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Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa
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| Zeitschriftentitel: | Journal of Bacteriology |
|---|---|
| Personen und Körperschaften: | , |
| In: | Journal of Bacteriology, 184, 2002, 6, S. 1503-1513 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
|
| Schlagwörter: |
| author_facet |
Zhao, Qixun Poole, Keith Zhao, Qixun Poole, Keith |
|---|---|
| author |
Zhao, Qixun Poole, Keith |
| spellingShingle |
Zhao, Qixun Poole, Keith Journal of Bacteriology Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa Molecular Biology Microbiology |
| author_sort |
zhao, qixun |
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Zhao, Qixun Poole, Keith 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.184.6.1503-1513.2002 <jats:title>ABSTRACT</jats:title><jats:p>Siderophore-mediated iron transport in<jats:italic>Pseudomonas aeruginosa</jats:italic>is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the<jats:italic>tonB1</jats:italic>gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>Escherichia coli</jats:italic>. Appending it to the N terminus of the nonfunctional (in<jats:italic>P. aeruginosa</jats:italic>)<jats:italic>Escherichia coli</jats:italic>TonB protein (TonB<jats:sub>Ec</jats:sub>) rendered TonB<jats:sub>Ec</jats:sub>weakly active in<jats:italic>P. aeruginosa</jats:italic>and did not compromise the activity of this protein in<jats:italic>E. coli</jats:italic>. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB<jats:sub>Ec</jats:sub>function in<jats:italic>E. coli</jats:italic>, was shown here to be essential for TonB1 activity in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in<jats:italic>P. aeruginosa</jats:italic>. Several TonB1-TonB<jats:sub>Ec</jats:sub>chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in<jats:italic>P. aeruginosa</jats:italic>, although chimeras possessing an<jats:italic>E. coli</jats:italic>C terminus were active in<jats:italic>E. coli</jats:italic>.</jats:p> Mutational Analysis of the TonB1 Energy Coupler of<i>Pseudomonas aeruginosa</i> Journal of Bacteriology |
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American Society for Microbiology, 2002 |
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2002 |
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American Society for Microbiology |
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Journal of Bacteriology |
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49 |
| title |
Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_unstemmed |
Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_full |
Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_fullStr |
Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_full_unstemmed |
Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_short |
Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_sort |
mutational analysis of the tonb1 energy coupler of<i>pseudomonas aeruginosa</i> |
| topic |
Molecular Biology Microbiology |
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http://dx.doi.org/10.1128/jb.184.6.1503-1513.2002 |
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2002 |
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1503-1513 |
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<jats:title>ABSTRACT</jats:title><jats:p>Siderophore-mediated iron transport in<jats:italic>Pseudomonas aeruginosa</jats:italic>is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the<jats:italic>tonB1</jats:italic>gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>Escherichia coli</jats:italic>. Appending it to the N terminus of the nonfunctional (in<jats:italic>P. aeruginosa</jats:italic>)<jats:italic>Escherichia coli</jats:italic>TonB protein (TonB<jats:sub>Ec</jats:sub>) rendered TonB<jats:sub>Ec</jats:sub>weakly active in<jats:italic>P. aeruginosa</jats:italic>and did not compromise the activity of this protein in<jats:italic>E. coli</jats:italic>. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB<jats:sub>Ec</jats:sub>function in<jats:italic>E. coli</jats:italic>, was shown here to be essential for TonB1 activity in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in<jats:italic>P. aeruginosa</jats:italic>. Several TonB1-TonB<jats:sub>Ec</jats:sub>chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in<jats:italic>P. aeruginosa</jats:italic>, although chimeras possessing an<jats:italic>E. coli</jats:italic>C terminus were active in<jats:italic>E. coli</jats:italic>.</jats:p> |
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| description | <jats:title>ABSTRACT</jats:title><jats:p>Siderophore-mediated iron transport in<jats:italic>Pseudomonas aeruginosa</jats:italic>is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the<jats:italic>tonB1</jats:italic>gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>Escherichia coli</jats:italic>. Appending it to the N terminus of the nonfunctional (in<jats:italic>P. aeruginosa</jats:italic>)<jats:italic>Escherichia coli</jats:italic>TonB protein (TonB<jats:sub>Ec</jats:sub>) rendered TonB<jats:sub>Ec</jats:sub>weakly active in<jats:italic>P. aeruginosa</jats:italic>and did not compromise the activity of this protein in<jats:italic>E. coli</jats:italic>. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB<jats:sub>Ec</jats:sub>function in<jats:italic>E. coli</jats:italic>, was shown here to be essential for TonB1 activity in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in<jats:italic>P. aeruginosa</jats:italic>. Several TonB1-TonB<jats:sub>Ec</jats:sub>chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in<jats:italic>P. aeruginosa</jats:italic>, although chimeras possessing an<jats:italic>E. coli</jats:italic>C terminus were active in<jats:italic>E. coli</jats:italic>.</jats:p> |
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| spelling | Zhao, Qixun Poole, Keith 0021-9193 1098-5530 American Society for Microbiology Molecular Biology Microbiology http://dx.doi.org/10.1128/jb.184.6.1503-1513.2002 <jats:title>ABSTRACT</jats:title><jats:p>Siderophore-mediated iron transport in<jats:italic>Pseudomonas aeruginosa</jats:italic>is dependent upon the cytoplasmic membrane-associated TonB1 energy coupling protein for activity. To assess the functional significance of the various regions of this molecule and to identify functionally important residues, the<jats:italic>tonB1</jats:italic>gene was subjected to site-directed mutagenesis, and the influence on iron acquisition was determined. The novel N-terminal extension of TonB1, which is absent in all other examples of TonB, was required for TonB1 activity in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>Escherichia coli</jats:italic>. Appending it to the N terminus of the nonfunctional (in<jats:italic>P. aeruginosa</jats:italic>)<jats:italic>Escherichia coli</jats:italic>TonB protein (TonB<jats:sub>Ec</jats:sub>) rendered TonB<jats:sub>Ec</jats:sub>weakly active in<jats:italic>P. aeruginosa</jats:italic>and did not compromise the activity of this protein in<jats:italic>E. coli</jats:italic>. Elimination of the membrane-spanning, presumed membrane anchor sequence of TonB1 abrogated TonB1 activity in<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>. Interestingly, however, a conserved His residue within the membrane anchor sequence, shown to be required for TonB<jats:sub>Ec</jats:sub>function in<jats:italic>E. coli</jats:italic>, was shown here to be essential for TonB1 activity in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. Several mutations within the C-terminal end of TonB1, within a region exhibiting the greatest similarity to other TonB proteins, compromised a TonB1 contribution to iron acquisition in both<jats:italic>P. aeruginosa</jats:italic>and<jats:italic>E. coli</jats:italic>, including substitutions at Tyr264, Glu274, Lys278, and Asp304. Mutations at Pro265, Gln293, and Val294 also impacted negatively on TonB1 function in<jats:italic>E. coli</jats:italic>but not in<jats:italic>P. aeruginosa</jats:italic>. The Asp304 mutation was suppressed by a second mutation at Glu274 of TonB1 but only in<jats:italic>P. aeruginosa</jats:italic>. Several TonB1-TonB<jats:sub>Ec</jats:sub>chimeras were constructed, and assessment of their activities revealed that substitutions at the N or C terminus of TonB1 compromised its activity in<jats:italic>P. aeruginosa</jats:italic>, although chimeras possessing an<jats:italic>E. coli</jats:italic>C terminus were active in<jats:italic>E. coli</jats:italic>.</jats:p> Mutational Analysis of the TonB1 Energy Coupler of<i>Pseudomonas aeruginosa</i> Journal of Bacteriology |
| spellingShingle | Zhao, Qixun, Poole, Keith, Journal of Bacteriology, Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa, Molecular Biology, Microbiology |
| title | Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_full | Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_fullStr | Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_full_unstemmed | Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_short | Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| title_sort | mutational analysis of the tonb1 energy coupler of<i>pseudomonas aeruginosa</i> |
| title_unstemmed | Mutational Analysis of the TonB1 Energy Coupler ofPseudomonas aeruginosa |
| topic | Molecular Biology, Microbiology |
| url | http://dx.doi.org/10.1128/jb.184.6.1503-1513.2002 |