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. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

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.

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. mrfI (encoding a putative recombinase) and mrfA (encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergent promoters. The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter. This region was shown to be capable of inversion, consistent with an ON/OFF regulation of the operon. In in vitro liquid cultures, both orientations were detected. Nevertheless, when we analyzed the expression of all of the genes in the mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater wax moth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvae at 32 h. In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection. Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase in inversion frequency at this stage. In both in vitro liquid cultures and in vivo larval infections, the distal genes of the operon were expressed at substantially lower levels than mrfA. These results indicate the complex regulation of the mrf cluster during infection.

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. Tca also appears in stationary phase, whereas Tcd is expressed earlier. These patterns seen in a culture flask are strikingly similar to those observed during insect infection. Thus, in an infected insect, bacteria grow exponentially until the time of insect death at approximately 48 h, when both light and the virulence factors Prt protease and Tca are produced. In contrast, Tcd appears much earlier in insect infection. However, at present, the biological significance of this difference in timing of the production of the two toxins in unclear. This is the first documentation of the expression of Tcs and Prt in an insect and highlights the malleability of Photorhabdus as a model system for bacterial infection.

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. ABC-dependent transport can be inwards across the membrane, or outwards to the cell exterior, and the ABC-ATPase can fuel transport through pathways which may involve a classical channel (CFTR), a "gateway" mechanism through a proteinacious chamber spanning the bilayer, or conceivably via a pathway at the protein-lipid interface of the outside of the membrane domain. This may be the case for drugs transported by Pgp, a multidrug resistance transporter. In this review, we try to identify the common fundamental principles which unite all ABC-transporters, including the basis of specificity for different transported compounds (allocrites), the interactions between the ATPase and membrane domains, activation of the ATPase and the coupling of consequent conformational changes, to the final movement of an allocrite through a given transport pathway. We discuss the so far limited structural information for the intact ABC-transporter complex and the exciting information from the first crystal structure of an ABC-ATPase. Finally, the action of specific transporters, CFTR (Cl- transport), Pgp, MRP and LmrA, all transporting many different drug molecules and HlyB transporting a large protein toxin are discussed.

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. HlyD is normally highly stable but in the absence of TolC, the steady-state level of HlyD is greatly reduced and the protein has a half-life at 37 degrees C of 36 min. In the absence of HlyB, HlyD is also unstable and specific degradation products are detected, which co-fractionate with the inner membrane, indicating in this case limited cleavage at specific sites. However, the effect of removing both HlyB and TolC is not additive. On the contrary, in the absence of both HlyB and TolC the half-life of HlyD is approximately 110 min. This result shows that in the presence of HlyB removal of TolC renders HlyD more unstable than it is in the absence of both HlyB and TolC. This suggests that the presence of HlyB induces a structural change in HlyD. In addition, HlyB itself appears to be less stable in the absence of HlyD. These results are consistent with an interaction between HlyD/TolC and HlyB/HlyD. A derivative of HlyD, HlyD22, lacking the 40 N-terminal residues of HlyD assembles into the inner membrane displaying the same stability with and without HlyB as wild type HlyD does. This N-terminal region therefore appears to play no role in stable localisation but is involved in secretion, since HlyD22 is completely secretion defective. Modification of the C-terminus on the other hand completely destabilised the molecule and HlyD was not detectable in the envelope. Secretion of active haemolysin is limited to a brief period during mid to late exponential phase. In contrast, HlyD is apparently synthesised constitutively throughout the growth phase, demonstrating that the production of this component of the translocator is not the limiting factor for growth phase-dependent secretion.

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. However, the number of bees responding in two of these areas were too low to be considered in the present study. One significant area of activity was associated with the major component, (E,E)-alpha-farnesene, whilst the others were associated with several minor components. Although all three groups of bees, irrespective of the treatments applied, showed some responses to the components eluting from the GC column, only bees subjected to paired conditioning consistently responded when re-tested to the mixture. In addition, paired conditioning increased the responsiveness of individuals in terms of the number of bees responding at least once to the effluent from the gas chromatograph. This work confirmed the occurrence of key compounds in floral volatile mixtures. Possible synergistic/inhibitory effects between components, relating to olfactory experience, are discussed.

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. Nevertheless, analysis of hlyB specific mRNA synthesis revealed that some constructs showed at least a 50-fold increase in mRNA levels, indicating that expression of HlyB may be limited at the translational level. When HlyB was expressed as a hybrid, downstream of LacZ, extremely high level overproduction was then detected in total cell extracts. When the expression of HlyB or HlyB fragments expressed from a T7 promoter was examined, the C-terminal ATPase domain was dramatically overexpressed but the production of fragments encompassing the N-terminal membrane domain, was reduced at least 1000-fold. These results indicate that mRNA structures corresponding to the membrane domain of HlyB greatly limit the post-transcriptional expression of HlyB. When such structures are deleted, or disrupted when part of a larger mRNA, HlyB or the HlyB ATPase domain can be overproduced in milligram quantities and this has facilitated the production of high titre antibodies to HlyB.

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. The electrophysiological activity of the identified compounds was confirmed by EAG using a physiologically discriminating dose, and by SCR studies. The importance of the combined use of the EAG and SCR techniques was demonstrated, since specific olfactory cells were located for five compounds that did not elicit significant EAG responses. The majority of the olfactory cells from which single cell recordings were obtained showed very high specificity, and in numerous recordings there were consistent pairings of specific cell types.

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli.

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Gly10Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the lambda Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.

Heterologous protein secretion and the versatile Escherichia coli haemolysin translocator.

Heterologous proteins synthesized in the Gram-negative bacterium Escherichia coli in bioreactor culture may accumulate in one of three 'compartments':the cytoplasm, the periplasm, or the extracellular medium. Many overexpressed proteins from various origins have been purified from each of these locations. However, to date, each system has required specific tailoring to meet the stringent requirements for each protein product to ensure correct folding, activity and appropriate yield. The E. coli haemolysin secretion system appears to provide a flexible mechanism with which to secrete a wide variety of heterologous fusion proteins into the extracellular medium.

Protein secretion pathway in Escherichia coli.

The export of proteins to the Escherichia coli periplasm is a well established system for heterologous protein production. With a better understanding of the protein export (SecA, Y-dependent) process and a greater awareness of the conditions necessary for correct folding of proteins in the periplasm, serious efforts are now being made to manipulate this system to achieve substantial increases in the yield of authentically folded proteins. Further advances in the development of methods for the recovery of recombinant proteins from the culture medium have made the use of fusion proteins secreted by the protein A or haemolysin pathways a more attractive option. Recent studies of the haemolysin system indicate its ability to secrete a wide range of polypeptides, including normally cytoplasmic proteins. As their features and potential applications become much clearer, a rapidly expanding number of protein-secretion mechanisms in Gram-negative bacteria are becoming available for heterologous protein expression. Most, if not all, of these systems can be successfully transplanted into E. coli, providing a wider choice of systems for the future.

Discrimination of oilseed rape volatiles by honey bee: Novel combined gas chromatographic-electrophysiological behavioral assay.

A novel technique for the simultaneous monitoring of electroan-tennogram (EAG) and conditioned proboscis extension (CPE) responses of honey bees to the effluent from a gas chromatograph (GC) was developed to locate biologically active components in blends of plant volatiles and to investigate odor recognition at the peripheral and behavioral levels. A six-component mixture, comprising compounds previously identified as oilseed rape floral volatiles, was used as the stimulus. Standard CPE and EAG recordings were done as a reference. EAG responses were elicited from unconditioned bees by all the components presented either in the coupled or the standard mode. Conditioned bees gave larger EAG responses than unconditioned bees, suggesting that antennal sensitivity is enhanced by conditioning. At the behavioral level, in both the standard and the coupled modes, only conditioned bees showed the proboscis extension response, with the majority of individuals responding to linalool, 2-phenylethanol, and benzyl alcohol.

Oilseed rape--a new allergen?

Oilseed rape (Brassica napus) is a commonly grown crop in Europe and it has been suggested that its pollen may be a potent new allergen. The prevalence of sensitization in a normal exposed population and an objective study of those patients found to be allergic to the rape pollen is described. The results show a low prevalence of allergy to oilseed rape pollen (less than 0.2%) unless the subjects were occupationally exposed. Those affected, with one exception, were already atopic and allergic to other pollens. The role of volatile materials given off by the plant remains to be elucidated.

Characterization of the hemolysin transporter, HlyB, using an epitope insertion.

The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.

Analysis of the membrane organization of an Escherichia coli protein translocator, HlyB, a member of a large family of prokaryote and eukaryote surface transport proteins.

Haemolysin B (HlyB) is essential for secretion of the 107 x 10(3) Mr haemolysin A protein from Escherichia coli and is a member of a family of highly conserved, apparently ATP-dependent surface proteins in many organisms. We have shown in this study that both HlyB and HlyD fractionate primarily with the cytoplasmic membrane of E. coli and are accessible to proteases after removal of the outer membrane. We have measured experimentally the topological organization of HlyB within the membrane by construction of fusions to beta-lactamase as a reporter. The predicted folding of HlyB, with a minimum of six transmembrane segments, does not always coincide with regions of highest average hydrophobicity. This suggests that HlyB may have a novel organization within the bilayer. From our data and comparative sequence analysis, we have been able to predict very similar topological models for the other members of the HlyB family.

The mechanism of secretion of hemolysin and other polypeptides from gram-negative bacteria.

In the secretion of polypeptides from Gram-negative bacteria, the outer membrane constitutes a specific barrier which has to be circumvented. In the majority of systems, secretion is a two-step process, with initial export to the periplasm involving an N-terminal signal sequence. Transport across the outer membrane then involves a variable number of ancillary polypeptides including both periplasmic and outer membrane. While such ancillary proteins are probably specific for each secreted protein, the mechanism of movement across the outer membrane is unknown. In contrast to these systems, secretion of the E. coli hemolysin (HlyA) has several distinctive features. These include a novel targeting signal located within the last 50 or so C-terminal amino acids, the absence of any periplasmic intermediates in transfer, and a specific membrane-bound translocator, HlyB, with important mammalian homologues such as P-glycoprotein (Mdr) and the cystic fibrosis protein. In this review we discuss the nature of the HlyA targeting signal, the structure and function of HlyB, and the probability that HlyA is secreted directly to the medium through a trans-envelope complex composed of HlyB and HlyD.

Structure and function of haemolysin B,P-glycoprotein and other members of a novel family of membrane translocators.

Recent studies have identified two sub-families of highly conserved polypeptides in a wide variety of organisms concerned with the transport of many different compounds, specific for each transport protein. Both families, represented by HisP and HlyB, respectively, have in common a highly conserved, approximately 25 kD domain, containing an ATP-binding site. The HisP sub-family essentially consists of cytoplasmic proteins which couple energy to the import of small substrates through cytoplasmic membrane permeases in Gram-negative bacteria. The HlyB (P-glycoprotein) sub-family, on the other hand, contains a second large domain which apparently acts as the transmembrane translocator itself, which in most cases drives the secretion of a variety of compounds. These membrane domains share a number of structural features which also serve to distinguish these proteins as a closely related group. Nevertheless, the compounds secreted by the HlyB sub-family include large polypeptides, polysaccharides and a variety of anti-tumour drugs. We describe here the properties of each of these remarkable proteins and we speculate on their possible mechanism of action.