Cell physiology and protein secretion of Bacillus licheniformis compared to Bacillus subtilis

The genome sequence of Bacillus subtilis was published in 1997 and since then many other bacterial genomes have been sequenced, among them Bacillus licheniformis in 2004. B. subtilis and B. licheniformis are closely related and feature similar saprophytic lifestyles in the soil. Both species can secrete numerous proteins into the surrounding medium enabling them to use high-molecular-weight substances, which are abundant in soils, as nutrient sources. The availability of complete genome sequences allows for the prediction of the proteins containing signals for secretion into the extracellular milieu and also of the proteins which form the secretion machinery needed for protein translocation through the cytoplasmic membrane. To confirm the predicted subcellular localization of proteins, proteomics is the best choice. The extracellular proteomes of B. subtilis and B. licheniformis have been analyzed under different growth conditions allowing comparisons of the extracellular proteomes and conclusions regarding similarities and differences of the protein secretion mechanisms between the two species.     

The Canonical Twin-Arginine Translocase Components Are Not Required for Secretion of Folded Green Fluorescent Protein from the Ancestral Strain of Bacillus subtilis

Cellular processes, such as the digestion of macromolecules, phosphate acquisition, and cell motility, require bacterial secretion systems. In Bacillus subtilis, the predominant protein export pathways are Sec (generalized secretory pathway) and Tat (twin-arginine translocase). Unlike Sec, which secretes unfolded proteins, the Tat machinery secretes fully folded proteins across the plasma membrane and into the medium. Proteins are directed for Tat-dependent export by N-terminal signal peptides that contain a conserved twin-arginine motif. Thus, utilizing the Tat secretion system by fusing a Tat signal peptide is an attractive strategy for the production and export of heterologous proteins. As a proof of concept, we expressed green fluorescent protein (GFP) fused to the PhoD Tat signal peptide in the laboratory and ancestral strains of B. subtilis. Secretion of the Tat-GFP construct, as well as secretion of proteins in general, was substantially increased in the ancestral strain. Furthermore, our results show that secreted, fluorescent GFP could be purified directly from the extracellular medium. Nonetheless, export was not dependent on the known Tat secretion components or the signal peptide twin-arginine motif. We propose that the ancestral strain contains additional Tat components and/or secretion regulators that were abrogated following domestication.     

Selective contribution of the twin-arginine translocation pathway to protein secretion in Bacillus subtilis

The availability of the complete genome sequence of Bacillus subtilis has allowed the prediction of all exported proteins of this Gram-positive eubacterium. Recently, approximately 180 secretory and 114 lipoprotein signal peptides were predicted to direct protein export from the cytoplasm. Whereas most exported proteins appear to use the Sec pathway, 69 of these proteins could potentially use the Tat pathway, as their signal peptides contain RR- or KR-motifs. In the present studies, proteomic techniques were applied to verify how many extracellular B. subtilis proteins follow the Tat pathway. Strikingly, the extracellular accumulation of 13 proteins with potential RR/KR-signal peptides was Tat-independent, showing that their RR/KR-motifs are not recognized by the Tat machinery. In fact, only the phosphodiesterase PhoD was shown to be secreted in a strictly Tat-dependent manner. Sodium azide-inhibition of SecA strongly affected the extracellular appearance of de novo synthesized proteins, including the lipase LipA and two other proteins with predicted RR/KR-signal peptides. The SecA-dependent export of pre-LipA is particularly remarkable, because its RR-signal peptide conforms well to stringent criteria for the prediction of Tat-dependent export in Escherichia coli. Taken together, our observations show that the Tat pathway makes a highly selective contribution to the extracellular proteome of B. subtilis.     

Protein traffic for secretion and related machinery of Bacillus subtilis

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.     

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。     

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.     

A proteomic view of cell physiology of Bacillus licheniformis

The still ongoing sequencing of Bacillus licheniformis at the G?ttingen Sequencing Laboratory provides the basis for proteome studies of the bacterium. By using two-dimensional (2-D) electrophoresis and protein identification by mass spectrometry, we were able to create master gels for B. licheniformis cells grown either in minimal medium or in complex medium containing about 300 and 180 entries, respectively. With the DECODON Delta 2D software we identified the most abundant protein spots on the gels, which were shown to perform mainly basic metabolic functions in the cell such as translation, amino acid metabolism, glycolysis, and tricarboxylic acid (TCA) cycle. Based on the master gels, we were able to study the regulation of metabolic pathways such as glycolysis and TCA cycle. In cells grown in the presence of glucose a significant increase of the amount of some glycolytic enzymes (TpiA, GapA, Pgk, Pgm, Eno, Pyk) and of the pyruvate dehydrogenase (PdhA-D) was found. At the same time, there is a strong repression of almost all TCA cycle enzymes and of the ATP synthase. Glucose also stimulates the acetate kinase (AckA) and the phosphotransacetylase (Pta) which are known to be involved in the overflow metabolism in B. subtilis. Furthermore, we began developing proteomic signatures for growth of B. licheniformis in complex medium. For this purpose, we compared the proteome pattern of exponentially growing cells with that of cells in different stages during stationary phase. The most obvious proteomic signature indicates that cells during stationary phase are subjected to a severe oxidative stress and a resulting protein stress. Furthermore, the level of many vegetative proteins is strongly reduced when the growth is arrested after entry into stationary phase. The data indicate that proteomics can be a valuable tool to describe the physiological state of B. licheniformis cell populations, e.g., of cells growing in a bioreactor.     

Experimental and computational analysis of the secretome of the hyperthermophilic archaeon Pyrococcus furiosus

Although Pyrococcus furiosus is one of the best studied hyperthermophilic archaea, to date no experimental investigation of the extent of protein secretion has been performed. We describe experimental verification of the extracellular proteome of P. furiosus grown on starch. LC–MS/MS-based analysis of culture supernatants led to the identification of 58 proteins. Fifteen of these proteins had a putative N-terminal signal peptide (SP), tagging the proteins for translocation across the membrane. The detected proteins with predicted SPs and known function were almost exclusively involved in important extracellular functions, like substrate degradation or transport. Most of the 43 proteins without predicted N-terminal signal sequences are known to have intracellular functions, mainly (70 %) related to intracellular metabolism. In silico analyses indicated that the genome of P. furiosus encodes 145 proteins with N-terminal SPs, including 21 putative lipoproteins and 17 with a class III peptide. From these we identified 15 (10 %; 7 SPI, 3 SPIII and 5 lipoproteins) under the specific growth conditions of this study. The putative lipoprotein signal peptides have a unique sequence motif, distinct from the motifs in bacteria and other archaeal orders.     

Genetic Analysis of the Colicin V Secretion Pathway

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.     

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide.

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.     

Digging deeper into the plant cell wall proteome

The proteome of the plant cell wall/apoplast is less well characterized than those of other subcellular compartments. This largely reflects the many technical challenges involved in extracting and identifying extracellular proteins, many of which resist isolation and identification, and in capturing a population that is both comprehensive and relatively uncontaminated with intracellular proteins. However, a range of disruptive techniques, involving tissue homogenization and subsequent sequential extraction and non-disruptive approaches has been developed. These approaches have been complemented more recently by other genome-scale screens, such as secretion traps that reveal the genes encoding proteins with N-terminal signal peptides that are targeted to the secretory pathway, many of which are subsequently localized in the wall. While the size and complexity of the wall proteome is still unresolved, the combination of experimental tools and computational prediction is rapidly expanding the catalog of known wall-localized proteins, suggesting the unexpected extracellular localization of other polypeptides and providing the basis for further exploration of plant wall structure and function.     

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.     

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.     

Secretion of hybrid proteins by the Yersinia Yop export system.

After incubation at 37 degrees C in the absence of Ca2+ ions, pathogenic strains of Yersinia spp. release large amounts of a set of plasmid-encoded proteins called Yops. The secretion of these proteins, involved in pathogenicity, occurs via a mechanism that involves neither the removal of a signal sequence nor the recognition of a C-terminal domain. Analysis of deletion mutants allowed the secretion recognition domain to be localized within the 48 N-terminal amino acids of protein YopH, within the 98 N-terminal residues of protein YopE, and within the 76 N-terminal residues of YopQ. Comparison of these regions failed to reveal any sequence similarity, suggesting that the secretion signal of Yop proteins is conformational rather than sequential. Hybrid proteins containing the amino-terminal part of YopH fused to either the alpha-peptide of beta-galactosidase or to alkaline phosphatase deprived of its signal sequence were efficiently secreted to the Yersinia culture medium. This observation opens new prospects in using Yersinia spp. as chimeric-protein producers and as potential live carriers for foreign antigens.     

The extracellular and cytoplasmic proteomes of the non-virulent Bacillus anthracis strain UM23C1-2

The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.     

Comprehensive analysis of the extracellular proteins from Xanthomonas campestris pv. campestris B100

The extracellular proteome of Xanthomonas campestris pv. campestris (Xcc) cultivated in minimal medium was isolated from the cell-free culture supernatant and separated by two-dimensional gel electrophoresis. This technique resolved 97 clearly visible protein spots, which were excised, digested with trypsin and identified on the basis of their peptide mass fingerprints generated by matrix assisted laser desorption/ionisation-time of flight-mass spectrometry. Using this approach 87 different proteins could be distinguished. The Signal P software predicted putative signal peptides for 53% of the extracellular proteins. These proteins are probably transported over the inner membrane and are localized in the periplasm, the outer membrane or secreted into the extracellular space. Among the secreted proteins are 11 degradative enzymes, which are involved in pathogenesis of Xcc. The proteins without obvious secretion signals are known to serve functions in the cytosol. How the cytosolic proteins are delivered to the extracellular space remains unclear.     

Enhanced production and secretion of streptokinase into extracellular medium in Escherichia coli by removal of 13 N-terminal amino acids

The production and secretion of streptokinase using OmpA signal sequence in E. coli was enhanced by removing the 13 N-terminal amino acids (SK(N13). The secretion level of SK(N13 protein into the extracellular medium was two times higher than that of wild-type streptokinase. About 4500 IU of SK(N13 protein per 1 ml LB-ampicillin medium was secreted into extracellular medium at 12 hours after induction. Fully active and enhanced extracellular preparation of the mutant streptokinase may be a potential alternative source for the simple downstream processing     

Glyceride-cysteine lipoproteins and secretion by Gram-positive bacteria.

The membrane penicillinases of Bacillus licheniformis and Bacillus cereus are lipoproteins with N-terminal glyceride thioether modification identical to that of the Escherichia coli outer membrane lipoprotein. They are readily labeled with [3H]palmitate present during exponential growth. At the same time, a few other proteins in each organism become labeled and can be detected by fluorography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total membrane proteins. We distinguish these proteins from the O-acyl proteolipids by demonstrating the formation of glyceryl cysteine sulfone after performic acid oxidation and hydrolysis of the protein. By this criterion, B. licheniformis and B. cereus contain sets of lipoproteins larger in average molecular weight than that of E. coli. Members of the sets probably are under a variety of physiological controls, as indicated by widely differing relative labeling intensity in different media. The set in B. licheniformis shares with membrane penicillinase a sensitivity to release from protoplasts by mild trypsin treatment, which suggests similar orientation on the outside of the membrane. At least one protein is the membrane-bound partner of an extracellular hydrophilic protein, the pair being related as membrane and exopenicillinases are. We propose that the lipoproteins of gram-positive organisms are the functional equivalent of periplasmic proteins in E. coli and other gram-negative bacteria, prevented from release by anchorage to the membrane rather than by a selectively impermeable outer membrane.     

DelGEF,a homologue of the Ran guanine nucleotide exchange factor RanGEF,binds to the exocyst component Sec5 and modulates secretion

In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two-hybrid system. The human homologue of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner. The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP). Downregulation of endogenous DelGEF in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process.