In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3′-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3′-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm. Received: 7 April 1997 / Accepted: 28 May 1997     

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° 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. Received: 10 July 1998 / Accepted: 19 October 1998     

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.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmicβ-galactosidase (β-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that allβ-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

Identification and characterization of two functional domains of the hemolysin translocator protein HlyD

Secretion of Escherichia coli hemolysin is mediated by a sec-independent pathway which requires the products of at least three genes, hlyB, hlyD and tolC. Two regions of HlyD were studied. The first region (region A), consisting of the 33-amino acid, C-terminal part of the HlyD protein, is predicted to form a potential helix-loop-helix structure. This sequence is conserved among HlyD analogues of similar transport systems of other bacterial species. Using site-directed mutagenesis, we showed that the amino acids Leu475, Glu477 and Arg478 of this region are essential for HlyD function. The last amino acid of HlyD, Arg478, is possibly involved in the release of the HlyA protein, since cells bearing a hlyD gene mutant at this position produce similar amounts of HlyA to the wild-type strain, but most of the protein remains cell-associated. Competition experiments between wild-type and mutant HlyD proteins indicate that region A interacts directly with a component of the secretion apparatus. The second region of HIyD (region B), located between amino acids Leul27 and Leu170, is highly homologous to the otherwise unrelated outer membrane protein TolC. Deletion of this region abolishes secretion of hemolysin. This sequence of HlyD also seems to interact with a component of the hemolysin secretion machinery since a hybrid HIyD protein carrying the corresponding TolC sequence, although inactive in the transport of HlyA, is able to displace wild-type HlyD from the secretion apparatus.     

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.     

Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli.

A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex. This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment. HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59). Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase. The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC. Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate. The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential. Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function. The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it. In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e. TolC recruitment was not triggered. Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex.     

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.     

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 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.     

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.     

Involvement of lipopolysaccharide in the secretion of Escherichia coli α haemolysin and Erwinia chrysanthemi proteases

The presence of the α-haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram-negative envelope (owing to its large size) (M_r 1400). The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non-haemolytic mutants. In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained. However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis. Mutations in rfaH and galU strongly reduced α-haemolysin secretion as weli as the secretion of Erwinia chrysanthemi proteases in E. coli without affecting their synthesis. These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion. These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmic-galactosidase (-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

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.     

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 (HlyA₂₁₈) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 × 10⁴ 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°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA₂₁₈ fusion protein at 23 and 30°C but unexpectedly not at 37°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-HlyA₂₁₈ was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA₂₁₈ fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.     

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.     

A topological model for the haemolysin translocator protein HlyD

Summary A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active H1yD-LacZ fusion proteins were only generated when lacZ was fused to hlyD. within the first 180 by (60 amino acids). H1yD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD. between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD Active insertions of phoA into the middle region of hlyD. were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA⁺ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA⁺ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus. Neither the HlyD fusion proteins obtained nor a mutant HlyD protein that had lost the last 10 amino acids from the C-terminus of HlyD exhibited translocator activity for HlyA or other reporter proteins carrying the HlyA signal sequence. The C-terminal 10 amino acids of HlyD showed significant similarity with the corresponding sequences of other HlyD-related proteins involved in protein secretion.     

On the role of TolC in multidrug efflux: the function and assembly of AcrAB–TolC tolerate significant depletion of intracellular TolC protein

TolC channel provides a route for the expelled drugs and toxins to cross the outer membrane of Escherichia coli. The puzzling feature of TolC structure is that the periplasmic entrance of the channel is closed by dense packing of 12 α‐helices. Efflux pumps exemplified by AcrAB are proposed to drive the opening of TolC channel. How interactions with AcrAB promote the close‐to‐open transition in TolC remains unclear. In this study, we investigated in vivo the functional and physical interactions of AcrAB with the closed TolC and its conformer opened by mutations in the periplasmic entrance. We found that the two conformers of TolC are readily distinguishable in vivo by characteristic drug susceptibility, thiol modification and proteolytic profiles. However, these profiles of TolC variants respond neither to the in vivo stoichiometry of AcrAB:TolC nor to the presence of vancomycin, which is used often to assess the permeability of TolC channel. We further found that the activity and assembly of AcrAB–TolC tolerates significant changes in amounts of TolC and that only a small fraction of intracellular TolC is likely used to support efflux needs of E. coli. Our findings explain why TolC is not a good target for inhibition of multidrug efflux.     

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.     

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments

Humanized Fab′ fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab′ yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab′ expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab′, DsbC had the most significant impact, increasing humanized Fab′ production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored “wild type” cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD₆₀₀ 105), 40 h post Fab′ induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab′ fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212–220, 2017