Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin

Summary Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 ins...

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Veröffentlicht in:Molecular microbiology 1993-02, Vol.7 (3), p.461-469
Hauptverfasser: Henderson, Douglas P., Payne, Shelley M.
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description Summary Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS‐PAGE of protein fractions indicate that pHUT10 (a subclone of p>HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem‐iron utilization genes allow transport of the entire haem moiety into the cell.
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Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS‐PAGE of protein fractions indicate that pHUT10 (a subclone of p&gt;HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. 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Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS‐PAGE of protein fractions indicate that pHUT10 (a subclone of p&gt;HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem‐iron utilization genes allow transport of the entire haem moiety into the cell.</description><subject>Aldehyde Oxidoreductases - genetics</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacteriology</subject><subject>beta-Galactosidase - biosynthesis</subject><subject>beta-Galactosidase - genetics</subject><subject>Biological and medical sciences</subject><subject>Cloning, Molecular</subject><subject>DNA Transposable Elements</subject><subject>DNA, Recombinant - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Fundamental and applied biological sciences. 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Psychology</topic><topic>Genes, Bacterial - genetics</topic><topic>Genetic Complementation Test</topic><topic>Heme - metabolism</topic><topic>Hemin - metabolism</topic><topic>Hemoglobins - metabolism</topic><topic>Iron - metabolism</topic><topic>Membrane Proteins - genetics</topic><topic>Microbiology</topic><topic>Mutagenesis, Insertional</topic><topic>Permeability, membrane transport, intracellular transport</topic><topic>Restriction Mapping</topic><topic>Siderophores - genetics</topic><topic>Transformation, Genetic</topic><topic>Vibrio cholerae</topic><topic>Vibrio cholerae - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Henderson, Douglas P.</creatorcontrib><creatorcontrib>Payne, Shelley M.</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>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Molecular microbiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Henderson, Douglas P.</au><au>Payne, Shelley M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin</atitle><jtitle>Molecular microbiology</jtitle><addtitle>Mol Microbiol</addtitle><date>1993-02</date><risdate>1993</risdate><volume>7</volume><issue>3</issue><spage>461</spage><epage>469</epage><pages>461-469</pages><issn>0950-382X</issn><eissn>1365-2958</eissn><abstract>Summary Vibrio cholerae can utilize haemin or haemoglobin as its sole source of iron. Four haem utilization mutants of a classical strain of V. cholerae were isolated. These mutations were complemented with pHUT1, a cosmid clone isolated from a library of wild‐type CA401 DNA. Two independent Tn5 insertions into the cloned sequence disrupted function in all of the complemented mutants. Escherichia coli 1017 transformed with pHUT1 failed to utilize haemin as an iron source; a second plasmid containing a different cloned fragment of V. cholerae DNA (pHUT3) was required in addition to pHUT1 to reconstitute the system in E. coli. Minicell analysis and SDS‐PAGE of protein fractions indicate that pHUT10 (a subclone of p&gt;HUT1) encodes a 26 kDa inner membrane protein, and pHUT3 encodes a 77 kDa outer membrane protein. Loss of either protein by Tn5 mutagenesis abolishes haem utilization in E. coli. An E. coli hemA mutant that cannot synthesize porphyrins was transformed with the recombinant plasmids to determine whether the plasmids encoded the ability to transport the porphyrin as well as the iron. The transformants grew aerobically in media containing haemin, whereas the parental strain was unable to grow under these conditions. This indicates that V. cholerae haem‐iron utilization genes allow transport of the entire haem moiety into the cell.</abstract><cop>Oxford, UK</cop><pub>Blackwell Publishing Ltd</pub><pmid>8384684</pmid><doi>10.1111/j.1365-2958.1993.tb01137.x</doi><tpages>9</tpages></addata></record>
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subjects Aldehyde Oxidoreductases - genetics
Bacterial Proteins - genetics
Bacteriology
beta-Galactosidase - biosynthesis
beta-Galactosidase - genetics
Biological and medical sciences
Cloning, Molecular
DNA Transposable Elements
DNA, Recombinant - genetics
Escherichia coli - metabolism
Fundamental and applied biological sciences. Psychology
Genes, Bacterial - genetics
Genetic Complementation Test
Heme - metabolism
Hemin - metabolism
Hemoglobins - metabolism
Iron - metabolism
Membrane Proteins - genetics
Microbiology
Mutagenesis, Insertional
Permeability, membrane transport, intracellular transport
Restriction Mapping
Siderophores - genetics
Transformation, Genetic
Vibrio cholerae
Vibrio cholerae - genetics
title Cloning and characterization of the Vibrio cholerae genes encoding the utilization of iron from haemin and haemoglobin
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