Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1 O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS
The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, bot...
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| Veröffentlicht in: | Microbial pathogenesis 1996, Vol.20 (1), p.11-30 |
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| creator | FÄLT, I. C MILLS, D SCHWEDA, E. K. H TIMMIS, K. N LINDBERG, A. A |
| description | The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced. All derivatives but one (SL3235) stably inherited the new trait. Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS. Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccharide with the following structure, [formula: see text] demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core. |
| doi_str_mv | 10.1006/mpat.1996.0002 |
| format | Article |
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C ; MILLS, D ; SCHWEDA, E. K. H ; TIMMIS, K. N ; LINDBERG, A. A</creator><creatorcontrib>FÄLT, I. C ; MILLS, D ; SCHWEDA, E. K. H ; TIMMIS, K. N ; LINDBERG, A. A</creatorcontrib><description>The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced. All derivatives but one (SL3235) stably inherited the new trait. Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS. Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccharide with the following structure, [formula: see text] demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core.</description><identifier>ISSN: 0882-4010</identifier><identifier>EISSN: 1096-1208</identifier><identifier>DOI: 10.1006/mpat.1996.0002</identifier><identifier>PMID: 8692007</identifier><identifier>CODEN: MIPAEV</identifier><language>eng</language><publisher>Oxford: Elsevier</publisher><subject>3-Phosphoshikimate 1-Carboxyvinyltransferase ; Alkyl and Aryl Transferases ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Bacteriology ; Bacteriophage P22 - genetics ; Biological and medical sciences ; Carbohydrate Conformation ; Carbohydrate Sequence ; DNA Transposable Elements - genetics ; Fundamental and applied biological sciences. Psychology ; Lipid A - biosynthesis ; Lipid A - chemistry ; Lipopolysaccharides - biosynthesis ; Lipopolysaccharides - chemistry ; Microbiology ; Molecular Sequence Data ; Morphology, structure, chemical composition ; Nuclear Proteins - genetics ; Nuclear Proteins - metabolism ; O Antigens - biosynthesis ; O Antigens - immunology ; Recombinant Proteins - metabolism ; Salmonella - genetics ; Salmonella - metabolism ; Salmonella dublin ; Salmonella typhimurium ; Salmonella typhimurium - genetics ; Salmonella typhimurium - immunology ; Salmonella typhimurium - metabolism ; Shigella dysenteriae ; Shigella dysenteriae - genetics ; Shigella dysenteriae - immunology ; Transferases - genetics</subject><ispartof>Microbial pathogenesis, 1996, Vol.20 (1), p.11-30</ispartof><rights>1996 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,777,781,4010,27904,27905,27906</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3039590$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8692007$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>FÄLT, I. C</creatorcontrib><creatorcontrib>MILLS, D</creatorcontrib><creatorcontrib>SCHWEDA, E. K. H</creatorcontrib><creatorcontrib>TIMMIS, K. N</creatorcontrib><creatorcontrib>LINDBERG, A. A</creatorcontrib><title>Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1 O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS</title><title>Microbial pathogenesis</title><addtitle>Microb Pathog</addtitle><description>The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced. All derivatives but one (SL3235) stably inherited the new trait. Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS. Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccharide with the following structure, [formula: see text] demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core.</description><subject>3-Phosphoshikimate 1-Carboxyvinyltransferase</subject><subject>Alkyl and Aryl Transferases</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Bacteriology</subject><subject>Bacteriophage P22 - genetics</subject><subject>Biological and medical sciences</subject><subject>Carbohydrate Conformation</subject><subject>Carbohydrate Sequence</subject><subject>DNA Transposable Elements - genetics</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Lipid A - biosynthesis</subject><subject>Lipid A - chemistry</subject><subject>Lipopolysaccharides - biosynthesis</subject><subject>Lipopolysaccharides - chemistry</subject><subject>Microbiology</subject><subject>Molecular Sequence Data</subject><subject>Morphology, structure, chemical composition</subject><subject>Nuclear Proteins - genetics</subject><subject>Nuclear Proteins - metabolism</subject><subject>O Antigens - biosynthesis</subject><subject>O Antigens - immunology</subject><subject>Recombinant Proteins - metabolism</subject><subject>Salmonella - genetics</subject><subject>Salmonella - metabolism</subject><subject>Salmonella dublin</subject><subject>Salmonella typhimurium</subject><subject>Salmonella typhimurium - genetics</subject><subject>Salmonella typhimurium - immunology</subject><subject>Salmonella typhimurium - metabolism</subject><subject>Shigella dysenteriae</subject><subject>Shigella dysenteriae - genetics</subject><subject>Shigella dysenteriae - immunology</subject><subject>Transferases - genetics</subject><issn>0882-4010</issn><issn>1096-1208</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1996</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkT2P1DAQhi0EOpaDlg7JBaLL3jhO_FGeVseHtNIhHdSriWPfGiV2sJ0i_Cv-IVlu2ZZqivfx887IhLxlsGUA4macsGyZ1mILAPUzsmGgRcVqUM_JBpSqqwYYvCSvcv6xErrh-opcKaFrALkhv3cx5JJmU3wMNDqarIlj5wOGQjHFW5pxGGOww4CW5oLdsNApxX42PjzScrT04egfTzHtl2xDscmfSJtiWSZLGb2vVteKBIqhp09lc8KBmiMmNKcHv_Bf_V_hpfHm4j4uXfI93X99eE1eOByyfXOe1-T7x7tvu8_V_v7Tl93tvppqIUrFOdhWttAYx6TlrutYr1A1XDrGRcMYGFSua6x2shW1ca3UnRBoley56S2_Jh-evOu1P2eby2H02ZyWCTbO-SAV460W9X9BJqFpGWcr-O4Mzt1o-8OU_IhpOZw_Y83fn3PMBgeXMBifLxgHrlsN_A_gY504</recordid><startdate>1996</startdate><enddate>1996</enddate><creator>FÄLT, I. C</creator><creator>MILLS, D</creator><creator>SCHWEDA, E. K. H</creator><creator>TIMMIS, K. N</creator><creator>LINDBERG, A. A</creator><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QL</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>1996</creationdate><title>Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1 O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS</title><author>FÄLT, I. C ; MILLS, D ; SCHWEDA, E. K. H ; TIMMIS, K. N ; LINDBERG, A. A</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p266t-330e57504cf17e3fbb1d8a8437f1364110ca8fb4e9f7562cf579b66ae87d3cde3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1996</creationdate><topic>3-Phosphoshikimate 1-Carboxyvinyltransferase</topic><topic>Alkyl and Aryl Transferases</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Bacteriology</topic><topic>Bacteriophage P22 - genetics</topic><topic>Biological and medical sciences</topic><topic>Carbohydrate Conformation</topic><topic>Carbohydrate Sequence</topic><topic>DNA Transposable Elements - genetics</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Lipid A - biosynthesis</topic><topic>Lipid A - chemistry</topic><topic>Lipopolysaccharides - biosynthesis</topic><topic>Lipopolysaccharides - chemistry</topic><topic>Microbiology</topic><topic>Molecular Sequence Data</topic><topic>Morphology, structure, chemical composition</topic><topic>Nuclear Proteins - genetics</topic><topic>Nuclear Proteins - metabolism</topic><topic>O Antigens - biosynthesis</topic><topic>O Antigens - immunology</topic><topic>Recombinant Proteins - metabolism</topic><topic>Salmonella - genetics</topic><topic>Salmonella - metabolism</topic><topic>Salmonella dublin</topic><topic>Salmonella typhimurium</topic><topic>Salmonella typhimurium - genetics</topic><topic>Salmonella typhimurium - immunology</topic><topic>Salmonella typhimurium - metabolism</topic><topic>Shigella dysenteriae</topic><topic>Shigella dysenteriae - genetics</topic><topic>Shigella dysenteriae - immunology</topic><topic>Transferases - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>FÄLT, I. C</creatorcontrib><creatorcontrib>MILLS, D</creatorcontrib><creatorcontrib>SCHWEDA, E. K. H</creatorcontrib><creatorcontrib>TIMMIS, K. N</creatorcontrib><creatorcontrib>LINDBERG, A. A</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Microbial pathogenesis</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>FÄLT, I. C</au><au>MILLS, D</au><au>SCHWEDA, E. K. H</au><au>TIMMIS, K. N</au><au>LINDBERG, A. A</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1 O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS</atitle><jtitle>Microbial pathogenesis</jtitle><addtitle>Microb Pathog</addtitle><date>1996</date><risdate>1996</risdate><volume>20</volume><issue>1</issue><spage>11</spage><epage>30</epage><pages>11-30</pages><issn>0882-4010</issn><eissn>1096-1208</eissn><coden>MIPAEV</coden><abstract>The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin. In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced. All derivatives but one (SL3235) stably inherited the new trait. Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS. Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit). The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain. This hybrid organism produced a polysaccharide with the following structure, [formula: see text] demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core.</abstract><cop>Oxford</cop><pub>Elsevier</pub><pmid>8692007</pmid><doi>10.1006/mpat.1996.0002</doi><tpages>20</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0882-4010 |
| ispartof | Microbial pathogenesis, 1996, Vol.20 (1), p.11-30 |
| issn | 0882-4010 1096-1208 |
| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | 3-Phosphoshikimate 1-Carboxyvinyltransferase Alkyl and Aryl Transferases Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacteriology Bacteriophage P22 - genetics Biological and medical sciences Carbohydrate Conformation Carbohydrate Sequence DNA Transposable Elements - genetics Fundamental and applied biological sciences. Psychology Lipid A - biosynthesis Lipid A - chemistry Lipopolysaccharides - biosynthesis Lipopolysaccharides - chemistry Microbiology Molecular Sequence Data Morphology, structure, chemical composition Nuclear Proteins - genetics Nuclear Proteins - metabolism O Antigens - biosynthesis O Antigens - immunology Recombinant Proteins - metabolism Salmonella - genetics Salmonella - metabolism Salmonella dublin Salmonella typhimurium Salmonella typhimurium - genetics Salmonella typhimurium - immunology Salmonella typhimurium - metabolism Shigella dysenteriae Shigella dysenteriae - genetics Shigella dysenteriae - immunology Transferases - genetics |
| title | Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1 O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS |
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