Quorum sensing and the regulation of multicellular behaviour in Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a human enteropathogen and is the direct ancestor of Yersinia pestis, the causative agent of plague. For its pathogenicity Y. pseudotuberculosis harbours a virulence plasmid (pYV) encoding genes for a type-III-secretion system (T3SS) which inhibits host phagocytosis and inflammatory responses. Y. pseudotuberculosis regulates key phenotypes, including T3S, motility, aggregation and biofilm formation on Caenorhabditis elegans via quorum sensing (QS). This involves two LuxR/I type QS systems termed YpsR/I and YtbR/I and multiple N-acylhomoserine lactone (AHL) signal molecules. Recently, the T3S system was shown to promote biofilm formation on the surface of the nematode Caenorhabditis elegans at 22ºC and in turn QS and flagellar regulatory components were found to repress the T3S system via a regulatory hierarchy. To further expand this network, this study then set out to investigate the regulation of the aggregation phenotype at 37ºC. Aggregation experiments using the parent and QS mutants revealed that aggregation is downregulated by QS, since aggregation was enhanced in strains lacking ypsI/ytbI and ypsR/ytbR. Cultures lacking pYV or treated with proteinase K did not aggregate, suggesting that QS is regulating aggregation by repressing a pYV encoded and surface located aggregation factor(s). Tn5 transposon mutagenesis of pYV together with targeted mutagenesis of the key structural T3S injectisome components YscF and YscV revealed that aggregation is attenuated when cells are unable to express the injectisome, demonstrating that the aggregation factor is the T3S injectisome and that QS reduces its expression. N-Acetyl-D-glucosamine (GlcNAc) is known to play diverse roles in bacterial cells for signalling pathways that impact on virulence and is the major component of the extracellular matrix of Yersinia biofilms. The repressor of the GlcNAc metabolism, NagC, may also impact on QS in Y. pseudotuberculosis since it was found to bind to the ypsR promoter in pull-down experiments. By using QS gene and nagC promoter lux-fusions, NagC was revealed as a repressor of both ytbI and ypsI, as an early activator of ypsR and as an activator of ytbR at 37ºC. It was further demonstrated that there is reciprocal regulation by QS. These observations were extended by phenotypic assays showing that NagC represses the aggregation phenotype and may affect T3S and motility by regulating QS. In contrast the biofilm phenotype appears to be directly regulated by NagC. Given that QS promotes biofilm formation, the upregulation of ypsI and ytbI in a nagC mutant should not lead to reduction of biofilm. However, biofilm formation in the nagC mutant was attenuated and addition of 5 mM GlcNAc could restore this phenotype, suggesting that attenuation of biofilm formation is caused by an enhanced degradation of external GlcNAc by the nag-operon. Addition of GlcNAc or N-acetylmuramic acid to strains containing QS gene promoter lux fusions repressed QS gene expression, revealing that Y. pseudotuberculosis may modify its behaviour in presence of other bacteria by sensing cell wall turnover. Taken together these results demonstrate that GlcNAc and/or NagC impacts on T3S and injectisome mediated aggregation by regulating QS and that the injectisome of the T3SS is the aggregation factor, which may help and protect the bacteria in the early stages of host infection

NIST Interlaboratory Study on Glycosylation Analysis of Monoclonal Antibodies: Comparison of Results from Diverse Analytical Methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submit- Avenue, Silver Spring, Maryland 20993; 22Glycoscience Research Laboratory, Genos, Borongajska cesta 83h, 10 000 Zagreb, Croatia; 23Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacˇ ic´ a 1, 10 000 Zagreb, Croatia; 24Department of Chemistry, Georgia State University, 100 Piedmont Avenue, Atlanta, Georgia 30303; 25glyXera GmbH, Brenneckestrasse 20 * ZENIT / 39120 Magdeburg, Germany; 26Health Products and Foods Branch, Health Canada, AL 2201E, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, K1A 0K9 Canada; 27Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama Higashi-Hiroshima 739–8530 Japan; 28ImmunoGen, 830 Winter Street, Waltham, Massachusetts 02451; 29Department of Medical Physiology, Jagiellonian University Medical College, ul. Michalowskiego 12, 31–126 Krakow, Poland; 30Department of Pathology, Johns Hopkins University, 400 N. Broadway Street Baltimore, Maryland 21287; 31Mass Spec Core Facility, KBI Biopharma, 1101 Hamlin Road Durham, North Carolina 27704; 32Division of Mass Spectrometry, Korea Basic Science Institute, 162 YeonGuDanji-Ro, Ochang-eup, Cheongwon-gu, Cheongju Chungbuk, 363–883 Korea (South); 33Advanced Therapy Products Research Division, Korea National Institute of Food and Drug Safety, 187 Osongsaengmyeong 2-ro Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, 363–700, Korea (South); 34Center for Proteomics and Metabolomics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands; 35Ludger Limited, Culham Science Centre, Abingdon, Oxfordshire, OX14 3EB, United Kingdom; 36Biomolecular Discovery and Design Research Centre and ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, North Ryde, Australia; 37Proteomics, Central European Institute for Technology, Masaryk University, Kamenice 5, A26, 625 00 BRNO, Czech Republic; 38Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany; 39Department of Biomolecular Sciences, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany; 40AstraZeneca, Granta Park, Cambridgeshire, CB21 6GH United Kingdom; 41Merck, 2015 Galloping Hill Rd, Kenilworth, New Jersey 07033; 42Analytical R&D, MilliporeSigma, 2909 Laclede Ave. St. Louis, Missouri 63103; 43MS Bioworks, LLC, 3950 Varsity Drive Ann Arbor, Michigan 48108; 44MSD, Molenstraat 110, 5342 CC Oss, The Netherlands; 45Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5–1 Higashiyama, Myodaiji, Okazaki 444–8787 Japan; 46Graduate School of Pharmaceutical Sciences, Nagoya City University, 3–1 Tanabe-dori, Mizuhoku, Nagoya 467–8603 Japan; 47Medical & Biological Laboratories Co., Ltd, 2-22-8 Chikusa, Chikusa-ku, Nagoya 464–0858 Japan; 48National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG United Kingdom; 49Division of Biological Chemistry & Biologicals, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158–8501 Japan; 50New England Biolabs, Inc., 240 County Road, Ipswich, Massachusetts 01938; 51New York University, 100 Washington Square East New York City, New York 10003; 52Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom; 53GlycoScience Group, The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland; 54Department of Chemistry, North Carolina State University, 2620 Yarborough Drive Raleigh, North Carolina 27695; 55Pantheon, 201 College Road East Princeton, New Jersey 08540; 56Pfizer Inc., 1 Burtt Road Andover, Massachusetts 01810; 57Proteodynamics, ZI La Varenne 20–22 rue Henri et Gilberte Goudier 63200 RIOM, France; 58ProZyme, Inc., 3832 Bay Center Place Hayward, California 94545; 59Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1 Nishinokyo Kuwabara-cho Nakagyo-ku, Kyoto, 604 8511 Japan; 60Children’s GMP LLC, St. Jude Children’s Research Hospital, 262 Danny Thomas Place Memphis, Tennessee 38105; 61Sumitomo Bakelite Co., Ltd., 1–5 Muromati 1-Chome, Nishiku, Kobe, 651–2241 Japan; 62Synthon Biopharmaceuticals, Microweg 22 P.O. Box 7071, 6503 GN Nijmegen, The Netherlands; 63Takeda Pharmaceuticals International Co., 40 Landsdowne Street Cambridge, Massachusetts 02139; 64Department of Chemistry and Biochemistry, Texas Tech University, 2500 Broadway, Lubbock, Texas 79409; 65Thermo Fisher Scientific, 1214 Oakmead Parkway Sunnyvale, California 94085; 66United States Pharmacopeia India Pvt. Ltd. IKP Knowledge Park, Genome Valley, Shamirpet, Turkapally Village, Medchal District, Hyderabad 500 101 Telangana, India; 67Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 68Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada; 69Department of Chemistry, University of California, One Shields Ave, Davis, California 95616; 70Horva´ th Csaba Memorial Laboratory for Bioseparation Sciences, Research Center for Molecular Medicine, Doctoral School of Molecular Medicine, Faculty of Medicine, University of Debrecen, Debrecen, Egyetem ter 1, Hungary; 71Translational Glycomics Research Group, Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Veszprem, Egyetem ut 10, Hungary; 72Delaware Biotechnology Institute, University of Delaware, 15 Innovation Way Newark, Delaware 19711; 73Proteomics Core Facility, University of Gothenburg, Medicinaregatan 1G SE 41390 Gothenburg, Sweden; 74Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Institute of Biomedicine, Sahlgrenska Academy, Medicinaregatan 9A, Box 440, 405 30, Gothenburg, Sweden; 75Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska Academy at the University of Gothenburg, Bruna Straket 16, 41345 Gothenburg, Sweden; 76Department of Chemistry, University of Hamburg, Martin Luther King Pl. 6 20146 Hamburg, Germany; 77Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2; 78Laboratory of Mass Spectrometry of Interactions and Systems, University of Strasbourg, UMR Unistra-CNRS 7140, France; 79Natural and Medical Sciences Institute, University of Tu¨ bingen, Markwiesenstrae 55, 72770 Reutlingen, Germany; 80Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; 81Division of Bioanalytical Chemistry, Amsterdam Institute for Molecules, Medicines and Systems, Vrije Universiteit Amsterdam, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands; 82Department of Chemistry, Waters Corporation, 34 Maple Street Milford, Massachusetts 01757; 83Zoetis, 333 Portage St. Kalamazoo, Michigan 49007 Author’s Choice—Final version open access under the terms of the Creative Commons CC-BY license. Received July 24, 2019, and in revised form, August 26, 2019 Published, MCP Papers in Press, October 7, 2019, DOI 10.1074/mcp.RA119.001677 ER: NISTmAb Glycosylation Interlaboratory Study 12 Molecular & Cellular Proteomics 19.1 Downloaded from https://www.mcponline.org by guest on January 20, 2020 ted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide communityderived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Molecular & Cellular Proteomics 19: 11–30, 2020. DOI: 10.1074/mcp.RA119.001677.L

Quorum sensing and the regulation of multicellular behaviour in Yersinia pseudotuberculosis

Yersinia pseudotuberculosis is a human enteropathogen and is the direct ancestor of Yersinia pestis, the causative agent of plague. For its pathogenicity Y. pseudotuberculosis harbours a virulence plasmid (pYV) encoding genes for a type-III-secretion system (T3SS) which inhibits host phagocytosis and inflammatory responses. Y. pseudotuberculosis regulates key phenotypes, including T3S, motility, aggregation and biofilm formation on Caenorhabditis elegans via quorum sensing (QS). This involves two LuxR/I type QS systems termed YpsR/I and YtbR/I and multiple N-acylhomoserine lactone (AHL) signal molecules. Recently, the T3S system was shown to promote biofilm formation on the surface of the nematode Caenorhabditis elegans at 22ºC and in turn QS and flagellar regulatory components were found to repress the T3S system via a regulatory hierarchy. To further expand this network, this study then set out to investigate the regulation of the aggregation phenotype at 37ºC. Aggregation experiments using the parent and QS mutants revealed that aggregation is downregulated by QS, since aggregation was enhanced in strains lacking ypsI/ytbI and ypsR/ytbR. Cultures lacking pYV or treated with proteinase K did not aggregate, suggesting that QS is regulating aggregation by repressing a pYV encoded and surface located aggregation factor(s). Tn5 transposon mutagenesis of pYV together with targeted mutagenesis of the key structural T3S injectisome components YscF and YscV revealed that aggregation is attenuated when cells are unable to express the injectisome, demonstrating that the aggregation factor is the T3S injectisome and that QS reduces its expression. N-Acetyl-D-glucosamine (GlcNAc) is known to play diverse roles in bacterial cells for signalling pathways that impact on virulence and is the major component of the extracellular matrix of Yersinia biofilms. The repressor of the GlcNAc metabolism, NagC, may also impact on QS in Y. pseudotuberculosis since it was found to bind to the ypsR promoter in pull-down experiments. By using QS gene and nagC promoter lux-fusions, NagC was revealed as a repressor of both ytbI and ypsI, as an early activator of ypsR and as an activator of ytbR at 37ºC. It was further demonstrated that there is reciprocal regulation by QS. These observations were extended by phenotypic assays showing that NagC represses the aggregation phenotype and may affect T3S and motility by regulating QS. In contrast the biofilm phenotype appears to be directly regulated by NagC. Given that QS promotes biofilm formation, the upregulation of ypsI and ytbI in a nagC mutant should not lead to reduction of biofilm. However, biofilm formation in the nagC mutant was attenuated and addition of 5 mM GlcNAc could restore this phenotype, suggesting that attenuation of biofilm formation is caused by an enhanced degradation of external GlcNAc by the nag-operon. Addition of GlcNAc or N-acetylmuramic acid to strains containing QS gene promoter lux fusions repressed QS gene expression, revealing that Y. pseudotuberculosis may modify its behaviour in presence of other bacteria by sensing cell wall turnover. Taken together these results demonstrate that GlcNAc and/or NagC impacts on T3S and injectisome mediated aggregation by regulating QS and that the injectisome of the T3SS is the aggregation factor, which may help and protect the bacteria in the early stages of host infection

Energy conservation in the acetogenic bacterium clostridium aceticum

In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum

Homologous production, one-step purification, and proof of Na+ transport by the Rnf complex from Acetobacterium woodii, a model for acetogenic conversion of C1 substrates to biofuels

Background: Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study. Results: We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis. Discussion: We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme

The Rnf complex is an energy-coupled transhydrogenase essential to reversibly link cellular NADH and ferredoxin pools in the acetogen acetobacterium woodii

© 2018 American Society for Microbiology. The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD + , and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H 2 plus CO 2 , nor did it produce acetate or ATP from H 2 plus CO 2 , and ferredoxin:NAD + oxidoreductase activity and Na + translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD + , and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO 2 reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H 2 to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates. IMPORTANCE Ferredoxin and NAD + are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD + couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD + . When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD + generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO 2 reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria

It does not always take two to tango: "Syntrophy" via hydrogen cycling in one bacterial cell

Interspecies hydrogen transfer in anoxic ecosystems is essential for the complete microbial breakdown of organic matter to methane. Acetogenic bacteria are key players in anaerobic food webs and have been considered as prime candidates for hydrogen cycling. We have tested this hypothesis by mutational analysis of the hydrogenase in the model acetogen Acetobacterium woodii. Hydrogenase-deletion mutants no longer grew on H2 + CO2 or organic substrates such as fructose, lactate, or ethanol. Heterotrophic growth could be restored by addition of molecular hydrogen to the culture, indicating that hydrogen is an intermediate in heterotrophic growth. Indeed, hydrogen production from fructose was detected in a stirred-tank reactor. The mutant grew well on organic substrates plus caffeate, an alternative electron acceptor that does not require molecular hydrogen but NADH as reductant. These data are consistent with the notion that molecular hydrogen is produced from organic substrates and then used as reductant for CO2 reduction. Surprisingly, hydrogen cycling in A. woodii is different from the known modes of interspecies or intraspecies hydrogen cycling. Our data are consistent with a novel type of hydrogen cycling that connects an oxidative and reductive metabolic module in one bacterial cell, "intracellular syntrophy.

Revealing formate production from carbon monoxide in wild type and mutants of Rnf‐ and Ech‐containing acetogens, Acetobacterium woodii and Thermoanaerobacter kivui

Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar‐based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many acetogens. In this study, we focused on the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified acetogens harbouring a H2‐dependent CO2 reductase and furthermore belong to different classes of ‘Rnf’‐ and ‘Ech‐acetogens’. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood–Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g−1 h−1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO‐based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms