Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus

Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unusually diverse. In addition to flagellin, PibD cleaves three sugar-binding proteins unique to this species and a number of proteins with unknown function. Here we demonstrate that PibD contains two aspartic acid residues that are essential for cleavage activity. An additional pair of aspartic acids in a large cytoplasmic loop is also important for function and is possibly involved in leader peptide recognition. Combining the results of transmembrane segment predictions and cysteine-labeling experiments, we suggest a membrane topology model for PibD with the active-site aspartic acid residues exposed to the cytosol.     

Dissection of key determinants of cleavage activity in signal peptidase III (SPaseIII) PibD

In Archaea, type IV prepilins and prearchaellins are processed by designated signal peptidase III (SPaseIII) prior to their incorporation into pili and the archaellum, respectively. These peptidases belong to the family of integral membrane aspartic acid proteases that contain two essential aspartate residues of which the second aspartate is located in a conserved GxGD motif. To this group also bacterial type IV prepilin peptidases, Alzheimer disease-related secretases, signal peptide peptidases and signal peptide peptidase-like proteases in humans belong. Here we have performed detailed in vivo analyses to understand the cleavage activity of PibD, SPaseIII from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Using an already established in vivo heterologous system cleavage assay, we could successfully identify the key amino acid residues essential for catalysis of PibD. Furthermore, in trans complementation of a pibD S. acidocaldarius deletion mutant with PibD variants having substituted key amino acids has consolidated our observations of the importance of these residues in catalysis. Based on our data, we propose to re-define class III peptidases/type IV prepilin/prearchaellin peptidases as GxHyD group (rather than GxGD) of proteases [Hy-hydrophobic amino acid].     

Cleavage of preflagellins by an aspartic acid signal peptidase is essential for flagellation in the archaeon Methanococcus voltae

The differences between archaeal and bacterial flagella are becoming more apparent as research on the archaeal structure progresses. One crucial difference is the presence of a leader peptide on archaeal preflagellins, which is removed from the flagellin prior to its incorporation into the flagellar filament. The enzyme responsible for the removal of the flagellin leader peptide was identified as FlaK. FlaK of Methanococcus voltae retains its preflagellin peptidase activity when expressed in Escherichia coli and used in an in vitro assay. Homologous recombination of an integration vector into the chromosomal copy of flaK resulted in a non-motile, non-flagellated phenotype. The flagellins of the mutant had larger molecular weights than their wild-type counterparts, as expected if they retained their 11- to 12-amino-acid leader peptide. Membranes of the flaK mutant were unable to process preflagellin in the in vitro assay. Site-directed mutagenesis demonstrated that two aspartic acid residues conserved with ones in type IV prepilin peptidases were necessary for proper recognition or processing of the preflagellin. As bacterial flagellins lack a leader peptide and a peptidase is not required for export and assembly, the requirement for FlaK further emphasizes the similarity archaeal flagella have with type IV pili, rather than with bacterial flagella.     

Identification of a system required for the functional surface localization of sugar binding proteins with class III signal peptides in Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus contains an unusual large number of sugar binding proteins that are synthesized as precursors with a class III signal peptide. Such signal peptides are commonly used to direct archaeal flagellin subunits or bacterial (pseudo)pilins into extracellular macromolecular surface appendages. Likewise, S. solfataricus binding proteins have been suggested to assemble in higher ordered surface structures as well, tentatively termed the bindosome. Here we show that S. solfataricus contains a specific system that is needed for the functional surface localization of sugar binding proteins. This system, encoded by the bas (bindosome assembly system) operon, is composed of five proteins: basABC, three homologues of so-called bacterial (pseudo)pilins; BasE, a cytoplasmic ATPase; and BasF, an integral membrane protein. Deletion of either the three (pseudo)pilin genes or the basEF genes resulted in a severe defect of the cells to grow on substrates which are transported by sugar binding proteins containing class III signal peptides, while growth on glucose and maltose was restored when the corresponding genes were reintroduced in these cells. Concomitantly, DeltabasABC and DeltabasEF cells were severely impaired in glucose uptake even though the sugar binding proteins were normally secreted across the cytoplasmic membrane. These data underline the hypothesis that the bas operon is involved in the functional localization of sugar binding proteins at the cell surface of S. solfataricus. In contrast to surface structure assembly systems of Gram-negative bacteria, the bas operon seems to resemble an ancestral simplified form of these machineries.     

Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions.

Pili of Pseudomonas aeruginosa are assembled from monomers of the structural subunit, pilin, after secretion of this protein across the bacterial membrane. These subunits are initally synthesized as precursors (prepilin) with a six-amino-acid leader peptide that is cleaved off during or after membrane traversal, followed by methylation of the amino-terminal phenylalanine residue. This report demonstrates that additional sequences from the N terminus of the mature protein are necessary for membrane translocation. Gene fusions were made between amino-terminal coding sequences of the cloned pilin gene (pilA) and the structural gene for Escherichia coli alkaline phosphatase (phoA) devoid of a signal sequence. Fusions between at least 45 amino acid residues of the mature pilin and alkaline phosphatase resulted in translocation of the fusion proteins across the cytoplasmic membranes of both P. aeruginosa and E. coli strains carrying recombinant plasmids, as measured by alkaline phosphatase activity and Western blotting. Fusion proteins constructed with the first 10 amino acids of prepilin (including the 6-amino-acid leader peptide) were not secreted, although they were detected in the cytoplasm. Therefore, unlike that of the majority of secreted proteins that are synthesized with transient signal sequences, the membrane traversal of pilin across the bacterial membrane requires the transient six-amino-acid leader peptide as well as sequences contained in the N-terminal region of the mature pilin protein.     

Cellulose degradation by Sulfolobus solfataricus requires a cell-anchored endo-β-1-4-glucanase

A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence of Sulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences in Escherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS of S. solfataricus that are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system for Sulfolobus and on expression control by the promoter of the S. solfataricus gene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed in S. solfataricus was stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.     

Recent advances in the structure and assembly of the archaeal flagellum

Archaeal motility occurs through the rotation of flagella that are distinct from the flagella found on bacteria. The differences between the two structures include the multi-flagellin nature of the archaeal filament, the widespread posttranslational modification of the flagellins and the presence of a short signal peptide on each flagellin that is cleaved by a specific signal peptidase prior to the incorporation of the mature flagellin into the flagellar filament. Research has revealed similarities between the archaeal flagellum and the type IV pilus, including the presence of similar unusual signal peptides on the flagellins and pilins, similarities in the amino acid sequences of the major structural proteins themselves, as well as similarities between potential assembly and processing components. The recent suggestion that type IV pili are part of a family of cell surface complexes, coupled with the similarities between type IV pili and archaeal flagella, raise questions about the evolution of these systems and possible inclusion of archaeal flagella into this surface complex family.     

Archaeal flagella, bacterial flagella and type IV pili: a comparison of genes and posttranslational modifications

The archaeal flagellum is a unique motility organelle. While superficially similar to the bacterial flagellum, several similarities have been reported between the archaeal flagellum and the bacterial type IV pilus system. These include the multiflagellin nature of the flagellar filament, N-terminal sequence similarities between archaeal flagellins and bacterial type IV pilins, as well as the presence of homologous proteins in the two systems. Recent advances in archaeal flagella research add to the growing list of similarities. First, the preflagellin peptidase that is responsible for processing the N-terminal signal peptide in preflagellins has been identified. The preflagellin peptidase is a membrane-bound enzyme topologically similar to its counterpart in the type IV pilus system (prepilin peptidase); the two enzymes are demonstrated to utilize the same catalytic mechanism. Second, it has been suggested that the archaeal flagellum and the bacterial type IV pilus share a similar mode of assembly. While bacterial flagellins and type IV pilins can be modified with O-linked glycans, N-linked glycans have recently been reported on archaeal flagellins. This mode of glycosylation, as well as the observation that the archaeal flagellum lacks a central channel, are both consistent with the proposed assembly model. On the other hand, the failure to identify other genes involved in archaeal flagellation by homology searches likely implies a novel aspect of the archaeal flagellar system. These interesting features remain to be deciphered through continued research. Such knowledge would be invaluable to motility and protein export studies in the Archaea.     

Overexpression of Methanococcus voltae Flagellin Subunits in Escherichia coli and Pseudomonas aeruginosa: a Source of Archaeal Preflagellin

Methanococcus voltae is a flagellated member of the Archaea. Four highly similar flagellin genes have previously been cloned and sequenced, and the presence of leader peptides has been demonstrated. While the flagellins of M. voltae are predicted from their gene sequences to be approximately 22 to 25 kDa, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified flagella revealed flagellin subunits with apparent molecular masses of 31 and 33 kDa. Here we describe the expression of a M. voltae flagellin in the bacteria Escherichia coli and Pseudomonas aeruginosa. Both of these systems successfully generated a specific expression product with an apparently uncleaved leader peptide migrating at approximately 26.5 kDa. This source of preflagellin was used to detect the presence of preflagellin peptidase activity in the membranes of M. voltae. In addition to the native flagellin, a hybrid flagellin gene containing the sequence encoding the M. voltae FlaB2 mature protein fused to the P. aeruginosa pilin (PilA) leader peptide was constructed and transformed into both wild-type P. aeruginosa and a prepilin peptidase (pilD) mutant of P. aeruginosa. Based on migration in SDS-PAGE, the leader peptide appeared to be cleaved in the wild-type cells. However, the archaeal flagellin could not be detected by immunoblotting when expressed in the pilD mutant, indicating a role of the peptidase in the ultimate stability of the fusion product. When the +5 position of the mature flagellin portion of the pilin-flagellin fusion was changed from glycine to glutamic acid (as in the P. aeruginosa pilin) and expressed in both wild-type and pilD mutant P. aeruginosa, the product detected by immunoblotting migrated slightly more slowly in the pilD mutant, indicating that the fusion was likely processed by the prepilin peptidase present in the wild type. Potential assembly of the cleaved fusion product by the type IV pilin assembly system in a P. aeruginosa PilA-deficient strain was tested, but no filaments were noted on the cell surface by electron microscopy.     

Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydrophila.

The exeE gene of Aeromonas hydrophila has been shown to be required for the secretion of most if not all of the extracellular proteins produced by this bacterium. In addition, an exeE::Tn5-751 insertion mutant of A. hydrophila was found to be deficient in the amounts of a number of its major outer membrane proteins (B. Jiang and S. P. Howard, J. Bacteriol. 173:1241-1249, 1991). The exeE gene and the exeF gene were previously isolated as part of a fragment which complemented this mutant. In this study, we have isolated and sequenced a further eight exe genes, exeG through exeN, which constitute the 3' end of the exe operon. These genes have a high degree of similarity with the extracellular secretion operons of a number of different gram-negative bacteria. Marker exchange mutagenesis was used to insert kanamycin resistance cassettes into three different regions of the exe operon. The phenotypes of these mutants showed that in A. hydrophila this operon is required not only for extracellular protein secretion but also for normal assembly of the outer membrane, in particular with respect to the quantities of the major porins. Five of the Exe proteins contain type IV prepilin signal sequences, although the prepilin peptidase gene does not appear to form part of the exe operon. Limited processing of the ExeG protein was observed when it was expressed in Escherichia coli, and this processing was greatly accelerated in the presence of the prepilin peptidase of Pseudomonas aeruginosa.     

Signal peptides of secreted proteins of the archaeon Sulfolobus solfataricus: a genomic survey

Analysis of the recently completed genome sequence of the thermoacidophilic archaeon Sulfolobus solfataricus reveals that about 4.2% of its proteome consists of putative secretory proteins with signal peptides. This includes members of the four major classes of signal peptides: secretory signal peptides, twin-arginine signal peptides, possible lipoprotein precursors, and type IV pilin signal peptides. The latter group is surprisingly large compared to the size of the groups in other organisms and seems to be used predominately for a subset of extracellular substrate-binding proteins.     

A second prepilin peptidase gene in Escherichia coli K-12

Escherichia coli K-12 strains grown at 37°C or 42°C, but not at 30°C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase-dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position −1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non-specific proteases. These data suggested that E . coli K-12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E . coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E . coli when expressed from high-copy-number plasmids but not when present in a single chromosomal copy. The analysis of pppA–lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E . coli isolates.     

An Aeromonas salmonicida type IV pilin is required for virulence in rainbow trout Oncorhynchus mykiss

Aeromonas salmonicida expresses a large number of proven and suspected virulence factors including bacterial surface proteins, extracellular degradative enzymes, and toxins. We report the isolation and characterization of a 4-gene cluster, tapABCD, from virulent A. salmonicida A450 that encodes proteins homologous to components required for type IV pilus biogenesis. One gene, tapA, encodes a protein with high homology to type IV pilus subunit proteins from many gram-negative bacterial pathogens, including Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio vulnificus. A survey of A. salmonicida isolates from a variety of sources shows that the tapA gene is as ubiquitous in this species as it is in other members of the Aeromonads. Immunoblotting experiments demonstrate that it is expressed in vitro and is antigenically conserved among the A. salmonicida strains tested. A mutant A. salmonicida strain defective in expression of TapA was constructed by allelic exchange and found to be slightly less pathogenic for juvenile Oncorhynchus mykiss (rainbow trout) than wild type when delivered by intraperitoneal injection. In addition, fish initially challenged with a high dose of wild type were slightly more resistant to rechallenge with wild type than those initially challenged with the tapA mutant strain, suggesting that presence of TapA contributes to immunity. Two of the other three genes identified, tapB and tapC, encode proteins with homology to factors known to be required for type IV pilus assembly in P. aeruginosa, but in an as yet unidentified manner. TapB is a member of the ABC-transporter family of proteins that contain characteristic nucleotide-binding regions, and which may provide energy for type IV pilus assembly through the hydrolysis of ATP. TapC homologs are integral cytoplasmic membrane proteins that may play a role in pilus anchoring or initiation of assembly. The fourth gene, tapD, encodes a product that shares homology with a family of proteins with a known biochemical function, namely, the type IV prepilin leader peptidases. These bifunctional enzymes proteolytically cleave the leader peptide from the pilin precursor (prepilin) and then N-methylate the newly exposed N-terminal amino acid prior to assembly of the subunits into the pilus structure. We demonstrate that A. salmonicida TapD is able to restore type IV pilus assembly and type II secretion in a P. aeruginosa strain carrying a mutation in its type IV peptidase gene, suggesting that it plays the same role in A. salmonicida.     

Xylanase attachment to the cell wall of the hyperthermophilic bacterium Thermotoga maritima

The cellular localization and processing of the endo-xylanases (1,4-beta-D-xylan-xylanohydrolase; EC 3.2.1.8) of the hyperthermophile Thermotoga maritima were investigated, in particular with respect to the unusual outer membrane ("toga") of this gram-negative bacterium. XynB (40 kDa) was detected in the periplasmic fraction of T. maritima cells and in the culture supernatant. XynA (120 kDa) was partially released to the surrounding medium, but most XynA remained cell associated. Immunogold labeling of thin sections revealed that cell-bound XynA was localized mainly in the outer membranes of T. maritima cells. Amino-terminal sequencing of purified membrane-bound XynA revealed processing of the signal peptide after the eighth residue, thereby leaving the hydrophobic core of the signal peptide attached to the enzyme. This mode of processing is reminiscent of type IV prepilin signal peptide cleavage. Removal of the entire XynA signal peptide was necessary for release from the cell because enzyme purified from the culture supernatant lacked 44 residues at the N terminus, including the hydrophobic part of the signal peptide. We conclude that toga association of XynA is mediated by residues 9 to 44 of the signal peptide. The biochemical and electron microscopic localization studies together with the amino-terminal processing data indicate that XynA is held at the cell surface of T. maritima via a hydrophobic peptide anchor, which is highly unusual for an outer membrane protein.     

An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca

The last gene (pulO) of the pulC-O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC-O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post-translationally. The products of two pulG derivatives with internal inframe deletions were also processed by PulO, but the three PulG-PhoA hybrids, two PulJ-PhoA hybrids and the single PulH-PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction experiments showed that both precursor and mature forms of PulG appear to be associated with low-density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.     

The bindosome is a structural component of the <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> cell envelope

Sugar binding proteins of the thermoacidophile Sulfolobus solfataricus function together with ABC transporters in the uptake of sugars. They are synthesized as precursors with a class III signal peptide that are normally found in archaeal flagellins and bacterial type IV pilins. The functional expression of sugar binding proteins at the cell surface is dependent on the bindosome assembly system (Bas) that is homologous to bacterial type IV pilin assembly systems. The Bas system consists of an assembly ATPase, BasE; a membrane anchoring protein, BasF; and three small class III signal peptide containing proteins BasABC. Expression of BasEF in a S. solfataricus ΔbasEF strain restored the uptake of glucose, while an ATPase mutant of BasE was unable to complement. BasEF was detergent-extracted from S. solfataricus membranes as a stable protein complex. Solute binding proteins can be extracted from the cell surface as two high molecular mass complexes of 600 and 400 kDa, wherein the largest complex also contains the main S-layer protein SlaA. Electron microscopic analysis of the cell surface of the wild-type and ΔbasEF strain indicates that the absence of the BasEF complex causes an alteration in cell morphology and the corrugation of the S-layer pattern that is reversed by complementation with the BasEF complex. These results suggest an interaction between the S-layer and the sugar binding proteins that contribute to cell shape.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.     

细菌三型分泌系统输出蛋白分泌信号研究进展

自提出三型分泌系统的概念以来,相关分子机制的研究让人们对其有了更深入的了解。与依赖信号肽分泌途径形成鲜明对比的是,蛋白通过细菌三型分泌系统分泌或者转运时没有可识别的保守信号序列。近期对三型分泌蛋白的研究发现了多种可以引导其分泌的分泌信号。本文分别介绍了细菌三型分泌系统的种类,分泌系统分泌蛋白的种类,并着重阐述了分泌信号的分子特性及其机制,以期为新型抗菌药物的研发提供新的思路。     

Processing and methylation of PulG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca

The signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the pulO gene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PulG signal sequence. Like most type IV pilins, processed PuiG was found to have a methylated amino-terminal phenylaianine residue. Site-directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11 (+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently IV-methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the car-boxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.     

地衣芽孢杆菌DSM13分泌蛋白信号肽分析

地衣芽孢杆菌是重要的工业微生物,对于其分泌途径及信号肽进行预测和分析,有助于改善影响蛋白分泌的关键因素,高效生产异源蛋白。本研究首次在全基因范围内,利用SignalPv3.0等方法识别了地衣芽孢杆菌DSM13中各种分泌蛋白的信号肽。DSM13信号肽类型包括分泌型Sec信号肽、双精氨酸Tat信号肽、脂蛋白信号肽、IV型纤毛结构信号肽及生物信息素信号肽。同时分析了分泌途径组成,信号肽长度,氨基酸组成,各分泌信号肽特征,与枯草芽孢杆菌的异同以及重要工业酶制剂的分泌途径。该研究对使DSM13成为更有效分泌表达外源蛋白表达系统,具有重要的理论指导意义。