Analysis of the in vivo activation of hemolysin (HlyA) from Escherichia coli.
Albrecht Ludwig,Fernando García,Susanne Bauer,Thomas Jarchau,Roland Benz,J Hoppe,Werner Goebel +6 more
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The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.Abstract:
Hemolysin (HlyA) from Escherichia coli containing the hlyCABD operon separated from the nonhemolytic pro-HlyA upon two-dimensional (2-D) polyacrylamide gel electrophoresis. The migration distance indicated a net loss of two positive charges in HlyA as a result of the HlyC-mediated activation (modification). HlyA activated in vitro in the presence of [U-14C]palmitoyl-acyl carrier protein comigrated with in vivo-activated hemolysin on 2-D gels and was specifically labelled, in agreement with the assumption that the activation is accomplished in vitro and in vivo by covalent fatty acid acylation. The in vivo-modified amino acid residues were identified by peptide mapping and 2-D polyacrylamide gel electrophoresis of mutant and truncated HlyA derivatives, synthesized in E. coli in the presence and absence of HlyC. These analyses indicated that the internal residues Lys-564 and Lys-690 of HlyA, which have recently been shown by others to be fatty acid acylated by HlyC in vitro, are also the only modification sites in vivo. HlyA activated in E. coli was quantitatively fatty acid acylated at both sites, and the double modification was required for wild-type hemolytic activity. Single modifications in mutant and truncated HlyA derivatives suggested that both lysine residues are independently fatty acid acylated by a mechanism requiring additional sequences or structures flanking the corresponding acylation site. The intact repeat domain of HlyA was not required for the activation. The pore-forming activities of pro-HlyA and singly modified HlyA mutants in planar lipid bilayer membranes suggested that the activation is not essential for transmembrane pore formation but rather required for efficient binding of the toxin to target membranes.read more
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Journal ArticleDOI
RTX proteins: a highly diverse family secreted by a common mechanism.
Irena Linhartova,Ladislav Bumba,Jiří Mašín,Marek Basler,Radim Osicka,Jana Kamanova,Kateřina Procházková,Irena Adkins,Jana Hejnová-Holubová,Lenka Sadilkova,Jana Morova,Peter Sebo +11 more
TL;DR: This review summarizes the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins and discusses the so far characterized RTX family members.
Book ChapterDOI
RTX toxin structure and function: a story of numerous anomalies and few analogies in toxin biology.
TL;DR: It can be agreed that RTX toxins contribute to the pathogenesis of different diseases by causing dysfunction of the general cellular reactions of the immune response, but precise and satisfactory answers to the following questions are not yet available.
Journal ArticleDOI
Acylation of Escherichia coli Hemolysin: A Unique Protein Lipidation Mechanism Underlying Toxin Function
TL;DR: The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity and is related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors.
Journal ArticleDOI
Acyltransferases in Bacteria
TL;DR: This review provides a detailed survey of the wide spectrum of bacterial acyltransferases and compares different enzyme families in regard to their catalytic mechanisms.
Journal ArticleDOI
Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12.
TL;DR: Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation‐selective transmembrane pores that have a diameter of about 2.5–3 nm.
References
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Journal Article
Cleavage of structural proteins during the assemble of the head of bacterio-phage T4
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DNA sequencing with chain-terminating inhibitors
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Journal ArticleDOI
The gapped duplex DNA approach to oligonucleotide-directed mutation construction
Wilfried Kramer,Valerji Drutsa,Hans-W. Jansen,Barbara Kramer,Monika Pflugfelder,Hans-Joachim Fritz +5 more
TL;DR: It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70% and a rigorous selection can be applied for phage carrying the markers of the (-) strand.