The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase.

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing.

Sense of Community on Twitter and Instagram: Exploring the Roles of Motives and Parasocial Relationships.

Although research has explored the ways in which people form virtual communities to converse about media figures, television shows, and similar topics, little research has examined the link between virtual communities and the parasocial relationships (PSRs) that are often the focus of these conversations and users' experiences in those virtual communities. We examined sense of community (SOC) on Twitter and Instagram as a function of users' motives for use and users' PSR on the sites. In addition to examining the relative importance of different motives for using Twitter and Instagram, we predicted that PSR would mediate the association between motives for use and SOC.

An Exploratory Investigation of Communication Technologies to Alleviate Communicative Difficulties and Depression in Older Adults.

Objective: To explore associations between older adults' communicative difficulties, depressive symptoms, and e-mail and telephone use with adult children.

Method: We examined these associations using data from 1,634 participants (age M = 71.16, SD = 0.

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.

From insects to human hosts: Identification of major genomic differences between entomopathogenic strains of Photorhabdus and the emerging human pathogen Photorhabdus asymbiotica.

Pathogenic bacteria of the genus Photorhabdus are naturally found in symbiotic association with soil entomopathogenic nematodes, and are of increasing economic interest in view of their potential for the development of novel biopesticides. This bipartite natural system is currently used for the biological control of crop pests in several countries. However, an increasing number of Photorhabdus strains have recently been isolated from human clinical specimens in both the United States and Australia, associated with locally invasive soft tissue infections and disseminated bacteraemia.

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.

Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model.

Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P.

Evidence of oral toxicity of Photorhabdus temperata strain K122 against Prays oleae and its improvement by heterologous expression of Bacillus thuringiensis cry1Aa and cry1Ia genes.

Photorhabdus temperata strain K122 exhibited oral toxicity against Prays oleae with an LC50 of 58.1 x 10(6) cells ml(-1). Recombinant P.

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.

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.

Orientation and feeding responses of the pollen beetle, Meligethes aeneus, to candytuft, Iberis amara.

The pollen beetle, Meligethes aeneus, which is an important pest of oilseed rape, Brassica napus, and turnip rape, B. rapa var. campestris, does not oviposit in all species of the Brassicaceae.

In vivo expression of the mannose-resistant fimbriae of Photorhabdus temperata K122 during insect infection.

Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes of the family Heterorhabditidae: Surface fimbriae are important for the colonization of many pathogenic bacteria, and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding the mannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organization similar to that of the mrp locus from Proteus mirabilis.

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.

Measuring virulence factor expression by the pathogenic bacterium Photorhabdus luminescens in culture and during insect infection.

During insect infection Photorhabdus luminescens emits light and expresses virulence factors, including insecticidal toxin complexes (Tcs) and an RTX-like metalloprotease (Prt). Using quantitative PCR and protein assays, we describe the expression patterns of these factors both in culture and during insect infection and compare them to the associated bacterial growth curves. In culture, light and active Prt protease are produced in stationary phase.

ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans.

The approximately 27 kDa ABC-ATPase, an extraordinarily conserved, unique type of ATPase, acts as a machine to fuel the movement across membranes of almost any type of molecule, from large polypeptides to small ions, via many different membrane-spanning proteins. A particular ABC-ATPase must therefore be tailor-made to function in a complex with its cognate membrane protein, forming a transport pathway appropriate for a specific type of molecule, or in the case of some ABC-transporters, several types of molecule. Molecules to be transported recognise their own transporter, bind and switch on the ATPase, which in turn activates or opens the transport pathway.

Antibody analysis of the localisation, expression and stability of HlyD, the MFP component of the E. coli haemolysin translocator.

HlyD has a single transmembrane domain (residues 59-80) and a large periplasmic domain, and is essential for the secretion of haemolysin from Escherichia coli. Using an antibody raised against HlyD, the protein was localised to the cell envelope by immunofluorescence and to the cytoplasmic membrane by sucrose gradient analysis. We have examined the stability of this protein in the presence and absence of other putative components of the translocator, HlyB and TolC.

Effect of conditioning on discrimination of oilseed rape volatiles by the honeybee: use of a combined gas chromatography-proboscis extension behavioural assay.

The coupled gas chromatography-proboscis extension assay technique was used on restrained worker bees to study responses to components of an extract of oilseed rape floral volatiles. Bees were stimulated with the effluent from the gas chromatograph after either paired or unpaired conditioning to the extract, or after a control treatment. Proboscis extension activity was elicited in six areas of the chromatogram.

Antennal perception of oilseed rape,Brassica napus (Brassicaceae), volatiles by the cabbage seed weevilCeutorhynchus assimilis (Coleoptera, Curculionidae).

The response of theCeutorhynchus assimilis antenna to volatiles in air entrainment-derived extracts of oilseed rape,Brassica napus, was studied using coupled gas chromatography (GC)-electroantennography (EAG) and coupled GC-single cell recording (SCR). By means of these techniques and coupled gas chromatography-mass spectrometry (GC-MS), 25 active compounds were identified, including isoprenoids and compounds derived from fatty acids and amino acids. Some of the latter, the isothiocyanates and goitrin, and probably indole and benzyl cyanide, are catabolites of glucosinolates.

Evidence for post-transcriptional regulation of the synthesis of the Escherichia coli HlyB haemolysin translocator and production of polyclonal anti-HlyB antibody.

Extensive attempts were made to overexpress the Escherichia coli haemolysin translocator protein HlyB, and HlyB fragments, utilising high copy number plasmids or hlyB expressed from strong promoters including lambda PR, ptrp and the T7 promoter. Analysis of both cytoplasmic and membrane fractions failed to detect any overexpression of the protein, although all the constructs showed biological activity and there was no evidence of HlyB-induced toxicity. In some constructs, the effect of removing a stem-loop structure, immediately upstream of the start codon and implicated in rho-independent termination of transcription, was tested but this did not lead to over-expression.