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.     

Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Effect of signal peptide changes on the extracellular processing of streptokinase from Escherichia coli : requirement for secondary structure at the cleavage junction

Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfils the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction.     

The majority of the matrix protein TapA is dispensable for Bacillus subtilis colony biofilm architecture

Biofilm formation is a co-operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease-resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA-fibre formation but is dispensable for in vitro TasA-fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253-amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.     

Proteolytic processing of <Emphasis Type="Italic">Escherichia coli</Emphasis> twin-arginine signal peptides by LepB

The twin-arginine translocation (Tat) apparatus is a protein targeting system found in the cytoplasmic membranes of many prokaryotes. Substrate proteins of the Tat pathway are synthesised with signal peptides bearing SRRxFLK ‘twin-arginine’ amino acid motifs. All Tat signal peptides have a common tripartite structure comprising a polar N-terminal region, followed by a hydrophobic region of variable length and a polar C-terminal region. In Escherichia coli, Tat signal peptides are proteolytically cleaved after translocation. The signal peptide C-terminal regions contain conserved AxA motifs, which are possible recognition sequences for leader peptidase I (LepB). In this work, the role of LepB in Tat signal peptide processing was addressed directly. Deliberate repression of lepB expression prevented processing of all Tat substrates tested, including SufI, AmiC, and a TorA-23K reporter protein. In addition, electron microscopy revealed gross defects in cell architecture and membrane integrity following depletion of cellular LepB protein levels.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> signal peptidase recognizes and cleaves archaeal signal sequence

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.     

An accessory protein required for anchoring and assembly of amyloid fibres in B. subtilis biofilms

Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibres, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibres. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibres to the cell wall and to assemble TasA into fibres. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibres can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibres. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibres that are not anchored to the cell.     

Characterization of the cleavage of signal peptide at the C-terminus of hepatitis C virus core protein by signal peptide peptidase

Production of hepatitis C virus (HCV) core protein requires the cleavages of polyprotein by signal peptidase and signal peptide peptidase (SPP). Cleavage of signal peptide at the C-terminus of HCV core protein by SPP was characterized in this study. The spko mutant (mutate a.a. 189–193 from ASAYQ to PPFPF) is more efficient than the A/F mutant (mutate a.a 189 and 191 from A to F) in blocking the cleavage of signal peptide by signal peptidase. The cleavage efficiency of SPP is inversely proportional to the length of C-terminal extension of the signal peptide: the longer the extension, the less efficiency the cleavage is. Thus, reducing the length of C-terminal extension of signal peptide by signal peptidase cleavage could facilitate further cleavage by SPP. The recombinant core protein fused with signal peptide from the C-terminus of p7 protein, but not those from the C-termini of E1 and E2, could be cleaved by SPP. Therefore, the sequence of the signal peptide is important but not the sole determinant for its cleavage by SPP. Replacement of the HCV core protein E.R.-associated domain (a.a. 120–150) with the E.R.-associated domain (a.a.1–50) of SARS-CoV membrane protein results in the failure of cleavage of this recombinant protein by SPP, though this protein still is E.R.-associated. This result suggests that not only E.R.-association but also specific protein sequence is important for the HCV core protein signal peptide cleavage by SPP. Thus, our results suggest that both sequences of the signal peptide and the E.R.-associated domain are important for the signal peptide cleavage of HCV core protein by SPP. Electronic Supplementary MaterialThe online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Escherichia coli signal peptidase recognizes and cleaves the signal sequence of α-amylase originating from Bacillus licheniformis

The gene encoding the α-amylase from Bacillus licheniformis was cloned, with and without the native signal sequence, and expressed in Escherichia coli, resulting in the production of the recombinant protein in the cytoplasm as insoluble but enzymatically active aggregates. Expression with a low concentration of the inducer at low temperature resulted in the production of the recombinant protein in soluble form in a significantly higher amount. The protein produced with signal sequence was exported to the extracellular medium, whereas there was no export of the protein produced from the gene without the signal sequence. Similarly, the α-amylase activity in the culture medium increased with time after induction in case of the protein produced with signal sequence. Molecular mass determinations by MALDI-TOF mass spectrometry and N-terminal amino acid sequencing of the purified recombinant α-amylase from the extracellular medium revealed that the native signal peptide was cleaved by E. coli signal peptidase between Ala28 and Ala29. It seems possible that the signal peptide of α-amylase from B. licheniformis can be used for the secretion of other recombinant proteins produced using the E. coli expression system.     

Plastidic type I signal peptidase 1 is a redox‐dependent thylakoidal processing peptidase

Thylakoids are the photosynthetic membranes in chloroplasts and cyanobacteria. The aqueous phase inside the thylakoid known as the thylakoid lumen plays an essential role in the photosynthetic electron transport. The presence and significance of thiol‐disulfide exchange in this compartment have been recognized but remain poorly understood. All proteins found free in the thylakoid lumen and some proteins associated to the thylakoid membrane require an N‐terminal targeting signal, which is removed in the lumen by a membrane‐bound serine protease called thylakoidal processing peptidase (TPP). TPP is homologous to Escherichia coli type I signal peptidase (SPI) called LepB. Genetic data indicate that plastidic SPI 1 (Plsp1) is the main TPP in Arabidopsis thaliana (Arabidopsis) although biochemical evidence had been lacking. Here we demonstrate catalytic activity of bacterially produced Arabidopsis Plsp1. Recombinant Plsp1 showed processing activity against various TPP substrates at a level comparable to that of LepB. Plsp1 and LepB were also similar in the pH optima, sensitivity to arylomycin variants and a preference for the residue at ?3 to the cleavage site within a substrate. Plsp1 orthologs found in angiosperms contain two unique Cys residues located in the lumen. Results of processing assays suggested that these residues were redox active and formation of a disulfide bond between them was necessary for the activity of recombinant Arabidopsis Plsp1. Furthermore, Plsp1 in Arabidopsis and pea thylakoids migrated faster under non‐reducing conditions than under reducing conditions on SDS‐PAGE. These results underpin the notion that Plsp1 is a redox‐dependent signal peptidase in the thylakoid lumen.     

Effect of signal peptide changes on the extracellular processing of streptokinase from Escherichia coli : requirement for secondary structure at the cleavage junction

Streptokinase (SK), an extracellular protein from Streptococcus equisimilis, is secreted post-translationally by Escherichia coli using both its native and E. coli-derived transport signals. In this communication we report that cleavage specificity of signal peptidase I, and thus efficiency of secretion, varies in E. coli when SK export is directed by different transport signals. The native (+1) N-terminus of mature SK was retained when it was transported under the control of its own, PelB or LamB signal peptide. However, when translocation of SK was controlled by the OmpA or MalE signal peptide, Ala2 of mature SK was preferred as a cleavage site for the pre-SK processing. Our results indicate that compatibility of the leader peptide with the mature sequences of SK, which fulfils the requirement for a given secondary structure within the cleavage region, is essential for maintaining the correct processing of pre-SK. An OmpA-SK fusion, which results in the deletion of two N-terminal amino acid residues of mature SK, was further studied with respect to the recognition of alternative cleavage site in E. coli. The alanine at +2 in mature SK was changed to glycine or its relative position was changed to +3 by introducing a methionine residue at the +1 position. Both alterations resulted in the correct cleavage of pre-SK at the original OmpA fusion site. In contrast, introduction of an additional alanine at +4, creating three probable cleavage sites (Ala-x-Ala-x-Ala-x-Ala), resulted in the recognition of all three target sites for cleavage, with varying efficiency. The results indicate that the nature of the secondary structure generated at the cleavage junction of pre-SK, resulting from the fusion of different signal peptides, modulates the cleavage specificity of signal peptidase I during extracellular processing of SK. Based on these findings it is proposed that flexibility in the interaction of the active site of signal peptidase I with the cleavage sites of signal peptides may occur when it encounters two or more juxtaposed cleavage sites. Preference for one cleavage site over another, then, may depend on fulfillment of secondary structure requirements in the vicinity of the pre-protein cleavage junction. Received: 22 September 1997 / Accepted: 17 December 1997     

Identification of Bacillus subtilis SipW as a bifunctional signal peptidase that controls surface-adhered biofilm formation

Biofilms of microbial cells encased in an exopolymeric matrix can form on solid surfaces, but how bacteria sense a solid surface and upregulate biofilm genes is largely unknown. We investigated the role of the Bacillus subtilis signal peptidase, SipW, which has a unique role in forming biofilms on a solid surface and is not required at an air-liquid interface. Surprisingly, we found that the signal peptidase activity of SipW was not required for solid-surface biofilms. Furthermore, a SipW mutant protein was constructed that lacks the ability to form a solid-surface biofilm but still retains signal peptidase activity. Through genetic and gene expression tests, the non-signal peptidase role of SipW was found to activate biofilm matrix genes specifically when cells were on a solid surface. These data provide the first evidence that a signal peptidase is bifunctional and that SipW has a regulatory role in addition to its role as a signal peptidase.     

SipA is required for pilus formation in Streptococcus pyogenes serotype M3

Pili are a major surface feature of the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). The T3 pilus is composed of a covalently linked polymer of protein T3 (formerly Orf100 or Fct3) with an ancillary protein, Cpa, attached. A putative signal peptidase, SipA (also called LepA), has been identified in several pilus gene clusters of GAS. We demonstrate that the SipA2 allele of a GAS serotype M3 strain is required for synthesis of T3 pili. Heterologous expression in Escherichia coli showed that SipA2, along with the pilus backbone protein T3 and the sortase SrtC2, is required for polymerization of the T3 protein. In addition, we found that SipA2 is also required for linkage of the ancillary pilin protein Cpa to polymerized T3. Despite partial conservation of motifs of the type I signal peptidase family proteins, SipA lacks the highly conserved and catalytically important serine and lysine residues of these enzymes. Substitution of alanine for either of the two serine residues closest to the expected location of an active site serine demonstrated that these serine residues are both dispensable for T3 polymerization. Therefore, it seems unlikely that SipA functions as a signal peptidase. However, a T3 protein mutated at the P-1 position of the signal peptide cleavage site (alanine to arginine) was unstable in the presence of SipA2, suggesting that there is an interaction between SipA and T3. A possible chaperone-like function of SipA2 in T3 pilus formation is discussed.     

Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidases

Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B. subtilis and in Escherichia coli. In its natural host B. subtilis, YvaY was transiently synthesised at the end of the exponential growth phase. It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein. In E. coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired. To uncover which elements of E. coli and B. subtilis translocation systems are responsible for the observed substrate specificity, components of the B. subtilis Sec-system were co-expressed besides yvaY in E. coli. Expression of B. subtilis secA or secYEG genes did not affect processing, but expression of B. subtilis signal peptidase genes significantly enhanced processing of preYvaY in E. coli. While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E. coli. These results reveal that targeting and translocation of preYvaY is mediated by the E. coli Sec proteins but processing of preYvaY is not performed by E. coli signal peptidase LepB. Thus, differences in substrate specificities of E. coli LepB and the B. subtilis Sip proteins provide the bottleneck for export of YvaY in E. coli. Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B. subtilis precursor.     

Effect of codon-optimized E. coli signal peptides on recombinant Bacillus stearothermophilus maltogenic amylase periplasmic localization,yield and activity

Recombinant proteins can be targeted to the Escherichia coli periplasm by fusing them to signal peptides. The popular pET vectors facilitate fusion of target proteins to the PelB signal. A systematic comparison of the PelB signal with native E. coli signal peptides for recombinant protein expression and periplasmic localization is not reported. We chose the Bacillus stearothermophilus maltogenic amylase (MA), an industrial enzyme widely used in the baking and brewing industry, as a model protein and analyzed the competence of seven, codon-optimized, E. coli signal sequences to translocate MA to the E. coli periplasm compared to PelB. MA fusions to three of the signals facilitated enhanced periplasmic localization of MA compared to the PelB fusion. Interestingly, these three fusions showed greatly improved MA yields and between 18- and 50-fold improved amylase activities compared to the PelB fusion. Previously, non-optimal codon usage in native E. coli signal peptide sequences has been reported to be important for protein stability and activity. Our results suggest that E. coli signal peptides with optimal codon usage could also be beneficial for heterologous protein secretion to the periplasm. Moreover, such fusions could even enhance activity rather than diminish it. This effect, to our knowledge has not been previously documented. In addition, the seven vector platform reported here could also be used as a screen to identify the best signal peptide partner for other recombinant targets of interest.     

Cotranslational Translocation and Folding of a Periplasmic Protein Domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45–50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.