Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or β-galactosidase fused to the Hly C-terminal signal domain

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.     

H662 is the linchpin of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB

The ABC transporter HlyB is a central element of the HlyA secretion machinery, a paradigm of Type I secretion. Here, we describe the crystal structure of the HlyB-NBD (nucleotide-binding domain) with H662 replaced by Ala in complex with ATP/Mg2+. The dimer shows a composite architecture, in which two intact ATP molecules are bound at the interface of the Walker A motif and the C-loop, provided by the two monomers. ATPase measurements confirm that H662 is essential for activity. Based on these data, we propose a model in which E631 and H662, highly conserved among ABC transporters, form a catalytic dyad. Here, H662 acts as a 'linchpin', holding together all required parts of a complicated network of interactions between ATP, water molecules, Mg2+, and amino acids both in cis and trans, necessary for intermonomer communication. Based on biochemical experiments, we discuss the hypothesis that substrate-assisted catalysis, rather than general base catalysis might operate in ABC-ATPases.     

Type 1 protein secretion in bacteria,the ABC-transporter dependent pathway (Review)

The relatively simple type 1 secretion system in Gram-negative bacteria is nevertheless capable of transporting polypeptides of up to 800 kDa across the cell envelope in a few seconds. The translocator is composed of an ABC-transporter, providing energy through ATP hydrolysis (and perhaps the initial channel across the inner membrane), linked to a multimeric Membrane Fusion Protein (MFP) spanning the initial part of the periplasm and forming a continuous channel to the surface with an outer membrane trimeric protein. Proteins targeted to the translocator carry an (uncleaved), poorly conserved secretion signal of approximately 50 residues. In E. coli the HlyA toxin interacts with both the MFP (HlyD) and the ABC protein HlyB, (a half transporter) triggering, via a conformational change in HlyD, recruitment of the third component, TolC, into the transenvelope complex. In vitro, HlyA, through its secretion signal, binds to the nucleotide binding domain (NBD or ABC-ATPase) of HlyB in a reaction reversible by ATP that may mimic initial movement of HlyA into the translocation channel. HlyA is then transported rapidly, apparently in an unfolded form, to the cell surface, where folding and release takes place. Whilst recent structural studies of TolC and MFP-like proteins are providing atomic detail of much of the transport path, structural analysis of the HlyB NBD and other ABC ATPases, have revealed details of the catalytic cycle within an NBD dimer and a glimpse of how the action of HlyB is coupled to the translocation of HlyA.     

Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review)

The relatively simple type 1 secretion system in gram-negative bacteria is nevertheless capable of transporting polypeptides of up to 800 kDa across the cell envelope in a few seconds. The translocator is composed of an ABC-transporter, providing energy through ATP hydrolysis (and perhaps the initial channel across the inner membrane), linked to a multimeric Membrane Fusion Protein (MFP) spanning the initial part of the periplasm and forming a continuous channel to the surface with an outer membrane trimeric protein. Proteins targeted to the translocator carry an (uncleaved), poorly conserved secretion signal of approximately 50 residues. In E. coli the HlyA toxin interacts with both the MFP (HlyD) and the ABC protein HlyB, (a half transporter) triggering, via a conformational change in HlyD, recruitment of the third component, TolC, into the transenvelope complex. In vitro, HlyA, through its secretion signal, binds to the nucleotide binding domain (NBD or ABC-ATPase) of HlyB in a reaction reversible by ATP that may mimic initial movement of HlyA into the translocation channel. HlyA is then transported rapidly, apparently in an unfolded form, to the cell surface, where folding and release takes place. Whilst recent structural studies of TolC and MFP-like proteins are providing atomic detail of much of the transport path, structural analysis of the HlyB NBD and other ABC ATPases, have revealed details of the catalytic cycle within an NBD dimer and a glimpse of how the action of HlyB is coupled to the translocation of HlyA.     

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 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 theE. 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.     

Identification of individual amino acids required for secretion within the haemolysin (HlyA) C-terminal targeting region

The release of haemolysin from Escherichia coli involves direct secretion across both the inner and outer membranes. Secretion of HlyA is dependent upon a specific membrane export complex composed of HlyB, -D and possibly TolC. HlyA is targeted to the medium via the membrane translocation complex, by a novel C-terminal secretion signal. Previous studies involving deletion and fusion analyses have given contradictory results for the minimal length (20-60 residues) of this HlyA signal region and little is known of the nature of the specific residues and structural features required for function. In this study we have analysed, quantitatively, the effect upon secretion of many point mutations introduced into the HlyA C-terminus. The results indicate the presence of a minimal secretion signal domain whose proximal boundary extends to at least residue -46 and which contains at least four individual residues essential for maximal secretion levels. We propose that such residues act co-operatively, forming multiple contact points with the translocator proteins, with the 'best fit' promoting maximal levels of secretion.     

Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins

Summary We studied the efficiency of the pHly152-derived haemolysin transport system using PhoA-HlyA fusion proteins and different constructs which provide HlyB/HlyD in trans. The optimal C-terminal HlyA signal consists of the last 60 amino acids. Longer stretches of HlyA do not improve the transport efficiency of PhoA-HlyA fusion proteins. The introduction of deletions and/or replacements in the 60 amino acid HlyA signal domain revealed at least three functional regions with different degrees of specificity. Amino acids 1–21 (numbered from the N-terminal part of the 60 amino acid HlyA signal), termed region I, could be replaced by a Pro-containing peptide. The other two regions II and III (amino acids 22–40 and 41–60, respectively) seem to interact directly with the HlyB/HlyD translocator since a PhoA fusion protein which contains either of the two regions was still secreted in a HlyB/HlyD-dependent mode, albeit at low efficiency. An efficient trans-complementing HlyB/HlyD system was only obtained from the pHLy152-encoded hly determinant when the regulatory hlyR element was provided in cis. Secretion of the PhoA-HlyA fusion protein did not interfere with the secretion of HlyA even when the fusion protein was induced to a high level. This suggests that the capacity of the HlyB/HlyD translocation system is high and not normally saturated by its natural HlyA substrate.Dedicated to Prof., Dr. F. Lingens on the occasion of his 65th birthday     

A heterologous membrane protein domain fused to the C-terminal ATP-binding domain of HlyB can export Escherichia coli hemolysin.

The hydrophobic-rich NH2-terminal 34 amino acids of a tetracycline resistance determinant (TetC) were fused to the COOH-terminal 240 amino acids of the hemolysin transporter, HlyB, which contains a putative ATP-binding domain. This hybrid protein replaced the NH2-terminal 467-amino-acid portion of HlyB and could still export the Escherichia coli hemolysin (HlyA). Export by the hybrid protein was approximately 10% as efficient as transport by HlyB. Extracellular secretion of HlyA by the TetC-HlyB hybrid required HlyD and TolC. The extracellular and periplasmic levels of beta-galactosidase and beta-lactamase in strains that produced the hybrid were similar to the levels in controls. Thus, HlyA transport was specific and did not appear to be due to leakage of cytoplasmic contents alone. Antibodies raised against the COOH terminus of HlyB reacted with the hybrid protein, as well as HlyB. HlyB was associated with membrane fractions, while the hybrid protein was found mainly in soluble extracts. Cellular fractionation studies were performed to determine whether transport by the hybrid occurred simultaneously across both membranes like wild-type HlyA secretion. However, we found that HlyA was present in the periplasm of strains that expressed the TetC-HlyB hybrid. HlyA remained in the periplasm unless the hlyD and tolC gene products were present in addition to the hybrid.     

Change in the cellular localization of alkaline phosphatase by alteration of its carboxy-terminal sequence

Summary Alkaline phosphatase (AP) is secreted into the medium when the carboxy-terminal 25 amino acids are replaced by the 60 amino acid carboxy-terminal signal peptide (HlyA_s) ofEscherichia coli haemolysin (HlyA). Secretion of the AP-HlyA_s fusion protein is dependent on HlyB and HlyD but independent of SecA and SecY. The efficiency of secretion by HlyB/HlyD is decreased when AP carries its own N-terminal signal peptide. Translocation of this fusion protein into the periplasm is not observed even in the absence of HlyB/HlyD. The failure of the Sec export machinery to transport the latter protein into the periplasm seems to be due in part to the loss of the carboxy-terminal sequence of AP since even AP derivatives which do not carry the HlyA signal peptide but lack the 25 C-terminal amino acids of AP are localized in the membrane but not translocated into the periplasm.     

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: Glyl0Arg 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 λ 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.     

Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli haemolysin

We have carried out an extensive mutational analysis of the C-terminal signal which targets the export of the 1024-residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Over 60 variants of the HlyA C-terminal 53-amino-acid sequence were created by oligonucleotide-directed mutagenesis and fused to the HlyA N-terminal 830 residues. Transport of the HlyA derivatives by the HlyB/HlyD system was compared with the wild-type level and the data indicate that the HlyA C-terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13-amino-acid uncharged region from residue T,997 to S,1009; and an 8-amino-acid hydroxylated tail at the extreme C-terminus. Analogous features were found in the C-terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD-type translocators. In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices. These results suggest a possible role for multiple regions of the HlyA C-terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm.     

Membrane fusion proteins of type I secretion system and tripartite efflux pumps share a binding motif for TolC in gram-negative bacteria

The Hly translocator complex of Escherichia coli catalyzes type I secretion of the toxin hemolysin A (HlyA). In this complex, HlyB is an inner membrane ABC (ATP Binding Cassette)-type transporter, TolC is an outer membrane channel protein, and HlyD is a periplasmic adaptor anchored in the inner membrane that bridges HlyB to TolC. This tripartite organization is reminiscent of that of drug efflux systems such as AcrA-AcrB-TolC and MacA-MacB-TolC of E. coli. We have previously shown the crucial role of conserved residues located at the hairpin tip region of AcrA and MacA adaptors during assembly of their cognate systems. In this study, we investigated the role of the putative tip region of HlyD using HlyD mutants with single amino acid substitutions at the conserved positions. In vivo and in vitro data show that all mutations abolished HlyD binding to TolC and resulted in the absence of HlyA secretion. Together, our results suggest that, similarly to AcrA and MacA, HlyD interacts with TolC in a tip-to-tip manner. A general model in which these conserved interactions induce opening of TolC during drug efflux and type I secretion is discussed.     

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: Glyl0Arg 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 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.     

Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli

Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA(218)) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 x 10(4) E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23 degrees C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA(218) fusion protein at 23 and 30 degrees C but unexpectedly not at 37 degrees C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA(218) was detected only in the L448F mutant (AE104F) at 23 degrees C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA(218) fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37 degrees C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

Translocation and compartmentalization of Escherichia coli hemolysin (HlyA).

Hemolysin plasmids were constructed with mutations in hlyB, hlyD, or both transport genes. The localization of hemolysin activity and HlyA protein in these mutants was analyzed by biochemical and immunological methods. It was found that mutants defective in hlyB accumulated internal hemolysin, part of which was associated with the inner membrane and was degraded in the late logarithmic growth phase. In an HlyB+ HlyD- mutant, hemolysin was predominantly localized in the membrane compartment. Labeling of these Escherichia coli cells with anti-HlyA antibody indicated that part of HlyA, presumably the C-terminal end but not the pore-forming domains, was already transported to the cellular surface. This finding suggests that HlyB is able to recognize the C-terminal signal of the HlyA protein and to initiate its translocation across the membranes.     

Substrate-induced assembly of a contiguous channel for protein export from E.coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore.

The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner-membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer-membrane (OM) porin-like TolC. These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross-linked in vivo complexes were isolated before and after substrate engagement. HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate. Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components. The IM-OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC. The bridge was transient, components reverting to IM and OM states after translocation. Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel. A similar picture was evident when the HlyD C-terminus was masked. Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate-induced, reversible bridging of the IM translocase to the OM export pore.     

Functional characterization and ATP-induced dimerization of the isolated ABC-domain of the haemolysin B transporter

Nucleotide-binding domains (NBD) are highly conserved constituents of ATP-binding cassette (ABC) transporters. Members of this family couple ATP hydrolysis to the transfer of various molecules across cell membranes. The NBD of the HlyB transporter, HlyB-NBD, was characterized with respect to its uncoupled ATPase activity, oligomeric state, and stability in solution. Experimental data showed that both the nature and pH of an assay buffer influenced the level of protein activity. Comparative analysis of protein stability and ATPase activity in various buffers suggests an inverse relationship between the two. The highest ATPase activity was detected in HEPES, pH 7.0. A kinetic analysis of the ATPase activity in this buffer revealed an enzyme concentration dependence and ATP-induced protein oligomerization. Assuming that the dimer is the active form of enzyme, at least half of the purified HlyB-NBD was estimated to be a dimer at 1.2 microM under the most optimal conditions for ATP hydrolysis. This is about 2 orders of magnitude lower than reported for other canonical ABC-ATPases. The maximum reaction velocity of 0.6 micromol/mg x min at 22 degrees C and the apparent kinetic constant K(app)(0.5) of 0.26 mM for ATP were determined for the dimerized HlyB-NBD. Gel filtration experiments with the wild-type protein and HlyB-NBD mutated in a key catalytic residue, H662A, provided further evidence for ATP-induced protein dimerization. ATPase activity experiments with protein mixtures composed of wild-type and the ATPase-deficient H662A mutant demonstrated that one intact NBD within a dimer is sufficient for ATP hydrolysis. This single site turnover might suggest a sequential mechanism of ATP hydrolysis in the intact HlyB transporter.     

Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes.

The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB. In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp). Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi. The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion. This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components. However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited. HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH. The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction. Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes. This may reflect its close association with the inner membrane or a membrane translocation complex.     

Topological and functional studies on HlyB of Escherichia coli

Summary The topology of HlyB, a protein located in the inner membrane of Escherichia coli and involved in the secretion of -haemolysin (HlyA), was determined by the generation of HlyB-PhoA and HlyB-LacZ fusion proteins. The data obtained by this biochemical method together with computer predictions suggest that HlyB is inserted in the cytoplasmic membrane by six stable hydrophobic, -helical transmembrane segments. These segments extend from amino acid positions 158 to 432 of HlyB. The cytoplasmic loops between these transmembrane segments are relatively large and carry an excess of positively charged amino acids, while the periplasmic loops are rather small. In addition to these six transmembrane segments, two additional regions in the 78 N-terminal amino acids of HlyB appear to be also inserted in the cytoplasmic membrane. However, the association of these two segments with the cytoplasmic membrane seems to be less tight, since active PhoA and LacZ fusions were obtained by insertion into the same positions of these segments. A LacZ-HlyAs fusion protein carrying, at the C-terminus of LacZ, the 60-amino acid signal sequence of HlyA was not secreted in the presence of HlyB/HlyD. However, transport of this fusion protein into the cytoplasmic membrane appeared to be initiated, as suggested by the tight association of this protein with the inner membrane. A similar close association of LacZ-HlyA_s with the inner membrane was also observed in the presence of HlyB alone but not in its absence. These data suggest that HlyB recognizes the HlyA signal sequence and initiates the transport of HlyA into the membrane.     

Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HIyB/HIyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA

Escherichia coli hemolysin (HlyA) is secreted by a specific export machinery which recognizes a topogenic secretion signal located at the C-terminal end of HlyA. This signal sequence has been variously defined as comprising from 27 to about 300 amino acids at the C-terminus of HlyA. We have used here a combined genetic and immunological approach to select for C-terminal HlyA peptides that are still secretion-component. A deletion library of HlyA mutant proteins was generated in vitro by successive degradation of hy1A from the 5′ end with exonuclease III. Secretion competence was tested by immunoblotting of the supernatant of each clone with an antiserum raised against a C-terminal portion of hemolysin. It was found that the hemolysin secretion system has no apparent size limitation for HlyA proteins over a range from 1024 to 62 amino acids. The smallest autonomously secretable peptide isolated in this selection procedure consists of the C-terminal 62 amino acids of HlyA. This sequence is shared by all secretion-competent, truncated HlyA proteins, which suggests that secretion of the E.coli hemolysin is strictly post-translational. The capacity of the hemolysin secretion machinery was found to be unsaturated by the steady-state level of its natural HlyA substrate and large amounts of truncated HlyA derivatives could still be secreted in addition to full-length HlyA.