Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants
The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important am...
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| Veröffentlicht in: | Journal of bacteriology 2015-01, Vol.197 (1), p.108-119 |
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| creator | Nath, Pratiti Tran, Elizabeth Ngoc Hoa Morona, Renato |
| description | The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability. |
| doi_str_mv | 10.1128/JB.01885-14 |
| format | Article |
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S.</contributor><creatorcontrib>Nath, Pratiti ; Tran, Elizabeth Ngoc Hoa ; Morona, Renato ; Parkinson, J. S.</creatorcontrib><description>The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.</description><identifier>ISSN: 0021-9193</identifier><identifier>EISSN: 1098-5530</identifier><identifier>DOI: 10.1128/JB.01885-14</identifier><identifier>PMID: 25313393</identifier><identifier>CODEN: JOBAAY</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Amino Acid Sequence ; Amino acids ; Antigens ; Bacterial proteins ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Bacteriology ; bacteriophages ; Biosynthesis ; Cloning, Molecular ; colicins ; Gene Expression Regulation, Bacterial - physiology ; Gene Expression Regulation, Enzymologic - physiology ; genes ; Genetic Complementation Test ; Glycosyltransferases - genetics ; Glycosyltransferases - metabolism ; Gram-negative bacteria ; Green Fluorescent Proteins ; lipopolysaccharides ; Molecular Sequence Data ; Mutagenesis ; Mutants ; Mutation ; Plasmids ; polymerization ; Protein Conformation ; screening ; Shigella flexneri ; Shigella flexneri - enzymology ; virulence</subject><ispartof>Journal of bacteriology, 2015-01, Vol.197 (1), p.108-119</ispartof><rights>Copyright © 2015, American Society for Microbiology. All Rights Reserved.</rights><rights>Copyright American Society for Microbiology Jan 2015</rights><rights>Copyright © 2015, American Society for Microbiology. All Rights Reserved. 2015 American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c499t-761b5c06cb4fe56f47b9a90da41fdc02be3724b35afec827097cb74a84c859fc3</citedby><cites>FETCH-LOGICAL-c499t-761b5c06cb4fe56f47b9a90da41fdc02be3724b35afec827097cb74a84c859fc3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288684/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288684/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27903,27904,53768,53770</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25313393$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Parkinson, J. S.</contributor><creatorcontrib>Nath, Pratiti</creatorcontrib><creatorcontrib>Tran, Elizabeth Ngoc Hoa</creatorcontrib><creatorcontrib>Morona, Renato</creatorcontrib><title>Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants</title><title>Journal of bacteriology</title><addtitle>J Bacteriol</addtitle><description>The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.</description><subject>Amino Acid Sequence</subject><subject>Amino acids</subject><subject>Antigens</subject><subject>Bacterial proteins</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Bacteriology</subject><subject>bacteriophages</subject><subject>Biosynthesis</subject><subject>Cloning, Molecular</subject><subject>colicins</subject><subject>Gene Expression Regulation, Bacterial - physiology</subject><subject>Gene Expression Regulation, Enzymologic - physiology</subject><subject>genes</subject><subject>Genetic Complementation Test</subject><subject>Glycosyltransferases - genetics</subject><subject>Glycosyltransferases - metabolism</subject><subject>Gram-negative bacteria</subject><subject>Green Fluorescent Proteins</subject><subject>lipopolysaccharides</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Plasmids</subject><subject>polymerization</subject><subject>Protein Conformation</subject><subject>screening</subject><subject>Shigella flexneri</subject><subject>Shigella flexneri - enzymology</subject><subject>virulence</subject><issn>0021-9193</issn><issn>1098-5530</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNks9vFCEcxYnR2G315F1JvJg0U2GAAXow2dYfbVNTk9r0SBgWdmlmhxVmGnf_-kK3bdSTF0h4nzy-vAcAbzA6wLgWH8-ODhAWglWYPgMTjKSoGCPoOZggVONKYkl2wG5KNwhhSln9EuzUjGBCJJmA-H0c9OBDrzs4zcs6-QSDg8PCwsuFn9uu09B19ndvo4cX1bQf8mEPf4RuvbRRJwuvN-tDeDqzWXHe3JsVh-vNpvpsV7YvSoFguaof0ivwwuku2dcP-x64-vrl5_FJdX7x7fR4el4ZKuVQ8Qa3zKDGtNRZ1jjKW6klmmmK3cygurWE17QlTDtrRM2R5KblVAtqBJPOkD3waeu7GtulnZk8RtSdWkW_1HGtgvbqb6X3CzUPt4rWQjSCZoMPDwYx_BptGtTSJ1MS6W0Yk8INF6yRgqD_QBltJKOMZ_T9P-hNGGOOvlA0t0cajjO1v6VMDClF657mxkiV2tXZkbqvXeEy6ds_n_rEPvacgXdbwOmg9Dz6pK4ua4RZ-RM5RkbuAPBSslQ</recordid><startdate>20150101</startdate><enddate>20150101</enddate><creator>Nath, Pratiti</creator><creator>Tran, Elizabeth Ngoc Hoa</creator><creator>Morona, Renato</creator><general>American Society for Microbiology</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20150101</creationdate><title>Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants</title><author>Nath, Pratiti ; Tran, Elizabeth Ngoc Hoa ; Morona, Renato</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c499t-761b5c06cb4fe56f47b9a90da41fdc02be3724b35afec827097cb74a84c859fc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Amino Acid Sequence</topic><topic>Amino acids</topic><topic>Antigens</topic><topic>Bacterial proteins</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Bacteriology</topic><topic>bacteriophages</topic><topic>Biosynthesis</topic><topic>Cloning, Molecular</topic><topic>colicins</topic><topic>Gene Expression Regulation, Bacterial - physiology</topic><topic>Gene Expression Regulation, Enzymologic - physiology</topic><topic>genes</topic><topic>Genetic Complementation Test</topic><topic>Glycosyltransferases - genetics</topic><topic>Glycosyltransferases - metabolism</topic><topic>Gram-negative bacteria</topic><topic>Green Fluorescent Proteins</topic><topic>lipopolysaccharides</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>Mutants</topic><topic>Mutation</topic><topic>Plasmids</topic><topic>polymerization</topic><topic>Protein Conformation</topic><topic>screening</topic><topic>Shigella flexneri</topic><topic>Shigella flexneri - enzymology</topic><topic>virulence</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nath, Pratiti</creatorcontrib><creatorcontrib>Tran, Elizabeth Ngoc Hoa</creatorcontrib><creatorcontrib>Morona, Renato</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of bacteriology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nath, Pratiti</au><au>Tran, Elizabeth Ngoc Hoa</au><au>Morona, Renato</au><au>Parkinson, J. S.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants</atitle><jtitle>Journal of bacteriology</jtitle><addtitle>J Bacteriol</addtitle><date>2015-01-01</date><risdate>2015</risdate><volume>197</volume><issue>1</issue><spage>108</spage><epage>119</epage><pages>108-119</pages><issn>0021-9193</issn><eissn>1098-5530</eissn><coden>JOBAAY</coden><abstract>The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant of Shigella flexneri and is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSf and pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on the wzySf gene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySf proteins in the wzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySf and WzzSf was investigated by transforming these mutated wzySf plasmids into a wzySf- and wzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzy and Δwzy Δwzz backgrounds identified WzySf mutants whose polymerization activities were WzzSf dependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzy and Δwzy Δwzz backgrounds identified a role for WzzSf in WzySf stability. Hence, in addition to its role in regulating Oag modal chain length, WzzSf also affects WzySf activity and stability.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>25313393</pmid><doi>10.1128/JB.01885-14</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Amino acids Antigens Bacterial proteins Bacterial Proteins - genetics Bacterial Proteins - metabolism Bacteriology bacteriophages Biosynthesis Cloning, Molecular colicins Gene Expression Regulation, Bacterial - physiology Gene Expression Regulation, Enzymologic - physiology genes Genetic Complementation Test Glycosyltransferases - genetics Glycosyltransferases - metabolism Gram-negative bacteria Green Fluorescent Proteins lipopolysaccharides Molecular Sequence Data Mutagenesis Mutants Mutation Plasmids polymerization Protein Conformation screening Shigella flexneri Shigella flexneri - enzymology virulence |
| title | Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants |
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