Export and processing analysis of a fusion between the extracellular heat-stable enterotoxin and the periplasmic B subunit of the heat-labile enterotoxin in Escherichia coli

As an initial approach in the study of the mechanism of secretion of the extracellular heat-stable enterotoxin of Escherichia coli (STA), and in order to use this polypeptide as an extracellular carrier we previously constructed a fusion between the complete STA toxin (pre-pro-STA) and the mature B subunit of the periplasmic heat-labile enterotoxin (LTB); the resulting STA-LTB hybrid was not secreted to the extracellular environment, and cells expressing the hybrid lysed at temperatures above 35 degrees C. In this work we have established that the hybrid is initially detected as pre-pro-STA-LTB and converted to pro-STA-LTB, which lacks the 19 amino acids that share the properties of a signal peptide; the sequenced 17 amino-terminal residues of pro-STA-LTB defined the processing site of pre-pro-STA-LTB at pro-3phe-2ala-1 decreases gln+1. This process was sensitive to an energy uncoupler (CCCP) and was correlated with translocation of pro-STA-LTB across the inner membrane. Additionally, we are able to show that although pre-pro-STA-LTB is processed at 37 degrees C and 29 degrees C, it is more efficiently processed at the latter temperature. At 37 degrees C, pro-STA-LTB was poorly released into the periplasm, resulting in accumulation of this protein, pre-pro-STA-LTB, and pre-beta-lactamase in the inner membrane, and in cell lysis. In contrast, at 29 degrees C pro-STA-LTB was localized in the periplasm and in the inner membrane, and pre-pro-STA-LTB and pre-beta-lactamase did not accumulate; however, translocation of periplasmic pro-STA-LTB across the outer membrane still did not occur, and a second processing step that would eliminate the pro segment from pro-STA-LTB was never observed. Thus, the fusion of pre-pro-STA and LTB resulted in a polypeptide that, while incompatible with secretion to the extracellular medium, is exported to the periplasm in a temperature-conditional fashion. This latter observation is consistent with an STA secretion pathway whereby pre-pro-STA is first processed to periplasmic pro-STA by the removal of a 19-amino-acid signal peptide.     

Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1–19) and Pro (aa 20–53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.     

Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae.

Cholera toxin and heat-labile enterotoxin (LT) are structurally similar oligomeric proteins which are capable of being efficiently secreted from Vibrio cholerae. Here we report that these proteins transiently enter the periplasm of V. cholerae as they traverse the cell envelope to reach the extracellular milieu. Pulse-chase experiments on V. cholerae TRH7000 harboring an LT-encoding plasmid revealed that radiolabeled LT A and B subunits entered the periplasm rapidly, followed by their slow efflux (half-time, 13 min) into the medium. LT B-subunit efflux from the periplasm was calculated to be at a rate of ca. 170 monomers per min per cell (which is equivalent to 34 assembled LT holotoxin molecules per min per cell). These values were estimated to be sufficient to account for the increase in extracellular enterotoxin concentration during exponential cell growth. Thus, all enterotoxin subunits which are secreted into the medium can be assumed to be channelled via the periplasm. These findings led to an improved model of the pathway of toxin secretion by V. cholerae.     

High cell density fermentation of recombinant Vibrio cholerae for the production of B subunit of Escherichia coli enterotoxin

High cell density fermentation studies were performed to produce the B subunit of Escherichia coli heat-labile enterotoxin (LTB) from a Vibrio cholerae culture that carries a recombinant plasmid with an ampicillin resistance gene, tac promoter, and the gene encoding LTB. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) the culture secreted the protein into the extracellular milieu. Fed-batch fermentation with stepwise addition of a total of 5 mM of IPTG during the active growth phase of the organism resulted in the production of 400 mg/L of LTB in 9 h and a cell optical density (OD) of 24. The LTB was purified to homogeneity with 70% recovery from the fermentation broth and was found to be chemically and biologically identical to the native protein by N-terminal amino acid sequencing and receptor binding assay. (c) 1995 John Wiley & Sons, Inc.     

A novel method for increasing production of mature proteins in the periplasm of Escherichia coli.

A novel strategy to obtain high-level production of mature proteins exported to the periplasm of Escherichia coli is described. It is based on a modified signal sequence generated by insertion of a coding sequence of the polypeptide precursor of interest at the BamHI site of the commercial vector pQE-30 resulting in an addition of a dodeca-peptide (MRGSH6GS) at the N-terminus of the precursor. The modification does not affect correct processing of the modified signal nor proper folding of the target protein, resulting in an untagged native product. The method is simple for avoiding onerous optimization of translation initiation and screening of host stains. The usefulness of this method is illustrated by overexpression of DsbC and DsbA. Induced by 0.01 mM IPTG at 37 degrees C, proteins were overproduced to comprise 20-30% of the total cellular proteins, and more than 95% of the expressed proteins were correctly processed and exported into the periplasm with yields of more than 100 mg per liter culture.     

Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane.

The plant pathogenic enterobacterium Erwinia chrysanthemi EC16 secretes several extracellular, plant cell wall-degrading enzymes, including pectate lyase isozyme PelE. Secretion kinetics of 35S-labeled PelE indicated that the precursor of PelE was rapidly processed by the removal of the amino-terminal signal peptide and that the resulting mature PelE remained cell bound for less than 60 s before being secreted to the bacterial medium. PelE-PhoA (alkaline phosphatase) hybrid proteins generated in vivo by TnphoA insertions were mostly localized in the periplasm of E. chrysanthemi, and one hybrid protein was observed to be associated with the outer membrane of E. chrysanthemi in an out gene-dependent manner. A gene fusion resulting in the substitution of the beta-lactamase signal peptide for the first six amino acids of the PelE signal peptide did not prevent processing or secretion of PelE in E. chrysanthemi. When pelE was overexpressed, mature PelE protein accumulated in the periplasm rather than the cytoplasm in cells of E. chrysanthemi and Escherichia coli MC4100 (pCPP2006), which harbors a functional cluster of E. chrysanthemi out genes. Removal of the signal peptide from pre-PelE was SecA dependent in E. coli MM52 even in the presence of the out gene cluster. These data indicate that the extracellular secretion of pectic enzymes by E. chrysanthemi is an extension of the Sec-dependent pathway for general export of proteins across the bacterial inner membrane.     

Artificial insertion of peptides between signal peptide and mature protein: effect on secretion and processing of hybrid thermostable alpha-amylases in Bacillus subtilis and Escherichia coli cells

To study the effect of inserted peptides on the secretion and processing of exported proteins in Bacillus subtilis and Escherichia coli, pBR322-derived DNA fragments coding for small peptides were inserted between the DNA coding for the 31 amino acid B. subtilis alpha-amylase signal peptide and that coding for the mature part of the extracellular thermostable alpha-amylase of B. stearothermophilus. Most of the inserted peptides (21 to 65 amino acids) decreased the production of the enzyme in B. subtilis and E. coli, the effect of each peptide being similar in the two strains. In contrast, with one peptide (a 21 amino acid sequence encoded by the extra DNA in pTUBE638), the production of alpha-amylase was enhanced more than 1.7-fold in B. subtilis in comparison with that of the parent strain. The molecular masses of the thermostable alpha-amylases in the periplasm of the E. coli transformants varied for each peptide insert, whereas those in the culture supernatants of the B. subtilis transformants had molecular masses similar to that of the mature enzyme. Based on the NH2-terminal amino acid sequence of the hybrid protein from pTUBE638, it was shown that in E. coli, the NH2-terminally extended thermostable alpha-amylase was translocated and remained in the periplasm after the 31 amino acid signal sequence was removed. In the case of B. subtilis, after the removal of a 34-amino acid signal sequence, the hybrid protein was secreted and processed to the mature form.     

The Secretion of an Intrinsically Disordered Protein with Different Secretion Signals in Bacillus subtilis

In this study, a naturally unsecretory intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was attempted to be secreted with different types of secretion signals in Bacillus subtilis. The results showed that Nsp can be secreted efficiently by all selected Sec-type signal peptides. Nsp was successfully exported when fused to Tat-type signal peptides but less efficient than Sec-type. The fusion protein with the non-classical extracellular proteins can be detected in the cell and extracellular milieu. This study further demonstrated that the mature protein plays an important role in protein secretion. Moreover, these results indicated that Nsp could be a useful tool to understand the individual roles of mature proteins and signal peptide in protein secretion, to evaluate the effect of conformation of mature proteins on their export pathway when coupled with Tat-type signal peptide, and to seek the signal of non-classical secretory proteins.     

The periplasmic chaperone PpiD interacts with secretory proteins exiting from the SecYEG translocon

The Sec translocon of Escherichia coli mediates the export of numerous secretory and membrane proteins. To dissect the passage of an exported protein across the Sec translocon into consecutive steps, we generated in vitro translocation intermediates of a polypeptide chain, which by its N-terminus is anchored in the membrane and by its C-terminus tethered to the ribosome. We find that in this situation, the motor protein SecA propagates translocation of a peptide loop across SecYEG prior to the removal of ribosomes. Upon SecA-driven exit from the translocon, this loop is brought into the immediate vicinity of the membrane-anchored, periplasmic chaperone PpiD. Consistent with a coupling between translocation across the SecYEG translocon and folding by periplasmic chaperones, a lack of PpiD retards the release of a translocating outer membrane protein into the periplasm.     

Efficient function of signal peptidase 1 of Escherichia coli is partly determined by residues in the mature N-terminus of exported proteins

Exported proteins require an N-terminal signal peptide to direct them from the cytoplasm to the periplasm. Once the protein has been translocated across the cytoplasmic membrane, the signal peptide is cleaved by a signal peptidase, allowing the remainder of the protein to fold into its mature state in the periplasm. Signal peptidase I (LepB) cleaves non-lipoproteins and recognises the sequence Ala-X-Ala. Amino acids present at the N-terminus of mature, exported proteins have been shown to affect the efficiency at which the protein is exported. Here we investigated a bias against aromatic amino acids at the second position in the mature protein (P2′). Maltose binding protein (MBP) was mutated to introduce aromatic amino acids (tryptophan, tyrosine and phenylalanine) at P2′. All mutants with aromatic amino acids at P2′ were exported less efficiently as indicated by a slight increase in precursor protein in vivo. Binding of LepB to peptides that encompass the MBP cleavage site were analysed using surface plasmon resonance. These studies showed peptides with an aromatic amino acid at P2′ had a slower off rate, due to a significantly higher binding affinity for LepB. These data are consistent with the accumulation of small amounts of preMBP in purified protein samples. Hence, the reason for the lack of aromatic amino acids at P2′ in E. coli is likely due to interference with efficient LepB activity. These data and previous bioinformatics strongly suggest that aromatic amino acids are not preferred at P2′ and this should be incorporated into signal peptide prediction algorithms.     

Vectorial transport and folding of an autotransporter virulence protein during outer membrane secretion

Autotransporter (AT) proteins are a large and diverse family of extracellular virulence proteins from Gram-negative bacteria, characterized by a central β-helix domain within the mature virulence protein. It is not clear how these proteins cross the outer membrane (OM) quickly and efficiently, without assistance from an external energy source such as ATP or a proton gradient. Conflicting results in the literature have led to several proposed mechanisms for AT OM secretion, including a concerted process, or vectorial secretion with different directionalities. We introduced pairs of cysteine residues into the passenger sequence of pertactin, an AT virulence protein from Bordetella pertussis , and show that OM secretion of the passenger domain stalls due to the formation of a disulphide bond. We further show that the C-terminus of the pertactin passenger domain β-helix crosses the OM first, followed by the N-terminal portions of the virulence protein. In vivo proteolytic digestion shows that the C-terminus is exposed to the extracellular milieu during stalling, and forms stable structure. These AT secretion and folding features can potentially facilitate efficient secretion.     

Role of DegP for two-partner secretion in Bordetella

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In Gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in β-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis , represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long β-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37°C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro , DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.     

Translocation of green fluorescent protein by comparative analysis with multiple signal peptides

Type I and II secretory pathways are used for the translocation of recombinant proteins from the cytoplasm of Escherichia coli. The purpose of this study was to evaluate four signal peptides (HlyA, TorA, GeneIII, and PelB), representing the most common secretion pathways in E. coli, for their ability to target green fluorescent protein (GFP) for membrane translocation. Signal peptide-GFP genetic fusions were designed in accordance with BioFusion standards (BBF RFC 10, BBF RFC 23). The HlyA signal peptide targeted GFP for secretion to the extracellular media via the type I secretory pathway, whereas TAT-dependent signal peptide TorA and Sec-dependent signal peptide GeneIII exported GFP to the periplasm. The PelB signal peptide was inefficient in translocating GFP. The use of biological technical standards simplified the design and construction of functional signal peptide-recombinant protein genetic devices for type I and II secretion in E. coli. The utility of the standardized parts model is further illustrated as constructed biological parts are available for direct application to other studies on recombinant protein translocation.     

Two regions of mature periplasmic maltose-binding protein of Escherichia coli involved in secretion.

Six mutations in malE, the structural gene for the periplasmic maltose-binding protein (MBP) from Escherichia coli, prevent growth on maltose as a carbon source, as well as release of the mutant proteins by the cold osmotic-shock procedure. These mutations correspond to insertion of an oligonucleotide linker, concomitant with a deletion. One of the mutations (malE127) affects the N-terminal extension (the signal peptide), whereas the five others lie within the mature protein. As expected, the export of protein MalE127 is blocked at an early stage. This protein is neither processed to maturity nor sensitive to proteinase K in spheroplasts. In contrast, in the five other mutants, the signal peptide is cleaved and the protein is accessible to proteinase K added to spheroplasts. This indicates that the five mutant proteins are, at least in part, exported through the inner membrane. We propose that the corresponding mutations define two regions of the mature protein (between residues 18 and 42 and between residues 280 and 306), which are important for release of the protein from the inner membrane into the periplasm. We discuss the results in terms of possible conformational changes at this late step of export to the periplasm.     

Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function

DipZ is a bacterial cytoplasmic membrane protein that transfers reducing power from the cytoplasm to the periplasm so as to facilitate the formation of correct disulphide bonds and c-type cytochromes in the latter compartment. Topological analysis using gene fusions between the Escherichia coli dipZ and either E. coli phoA or lacZ shows that DipZ has a highly hydrophobic central domain comprising eight transmembrane alpha-helices plus periplasmic globular N-terminal and C-terminal domains. The previously assigned translational start codon for the E. coli DipZ was shown to be incorrect and the protein to be larger than previously thought. The experimentally determined translational start position indicates that an additional alpha-helix at the N-terminus acts as a cleavable signal peptide so that the N-terminus of the mature protein is located in the periplasm. The newly assigned 5' end of the dipZ gene was shown to be preceded by a functional ribosome-binding site. The hydrophobic central domain and both of the periplasmic globular domains each have a pair of highly conserved cysteine residues, and it was shown by site directed mutagenesis that all six conserved cysteine residues contribute to DipZ function.     

Epigallocatechin gallate and gallocatechin gallate in green tea catechins inhibit extracellular release of Vero toxin from enterohemorrhagic Escherichia coli O157:H7

We studied the effects of six catechin derivatives (catechin, epigallocatechin, epicatechin, epicatechin gallate, epigallocatechin gallate (EGCg) and gallocatechin gallate (GCg)) in green tea on the production and extracellular release of Vero toxins (VTs) from enterohemorrhagic Escherichia coli (EHEC) cultured at 37 degrees C for 24 h. EGCg and GCg in the culture medium markedly inhibited extracellular VTs release from EHEC cells into the culture supernatant fluid at concentrations of 0.05 mg/ml or higher, as estimated by both the reversed passive latex agglutination assay and cytotoxic assay using Vero cells. Production and extracellular release of maltose binding protein, a periplasmic protein, into the culture supernatant were also inhibited by EGCg and GCg, indicating that their inhibitory effect on release from periplasm into the outer milieu is not specific to VTs, but general to the proteins accumulated in EHEC periplasm.     

The superoxide dismutase SodA is targeted to the periplasm in a SecA-dependent manner by a novel mechanism

The manganese/iron-type superoxide dismutase (SodA) of Rhizobium leguminosarum bv. viciae 3841 is exported to the periplasm of R. l. bv. viciae and Escherichia coli. However, it does not possess a hydrophobic cleaved N-terminal signal peptide typically present in soluble proteins exported by the Sec-dependent (Sec) pathway or the twin-arginine translocation (TAT) pathway. A tatC mutant of R. l. bv. viciae exported SodA to the periplasm, ruling out export of SodA as a complex with a TAT substrate as a chaperone. The export of SodA was unaffected in a secB mutant of E. coli, but its export from R. l. bv. viciae was inhibited by azide, an inhibitor of SecA ATPase activity. A temperature-sensitive secA mutant of E. coli was strongly reduced for SodA export. The 10 N-terminal amino acid residues of SodA were sufficient to target the reporter protein alkaline phosphatase to the periplasm. Our results demonstrate the export of a protein lacking a classical signal peptide to the periplasm by a SecA-dependent, but SecB-independent targeting mechanism. Export of the R. l. bv. viciae SodA to the periplasm was not limited to the genus Rhizobium, but was also observed in other proteobacteria.     

A signal sequence mutant defective in export of ribose-binding protein and a corresponding pseudorevertant isolated without imposed selection.

Ribose-binding protein is exported to the periplasmic compartment of Escherichia coli by a process that involves proteolytic cleavage of an amino-terminal extension of amino acids from the precursor form of the protein. In a collection of mutants isolated as defective in the Rbs transport system, a strain was identified that contained only precursor ribose-binding protein, none of which was exported to its normal location in the periplasm. The mutated rbsB contained a base substitution that results in a change of leucine to a proline at position-17 in the signal sequence. A pseudorevertant of the mutant contained proteolytically processed, active ribose-binding protein in the periplasm. The pseudorevertant rbsB carried a second mutation: serine at position-15 in the signal sequence was changed to phenylalanine. Isolation of a signal sequence mutant and a corresponding pseudorevertant without specific selection or site-directed mutagenesis emphasizes the possibility of obtaining export mutants without the use of procedures that could bias or limit the range of mutations found. Explanation of the extreme phenotype of the mutant and the effective correction of that phenotype in the pseudorevertant requires extension of current notions of critical features of signal sequences.     

Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane

Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions. The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA-deficient strain of E. coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.     

提高大肠杆菌分泌表达重组蛋白的研究进展

大肠杆菌是表达重组蛋白的常见宿主之一。重组蛋白分泌到周质空间或胞外培养基中较之在胞内以包含体形式表达有许多优势。主要讨论大肠杆菌Ⅰ、Ⅱ型分泌机制,并总结近年来在提高重组蛋白分泌表达的策略方面取得的进展。