Distinctive amino acid residue periodicities in terminal sequences of type III and type I secreted proteins from proteobacteria

The Fourier transform (FT) method was applied to specify the distribution of 14 predefined groups of amino acids (64 residues) at both termini of annotated type III and type I secreted proteins from proteobacteria. Type I proteins displayed a higher occurrence of significant periodicities at both C-and N-termini, indicating potent features to discriminate between secretion types, particularly by the use of variables selected from the full periodicity profiles at 19 orders of FT. The Fishers linear discriminant analysis, together with the stepwise selection of variables throughout equal pairs of combinations for all predefined groups of residues, revealed the C-terminal harmonics of aromatic (HFWY) and aliphatic (VLIA) residues as a set of strong predictor variables to classify both types of secreted proteins with an accuracy of 100% for original grouped cases and 96.4% for cross-validated grouped cases. The prediction accuracy of proposed discriminant function was estimated by repeated k-fold cross-validation procedures where the original data set was randomly divided into k subsets, with one of the k-subsets serving as the test set and the remaining data forming the training set. The average error rate computed across all k-trials and repeats did not exceed that of leave-one-out procedure. The proposed set of predictor variables could be used to assess the compatibility between secretion pathways and secretion substrates of proteobacteria by means of discriminant analysis.     

Discriminative features of type I and type III secreted proteins from Gram-negative bacteria

The amino acid composition of sequences and structural attributes (α-helices, β-sheets) of C-and N-terminal fragments (50 amino acids) were compared to annotated (SWISS-PROT/ TrEMBL) type I (20 sequences) and type III (22 sequences) secreted proteins of Gram-negative bacteria. The discriminant analysis together with the stepwise forward and backward selection of variables revealed the frequencies of the residues Arg, Glu, Gly, Ile, Met, Pro, Ser, Tyr, Val as a set of strong (1-P < 0.001) predictor variables to discriminate between the sequences of type I and type III secreted proteins with a cross-validated accuracy of 98.6–100 %. The internal and external validity of discriminant analysis was confirmed by multiple (15 repeats) test-retest procedures using a randomly split original set of proteins; this validation method demonstrated an accuracy of 100 % for 191 non-selected (retest) sequences. The discriminant analysis was also applied using selected variables from the propensities for β-sheets and polarity of C-terminal fragments. This approach produced the next highest and comparable cross-validated classification accuracy for randomly selected and retest proteins (85.4–86.0 % and 82.4–84.5 %, respectively). The proposed sets of predictor variables could be used to assess the compatibility between secretion substrates and secretion pathways of Gram-negative bacteria by means of discriminant analysis.     

Distinguishable codon usage and amino acid composition patterns among substrates of leaderless secretory pathways from proteobacteria

The combined set of codon usage frequencies (61 sense codons) from the 111 annotated sequences of leaderless secreted type I, type III, type IV, and type VI proteins from proteobacteria were subjected to the forward and backward selection to obtain a combination of most effective predictor variables for classification/prediction purposes. The group of 24 codon frequencies displayed a strong discriminatory power with an accuracy of 100% for originally grouped and 97.3 ± 1.6% for cross-validated (LOOCV) cases and an acceptable error rate (0.062 ± 0.012) in k-fold (k = 6) cross-validation (KCV). The summary frequencies of synonymous codons for ten amino acids as the alternative predictor variables revealed a comparable discriminatory power (92.8 ± 2.5% for LOOCV), however at somewhat lower levels of prediction accuracy (0.106 ± 0.015 of KCV). A number of significant (p < 0.001) differences were found among indices of codon usage and amino acid composition depending on a definite secretion type. About 60% of secretion substrates were characterized as apparently originated from horizontal gene transfer events or putative alien genes and found to be unequally allocated in respect of groups. The proposed prediction approaches could be used to specify secretome proteins from genomic sequences as well as to assess the compatibility between bacterial secretion pathways and secretion substrates.     

NClassG+: A classifier for non-classically secreted Gram-positive bacterial proteins

Background  

Most predictive methods currently available for the identification of protein secretion mechanisms have focused on classically secreted proteins. In fact, only two methods have been reported for predicting non-classically secreted proteins of Gram-positive bacteria. This study describes the implementation of a sequence-based classifier, denoted as NClassG+, for identifying non-classically secreted Gram-positive bacterial proteins.     

The turn of the screw: variations of the abundant beta-solenoid motif in passenger domains of Type V secretory proteins

Many virulence factors of gram-negative bacteria are secreted by the Type V secretion system via the autotransporter (AT) and two-partner secretion (TPS) pathways. AT proteins effect their own secretion. They comprise three domains: the amino-terminal leader sequence; the secreted passenger domain; and the translocator domain that forms the secretory channel. In the TPS pathway, the passenger and translocator domains are translated as separate proteins. In a previous publication, we proposed a beta-helical structure for the TPS passenger domain of the filamentous hemagglutinin (FHA) of Bordetella pertussis which contains two tracts, R1 and R2, of 19-residue sequence repeats and built molecular models for the R1 and R2 beta-helices. Here, we compare the structure predicted for R1 with the recently determined crystal structure of a fragment containing three R1 repeats and find close agreement, with an RMSD of 1.1A. In the interim, the number of known AT and TPS protein sequences has increased to >1000. To investigate the incidence of beta-helical structures among them, we carried out a sequence-based analysis and conclude that, despite wide diversity in the sizes and sequences of passenger domains, most of them contain beta-solenoids that we classify into thirteen types based on distinctive properties of their beta-coils (repeat length, numbers and lengths of beta-strands and turns, cross-sectional shape, presence of specific residues in certain positions) summarized in a 2D coil template. Some coil types are typical for conventional AT proteins, others, for TPS or trimeric AT proteins. Some beta-solenoids consist of stacked subdomains with coils of different types. To illustrate model-building from a coil template, we modeled a type-T4 beta-solenoid for TibA of Escherichia coli which is predicted to have two conserved polar residues, Thr and Gln, in interior positions.     

SecretP: Identifying bacterial secreted proteins by fusing new features into Chou’s pseudo-amino acid composition

Protein secretion plays an important role in bacterial lifestyles. Secreted proteins are crucial for bacterial pathogenesis by making bacteria interact with their environments, particularly delivering pathogenic and symbiotic bacteria into their eukaryotic hosts. Therefore, identification of bacterial secreted proteins becomes an important process for the study of various diseases and the corresponding drugs. In this paper, fusing several new features into Chou’s pseudo-amino acid composition (PseAAC), two support vector machine (SVM)-based ternary classifiers are developed to predict secreted proteins of Gram-negative and Gram-positive bacteria. For the two types of bacteria, the high accuracy of 94.03% and 94.36% are obtained in distinguishing classically secreted, non-classically secreted and non-secreted proteins by our method. In order to compare the practical ability of our method in identifying bacterial secreted proteins with those of six published methods, proteins in Escherichia coli and Bacillus subtilis are collected to construct the test sets of Gram-negative and Gram-positive bacteria, and the prediction results of our method are comparable to those of existing methods. When performed on two public independent data sets for predicting NCSPs, it also yields satisfactory results for Gram-negative bacterial proteins. The prediction server SecretP can be accessed at http://cic.scu.edu.cn/bioinformatics/secretPV2/index.htm.     

大肠埃希氏杆菌UTI89分泌蛋白组的预测分析

目的：从大肠埃希氏杆菌UTI89基因组中筛选出全部潜在的分泌蛋白并进行初步研究。方法：使用SignalP3.0、TatP1.0、 SecretomeP2.0等蛋白分析软件对5211个ORF进行预测;对筛选出的信号肽及分泌蛋白的基本特征进行统计学分析;使用Blast 2 Sequences进行同源性分析。结果：共筛选出432个sec途径分泌蛋白,19个Tat途径分泌蛋白,386个非经典分泌蛋白;信号肽、分泌蛋白平均长度分别为25.5aa、282.8aa;信号肽中出现频率最高的3种氨基酸依次为L、A、S;仅有两个信号肽的氨基酸序列完全相同,相应的分泌蛋白高度同源。结论：大肠埃希氏杆菌UTI89基因组中有837个ORF可能编码分泌蛋白;分泌蛋白集中在500aa以下;组成信号肽的氨基酸相对保守,多数为疏水氨基酸;信号肽变异性较大,含相同信号肽的蛋白可能由同源基因编码。     

Molecular characterization of type III secretion signals via analysis of synthetic N-terminal amino acid sequences

Yersinia species utilize a type III secretion system to inject toxins, called Yops (Yersinia outer proteins), into eukaryotic cells. The N-termini of the Yops serve as type III secretion signals, but they do not share a consensus sequence. To simplify the analysis of type III secretion signals, we replaced amino acids 2-8 of the secreted protein YopE with all permutations (27 or 128) of synthetic serine/isoleucine sequences. The results demonstrate that amphipathic N-terminal sequences, containing four or five serine residues, have a much greater probability than hydrophobic or hydrophilic sequences to target YopE for secretion. Multiple linear regression analysis of the synthetic sequences was used to obtain a model for N-terminal secretion signals. The model accurately classifies the N-terminal sequences of native type III substrates as efficient secretion signals.     

Effective Identification of Bacterial Type III Secretion Signals Using Joint Element Features

Type III secretion system (T3SS) plays important roles in bacteria and host cell interactions by specifically translocating type III effectors into the cytoplasm of the host cells. The N-terminal amino acid sequences of the bacterial type III effectors determine their specific secretion via type III secretion conduits. It is still unclear as to how the N-terminal sequences guide this specificity. In this work, the amino acid composition, secondary structure, and solvent accessibility in the N-termini of type III and non-type III secreted proteins were compared and contrasted. A high-efficacy mathematical model based on these joint features was developed to distinguish the type III proteins from the non-type III ones. The results indicate that secondary structure and solvent accessibility may make important contribution to the specific recognition of type III secretion signals. Analysis also showed that the joint feature of the N-terminal 6^th–10^th amino acids are especially important for guiding specific type III secretion. Furthermore, a genome-wide screening was performed to predict Salmonella type III secreted proteins, and 8 new candidates were experimentally validated. Interestingly, type III secretion signals were also predicted in gram-positive bacteria and yeasts. Experimental validation showed that two candidates from yeast can indeed be secreted through Salmonella type III secretion conduit. This research provides the first line of direct evidence that secondary structure and solvent accessibility contain important features for guiding specific type III secretion. The new software based on these joint features ensures a high accuracy (general cross-validation sensitivity of ∼96% at a specificity of ∼98%) in silico identification of new type III secreted proteins, which may facilitate our understanding about the specificity of type III secretion and the evolution of type III secreted proteins.     

Type III secretion of EspB in enterohemorrhagic Escherichia coli O157:H7

EspB of enterohemorrhagic Escherichia coli O157:H7 is one of the type III proteins, categorized as translocators, that are secreted in abundance. To define the secretion determinants, different fragments of EspB were fused in recombinant proteins and the proteins secreted into media analyzed by Western blot. The results indicated that the C-terminal 30 residues of EspB were dispensable for secretion whereas the N-terminal first 117 residues played a major role. However, this N-terminal segment alone was not sufficient to confer the secretion. To acquire basic activity, the EspB fusion protein had to contain the N-terminal segment and another segment consisting of either residues 118–190 or residues 191–282. It is possible that the N-terminal region may act as the primary component of the secretion signal while other determinants help to maintain a conformation of EspB favorable for secretion. However, alternative mechanisms cannot be completely excluded. Not withstanding this, the signal for the type III secretion of EspB is apparently distinct from those previously described for the secretion of effector proteins such as Yops in Yersinia.     

How are the Non-classically Secreted Bacterial Proteins Released into the Extracellular Milieu?

Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.     

Comparative and bioinformatics analyses of pathogenic bacterial secretomes identified by mass spectrometry in <Emphasis Type="Italic">Burkholderia</Emphasis> species

Secreted proteins (secretomes) play crucial roles during bacterial pathogenesis in both plant and human hosts. The identification and characterization of secretomes in the two plant pathogens Burkholderia glumae BGR1 and B. gladioli BSR3, which cause diseases in rice such as seedling blight, panicle blight, and grain rot, are important steps to not only understand the disease-causing mechanisms but also find remedies for the diseases. Here, we identified two datasets of secretomes in B. glumae BGR1 and B. gladioli BSR3, which consist of 118 and 111 proteins, respectively, using mass spectrometry approach and literature curation. Next, we characterized the functional properties, potential secretion pathways and sequence information properties of secretomes of two plant pathogens in a comparative analysis by various computational approaches. The ratio of potential non-classically secreted proteins (NCSPs) to classically secreted proteins (CSPs) in B. glumae BGR1 was greater than that in B. gladioli BSR3. For CSPs, the putative hydrophobic regions (PHRs) which are essential for secretion process of CSPs were screened in detail at their N-terminal sequences using hidden Markov model (HMM)–based method. Total 31 pairs of homologous proteins in two bacterial secretomes were indicated based on the global alignment (identity ≥ 70%). Our results may facilitate the understanding of the species-specific features of secretomes in two plant pathogenic Burkholderia species.     

The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming subgroups of this class. The significant homologies between types I, II and III occur almost exclusively in segments of the chain that have been identified as having a coiled-coil structure together with the relatively short sections connecting these segments. The non-coiled-coil segments at the C- and N-termini show no significant homology between types, nor is homology in these segments apparent in all members of one type. Component 8c-1 does not show homology in its terminal segments with the known sequence of any other filamentin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium

We analysed all major proteins secreted into culture media from Salmonella typhimurium. Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS-polyacrylamide gels and analysed by amino acid sequencing. Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a deltamot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented.     

A Multifunctional Region of the Shigella Type 3 Effector IpgB1 Is Important for Secretion from Bacteria and Membrane Targeting in Eukaryotic Cells

Type 3 secretion systems are complex nanomachines used by many Gram–negative bacteria to deliver tens of proteins (effectors) directly into host cells. Once delivered into host cells, effectors often target to specific cellular loci where they usurp host cell processes to their advantage. Here, using the yeast model system, we identify the membrane localization domain (MLD) of IpgB1, a stretch of 20 amino acids enriched for hydrophobic residues essential for the targeting of this effector to the plasma membrane. Embedded within these residues are ten that define the IpgB1 chaperone-binding domain for Spa15. As observed with dedicated class IA chaperones that mask hydrophobic MLDs, Spa15, a class IB chaperone, promotes IpgB1 stability by binding this hydrophobic region. However, despite being stable, an IpgB1 allele that lacks the MLD is not recognized as a secreted substrate. Similarly, deletion of the chaperone binding domains of IpgB1 and three additional Spa15-dependent effectors result in alleles that are no longer recognized as secreted substrates despite the presence of intact N-terminal secretion signal sequences. This is in contrast with MLD-containing effectors that bind class IA dedicated chaperones, as deletion of the MLD of these effectors alleviates the chaperone requirement for secretion. These observations indicate that at least for substrates of class IB chaperones, the chaperone-effector complex plays a major role in defining type 3 secreted proteins and highlight how a single region of an effector can play important roles both within prokaryotic and eukaryotic cells.     

Identification of potential type III secretion proteins via heterologous expression of Vibrio parahaemolyticus DNA

We employed a heterologous secretion assay to identify proteins potentially secreted by type III secretion systems (T3SSs) in Vibrio parahaemolyticus. N-terminal sequences from 32 proteins within T3SS genomic islands and seven proteins from elsewhere in the chromosome included proteins that were recognized for export by the Yersinia enterocolitica flagellar T3SS.     

A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78

Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant–bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.     

A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78

Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant–bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.     

根癌土壤杆菌C58 Cereon中分泌蛋白信号肽分析

利用SignalP3．0、LipoP1．0、TMHMM2．0和TargetP1．014种蛋白分析软件预测了Agrobacterium tumefaciens C58 Cereon菌株全部基因组的4554个ORF编码的蛋白信号肽,共发现203个信号肽,且它们的氨基酸残基相对保守。其中158条具分泌型信号肽,9条具RR-motif型信号肽,28条具信号肽酶Ⅱ型信号肽,8条具细菌素-信息素型信号肽,但只有分泌蛋白AGR-C-1878p和AGR-C-1880p的信号肽氨基酸残基完全相同,表明信号肽是高度变异的。