Membrane proteins of Escherichia coli K-12: two-dimensional polyacrylamide gel electrophoresis of inner and outer membranes.

Protein compositions of the inner and outer membranes of Escherichia coli K-12 have been analyzed by two-dimensional gel electrophoresis in which proteins are separated according to apparent isoelectric point (first dimension) and to apparent molecular weight (second dimension). Membrane proteins except for a pair of major outer membrane proteins (proteins Ia and Ib) were found to be solubilized effectively by lysis buffer containing urea, Triton X-100, ampholines and 2-mercaptoethanol. The latter two proteins could be solubilized after precipitation of membrane fraction with trichloroacetic acid; they formed a pair of spots at an acidic region on the electropherogram. Another major protein of the outer membrane, protein II, was also identified. Most of the inner and outer membrane proteins were shown to be focused at a pH range between 4 and 6.5. Specific protein patterns characteristic for both the inner and outer membranes could thous be visualized by the present system. At least 120 and 50 protein species were detected for the inner and outer membranes, respectively.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

Distribution of proteins similar to IIIManH and IIIManL of the Streptococcus salivarius phosphoenolpyruvate:mannose-glucose phosphotransferase system among oral and nonoral bacteria.

In Streptococcus salivarius, the phosphoenolpyruvate (PEP):mannose-glucose phosphotransferase system, which concomitantly transports and phosphorylates mannose, glucose, fructose, and 2-deoxyglucose, is composed of the general energy-coupling proteins EI and HPr, the specific membrane-bound IIIMan, and two forms of a protein called IIIMan, with molecular weights of 38,900 (IIIManH) and 35,200 (IIIManL), that are found in the cytoplasm as well as associated with the membrane. Several lines of evidence suggest that IIIManH and/or IIIManL are involved in the control of sugar metabolism. To determine whether other bacteria possess these proteins, we tested for their presence in 28 oral streptococcus strains, 3 nonoral streptococcus strains, 2 lactococcus strains, 2 enterococcus strains, 2 bacillus strains, 1 lactobacillus strain, Staphylococcus aureus, and Escherichia coli. Three approaches were used to determine whether the IIIMan proteins were present in these bacteria: (i) Western blot (immunoblot) analysis of cytoplasmic and membrane proteins, using anti-IIIManH and anti-IIIManH rabbit polyclonal antibodies; (ii) analysis of PEP-dependent phosphoproteins by polyacrylamide gel electrophoresis; and (iii) inhibition by anti-IIIMan antibodies of the PEP-dependent phosphorylation of 2-deoxyglucose (a mannose analog) by crude cellular extracts. Only the species S. salivarius and Streptococcus vestibularis possessed the two forms of IIIMan. Fifteen other streptococcal species possessed one protein with a molecular weight between 35,200 and 38,900 that cross-reacted with both antibodies. In the case of 9 species, a protein possessing the same electrophoretic mobility was phosphorylated at the expense of PEP. No such phosphoprotein, however, could be detected in the other six species. A III(Man)-like protein with a molecular weight of 35,500 was also detected in Lactobacillus casei by Western blot experiments as well as by PEP-dependent phosphoprotein analysis, and a protein with a molecular weight of 38,900 that cross-reacted with anti-III(Man) antibodies was detected in Lactococcus lactis. In several cases, the involvement of these putative III(Man) proteins in the PEP-dependent phosphorylation of 2-deoxyglucose was substantiated by the inhibition of phosphorylation activity of anti-III(Man) antibodies. No proteins cross-reacting with anti-III(Man) antibodies were detected in enterococci, bacilli, and E. coli. In S. aureus, a membrane protein with a molecular weight of 50,000 reacted strongly with the antibodies. This protein, however, was not phosphorylated at the expense of PEP.     

Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic analysis of Escherichia coli

Separation and relative quantitation of complex protein mixtures remain two of the most challenging aspects of proteomics. Here an advanced technique called fluorescence difference 2-D gel electrophoresis technology (2D-DIGE) has been applied to a model system study of the Escherichia coli proteome after benzoic acid treatment. The molecular weight and charge matched cyanine dyes enable pre-electrophoretic labelling of control and treated samples which are then mixed and run in the same gel. Pooled control and treated samples labelled with Cy trade mark 3 were used as an internal standard for both Cy5 labelled control and treated E. coli samples. Together with DeCyder trade mark imaging analysis software, more accurate quantitative analysis than conventional two-dimensional polyacrylamide gel electrophoresis was achieved. Using matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight mass spectrometry a total of 179 differentially expressed protein spots were identified. These included enzymes, stress related and substrate (e.g. amino acids, maltose, ribose and TRP repressor) binding proteins. Of the spots analysed, 77% contained only one protein species per spot, hence the change in protein expression measured was solely attributed to the identified protein. Many membrane proteins and protein isoforms were identified indicating both adequate solubilization of E. coli samples and potential post-translational modification. The results indicate that the regulatory mechanisms following benzoic acid treatment of E. coli are far more complicated than hitherto expected.     

The SecY membrane component of the bacterial protein export machinery: analysis by new electrophoretic methods for integral membrane proteins.

The product of the secY (prlA) gene (the SecY protein) involved in protein export in Escherichia coli was overproduced and localized in the cytoplasmic (inner) membrane. Because of its strong interaction with a non-ionic detergent (NP40), it partitioned into the detergent layer during electroblotting through a NP40-containing gel (detergent blotting), and it formed a horizontal streak in the O'Farrell two-dimensional gel electrophoretic system. Consequently, we developed an alternative two-dimensional gel procedure, which proved useful for analysis of integral membrane proteins, especially in combination with detergent blotting. SDS-gel electrophoresis was carried out successively through gels of lower (first dimension) and higher (second dimension) sieving effects. Many membrane proteins, unlike soluble proteins, formed spots off and above the diagonal line, and all of these spots partitioned exclusively into the detergent layer. A characteristic pattern of integral membrane proteins of E. coli was thus obtained and the spot of the SecY protein in the cytoplasmic membrane was identified even when it was not overproduced. These results show that the gene secY specifies an integral membrane component of the protein export machinery.     

Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

Study of human erythrocyte membrane proteins by 2-dimensional electrophoresis

Fractionation of human erythrocyte membrane proteins was performed using a modification of two-dimensional gel electrophoresis described by P. O'Farrel with isoelectric point plotted against molecular mass. All major erythrocyte proteins, including high molecular weight proteins, such as spectrin and band 3 protein, identified by one-dimensional sodium dodecyl sulfate gel electrophoresis, were visualized by silver staining of two-dimensional gels. All in all about 50 polypeptides were distinguished on two-dimensional electrophoretic patterns. Preliminary protein map was developed.     

Membrane protein biosynthesis in bacteriophage BF23-infected Escherichia coli.

When Escherichia coli is infected with bacteriophage BF23, two new proteins with molecular weights greater than 10,000, as indicated by polyacrylamide gel electrophoresis, are found associated with the cells' membranes. One of these, found associated with both the inner and outer membrane, has a molecular weight of about 55,000 and is regulated by the A1 gene of this phage, a gene found on the spontaneously injected 8% piece of BF23 DNA, DNA that codes for the synthesis of proteins necessary for the injection of the whole phage genome. The other protein, often undetected in whole membrane preparations, is found exclusively associated with the inner membrane. Evidence indicates that this protein is also regulated by the initially injected 8% piece of the DNA.     

Fractionation of the Providencia stuartii cell envelope

Cell envelopes (i.e. unfractionated inner and outer membranes) were obtained from Providencia stuartii by following procedures previously applied to the isolation of envelopes from Escherichia coli. The P. stuartii envelopes contained known inner membrane enzymes that included a variety of dehydrogenases and ATPase. The catalytic activity of the ATPase depended upon the concentration of magnesium ions, the substrate (ATP) level and the ratio of magnesium ions to ATP. Cell envelopes from P. stuartii were further fractionated to recover inner and outer membrane polypeptides by treatment with the detergent Sarkosyl. Proteins from the periplasmic region were recovered by a simple osmotic shock procedure also previously applied to E. coli. The purity of the various P. stuartii cell envelope fractions was assessed by a combination of techniques that included one- and two-dimensional gel electrophoresis of proteins, enzyme assays and detection of penicillin-binding proteins.     

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.     

Localization, annotation, and comparison of the Escherichia coli K-12 proteome under two states of growth

Here we describe a proteomic analysis of Escherichia coli in which 3,199 protein forms were detected, and of those 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation followed by two-dimensional gel electrophoresis and tandem mass spectrometry. Represented within this inventory were unique and modified forms corresponding to 575 different ORFs that included 151 proteins whose existence had been predicted from hypothetical ORFs, 76 proteins of completely unknown function, and 222 proteins currently without location assignments in the Swiss-Prot Database. Of the 575 unique proteins identified, 42% were found to exist in multiple forms. Using DIGE, we also examined the relative changes in protein expression when cells were grown in the presence and absence of amino acids. A total of 23 different proteins were identified whose abundance changed significantly between the two conditions. Most of these changes were found to be associated with proteins involved in carbon and amino acid metabolism, transport, and chemotaxis. Detailed information related to all 2,160 protein forms (protein and gene names, accession numbers, subcellular locations, relative abundances, sequence coverage, molecular masses, and isoelectric points) can be obtained upon request in either tabular form or as interactive gel images.     

Cross-linking of the proteins in the outer membrane of Escherichia coli.

1. The organization of the proteins in the outer membrane of Escherichia coli was examined by the use of cross-linking agents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment of protein A-peptidoglycan complexes with dithiobis(succinimidyl propionate) or glutaraldehyde produced the dimer, trimer, and higher oligomers of protein A. Both forms of this protein, proteins A1 and A2, produced similar cross-linking products. No cross-linking of protein A to the peptidoglycan was detected. 2. The proteins of the isolated outer membrane varied in their ease of cross-linking. The heat-modifiable protein, protein B, was readily cross-linked to give high molecular weight oligomers, while protein A formed mainly the dimer and trimer under the same conditions. The pronase resistant fragment, protein Bp, derived from protein B was not readily cross-linked. No linkage of protein A to protein B was detected. 3. Cross-linking of cell wall preparations, consisting of the outer membrane and peptidoglycan, showed that protein B and the free form of the lipoprotein, protein F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. 4. These results suggest that specific protein-protein interactions occur in the outer membrane.     

Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier.

A method for separating the outer and inner membranes of Pseudomonas aeruginosa PAO1 in the absence of added ethylenediaminetetraacetic acid was devised. The method yields two outer membrane fractions which show the same protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differ substantially in their relative contents of phospholipids. One of these outer membrane fractions and the inner membrane fraction are less than 4% cross-contaminated, as judged by the content of typical inner and outer membrane markers. The outer membrane contains four major protein bands with apparent molecular weights of 37,000, 35,000, 21,000 and 17,000. Vesicles reconstituted from lipopolysaccharide and phospholipids were impermeable to all saccharides included in the vesicles during vesicle formation. When the vesicles contained outer membrane proteins, they fully retained only those saccharides of greater than 9,000 molecular weight, suggesting that the exclusion limit of the outer membrane of P. aeruginosa for saccharides is substantially larger than the figure (500 to 600 daltons) obtained for certain enteric bacteria. The advantages and potential disadvantages of having an outer membrane with a higher exclusion limit for hydrophilic substances are discussed.     

Lysozyme-promoted association of protein I molecules in the outer membrane of Escherichia coli.

Incubation of whole envelopes prepared from sonically oscillated Escherichia coli K-12 cultures with lysozyme in vitro resulted in the appearance of a protein species with an apparent molecular weight double that of outer membrane protein I. Similar dimers were also detected in purified outer membranes and whole envelopes from lysozyme-induced spheroplasts of E. coli K-12. This was confirmed by two-dimensional electrophoresis in which the dimers were resolved in the second dimension to run as single polypeptides of protein I. Formation of dimers was correlated with peptidoglycan degradation, but the ability of protein I molecules to associate may vary between strains of E. coli, since dimers were found only in outer membranes from E. coli W7. We suggest that extensive degradation of peptidoglycan leads to nonspecific formation of protein I aggregates, but that these aggregates do not occur in vivo.     

Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme

The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Translocation of an outer membrane protein into prey cytoplasmic membranes by bdellovibrios.

Within minutes of Bdellovibrio bacteriovorus attack on prey cells, such as Escherichia coli, the cytoplasmic membrane of the prey is altered. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invaded prey cell (bdelloplast) membranes revealed the appearance of a noncytoplasmic membrane protein. This protein is not observed in preparations of noninvaded E. coli membranes and migrates in a manner similar to that of E. coli OmpF. Isoelectric focusing and two-dimensional gel electrophoresis of bdelloplast cytoplasmic membrane preparations also revealed the presence of a protein with electrophoretic properties similar to those of OmpF and the major Bdellovibrio outer membrane proteins. The protein appears in cytoplasmic membrane preparations within minutes of attack and persists throughout most of the intraperiplasmic developmental cycle. The appearance of this protein is consistent with our hypothesis that bdellovibrios translocate a pore protein into the bdelloplast cytoplasmic membrane to kill their prey and to gain access to the cytoplasmic contents for growth.     

Biochemical Identification of the Two Races of Radopholus similis by Polyacrylamide Gel Electrophoresis

Analysis of proteins of the banana and citrus race of Radopholus similis was carried out by several different types of polyacrylamide gel electrophoresis. These included standard slab gel, SDS slab gel, gradient slab gel, and two-ditnensional slab gel electrophoresis. A major band difference was detected between the two races by slab gel electrophoresis. However, several other poorly resolved but consistent hands of high molecular weight proteins near the gel origin also were considered as diagnostic. Resolution of protein bands was greatly improved by SDS and gradient slab gel electrophoresis, but no differences could be detected among the proteins resolved between the two rares with these techniques. Two-dimensional gels revealed a large number of proteins, but background staining obscured them hindering interpretation. When nematode races were reared on three different host plants, no differences in protein patterns were detected between them, indicating host preferences does not play a role in determining the types proteins occurring in these nematodes.     

Identification as Synapsin of a Synaptosomal Protein Immunoreacting with Anti-Myelin Basic Protein Antiserum

Rat brain proteins able to react with anti-myelin basic protein antiserum, raised under conditions to induce experimental allergic encephalomyelitis in rabbits, were examined by immunoblot methods after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Apart from the four forms of myelin basic protein present in rat brain, the antiserum detected other proteins of higher molecular weight. Subcellular fractionation shows that these high-molecular-weight proteins are relatively concentrated in a synaptosome-enriched fraction compared to a myelin fraction. A major protein fraction immunorelated to myelin basic protein migrated in the gels as a doublet with apparent molecular weights of approximately 80K and 86K; these proteins were tentatively identified as synapsin Ia and Ib. A purified synapsin preparation analyzed by immunoblot after two-dimensional gel electrophoresis also reacted with anti-myelin basic protein antisera. When the serum was purified by affinity chromatography on a myelin basic protein-conjugated Sepharose column the nonadsorbed material lost this activity whereas the eluted antibodies reacted with myelin basic protein and synapsin. In addition, sequence amino acid comparison of decapeptides showed some homology between these two proteins. A possible implication of immunological agents against myelin basic protein cross-reacting with extra-myelin proteins in the process of experimental allergic encephalomyelitis is considered.     

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.