The structure of the Haemophilus influenzae HMW1 pro-piece reveals a structural domain essential for bacterial two-partner secretion

In pathogenic Gram-negative bacteria, many virulence factors are secreted via the two-partner secretion pathway, which consists of an exoprotein called TpsA and a cognate outer membrane translocator called TpsB. The HMW1 and HMW2 adhesins are major virulence factors in nontypeable Haemophilus influenzae and are prototype two-partner secretion pathway exoproteins. A key step in the delivery of HMW1 and HMW2 to the bacterial surface involves targeting to the HMW1B and HMW2B outer membrane translocators by an N-terminal region called the secretion domain. Here we present the crystal structure at 1.92 A of the HMW1 pro-piece (HMW1-PP), a region that contains the HMW1 secretion domain and is cleaved and released during HMW1 secretion. Structural analysis of HMW1-PP revealed a right-handed beta-helix fold containing 12 complete parallel coils and one large extra-helical domain. Comparison of HMW1-PP and the Bordetella pertussis FHA secretion domain (Fha30) reveals limited amino acid homology but shared structural features, suggesting that diverse TpsA proteins have a common structural domain required for targeting to cognate TpsB proteins. Further comparison of HMW1-PP and Fha30 structures may provide insights into the keen specificity of TpsA-TpsB interactions.     

Channel properties of TpsB transporter FhaC point to two functional domains with a C-terminal protein-conducting pore

Integral outer membrane transporters of the Omp85/TpsB superfamily mediate the translocation of proteins across, or their integration into, the outer membranes of Gram-negative bacteria, chloroplasts, and mitochondria. The Bordetella pertussis FhaC/FHA couple serves as a model for the two-partner secretion pathway in Gram-negative bacteria, with the TpsB protein, FhaC, being the specific transporter of its TpsA partner, FHA, across the outer membrane. In this work, we have investigated the structure/function relationship of FhaC by analyzing the ion channel properties of the wild type protein and a collection of mutants with varied FHA secretion activities. We demonstrated that the channel is formed by the C-terminal two-thirds of FhaC most likely folding into a beta-barrel domain predicted to be conserved throughout the family. A C-proximal motif that represents the family signature appears essential for pore function. The N-terminal 200 residues of FhaC constitute a functionally distinct domain that modulates the pore properties and may participate in FHA recognition.     

Two-partner secretion of gram-negative bacteria: a single β-barrel protein enables transport across the outer membrane

The mechanisms of protein secretion by pathogenic bacteria remain poorly understood. In gram-negative bacteria, the two-partner secretion pathway exports large, mostly virulence-related "TpsA" proteins across the outer membrane via their dedicated "TpsB" transporters. TpsB transporters belong to the ubiquitous Omp85 superfamily, whose members are involved in protein translocation across, or integration into, cellular membranes. The filamentous hemagglutinin/FhaC pair of Bordetella pertussis is a model two-partner secretion system. We have reconstituted the TpsB transporter FhaC into proteoliposomes and demonstrate that FhaC is the sole outer membrane protein required for translocation of its cognate TpsA protein. This is the first in vitro system for analyzing protein secretion across the outer membrane of gram-negative bacteria. Our data also provide clear evidence for the protein translocation function of Omp85 transporters.     

Novel topological features of FhaC, the outer membrane transporter involved in the secretion of the Bordetella pertussis filamentous hemagglutinin

Many pathogenic Gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. The secretion of filamentous hemagglutinin (FHA) by Bordetella pertussis depends on the pore-forming outer membrane protein FhaC, which belongs to a growing family of protein transporters. Protein alignment and secondary structure predictions indicated that FhaC is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface loops and short periplasmic turns. The membrane topology of FhaC was investigated by random insertion of the c-Myc epitope and the tobacco etch virus protease-specific cleavage sequence. FhaC was fairly permissive to short linker insertions. Furthermore, FhaC appeared to undergo conformational changes upon FHA secretion. Surface detection of the inserted sequences indicated that several predicted loops in the C-terminal moiety as well as the N terminus of the protein are exposed. However, a large surface-predicted region in the N-terminal moiety of FhaC was inaccessible from the surface. In addition, the activity and the stability of the protein were affected by insertions in that region, indicating that it may have important structural and/or functional roles. The surface exposure of the N terminus and the presence of an odd number of beta-strands are novel features for beta-barrel outer membrane proteins.     

Structure–function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of Chaperone-Usher Pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a nonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (POlypeptide TRansport Associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4 (LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain.     

Secretion signal of the filamentous haemagglutinin, a model two-partner secretion substrate

The sorting of proteins to their proper subcellular compartment requires specific addressing signals that mediate interactions with ad hoc transport machineries. In Gram-negative bacteria, the widespread two-partner secretion (TPS) pathway is dedicated to the secretion of large, mostly virulence-related proteins. The secreted TpsA proteins carry a characteristic 250-residue-long N-terminal 'TPS domain' essential for secretion, while their TpsB transporters are pore-forming proteins that specifically recognize their respective TpsA partners and mediate their translocation across the outer membrane. However, the nature of the secretion signal has not been elucidated yet. The whooping cough agent Bordetella pertussis secretes its major adhesin filamentous haemagglutinin (FHA) via the TpsB transporter FhaC. In this work, we show specific interactions between an N-terminal fragment of FHA containing the TPS domain and FhaC by using two different techniques, an overlay assay and a pull-down of the complex. FhaC recognizes only non-native conformations of the TPS domain, corroborating the model that in vivo, periplasmic FHA is not yet folded. By generating single amino acid substitutions, we have identified interaction determinants forming the secretion signal. They are found unexpectedly far into the TPS domain and include both conserved and variable residues, which most likely explains the specificity of the TpsA-TpsB interaction. The N-terminal domain of FhaC is involved in the FHA-FhaC interaction, in agreement with its proposed function and periplasmic localization.     

System Specificity of the TpsB Transporters of Coexpressed Two-Partner Secretion Systems of Neisseria meningitidis

The two-partner secretion (TPS) systems of Gram-negative bacteria consist of a large secreted exoprotein (TpsA) and a transporter protein (TpsB) located in the outer membrane. TpsA targets TpsB for transport across the membrane via its ∼30-kDa TPS domain located at its N terminus, and this domain is also the minimal secretory unit. Neisseria meningitidis genomes encode up to five TpsAs and two TpsBs. Sequence alignments of TPS domains suggested that these are organized into three systems, while there are two TpsBs, which raised questions on their system specificity. We show here that the TpsB2 transporter of Neisseria meningitidis is able to secrete all types of TPS domains encoded in N. meningitidis and the related species Neisseria lactamica but not domains of Haemophilus influenzae and Pseudomonas aeruginosa. In contrast, the TpsB1 transporter seemed to be specific for its cognate N. meningitidis system and did not secrete the TPS domains of other meningococcal systems. However, TpsB1 did secrete the TPS2b domain of N. lactamica, which is related to the meningococcal TPS2 domains. Apparently, the secretion depends on specific sequences within the TPS domain rather than the overall TPS domain structure.     

Functional importance of a conserved sequence motif in FhaC, a prototypic member of the TpsB/Omp85 superfamily

In Gram-negative bacteria, the two-partner secretion pathway mediates the secretion of TpsA proteins with various functions. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their transport through a hydrophilic channel. The filamentous haemagglutinin adhesin (FHA)/FhaC pair represents a model two-partner secretion system, with the structure of the TpsB transporter FhaC providing the bases to decipher the mechanism of action of these proteins. FhaC is composed of a β-barrel preceded by two periplasmic polypeptide-transport-associated (POTRA) domains in tandem. The barrel is occluded by an N-terminal helix and an extracellular loop, L6, folded back into the FhaC channel. In this article, we describe a functionally important motif of FhaC. The VRGY tetrad is highly conserved in the TpsB family and, in FhaC, it is located at the tip of L6 reaching the periplasm. Replacement by Ala of the invariant Arg dramatically affects the secretion efficiency, although the structure of FhaC and its channel properties remain unaffected. This substitution affects the secretion mechanism at a step beyond the initial TpsA-TpsB interaction. Replacement of the conserved Tyr affects the channel properties, but not the secretion activity, suggesting that this residue stabilizes the loop in the resting conformation of FhaC. Thus, the conserved motif at the tip of L6 represents an important piece of two-partner secretion machinery. This motif is conserved in a predicted loop between two β-barrel strands in more distant relatives of FhaC involved in protein transport across or assembly into the outer membranes of bacteria and organelles, suggesting a conserved function in the molecular mechanism of transport.     

Substrate recognition by the POTRA domains of TpsB transporter FhaC

Widespread in Gram-negative bacteria, the two-partner secretion (TPS) pathway mediates the secretion of large, β-helical 'TpsA' proteins with various functions. TpsA proteins harbour a conserved, N-proximal TPS domain essential for secretion. TpsB transporters specifically recognize their TpsA partners in the periplasm and mediate their translocation across the outer membrane through a hydrophilic channel. The FHA/FhaC pair of Bordetella pertussis represents a model TPS system. FhaC is composed of a β barrel preceded by two periplasmic POTRA domains in tandem. Here we show that both POTRAs are involved in FHA recognition. Surface plasmon resonance analyses indicated an interaction of micromolar affinity between the POTRAs and the TPS domain with fast association and dissociation steps, consistent with the transient character of this interaction in vivo. Major interaction sites in POTRAs correspond to hydrophobic grooves formed by a β sheet edge and the flanking α helix, well-suited to accommodate extended, amphipathic strands of the substrate and consistent with β augmentation. The initial recruitment of the TPS domain to POTRAs appears to be facilitated by electrostatic attractions. A domain corresponding to the first part of the repeat-rich central region of FHA is also recognized by the POTRAs, suggesting successive interactions in the course of secretion.     

The Polypeptide Transport-associated (POTRA) Domains of TpsB Transporters Determine the System Specificity of Two-partner Secretion Systems

The two-partner secretion (TPS) systems of Gram-negative bacteria secrete large TpsA exoproteins by a dedicated TpsB transporter in the outer membrane. TpsBs contain an N-terminal module located in the periplasm that includes two polypeptide transport-associated (POTRA) domains. These are thought to initiate secretion of a TpsA by binding its N-terminal secretion signal, called the TPS domain. Neisseria meningitidis encodes up to five TpsA proteins that are secreted via only two TpsB transporters: TpsB1 and TpsB2. Of these two, the TpsB2 recognizes the TPS domains of all TpsAs, despite their sequence diversity. By contrast, the TpsB1 shows a limited recognition of a TPS domain that is shared by two TpsAs. The difference in substrate specificity of the TpsBs enabled us to investigate the role of the POTRA domains in the selection of TPS domains. We tested secretion of TPS domains or full-length TpsAs by TpsB mutants with deleted, duplicated, and exchanged POTRA domains. Exchanging the two POTRA domains of a TpsB resulted in a switch in specificity. Furthermore, exchanging a single POTRA domain showed that each of the two domains contributed to the cargo selection. Remarkably, the order of the POTRA domains could be reversed without affecting substrate selection, but this aberrant order did result in an alternatively processed secretion product. Our results suggest that secretion of a TpsA is initiated by engaging both POTRA domains of a TpsB transporter and that these select the cognate TpsAs for secretion.     

The TpsB translocator HMW1B of haemophilus influenzae forms a large conductance channel

The Haemophilus influenzae HMW1 adhesin is secreted via the two-partner secretion pathway and requires HMW1B for translocation across the outer membrane. HMW1B belongs to the Omp85-TpsB superfamily of transporters and consists of two structural domains, a C-terminal transmembrane beta-barrel and an N-terminal periplasmic domain. We investigated the electrophysiological properties of the purified full-length HMW1B and the C-terminal domain using planar lipid bilayers. Both the full-length and the truncated proteins formed conductive pores with a low open probability, two well defined conductance states, and other substates. The kinetic patterns of the two conductance states were distinct, with rapid and frequent transitions to the small conductance state and occasional and more prolonged openings to the large conductance state. The channel formed by the full-length HMW1B showed selectivity for cations, which decreased when measured at pH 5.2, suggesting the presence of acidic residues in the pore. The C-terminal domain of HMW1B was less stable and required reconstitution into liposomes prior to insertion in the bilayer. It formed a channel of smaller conductance but a similar gating pattern as the full-length protein, demonstrating the ability of the last 312 C-terminal amino acids to form a pore and suggesting that the periplasmic domain is not involved in occluding the pore, nor in controlling the inherent basal kinetics of the channel. The HMW1 pro-piece containing the secretion domain, although binding to the channel with high affinity, did not induce channel opening.     

Protein secretion through autotransporter and two-partner pathways

Two distinct protein secretion pathways, the autotransporter (AT) and the two-partner secretion (TPS) pathways are characterized by their apparent simplicity. Both are devoted to the translocation across the outer membrane of mostly large proteins or protein domains. As implied by their name, AT proteins contain their own transporter domain, covalently attached to the C-terminal extremity of the secreted passenger domain, while TPS systems are composed of two separate proteins, with TpsA being the secreted protein and TpsB its specific transporter. In both pathways, the secreted proteins are exported in a Sec-dependent manner across the inner membrane, after which they cross the outer membrane with the help of their cognate transporters. The AT translocator domains and the TpsB proteins constitute distinct families of protein-translocating, outer membrane porins of Gram-negative bacteria. Both types of transporters insert into the outer membrane as beta-barrel proteins possibly forming oligomeric pores in the case of AT and serve as conduits for their cognate secreted proteins or domains across the outer membrane. Translocation appears to be folding-sensitive in both pathways, indicating that AT passenger domains and TpsA proteins cross the periplasm and the outer membrane in non-native conformations and fold progressively at the cell surface. A major difference between AT and TPS pathways arises from the manner by which specificity is established between the secreted protein and its transporter. In AT, the covalent link between the passenger and the translocator domains ensures the translocation of the former without the need for a specific molecular recognition between the two modules. In contrast, the TPS pathway has solved the question of specific recognition between the TpsA proteins and their transporters by the addition to the TpsA proteins of an N-proximal module, the conserved TPS domain, which represents a hallmark of the TPS pathway.     

Secretion of the Haemophilus influenzae HMW1 and HMW2 adhesins involves a periplasmic intermediate and requires the HMWB and HMWC proteins

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H . influenzae HMW1 and HMW2 non-pilus adhesins mediate attachment to human epithelial cells, an essential step during colonization. In order to facilitate interaction with host cells, HMW1 and HMW2 are localized on the surface of the organism in a process that involves cleavage of a 441-amino-acid N-terminal fragment. In the present study, we investigated the pathway for the secretion of HMW1 and HMW2. Cell fractionation experiments and cryoimmunoelectron microscopy demonstrated that a periplasmic intermediate occurs, suggesting involvement of the Sec machinery. Additional analysis revealed that, ultimately, the proteins are partially released from the surface of the organism. Studies with Escherichia coli harbouring plasmid subclones extended earlier findings and suggested that the secretion of HMW1 requires accessory proteins designated HMW1B and HMW1C, while the secretion of HMW2 requires proteins called HMW2B and HMW2C. Further analysis established that HMW1B/HMW1C and HMW2B/HMW2C are interchangeable, an observation consistent with the high degree of homology between HMW1B and HMW2B and between HMW1C and HMW2C. Additional studies of the hmw1 locus indicated that HMW1B is located in the outer membrane and serves to translocate HMW1 across the outer membrane. In the absence of HMW1B, HMW1 remains unprocessed and is degraded in the periplasmic space, at least in part by the DegP protease. Mutagenesis of an HMW1 N-terminal motif shared with other secreted proteins resulted in diminished processing and extracellular release, suggesting interaction of this motif with the HMW1B protein. Continued investigation of the HMW1 and HMW2 adhesins may provide general insights into protein secretion and bacterial pathogenesis.     

EtpB Is a Pore-Forming Outer Membrane Protein Showing TpsB Protein Features Involved in the Two-Partner Secretion System

Attachment to host tissues is a critical step in the pathogenesis of most bacterial infections. Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in humans. The recent identification of additional ETEC surface molecules suggests that new targets may be exploited in vaccine development. The EtpA protein identified in ETEC H10407 is a large glycosylated adhesin secreted via the two-partner secretion system. EtpA requires its putative partner EtpB for translocation across the outer membrane (OM). We investigated the biochemical and electrophysiological properties of purified EtpB. We showed that EtpB is 65-kDa heat-modifiable protein localized to the OM. Electrophysiological experiments indicated that EtpB is able to form pores in planar lipid bilayer membranes with an asymmetric current, suggesting its functional asymmetry. The pore of EtpB frequently assumes an opened conformation and fluctuates between three well-defined conductance states. In silico analysis of the EtpB amino acid sequence and molecular modeling suggest that EtpB is similar to the well-known TpsB protein FhaC from Bordetella pertussis and has a C-terminal transmembrane β-barrel domain that is occluded by an N-terminal α-helix, an extracellular loop, and two periplasmic polypeptide-transport-associated (POTRA) domains. Together, these data confirm that EtpB is a pore-forming protein mainly folded into a β-barrel conformation and indicate that EtpB presents typical features of the OM TpsB proteins.     

The 'P-usher', a novel protein transporter involved in fimbrial assembly and TpsA secretion

We identified a new bacterial transporter, the Pseudomonas aeruginosa CupB3 protein, which is an outer membrane usher involved in pili assembly. In CupB3, the usher domain has fused during evolution with a POTRA (polypeptide-transport-associated)-like domain found in TpsB transporters of two-partner secretion systems. In TpsBs, the POTRA captures the TpsA passenger, which is then transported across the outer membrane through the TpsB beta-barrel. We named CupB3 a 'P-usher' for POTRA-like domain-containing usher. We showed that CupB3 assembles CupB1 fimbrial subunits into pili and secretes CupB5, a TpsA-like protein. The CupB3 usher domain has the function of a TpsB beta-barrel in CupB5 translocation. We revealed that the POTRA-like domain is neither essential for CupB1 fimbriae assembly nor for cell surface exposition of CupB5, but is crucial to coordinate bona fide transport of CupB1 and CupB5 through the usher domain. The P-usher defines a novel transport pathway involving a molecular machine made with old spare parts.     

Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins

A collection of large virulence exoproteins, including Ca2+-independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two-partner secretion (TPS) pathway in various Gram-negative bacteria. The hallmarks of the TPS pathway are the presence of an N-proximal module called the 'secretion domain' in the exoproteins that we have named the TpsA family, and the channel-forming beta-barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N-terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec-dependent fashion, and their signal peptide is probably processed by a LepB-type signal peptidase. The N-proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.     

Structure of the Haemophilus influenzae HMW1B translocator protein: evidence for a twin pore

Secretion of the Haemophilus influenzae HMW1 adhesin occurs via the two-partner secretion pathway and requires the HMW1B outer membrane translocator. HMW1B has been subjected to extensive biochemical studies to date. However, direct examination of the structure of HMW1B has been lacking, leaving fundamental questions about the oligomeric state, the membrane-embedded beta-barrel domain, the approximate size of the beta-barrel pore, and the mechanism of translocator activity. In the current study, examination of purified HMW1B by size exclusion chromatography and negative staining electron microscopy revealed that the predominant species was a dimer. In the presence of lipid, purified HMW1B formed two-dimensional crystalline sheets. Examination of these crystals by cryo-electron microscopy allowed determination of a projection structure of HMW1B to 10 A resolution. The native HMW1B structure is a dimer of beta-barrels, with each beta-barrel measuring 40 A by 50 A in the two orthogonal directions and appearing largely occluded, leaving only a narrow pore. These observations suggest that HMW1B undergoes a large conformational change during translocation of the 125-kDa HMW1 adhesin.     

Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin

The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism. It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC. In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80 kDa N-terminal FHA derivative, Fha44. In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated. In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled. A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B. pertussis or in Escherichia coli expressing FhaC. The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA^?E. coli. The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface. These results argue that the Fha44–CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated. The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines. We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane. Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane. Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B. pertussis.     

Extracellular secretion of pullulanase is unaffected by minor sequence changes but is usually prevented by adding reporter proteins to its N- or C-terminal end.

Linker insertions in the pullulanase structural gene (pulA) were examined for their effects on pullulanase activity and cell surface localization in Escherichia coli carrying the cognate secretion genes from Klebsiella oxytoca. Of the 23 insertions, 11 abolished pullulanase activity but none were found to prevent secretion. To see whether more drastic changes affected secretion, we fused up to five reporter proteins (E. coli periplasmic alkaline phosphatase, E. coli periplasmic maltose-binding protein, periplasmic TEM beta-lactamase, Erwinia chrysanthemi extracellular endoglucanase Z, and Bacillus subtilis extracellular levansucrase) to three different positions in the pullulanase polypeptide: close to the N terminus of the mature protein, at the C terminus of the protein, or at the C terminus of a truncated pullulanase variant lacking the last 256 amino acids. Only 3 of the 13 different hybrids were efficiently secreted: 2 in which beta-lactamase was fused to the C terminus of full-length or truncated pullulanase and 1 in which maltose-binding protein was fused close to the N terminus of pullulanase. Affinity-purified endoglucanase-pullulanase and pullulanase-endoglucanase hybrids exhibited apparently normal levels of pullulanase activity, indicating that the conformation of the pullulanase segment of the hybrid had not been dramatically altered by the presence of the reporter. However, pullulanase-endoglucanase hybrids were secreted efficiently if the endoglucanase component comprised only the 60-amino-acid, C-terminal cellulose-binding domain, suggesting that at least one factor limiting hybrid protein secretion might be the size of the reporter.     

Surface anchoring of a bacterial adhesin secreted by the two-partner secretion pathway

In Gram-negative bacteria, most surface-associated proteins are present as integral outer-membrane proteins. Exceptions include the Haemophilus influenzae HMW1 and HMW2 adhesins and a subset of other proteins secreted by the two-partner secretion system. In the present study we sought to determine the mechanism by which HMW1 is anchored to the bacterial surface. In initial experiments we found that HMW1 forms hair-like fibres on the bacterial surface and is usually present as pairs that appear to be joined together at one end. Further analysis established that HMW1 is anchored to the multimeric HMW1B outer membrane translocator, resulting in a direct correlation between the level of surface-associated HMW1 and the quantity of HMW1B in the outer membrane. Mutagenesis and polyethylene glycol maleimide labelling revealed that anchoring of HMW1 requires the C-terminal 20 amino acids of the protein and is dependent upon disulphide bond formation between two conserved cysteine residues in this region. Immunolabelling studies demonstrated that the immediate C-terminus of HMW1 is inaccessible to surface labelling, suggesting that it remains in the periplasm or is buried in HMW1B. Coexpression of HMW1 lacking the C-terminal 20 amino acids and wild-type HMW1 supported the conclusion that the C-terminus of HMW1 occupies the HMW1B pore. These observations may have broad relevance to proteins secreted by the two-partner secretion system, especially given the conservation of C-terminal cysteine residues among surface-associated proteins in this family.