transporters or transmembrane significant natural antibiotic fosfomycin, and the fungal natural proteins, while one encodes the essential peptidoglycan biosyn- product terreic acid, are electrophilic molecules that both target N thetic enzyme, UDP- -acetylglucosamine enolpyruvoyl transferase an essential nucleophilic cysteine residue in bacteria (8, 9). -
An Atpase Domain Common to Prokaryotic Cell Cycle Proteins
Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7290-7294, August 1992 Biochemistry An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp7O heat shock proteins (structural comparison/property pattern/remote homology) PEER BORK, CHRIS SANDER, AND ALFONSO VALENCIA European Molecular Biology Laboratory, D-6900 Heidelberg, Federal Republic of Germany Communicated by Russell F. Doolittle, March 6, 1992 ABSTRACT The functionally diverse actin, hexokinase, and hsp7O protein families have in common an ATPase domain of known three-dimensional structure. Optimal superposition ofthe three structures and alignment ofmany sequences in each of the three families has revealed a set of common conserved residues, distributed in five sequence motifs, which are in- volved in ATP binding and in a putative interdomain hinge. From the multiple sequence aliment in these motifs a pattern of amino acid properties required at each position is defined. The discriminatory power of the pattern is in part due to the use of several known three-dimensional structures and many sequences and in part to the "property" method ofgeneralizing from observed amino acid frequencies to amino acid fitness at each sequence position. A sequence data base search with the pattern significantly matches sugar kinases, such as fuco-, glucono-, xylulo-, ribulo-, and glycerokinase, as well as the prokaryotic cell cycle proteins MreB, FtsA, and StbA. These are predicted to have subdomains with the same tertiary structure as the ATPase subdomains Ia and Ha of hexokinase, actin, and Hsc7O, a very similar ATP binding pocket, and the capacity for interdomain hinge motion accompanying func- tional state changes. -
Comparison of Strand-Specific Transcriptomes Of
Landstorfer et al. BMC Genomics 2014, 15:353 http://www.biomedcentral.com/1471-2164/15/353 RESEARCH ARTICLE Open Access Comparison of strand-specific transcriptomes of enterohemorrhagic Escherichia coli O157:H7 EDL933 (EHEC) under eleven different environmental conditions including radish sprouts and cattle feces Richard Landstorfer1, Svenja Simon2, Steffen Schober3, Daniel Keim2, Siegfried Scherer1 and Klaus Neuhaus1* Abstract Background: Multiple infection sources for enterohemorrhagic Escherichia coli O157:H7 (EHEC) are known, including animal products, fruit and vegetables. The ecology of this pathogen outside its human host is largely unknown and one third of its annotated genes are still hypothetical. To identify genetic determinants expressed under a variety of environmental factors, we applied strand-specific RNA-sequencing, comparing the SOLiD and Illumina systems. Results: Transcriptomes of EHEC were sequenced under 11 different biotic and abiotic conditions: LB medium at pH4, pH7, pH9, or at 15°C; LB with nitrite or trimethoprim-sulfamethoxazole; LB-agar surface, M9 minimal medium, spinach leaf juice, surface of living radish sprouts, and cattle feces. Of 5379 annotated genes in strain EDL933 (genome and plasmid), a surprising minority of only 144 had null sequencing reads under all conditions. We therefore developed a statistical method to distinguish weakly transcribed genes from background transcription. We find that 96% of all genes and 91.5% of the hypothetical genes exhibit a significant transcriptional signal under at least one condition. Comparing SOLiD and Illumina systems, we find a high correlation between both approaches for fold-changes of the induced or repressed genes. The pathogenicity island LEE showed highest transcriptional activity in LB medium, minimal medium, and after treatment with antibiotics. -
Non-Homologous Isofunctional Enzymes: a Systematic Analysis Of
Omelchenko et al. Biology Direct 2010, 5:31 http://www.biology-direct.com/content/5/1/31 RESEARCH Open Access Non-homologousResearch isofunctional enzymes: A systematic analysis of alternative solutions in enzyme evolution Marina V Omelchenko, Michael Y Galperin*, Yuri I Wolf and Eugene V Koonin Abstract Background: Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. Results: We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. -
Generated by SRI International Pathway Tools Version 25.0, Authors S
An online version of this diagram is available at BioCyc.org. Biosynthetic pathways are positioned in the left of the cytoplasm, degradative pathways on the right, and reactions not assigned to any pathway are in the far right of the cytoplasm. Transporters and membrane proteins are shown on the membrane. Periplasmic (where appropriate) and extracellular reactions and proteins may also be shown. Pathways are colored according to their cellular function. Gcf_000238675-HmpCyc: Bacillus smithii 7_3_47FAA Cellular Overview Connections between pathways are omitted for legibility. -
Anti-Dicer (SAB4200087)
Anti-Dicer produced in rabbit, affinity isolated antibody Product Number SAB4200087 Product Description Precautions and Disclaimer Anti-Dicer is produced in rabbit using as the For R&D use only. Not for drug, household, or other immunogen a synthetic peptide corresponding to a uses. Please consult the Safety Data Sheet for fragment of human Dicer (Gene ID: 23405) conjugated information regarding hazards and safe handling to KLH. The corresponding sequence is identical in practices. mouse. The antibody is affinity-purified using the immunizing peptide immobilized on agarose. Storage/Stability Store at –20 C. For continuous use, store at 2–8 C for Anti-Dicer recognizes human Dicer. The antibody may up to one month. For extended storage, freeze in be used in several immunochemical techniques working aliquots at –20 C. Repeated freezing and including immunoblotting (218 kDa), immuno- thawing, or storage in “frost-free” freezers, is not precipitation, and immunofluorescence. Detection of recommended. If slight turbidity occurs upon prolonged the Dicer band by immunoblotting is specifically storage, clarify the solution by centrifugation before inhibited with the immunizing peptide. use. Working dilutions should be discarded if not used within 12 hours. Dicer, also known as Dicer1, Endoribonuclease Dicer, Helicase with RNase motif, and HERNA, is a member Product Profile of the RNase III family that catalyzes the first step in the Immunoblotting: a working antibody concentration of RNA interference (RNAi) pathway and initiates 3-6 g/mL is recommended using HeLa cell lysates. formation of the RNA-induced silencing complex (RISC). Dicer processes the dsRNA into small Immunoprecipitation: a working antibody amount of fragments called short interfering RNA (siRNA) or 2.5-5 g is recommended using HeLa cell lysates. -
JASON MARC GOLDSTEIN the Isolation, Characterization
JASON MARC GOLDSTEIN The Isolation, Characterization and Cloning of Three Novel Peptidases From Streptoccocus gordonii: Their Potential Roles in Subacute Bacterial Endocarditis (Under the Direction of JAMES TRAVIS) Streptococcus gordonii is generally considered a benign inhabitant of the oral microflora yet is a primary etiological agent in the development of subacute bacterial endocarditis (SBE), an inflammatory state that propagates thrombus formation and tissue damage on the surface of heart valves. Colonization and adherence mechanisms have been identified, yet factors necessary to sustain growth remain unidentified. Strain FSS2 produced three extracellular aminopeptidase activities during growth in neutral pH- controlled batch cultures. The first included a serine-class dipeptidyl-aminopeptidase, an x-Pro DPP (Sg-xPDPP) found as an 85 kDa monomer by SDS-PAGE while appearing as a homodimer under native conditions. Kinetic studies indicated a unique and stringent x- Pro specificity comparable to the DPPIV/CD26 and lactococcal x-Pro DPP families. Isolation of the full-length gene uncovered a 759-amino acid polypeptide with a mass of 87,115 Da and theoretical pI of 5.6. Significant homology was found with PepX gene family members from Lactobacillus ssp. and Lactococcus ssp., and putative streptococcal x-Pro DPPs. The second activity was a putative serine-class arginine aminopeptidase (Sg- RAP) with some cysteine-class characteristics. It was found as a protein monomer of 70 kDa under denaturing conditions. Nested PCR cloning enabled the isolation of a 324 bp- long DNA fragment encoding the protein’s 108 amino acid N-terminus. Culture activity profiles and N-terminal sequence analysis indicated the release of this protein from the cell surface. -
Lawrence Berkeley National Laboratory Recent Work
Lawrence Berkeley National Laboratory Recent Work Title What we can deduce about metabolism in the moderate halophile chromohalobacter salexigens from its genomic sequence Permalink https://escholarship.org/uc/item/1df4q5m5 Authors Csonka, Laszlo N. O'Connor, Kathleen Larimer, Frank et al. Publication Date 2005 eScholarship.org Powered by the California Digital Library University of California 1 Biodata of Laszlo N. Csonka, author of “What We Can Deduce about Metabolism in the Moderate Halophile Chromohalobacter salexigens from its Genomic Sequence” Dr. Laszlo Csonka is a professor in the Department of Biological Sciences at Purdue University. He received his Ph. D. in 1975 at Harvard Medical School in the laboratory of Dr. Dan Fraenkel, studying the pathways of NADPH formation in Escherichia coli. His main research interests are the analysis of the response to osmotic stress in bacteria and plants, the connection between osmotic adaptation and thermotolerance, and carbon- source metabolism in bacteria. E-mail: [email protected] 2 3 WHAT WE CAN DEDUCE ABOUT METABOLISM IN THE MODERATE HALOPHILE CHROMOHALOBACTER SALEXIGENS FROM ITS GENOMIC SEQUENCE LASZLO N. CSONKA1, KATHLEEN O’CONNOR1, FRANK LARIMER2, PAUL RICHARDSON3, ALLA LAPIDUS3, ADAM D. EWING4, BRADLEY W. GOODNER4 and AHARON OREN5 1Department of Biological Sciences, Purdue University, West Lafayette IN 47907-1392, USA; 2Genome Analysis and Systems Modeling, Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge TN 37831, USA; 3DOE Joint Genome Institute, Walnut Creek CA 94598, USA; 4Department of Biology, Hiram College, Hiram OH 44234, USA; 5The Institute of Life Science, and the Moshe Shilo Minerva Center for Marine Biogeochemistry, The Hebrew University of Jerusalem, 91904, Israel 1. -
Enzymatic Synthesis of L-Fucose from L-Fuculose Using a Fucose Isomerase
Kim et al. Biotechnol Biofuels (2019) 12:282 https://doi.org/10.1186/s13068-019-1619-0 Biotechnology for Biofuels RESEARCH Open Access Enzymatic synthesis of L-fucose from L-fuculose using a fucose isomerase from Raoultella sp. and the biochemical and structural analyses of the enzyme In Jung Kim1†, Do Hyoung Kim1†, Ki Hyun Nam1,2 and Kyoung Heon Kim1* Abstract Background: L-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using L-fucose isomerase, which interconverts L-fucose and L-fuculose, can be an efcient way of producing L-fucose for industrial applications. Here, we performed biochemical and structural analyses of L-fucose isomerase identifed from a novel species of Raoultella (RdFucI). Results: RdFucI exhibited higher enzymatic activity for L-fuculose than for L-fucose, and the rate for the reverse reaction of converting L-fuculose to L-fucose was higher than that for the forward reaction of converting L-fucose to L-fuculose. In the equilibrium mixture, a much higher proportion of L-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of L-fucose from L-fuculose. When biochemical analysis was conducted using L-fuculose as the substrate, the optimal conditions for RdFucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not afected by reaction temperature in the range 2 of 30 to 50 °C. Furthermore, RdFucI was found to be a metalloenzyme requiring Mn + as a cofactor. The comparative crystal structural analysis of RdFucI revealed the distinct conformation of α7–α8 loop of RdFucI. -
Table 6. Putative Genes Involved in the Utilization of Carbohydrates in G
Table 6. Putative genes involved in the utilization of carbohydrates in G. thermodenitrificans NG80-2 genome Carbohydrates* Enzymes Gene ID Glycerol Glycerol Kinase GT1216 Glycerol-3-phosphate dehydrogenase, aerobic GT2089 NAD(P)H-dependent glycerol-3-phosphate dehydrogenase GT2153 Enolase GT3003 2,3-bisphosphoglycerate-independentphosphoglycerate mutase GT3004 Triosephosphate isomerase GT3005 3-phosphoglycerate kinase GT3006 Glyceraldehyde-3-phosphate dehydrogenase GT3007 Phosphoglycerate mutase GT1326 Pyruvate kinase GT2663 L-Arabinose L-arabinose isomerase GT1795 L-ribulokinase GT1796 L-ribulose 5-phosphate 4-epimerase GT1797 D-Ribose Ribokinase GT3174 Transketolase GT1187 Ribose 5-phosphate epimerase GT3316 D-Xylose Xylose kinase GT1756 Xylose isomerase GT1757 D-Galactose Galactokinase GT2086 Galactose-1-phosphate uridyltransferase GT2084 UDP-glucose 4-epimerase GT2085 Carbohydrates* Enzymes Gene ID D-Fructose 1-phosphofructokinase GT1727 Fructose-1,6-bisphosphate aldolase GT1805 Fructose-1,6-bisphosphate aldolase type II GT3331 Triosephosphate isomerase GT3005 D-Mannose Mannnose-6 phospate isomelase GT3398 6-phospho-1-fructokinase GT2664 D-Mannitol Mannitol-1-phosphate dehydrogenase GT1844 N-Acetylglucosamine N-acetylglucosamine-6-phosphate deacetylase GT2205 N-acetylglucosamine-6-phosphate isomerase GT2204 D-Maltose Alpha-1,4-glucosidase GT0528, GT1643 Sucrose Sucrose phosphorylase GT3215 D-Trehalose Alpha-glucosidase GT1643 Glucose kinase GT2381 Inositol Myo-inositol catabolism protein iolC;5-dehydro-2- GT1807 deoxygluconokinase -
High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma
molecules Article High-Resolution Mass Spectrometry-Based Approaches for the Detection and Quantification of Peptidase Activity in Plasma Elisa Maffioli 1,2 , Zhenze Jiang 3, Simona Nonnis 1,2 , Armando Negri 1,2, Valentina Romeo 1, Christopher B. Lietz 3, Vivian Hook 3,4, Giuseppe Ristagno 5, Giuseppe Baselli 6, Erik B. Kistler 7,8 , Federico Aletti 9, Anthony J. O’Donoghue 3,* and Gabriella Tedeschi 1,2,* 1 Department of Veterinary Medicine, University of Milano, 20133 Milano, Italy; elisa.maffi[email protected] (E.M.); [email protected] (S.N.); [email protected] (A.N.); [email protected] (V.R.) 2 Centre for Nanostructured Materials and Interfaces (CIMAINA), University of Milano, 20133 Milano, Italy 3 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA; [email protected] (Z.J.); [email protected] (C.B.L.); [email protected] (V.H.) 4 Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093, USA 5 Department of Pathophysiology and Transplantation, University of Milan, 20133 Milan, Italy; [email protected] 6 Dipartimento di Elettronica, Informazione e Bioingegneria, Politecnico di Milano, 20133 Milan, Italy; [email protected] 7 Department of Anesthesiology & Critical Care, University of California San Diego, La Jolla, CA 92093, USA; [email protected] 8 Department of Anesthesiology & Critical Care, VA San Diego HealthCare System, San Diego, CA 92161, USA 9 Department of Bioengineering, University of California San Diego, La Jolla, CA 92093, USA; [email protected] * Correspondence: [email protected] (A.J.O.); [email protected] (G.T.); Tel.: +1-8585345360 (A.J.O.); +39-02-50318127 (G.T.) Academic Editor: Paolo Iadarola Received: 28 July 2020; Accepted: 4 September 2020; Published: 6 September 2020 Abstract: Proteomic technologies have identified 234 peptidases in plasma but little quantitative information about the proteolytic activity has been uncovered. -
A Novel Pseudomonas Geniculata AGE Family Epimerase/Isomerase and Its Application in D-Mannose Synthesis
foods Article A Novel Pseudomonas geniculata AGE Family Epimerase/Isomerase and Its Application in d-Mannose Synthesis Zhanzhi Liu 1,2, Ying Li 1,2, Jing Wu 1,2 and Sheng Chen 1,2,* 1 State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China; [email protected] (Z.L.); [email protected] (Y.L.); [email protected] (J.W.) 2 School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China * Correspondence: [email protected] Received: 29 September 2020; Accepted: 3 December 2020; Published: 6 December 2020 Abstract: d-mannose has exhibited excellent physiological properties in the food, pharmaceutical, and feed industries. Therefore, emerging attention has been applied to enzymatic production of d-mannose due to its advantage over chemical synthesis. The gene age of N-acetyl-d-glucosamine 2-epimerase family epimerase/isomerase (AGEase) derived from Pseudomonas geniculata was amplified, and the recombinant P. geniculata AGEase was characterized. The optimal temperature and pH of P. geniculata AGEase were 60 ◦C and 7.5, respectively. The Km, kcat, and kcat/Km of P. geniculata AGEase for d-mannose were 49.2 8.5 mM, 476.3 4.0 s 1, and 9.7 0.5 s 1 mM 1, respectively. ± ± − ± − · − The recombinant P. geniculata AGEase was classified into the YihS enzyme subfamily in the AGE enzyme family by analyzing its substrate specificity and active center of the three-dimensional (3D) structure. Further studies on the kinetics of different substrates showed that the P.