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Volume 153, Issue 2

Identification of amino acid residues required for ferric-anguibactin transport in the outer-membrane receptor FatA of Free

Abstract

775 is a fish pathogen that causes a disease characterized by a fatal haemorrhagic septicaemia. It harbours the 65 kbp pJM1 plasmid, which encodes an iron sequestering system specific for the siderophore anguibactin and is essential for virulence. The genes involved in the biosynthesis of anguibactin are located on both the pJM1 plasmid and the chromosome. However, the genes for the outer-membrane receptor FatA and the other transport proteins are only carried on the plasmid. With the aim of elucidating the mechanism of ferric-anguibactin transport mediated by FatA, this work focuses on the identification of FatA amino acid residues that play a role in the transport of ferric-anguibactin, by analysing the transport kinetics of site-directed mutants. The mutations studied were located in conserved residues of the lock region, which contains a cluster of ten residues belonging to the N-terminal and barrel domains, and of the channel region of FatA, which contains conserved glycines located in the 5-6 loop and a conserved arginine located in strand 11 of the -barrel. In the case of the FatA lock region, it is clear that although the residues analysed in this work (R95, K130, E505 and E550) are conserved among various outer-membrane receptors, their involvement in the transport process might differ among receptors. Furthermore, it was determined that in the FatA channel region double substitutions of the conserved glycines 131 and 143 with alanine resulted in a variant receptor unable to transport ferric-anguibactin. It was also shown that the conserved arginine 428 located in strand 11 is essential for transport. The results suggest that a conformational change or partial unfolding of the plug domain occurs during ferric-anguibactin transport.

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Keyword(s): EDDHA, ethylenediamine-di-(o-hydroxyphenylacetic) acid , ITB, iron transport biosynthesis , KD, dissociation constant , Km, Michaelis constant and SOE, splicing by overlap extension
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2007-02-01
2024-04-23
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Identification of amino acid residues required for ferric-anguibactin transport in the outer-membrane receptor FatA of Vibrio anguillarum
Microbiology 153, 570 (2007); https://doi.org/10.1099/mic.0.2006/001735-0
/content/journal/micro/10.1099/mic.0.2006/001735-0
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