Systematic identification of the subproteome of Escherichia coli cell envelope reveals the interaction network of membrane proteins and membrane-associated peripheral proteins

Membrane proteins of Gram-negative bacteria are key molecules that interface the cells with the environment. Despite recent proteomic identification of numerous oligomer proteins in the Escherichia coli cell envelope, the protein complex of E. coli membrane proteins and their peripherally associated proteins remain ill-defined. In the current study, we systematically analyze the subproteome of E. coli cell envelope enriched in sarcosine-insoluble fraction (SIF) and sarcosine-soluble fraction (SSF) by using proteomic methodologies. One hundred and four proteins out of 184 spots on 2D electrophoresis gels are identified, which includes 31 outer membrane proteins (OMPs). Importantly, our further proteomic studies reveal a number of previously unrecognized membrane-interacting protein complexes, such as the complex consisting of OmpW and fumarate reductase. This established complete proteomic profile of E. coli envelope also sheds new insight into the function(s) of E. coli outer envelope.     

凡纳滨对虾血蓝蛋白与病原菌凝集作用靶标的鉴定

血蓝蛋白是一种具有多种非特异性免疫学活性的多功能蛋白,以前的研究发现,血蓝蛋白具有凝集活性.本研究采用凝集抑制实验和亲和蛋白质组学等方法探索凡纳滨对虾血蓝蛋白与病原菌的凝集作用靶标.结果显示,大肠杆菌K12和副溶血弧菌外膜蛋白可以抑制血蓝蛋白对7种细菌的凝集活性,其中大肠杆菌K12中2种分子质量分别为16 kD、18 kD （命名为 p16、p18）的外膜蛋白可以与血蓝蛋白发生特异性的结合,经MALDI-TOF/MS鉴定,p16、p18 分别与大肠杆菌外膜蛋白OmpC、OmpX具有高度同源性.尤其是与大肠杆菌K12野生菌株相比,血蓝蛋白对 ΔOmpX 的凝集特异性明显降低,后者仅为前者的25％.由此推测,OmpX 应为血蓝蛋白与病原菌的凝集作用靶标.     

Bacterial expression systems for recombinant protein production: E. coli and beyond

Escherichia coli expression system continues to dominate the bacterial expression systems and remain to be the preferred system for laboratory investigations and initial development in commercial activities or as a useful benchmark for comparison among various expression platforms. Some new developments in overcoming its shortcomings are reviewed in this paper, including antibiotics-free selection plasmids, extracellular production, and posttranslational modifications. The ability for E. coli to make mg glycosylated proteins promises even broader applications of the E. coli system in the future. Significant progresses have also been made over the past few years in alternative bacterial expression systems. Notably, the Lactoccocus lactis system has proven to be a viable choice for membrane proteins. Additionally, several Pseudomonas systems were developed and achieved product titers comparable to E. coli systems. Other bacterial systems such as Streptomyces, coryneform bacteria, and halophilic bacteria offer advantages in some niche areas, providing more choices of bacterial expression systems for recalcitrant proteins.     

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.     

Novel proteomic tools reveal essential roles of SRP and importance of proper membrane protein biogenesis

The signal recognition particle (SRP), which mediates cotranslational protein targeting to cellular membranes, is universally conserved and essential for bacterial and mammalian cells. However, the current understanding of the role of SRP in cell physiology and pathology is still poor, and the reasons behind its essential role in cell survival remain unclear. Here, we systematically analyzed the consequences of SRP loss in E. coli using time-resolved quantitative proteomic analyses. A series of snapshots of the steady-state and newly synthesized proteome unveiled three stages of cellular responses to SRP depletion, and demonstrated essential roles of SRP in metabolism, membrane potential, and protein and energy homeostasis in both the membrane and cytoplasm. We also identified a group of periplasmic proteins, including key molecular chaperones, whose localization was impaired by the loss of SRP; this and additional results showed that SRP is crucial for protein homeostasis in the bacterial envelope. These results reveal the extensive roles that SRP plays in bacterial physiology, emphasize the importance of proper membrane protein biogenesis, and demonstrate the ability of time-resolved quantitative proteomic analysis to provide new biological insights.     

Characterization of Outer Membrane Proteins of Escherichia Coli in Response to Phenol Stress

Gram-negative bacteria are generally more tolerant to disinfectants than Gram-positive bacteria due to outer membrane (OM) barrier, but the tolerant mechanism is not well characterized. We have utilized comparative proteomic methodologies to characterize the OM proteins of E. coli K-12 K99+ in response to phenol stress and found that nine proteins were altered significantly. They were OM proteins OmpA, FadL, LamB, and OmpT, cytoplasmic-associated proteins AceA and EF-Tu, inner membrane protein AtpB, putative capsid protein Q8FewO, and unknown location protein Dps. They were reported here for the first time to be phenol-tolerant proteins. The alteration and functional characterization of the four OM proteins were further investigated using western blotting, genetically modified strains with gene deletion and gene complementation approaches. Our results characterized the functional OM proteins of E. coli in resistance to phenol, and provide novel insights into the mechanisms of bacterial disinfectant-tolerance and new drug targets for control of phenol-resistant bacteria.     

Comparative analysis of envelope proteomes in Escherichia coli B and K-12 strains

Recent genome comparisons of E. coli B and K-12 strains have indicated that the makeup of the cell envelopes in these two strains is quite different. Therefore, we analyzed and compared the envelope proteomes of E. coli BL21(DE3) and MG1655. A total of 165 protein spots, including 62 nonredundant proteins, were unambiguously identified by two-dimensional gel electrophoresis and mass spectrometry. Of these, 43 proteins were conserved between the two strains, whereas 4 and 16 strain-specific proteins were identified only in E. coli BL21(DE3) and MG1655, respectively. Additionally, 24 proteins showed more than 2-fold differences in intensities between the B and K-12 strains. The reference envelope proteome maps showed that E. coli envelope mainly contained channel proteins and lipoproteins. Interesting proteomic observations between the two strains were as follows: (i) B produced more OmpF porin with a larger pore size than K-12, indicating an increase in the membrane permeability; (ii) B produced higher amounts of lipoproteins, which facilitates the assembly of outer membrane beta-barrel proteins; and (iii) motility- (FliC) and chemotaxis-related proteins (CheA and CheW) were detected only in K-12, which showed that E. coli B is restricted with regard to migration under unfavorable conditions. These differences may influence the permeability and integrity of the cell envelope, showing that E. coli B may be more susceptible than K-12 to certain stress conditions. Thus, these findings suggest that E. coli K-12 and its derivatives will be more favorable strains in certain biotechnological applications, such as cell surface display or membrane engineering studies.     

Identification and antibody-therapeutic targeting of chloramphenicol-resistant outer membrane proteins in Escherichia coli

Bacterial resistance to an antibiotic may result from survival in a suddenly strong antibiotic or in sub-minimum inhibitory concentration of the drug. Their shared proteins responsible for the resistance should be potential targets for designing new drugs to inhibit the growth of the antibiotic-resistant bacteria. In the current study, comparative proteomic methodologies were used for identification of sharedly altered outer membrane proteins (OM proteins) that are responsible for chloramphenical (CAP)-resistant Escherichia coli and for survival in medium with suddenly strong CAP treatment. Six differential OM proteins and another protein with unknown location were determined to be sharedly CAP-resistant-related proteins with the use of 2-DE/MS, Western blotting and gene mutant methods, in which TolC, OmpT, OmpC, and OmpW were critically altered proteins and potential targets for designing of the new drugs. Furthermore, a novel method of specific antibody combating bacterial growth was developed on these OM proteins. Only anti-TolC showed a very significant inhibition on bacterial growth in medium with CAP when antisera to TolC, OmpC, OmpT, and OmpW were separately utilized. The growth of CAP-resistant E. coli and its original strain was completely inhibited when they bound with anti-TolC and survived in 1/8 MIC of CAP. This observed result is basically the same to the finding that DeltatolC was survived in the same concentration of the antibiotic. Our study demonstrates that the enhancement of expression of antibody target with antibiotic could be very effective approach compared to using a drug alone, which highlights a potential way for treatment of infection by antibiotic-resistant bacteria.     

Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit and does not represent a recent acquisition of the phage. The P1 and E. coli SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing E. coli host, and it is sufficient to promote bacterial growth in the absence of the E. coli SSB protein. Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary phase. These results reconciled the observed evolutionary conservation with the seemingly redundant presence of ssb genes in many bacteriophages and conjugative plasmids.     

Escherichia coli--a model system that benefits from and contributes to the evolution of proteomics

The large body of knowledge about Escherichia coli makes it a useful model organism for the expression of heterologous proteins. Proteomic studies have helped to elucidate the complex cellular responses of E. coli and facilitated its use in a variety of biotechnology applications. Knowledge of basic cellular processes provides the means for better control of heterologous protein expression. Beyond such important applications, E. coli is an ideal organism for testing new analytical technologies because of the extensive knowledge base available about the organism. For example, improved technology for characterization of unknown proteins using mass spectrometry has made two-dimensional electrophoresis (2DE) studies more useful and more rewarding, and much of the initial testing of novel protocols is based on well-studied samples derived from E. coli. These techniques have facilitated the construction of more accurate 2DE maps. In this review, we present work that led to the 2DE databases, including a new map based on tandem time-of-flight (TOF) mass spectrometry (MS); describe cellular responses relevant to biotechnology applications; and discuss some emerging proteomic techniques.     

Characterization and identification of vaccine candidate proteins through analysis of the group A Streptococcus surface proteome

We describe a proteomic approach for identifying bacterial surface-exposed proteins quickly and reliably for their use as vaccine candidates. Whole cells are treated with proteases to selectively digest protruding proteins that are subsequently identified by mass spectrometry analysis of the released peptides. When applied to the sequenced M1_SF370 group A Streptococcus strain, 68 PSORT-predicted surface-associated proteins were identified, including most of the protective antigens described in the literature. The number of surface-exposed proteins varied from strain to strain, most likely as a consequence of different capsule content. The surface-exposed proteins of the highly virulent M23_DSM2071 strain included 17 proteins, 15 in common with M1_SF370. When 14 of the 17 proteins were expressed in E. coli and tested in the mouse for their capacity to confer protection against a lethal dose of M23_DSM2071, one new protective antigen (Spy0416) was identified. This strategy overcomes the difficulties so far encountered in surface protein characterization and has great potential in vaccine discovery.     

Escherichia coli exhibits a membrane-related response to a small arginine- and tryptophan-rich antimicrobial peptide

Since multiresistant bacterial strains are more widespread and the victim numbers steadily increase, it is very important to possess a broad bandwidth of antimicrobial substances. Antibiotics often feature membrane-associated effect mechanisms. So, we present a membrane proteomic approach to shed light on the cellular response of Escherichia coli as model organism to the hexapeptide MP196, which is arginine and tryptophan rich. Analyzing integral membrane proteins are still challenging, although various detection strategies have been developed in the past. In particular, membrane proteomics in bacteria have been conducted very little due to the special physical properties of these membrane proteins. To obtain more information on the cellular response of the new compound group of small peptides, the tryptophan- and arginine-rich hexapeptide MP196 was subject to a comprehensive quantitative membrane proteomic study on E. coli by means of metabolic labeling in combination with membrane lipid analyses. This study provides in total 767 protein identifications including 185 integral membrane proteins, from which 624 could be quantified. Among these proteins, 134 were differentially expressed. Thereby, functional groups such as amino acid and membrane biosynthesis were affected, stress response could be observed, and the lipid composition of the membrane was significantly altered. Especially, the strong upregulation of the envelope stress induced protein. Spy indicates membrane damage, as well as the downregulation of the mechano-sensitive channel MscL beside others. Finally, the exceptional downregulation of transport systems strengthens these findings.     

Expression Analysis of Up-Regulated Genes Responding to Plumbagin in Escherichia coli

Plumbagin is found in many medicinal plants and has been reported to have antimicrobial activities. We examined the molecular responses of Escherichia coli to plumbagin by using a proteomic approach to search for bacterial genes up-regulated by the drug. The protein profile obtained was compared with that of E. coli without the plumbagin treatment. Subsequent analyses of the induced proteins by mass spectroscopy identified several up-regulated genes, including ygfZ, whose function has not been defined. Analyses of the 5'-flanking sequences indicate that most of these genes contain a marbox-like stretch, and several of them are categorized as members of the mar/sox regulon. Representatives of these genes were cloned into plasmids, and the marbox-like sequences were modified by site-directed mutagenesis. It was proven that mutations in these regions substantially repressed the level of proteins encoded by the downstream genes. Furthermore, plumbagin's early effect was demonstrated to robustly induce SoxS rather than MarA, an observation distinctly different from that seen with sodium salicylate.     

Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.     

Proteomic analysis of cell envelope from Staphylococcus xylosus C2a, a coagulase-negative staphylococcus

Staphylococcus xylosus is a saprophytic bacterium commonly found on skin of mammals but also used for its organoleptic properties in manufacturing of fermented meat products. This bacterium is able to form biofilms and to colonize biotic or abiotic surfaces, processes which are mediated, to a certain extent, by cell-envelope proteins. Thus, the present investigation aimed at evaluating and adapting different existing methods for cell-envelope subproteome analyses of the strain S. xylosus C2a. The protocol selected consisted initially of a lysostaphin treatment producing protoplasts and giving a fraction I enriched in cell wall proteins. A second fraction enriched in membrane proteins was then efficiently recovered by a procedure involving delipidation with a mixture of tributyl phosphate, methanol, and acetone and solubilization with a buffer containing ASB14. Proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). A total of 168 protein spots was identified corresponding to 90 distinct proteins. To categorize and analyze these proteomic data, a rational bioinformatic approach was carried out on proteins identified within cell envelope of S. xylosus C2a. Thirty-four proteins were predicted as membrane-associated with 91% present, as expected, within fraction II enriched in membrane proteins: 24 proteins were predicted as membranal, 3 as lipoproteins, and 7 as components of membrane protein complex. Eighteen out of 25 (72%) proteins predicted as secreted were indeed identified in fraction I enriched in cell wall proteins: 6 proteins were predicted as secreted via Sec translocon, and the remaining 19 proteins were predicted as secreted via unknown secretion system. Eighty-one percent (25/31) of proteins predicted as cytoplasmic were found in fraction II: 8 were clearly predicted as interacting temporarily with membrane components. By coupling conventional 2-DE and bioinformatic analysis, the approach developed allows fractionating, resolving, and analyzing a significant and important set of cell envelope proteins from a coagulase-negative staphylococcus, that is, S. xylosus C2a.     

Identification of the interactome between fish plasma proteins and Edwardsiella tarda reveals tissue-specific strategies against bacterial infection

Elucidating the complex pathogen-host interaction is essential for a comprehensive understanding of how these remarkable agents invade their hosts and how the hosts defend against these invaders. During the infection, pathogens interact intensively with host to enable their survival, which can be revealed through their interactome. Edwardsiella tarda is a Gram-negative bacterial pathogen causing huge economic loss in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. E. tarda is an ideal model for host-pathogen investigation as it infects fish in three distinct steps: entering the host, circulating through the blood and establishing infection. We adopted a previous established proteomic approach that inactivated E. tarda cells and covalent crosslink fish plasma proteins were used to capture plasma proteins and bacterial outer membrane proteins, respectively. By the combinatorial use of proteomic and biochemical approaches, six plasma proteins and seven outer membrane proteins (OMPs) were identified. Interactions among these proteins were validated with protein-array, far-Western blotting and co-immunoprecipitation. At last, seventeen plasma protein-bacteria protein⿿protein interaction were confirmed to be involved in the interaction network, forming a complex interactome. Compared to our previous results, different host proteins were detected, whereas some of the bacterial proteins were similar, which indicates that hosts adopt tissue-specific strategies to cope with the same pathogen during infection. Thus, our results provide a robust demonstration of both bacterial initiators and host receptors or interacting proteins to further explore infection and anti-infective mechanisms between hosts and microbes.     

HU-GFP and DAPI co-localize on the Escherichia coli nucleoid

The heterodimeric HU protein, one of the most abundant DNA binding proteins, plays a pleiotropic role in bacteria. Among others, HU was shown to contribute to the maintenance of DNA superhelical density in Escherichia coli. By its properties HU shares some traits with histones and HMG proteins. More recently, its specific binding to DNA recombination and repair intermediates suggests that HU should be considered as a DNA damage sensor. For all these reasons, it will be of interest to follow the localization of HU within the living bacterial cells. To this end, we constructed HU-GFP fusion proteins and compared by microscopy the GFP green fluorescence with images of the nucleoid after DAPI staining. We show that DAPI and HU-GFP colocalize on the E. coli nucleoid. HU, therefore, can be considered as a natural tracer of DNA in the living bacterial cell.     

The levanase operon of Bacillus subtilis expressed in Escherichia coli can substitute for the mannose permease in mannose uptake and bacteriophage lambda infection.

Bacteriophage lambda adsorbs to its Escherichia coli K-12 host by interacting with LamB, a maltose- and maltodextrin-specific porin of the outer membrane. LamB also serves as a receptor for several other bacteriophages. Lambda DNA requires, in addition to LamB, the presence of two bacterial cytoplasmic integral membrane proteins for penetration, namely, the IIC(Man) and IID(Man) proteins of the E. coli mannose transporter, a member of the sugar-specific phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS transporters for mannose of E. coli, for fructose of Bacillus subtilis, and for sorbose of Klebsiella pneumoniae were shown to be highly similar to each other but significantly different from other PTS transporters. These three enzyme II complexes are the only ones to possess distinct IIC and IID transmembrane proteins. In the present work, we show that the fructose-specific permease encoded by the levanase operon of B. subtilis is inducible by mannose and allows mannose uptake in B. subtilis as well as in E. coli. Moreover, we show that the B. subtilis permease can substitute for the E. coli mannose permease cytoplasmic membrane components for phage lambda infection. In contrast, a series of other bacteriophages, also using the LamB protein as a cell surface receptor, do not require the mannose transporter for infection.