Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin.

Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell.

Positive correlation between in vivo and in vitro assays for the evaluation of Pseudomonas virulence.

Some bacterial phenotypes measured in vitro can be used to access bacterial virulence, on the premise that they are positively correlated with data from in vivo experiments. We show here that in vitro assessment of bacterial phenotypes, such as adherence and cytotoxicity, are positively correlated with data from in vivo experiments in Drosophila and can be used to assess bacterial virulence in vivo. Manipulation of environmental parameters, such as iron availability, induced changes in the phenotypes measured in vitro that correlated with changes in vivo virulence of all strains tested.

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli.

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers.

In vitro identification of two adherence factors required for in vivo virulence of Pseudomonas fluorescens.

By enriching a random transposon insertion bank of Pseudomonas fluorescens for mutants affected in their adherence to the human extracellular matrix protein fibronectin, we isolated 23 adherence minus mutants. Mutants showed a defect in their ability to develop a biofilm on an abiotic surface and were impaired for virulence when tested in an in vivo virulence model in the fruit fly, Drosophila melanogaster. Molecular characterisation of these mutants showed that the transposon insertions localised to two distinct chromosomal locations, which were subsequently cloned and characterised from two mutants.

Crystal structure of the nucleotide-binding domain of the ABC-transporter haemolysin B: identification of a variable region within ABC helical domains.

The ABC-transporter haemolysin B is a central component of the secretion machinery that translocates the toxin, haemolysin A, in a Sec-independent fashion across both membranes of E. coli. Here, we report the X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB.

Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus.

Proteases play a key role in the interaction between pathogens and their hosts. The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects. Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect.

A specific interaction between the NBD of the ABC-transporter HlyB and a C-terminal fragment of its transport substrate haemolysin A.

A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB-NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence.

A zinc metalloprotease inhibitor, Inh, from the insect pathogen Photorhabdus luminescens.

The entomopathogen Photorhabdus luminescens secretes many proteins during the late stages of insect larvae infection and during in vitro laboratory culture. The authors have previously characterized and purified a 55 kDa zinc metalloprotease, PrtA, from culture supernatants of P. luminescens.

Crystallization and preliminary X-ray analysis of the ATP-binding domain of the ABC transporter haemolysin B from Escherichia coli.

Haemolysin B (HlyB) is a transmembrane protein which belongs to the superfamily of ABC transporters. In vivo, it mediates the non-classical translocation of the 107 kDa toxin HlyA across both membranes of Escherichia coli together with haemolysin D and the outer membrane protein TolC. The cytosolic ATP-binding domain of HlyB has been overexpressed and purified as an N-terminal His-tag fusion protein.