Characterization of proteins in different subcellular localizations for Escherichia coli K12

Knowing the comprehensive knowledge about the protein subcellular localization is an important step to understand the function of the proteins. Recent advances in system biology have allowed us to develop more accurate methods for characterizing the proteins at subcellular localization level. In this study, the analysis method was developed to characterize the topological properties and biological properties of the cytoplasmic proteins, inner membrane proteins, outer membrane proteins and periplasmic proteins in Escherichia coli (E. coli). Statistical significant differences were found in all topological properties and biological properties among proteins in different subcellular localizations. In addition, investigation was carried out to analyze the differences in 20 amino acid compositions for four protein categories. We also found that there were significant differences in all of the 20 amino acid compositions. These findings may be helpful for understanding the comprehensive relationship between protein subcellular localization and biological function     

Lactoperoxidase-125I localization of salt-extractable alkaline phosphatase on the cytoplasmic membrane of Bacillus licheniformis.

Previous histochemical and biochemical localizations of alkaline phosphatase in Bacillus licheniformis MC14 have shown that the membrane-associated form of the enzyme is located on the inner surface of the cytoplasmic membrane, and soluble forms are located in the periplasmic space and in the growth medium. The distribution of salt-extractable alkaline phosphatase on the surfaces of the cytoplasmic membrane of B. licheniformis MC14 was determined by using lactoperoxidase-125I labeling techniques. Cells harvested during rapid alkaline phosphatase production were converted to protoplasts or lysed protoplasts and labeled. Analysis of the data obtained indicated that 30% of the salt-extractable, membrane-associated alkaline phosphatase was located on the outer surface of the cytoplasmic membrane, whereas 70% of the membrane-associated enzyme was localized on the inner surface. Controls for protoplast integrity (release of tritiated thymidine or examination of cytoplasmic proteins for label content) indicated excellent protoplast stability. Controls indicated that chemical labeling was not a factor in the apparent distribution of alkaline phosphatase on the membrane. These results support the previously reported histochemical localization of alkaline phosphatase on the membrane inner surface. The presence of alkaline phosphatase on the membrane outer surface is reasonable, considering the soluble forms of the enzyme found in the periplasmic region and in the culture medium.     

Outer membrane vesicles from group B Neisseria meningitidis delta gna33 mutant: proteomic and immunological comparison with detergent-derived outer membrane vesicles

We compared the proteome of detergent-derived group B Neisseria meningitidis (MenB) outer membrane vesicles (DOMVs) with the proteome of outer membrane vesicles (m-OMVs) spontaneously released into culture supernatant by MenB delta gna33, a mutant in which the gene coding for a lytic transglycosylase homologous to the E. coli MltA was deleted. In total, 138 proteins were identified in DOMVs by 1- and 2-DE coupled with MS; 64% of these proteins belonged to the inner membrane and cytoplasmic compartments. By contrast, most of the 60 proteins of m-OMVs were classified by PSORT as outer membrane proteins. When tested for their capacity to elicit bactericidal antibodies, m-OMVs elicited a broad protective activity against a large panel of MenB strains. Therefore, the identification of mutations capable of conferring an OMV-releasing phenotype in bacteria may represent an attractive approach to study bacterial membrane composition and organization, and to design new efficacious vaccine formulations.     

A morphologic study of filamentous phage infection of Escherichia coli using biotinylated phages

Using biotinylated phage (BIO-phages), we observed the infection of filamentous phages into Escherichia coli JM109 morphologically. BIO-phages and BIO-phage-derived proteins, mainly pVIII, were detected in E. coli by using the avidin-biotin-peroxidase complex method with electron microscopy. Infected cells revealed positive staining on the outer and inner membranes and in the periplasmic space. Some cells showed specific or predominant staining of the outer membrane, whereas others showed predominant staining of the inner membrane or equivalent staining of the outer and inner membranes. The periplasmic spaces in some infected cells were expanded and filled with reaction products. Some cells showed wavy lines of positive staining in the periplasmic space. BIO-phages were detected as thick filaments or clusters covered with reaction products. The ends of the infecting phages were located on the surface of cells, in the periplasmic space, or on the inner membrane. These findings suggest that phage major coat proteins are integrated into the outer membrane and that phages cause periplasmic expansion during infection.     

Direct Interaction of the EpsL and EpsM Proteins of the General Secretion Apparatus in Vibrio cholerae

The general secretion pathway of gram-negative bacteria is responsible for extracellular secretion of a number of different proteins, including proteases and toxins. This pathway supports secretion of proteins across the cell envelope in two distinct steps, in which the second step, involving translocation through the outer membrane, is assisted by at least 13 different gene products. Two of these components, the cytoplasmic membrane proteins EpsL and EpsM of Vibrio cholerae, have been purified and characterized. Based on gel filtration analysis, both purified EpsM(His)6 and wild-type EpsL present in an Escherichia coli Triton X-100 extract are dimeric proteins. EpsL and EpsM were also found to interact directly and form a Triton X-100 stable complex that could be precipitated with either anti-EpsL or anti-EpsM antibodies. In addition, when the L and M proteins were coexpressed in E. coli, they formed a stable complex and protected each other from proteolytic degradation, indicating that these two proteins interact in vivo and that no other Eps protein is required for their association. Since EpsL is predicted to contain a large cytoplasmic domain, while EpsM is predominantly exposed on the periplasmic side, we speculate that these components might be part of a structure that is involved in bridging the inner and outer membranes. Furthermore, since EpsL has previously been shown to interact with the autophosphorylating cytoplasmic membrane protein EpsE, we hypothesize that this trimolecular complex might be involved in regulating the opening and closing of the secretion pore and/or transducing energy to the site of outer membrane translocation.     

Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli

Extracellular production of recombinant proteins in Escherichia coli has several advantages over cytoplasmic or periplasmic production. However, nonpathogenic laboratory strains of E. coli generally excrete only trace amounts of proteins into the culture medium under normal growth conditions. Here we report a systematic proteome-based approach for developing a system for high-level extracellular production of recombinant proteins in E. coli. First, we analyzed the extracellular proteome of an E. coli B strain, BL21(DE3), to identify naturally excreted proteins, assuming that these proteins may serve as potential fusion partners for the production of recombinant proteins in the medium. Next, overexpression and excretion studies were performed for the 20 selected fusion partners with molecular weights below 40 kDa. Twelve of them were found to allow fused proteins to excrete into the medium at considerable levels. The most efficient excreting fusion partner, OsmY, was used as a carrier protein to excrete heterologous proteins into the medium. E. coli alkaline phosphatase, Bacillus subtilis alpha-amylase, and human leptin used as model proteins could all be excreted into the medium at concentrations ranging from 5 to 64 mg/L during the flask cultivation. When only the signal peptide or the mature part of OsmY was used as a fusion partner, no such excretion was observed; this confirmed that these proteins were truly excreted rather than released by outer membrane leakage. The recombinant protein of interest could be recovered by cleaving off the fusion partner by enterokinase as demonstrated for alkaline phosphatase as an example. High cell density cultivation allowed production of these proteins to the levels of 250-700 mg/L in the culture medium, suggesting the good potential of this approach for the excretory production of recombinant proteins.     

MsbA-dependent translocation of lipids across the inner membrane of Escherichia coli

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.     

SOSUI-GramN: high performance prediction for sub-cellular localization of proteins in gram-negative bacteria

A predictive software system, SOSUI-GramN, was developed for assessing the subcellular localization of proteins in Gram-negative bacteria. The system does not require the sequence homology data of any known sequences; instead, it uses only physicochemical parameters of the N- and C-terminal signal sequences, and the total sequence. The precision of the prediction system for subcellular localization to extracellular, outer membrane, periplasm, inner membrane and cytoplasmic medium was 92.3%, 89.4%, 86.4%, 97.5% and 93.5%, respectively, with corresponding recall rates of 70.3%, 87.5%, 76.0%, 97.5% and 88.4%, respectively. The overall performance for precision and recall obtained using this method was 92.9% and 86.7%, respectively. The comparison of performance of SOSUI-GramN with that of other methods showed the performance of prediction for extracellular proteins, as well as inner and outer membrane proteins, was either superior or equivalent to that obtained with other systems. SOSUI-GramN particularly improved the accuracy for predictions of extracellular proteins which is an area of weakness common to the other methods.     

Signal peptide amino acid sequences in Escherichia coli contain information related to final protein localization. A multivariate data analysis

With few exceptions, the signal peptides from proteins inserted into, or translocated through, the membranes of gram-negative bacteria or the endoplasmic reticulum of eukaryotes have no sequence homologies. Therefore these signal peptides have not been considered to contain information related to the different final localizations of the proteins. In this study, 43 signal peptide amino acid sequences from proteins with different final localizations in Escherichia coli have been subjected to a multivariate data analysis. Each amino acid residue was characterized by 20 physico-chemical properties, yielding a multivariate property profile for each peptide. The similarities/dissimilarities in the property profiles for the signal peptides from different classes were compared with each other by generating few-dimensional partial least squares (PLS) discriminant plots. With this approach, signal peptides from proteins localized to the periplasmic space (PS), the outer membrane (OM), and the extracellular surroundings (excreted proteins), were separated into distinct groups. Signal peptides from pili proteins were not separated from the OM signal peptides and only partly from the PS signal peptides, but were clearly different from the signal peptides of the excreted proteins. Signal peptides from inner membrane proteins were similar to those of the PS peptides. The size and the hydrophobicity of different peptide segments were responsible for the separation of the signal peptide classes. For example, the hydrophobicity of the N-terminal segment of the signal peptides increased with an increased distance from the cytoplasm of the final localization for the corresponding proteins. Thus, many signal peptides from proteins with different final localizations in E. coli have different discernible physico-chemical profiles.     

Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation

Escherichia coli BL21 (DE3) and W3110 strains, belonging to the family B and K-12, respectively, have been most widely employed for recombinant protein production. During the excretory production of recombinant proteins by high cell density cultivation (HCDC) of these strains, other native E. coli proteins were also released. Thus, we analyzed the extracellular proteomes of E. coli BL21 (DE3) and W3110 during HCDC. E. coli BL21 (DE3) released more than twice the amount of protein compared with W3110 during HCDC. A total of 204 protein spots including 83 nonredundant proteins were unambiguously identified by 2-DE and MS. Of these, 32 proteins were conserved in the two strains, while 20 and 33 strain-specific proteins were identified for E. coli BL21 (DE3) and W3110, respectively. More than 70% of identified proteins were found to be of periplasmic origin. The outer membrane proteins, OmpA and OmpF, were most abundant. Two strains showed much different patterns in their released proteins. Also, cell density-dependent variations in the released proteins were observed in both strains. These findings summarized as reference proteome maps will be useful for studying protein release in further detail, and provide new strategies for enhanced excretory production of recombinant proteins.     

Purification of a GSH-affinity binding protein from Bacteroides fragilis devoid of glutathione transferase activity

For the location of the aminoglycoside-(3)-N-acetyltransferase isoenzyme II (AAC(3)-II) in the bacterial cell, two strains were studied: Escherichia coli HB101(pJV03), producing the 31-kDa AAC (3)-II enzyme, and E. coli HB101, which served as a control. From each strain five protein fractions were prepared: culture supernatant, and proteins occurring in the periplasm, cytoplasm, inner membrane and outer membrane. All fractions were tested for enzymatic activity of AAC(3)-II. Most of the acetylating activity was found in the cytoplasmic fraction. The distribution of marker enzymes showed a good separation between the periplasmic and the cytoplasmic fraction.     

Secretion and processing of the Bacillus subtilis endo-β-1,3-1,4-glucanase in Escherichia coli

The endo-beta-1,3-1,4-glucanase enzyme of Bacillus subtilis C120, when synthesized in Escherichia coli, is located mainly in the cytoplasm, but enzyme activity is also detected in the periplasmic space and in the extracellular medium. The proportion recovered in the extracellular medium is not altered by changes in the levels of synthesis of the enzyme. Lysis of E. coli cells is ruled out as the cause of the secretion by the normal localization of beta-galactosidase, an intracellular protein. However, beta-lactamase, which is normally found in the periplasmic space, is detected in the extracellular medium of E. coli transformants containing beta-glucanase plasmids, suggesting that the presence of beta-glucanase in the cell alters the permeability of the outer membrane. The beta-glucanase proteins found in the extracellular medium, the periplasmic space and the cytoplasm have the same electrophoretic mobilities as the secreted enzyme of B. subtilis. Amino-terminal sequencing has shown that the beta-glucanase enzyme in the intracellular fraction of E. coli is processed at a site two amino acids distant from the processing site used in B. subtilis.     

Analysis of curated and predicted plastid subproteomes of Arabidopsis. Subcellular compartmentalization leads to distinctive proteome properties

Carefully curated proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These curated proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional (low abundant) components of each membrane system. The assembly of rigorously curated subcellular proteomes is in itself also important as a parts list for plant and systems biology. Transmembrane and subcellular prediction strategies were evaluated using the curated data sets. The three curated proteomes differ strongly in average isoelectric point and protein size, as well as transmembrane distribution. Removal of the cleavable, N-terminal transit peptide sequences greatly affected isoelectric point and size distribution. Unexpectedly, the Cys content was much lower for the thylakoid proteomes than for the inner envelope. This likely relates to the role of the thylakoid membrane in light-driven electron transport and helps to avoid unwanted oxidation-reduction reactions. A rule of thumb for discriminating between the predicted integral inner envelope membrane and integral thylakoid membrane proteins is suggested. Using a combination of predictors and experimentally derived parameters, four plastid subproteomes were predicted from the fully annotated Arabidopsis genome. These predicted subproteomes were analyzed for their properties and compared to the curated proteomes. The sensitivity and accuracy of the prediction strategies are discussed. Data can be extracted from the new plastid proteome database (http://ppdb.tc.cornell.edu).     

Prediction of protein subcellular location using a combined feature of sequence

To understand the structure and function of a protein, an important task is to know where it occurs in the cell. Thus, a computational method for properly predicting the subcellular location of proteins would be significant in interpreting the original data produced by the large-scale genome sequencing projects. The present work tries to explore an effective method for extracting features from protein primary sequence and find a novel measurement of similarity among proteins for classifying a protein to its proper subcellular location. We considered four locations in eukaryotic cells and three locations in prokaryotic cells, which have been investigated by several groups in the past. A combined feature of primary sequence defined as a 430D (dimensional) vector was utilized to represent a protein, including 20 amino acid compositions, 400 dipeptide compositions and 10 physicochemical properties. To evaluate the prediction performance of this encoding scheme, a jackknife test based on nearest neighbor algorithm was employed. The prediction accuracies for cytoplasmic, extracellular, mitochondrial, and nuclear proteins in the former dataset were 86.3%, 89.2%, 73.5% and 89.4%, respectively, and the total prediction accuracy reached 86.3%. As for the prediction accuracies of cytoplasmic, extracellular, and periplasmic proteins in the latter dataset, the prediction accuracies were 97.4%, 86.0%, and 79.7, respectively, and the total prediction accuracy of 92.5% was achieved. The results indicate that this method outperforms some existing approaches based on amino acid composition or amino acid composition and dipeptide composition.     

Subcellular location and unique secretion of the hemolysin of Serratia marcescens

It is shown that Serratia marcescens exports a hemolysin to the cell surface and secretes it to the extracellular space. Escherichia coli containing the cloned hemolysin genes shlA and shlB exported and secreted the S. marcescens hemolysin. A nonhemolytic secretion-incompetent precursor of the hemolysin, designated ShlA*, was synthesized in a shlB deletion mutant and accumulated in the periplasmic space of E. coli. Immunogold-labeled ultrathin sections revealed ShlA* bound to the outer face of the cytoplasmic membrane and to the inner face of the outer membrane. A number of mutants carrying 3' deletions in the shlA gene secreted truncated polypeptides, the smallest of which contained only 261 of the 1578 amino acids of the mature ShlA hemolysin, showing that the information for export to the cell surface of E. coli and secretion into the culture medium is located in the NH2-terminal segment of the hemolysin. We propose a secretion pathway in which ShlA and ShlB are exported across the cytoplasmic membrane via a signal sequence-dependent mechanism. ShlB is integrated into the outer membrane. ShlA is translocated across the outer membrane with the help of ShlB. During the latter export process or at the cell surface, ShlA acquires the hemolytically active conformation and is released to the extracellular space. The hemolysin secretion pathway appears to be different from any other secretion system hitherto reported and involves only a single specific export protein.     

Proteome of the Escherichia coli envelope and technological challenges in membrane proteome analysis

The envelope of Escherichia coli is a complex organelle composed of the outer membrane, periplasm-peptidoglycan layer and cytoplasmic membrane. Each compartment has a unique complement of proteins, the proteome. Determining the proteome of the envelope is essential for developing an in silico bacterial model, for determining cellular responses to environmental alterations, for determining the function of proteins encoded by genes of unknown function and for development and testing of new experimental technologies such as mass spectrometric methods for identifying and quantifying hydrophobic proteins. The availability of complete genomic information has led several groups to develop computer algorithms to predict the proteome of each part of the envelope by searching the genome for leader sequences, beta-sheet motifs and stretches of alpha-helical hydrophobic amino acids. In addition, published experimental data has been mined directly and by machine learning approaches. In this review we examine the somewhat confusing available literature and relate published experimental data to the most recent gene annotation of E. coli to describe the predicted and experimental proteome of each compartment. The problem of characterizing integral versus membrane-associated proteins is discussed. The E. coli envelope proteome provides an excellent test bed for developing mass spectrometric techniques for identifying hydrophobic proteins that have generally been refractory to analysis. We describe the gel based and solution based proteome analysis approaches along with protein cleavage and proteolysis methods that investigators are taking to tackle this difficult problem.     

Extracellular recombinant protein production from an Escherichia coli lpp deletion mutant

E. coli is one of the most commonly used host strains for recombinant protein production. However, recombinant proteins are usually found intracellularly, in either cytoplasm or periplasmic space. Inadequate secretion to the extracellular environment is one of its limitations. This study addresses the outer membrane barrier for the translocation of recombinant protein directed to the periplasmic space. Specifically, using recombinant maltose binding protein (MalE), xylanase, and cellulase as model proteins, we investigated whether the lpp deletion could render the outer membrane permeable enough to allow extracellular protein production. In each case, significantly higher excretion of recombinant protein was observed with the lpp deletion mutant. Up to 90% of the recombinant xylanase activity and 70% of recombinant cellulase activity were found in the culture medium with the deletion mutant, whereas only 40-50% of the xylanase and cellulase activities were extracellular for the control strain. Despite the weakened outer membrane in the mutant strain, cell lysis did not occur, and increased excretion of periplasmic protein was not due to cell lysis. The lpp deletion is a simple method to generate an E. coli strain to effect significant extracellular protein production. The phenotype of extracellular protein production without cell lysis is useful in many biotechnological applications, such as bioremediation and plant biomass conversion.     

Killing of E coli cells by E group nuclease colicins

The process by which the endonuclease domain of colicin E9 is translocated across the outer membrane, the periplasmic space and the cytoplasmic membrane to reach the cytoplasm of E. coli cells, resulting in DNA degradation and cell death, is a unique event in prokaryotic biology. Although considerable information is known about the role of the BtuB outer membrane receptor, as well as the mostly periplasmic Tol proteins that are essential for the translocation process, the precise nature of the interactions between colicin E9 and these proteins remains to be elucidated. In this review, we consider our current understanding of the key events in this process, concentrating on recent findings concerning receptor-binding, translocation and the mechanism of cytotoxicity.