Mutations in HlyD, part of the type 1 translocator for hemolysin secretion, affect the folding of the secreted toxin.
HlyD, a member of the membrane fusion protein family, is essential for the secretion of the RTX hemolytic toxin HlyA from Escherichia coli. Random point mutations affecting HlyA secretion were obtained, distributed in most periplasmic regions of the HlyD molecule. Analysis of the secretion phenotypes of different mutants allowed the identification of regions in HlyD involved in different steps of HlyA translocation. Four mutants, V349-I, T85-I, V334-I and L165-Q, were conditionally defective, a phenotype shown to be linked to the presence of inhibitory concentrations of Ca2+ in extracellular medium. Hly mutant T85-I was defective at an early stage in secretion, while mutants V334-I and L165-Q appeared to accumulate HlyA in the cell envelope, indicating a block at an intermediate step. Mutants V349-I, V334-I, and L165-Q were only partially defective in secretion, allowing significant levels of HlyA to be transported, but in the case of V349-I and L165-Q the HlyA molecules secreted showed greatly reduced hemolytic activity. Hemolysin molecules secreted from V349-I and V334-I are defective in normal folding and can be reactivated in vitro to the same levels as HlyA secreted from the wild-type translocator. Both V349-I and V334-I mutations mapped to the C-terminal lipoyl repeat motif, involved in the switching from the helical hairpin to the extended form of HlyD during assembly of the functional transport channel. These results suggest that HlyD is an integral component of the transport pathway, whose integrity is essential for the final folding of secreted HlyA into its active form.
Pimenta
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AL
Université de Cergy Pontoise, Department of Biology, ERRMECe, 2 Av. Adolphe Chauvin, 95302Cergy-Pontoise cedex, France. pimenta@bio.u-cergy.fr
Racher
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K
Jamieson
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L
Blight
M A
MA
Holland
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IB
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002118:Calcium; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology* / Q000633:toxicity; D006460:Hemolysin Proteins; D026901:Membrane Transport Proteins / Q000235:genetics / Q000502:physiology*; D020125:Mutation, Missense; D017354:Point Mutation; D017510:Protein Folding; D021381:Protein Transport
D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C065231:HlyD protein, E coli; C036507:Hlya protein, E coli; D026901:Membrane Transport Proteins; D002118:Calcium