Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.     

The outer membrane of Treponema pallidum: biological significance and biochemical properties

Rabbits infected intravenously with Treponema pallidum were not markedly febrile, and the pyrogenicity of treponeme preparations administered intravenously to rabbits was negligible. The antibiotic polymyxin B did not induce any ultrastructural changes on the treponemal surface and was not lethal (immobilizing) for T. pallidum, which was, however, highly susceptible to detergents such as SDS. Extraction of treponemes with Triton X-100 removed the outer membrane (despite the presence of Mg2+) as shown by electron microscopy, and solubilized a limited number of proteins detectable by SDS-PAGE, including a dominant antigen (47 kDal) demonstrated by immunoblotting. None of the proteins were heat-modifiable. Periodic acid-silver staining of polyacrylamide gels for carbohydrate together with protease K digestion did not demonstrate major carbohydrate components in whole treponemes, or in the Triton-soluble fraction. Surface iodination of intact treponemes revealed very little surface exposure of treponemal proteins, although a protein which co-migrated with host albumin was labelled and appeared to be associated with the treponemal surface. Many treponemal proteins were, however, labelled when iodination was done in the presence of Triton. These observations, indicate that the outer membrane of T. pallidum differs significantly from those of many Gram-negative pathogens.     

Two-Dimensional Electrophoresis of Cerebrospinal Fluid and Ventricular Fluid Proteins, Identification of Enriched and Unique Proteins, and Comparison with Serum

The proteins of cerebrospinal fluid (CSF) and ventricular fluid have been analyzed by two-dimensional electrophoresis (2DE) and the patterns compared with autologous serum. Fourteen proteins were specifically identified by immunoprecipitation followed by 2DE, or by blotting 2DE gels to nitrocellulose and detection by peroxidase staining. Proteins in CSF and serum with high and low affinity for the ligands, protein A, Cibacron Blue, and concanavalin A, were also characterized by 2DE. The 2DE profiles of CSF and serum proteins were similar and indicated that a relatively nonselective filtration mechanism based on protein size is the major determinant for the overall pattern of CSF proteins. The classic CSF-enriched or CSF-specific proteins, beta-trace, prealbumin, transferrin, and beta-2-microglobulin, were identified according to 2DE coordinates. Charge differences between CSF and serum for transferrin and prealbumin were identified. In addition, a large number of additional CSF-enriched or CSF-specific proteins of high, intermediate, and low molecular weight, all predominantly anodic in mobility, were identified. Three acidic protein complexes, heterogeneous in charge and molecular weight, were characterized as constituents of normal CSF, and two of these are increased in patients with inflammatory diseases of the CNS. All three proteins and several other proteins unique to CSF bound to Cibacron Blue-Sepharose. The use of 2DE in conjunction with affinity chromatography and sensitive protein stains enlarged the number of proteins previously identified as unique to CSF. By a modified 2DE and silver staining procedure, most of these proteins were visible without prior concentration of CSF.     

TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein

Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.     

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.     

Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114.

The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 micron to 0.1-0.15 micron. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 degrees C were divided into aqueous and hydrophobic phases after incubation at 37 degrees C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organisms. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.     

Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete.

Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs). The resulting sequences enabled us to PCR amplify from T. pallidum DNA a 275-bp fragment of the corresponding gene. The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR. Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements. The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-amino-acid leader peptide terminated by a signal peptidase I cleavage site. The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins. Accordingly, the polypeptide was designated T. pallidum leucine-rich repeat protein (TpLRR). Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space. Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen. Lastly, the lrr(T. pallidum) gene was mapped to a 60-kb SfiI-SpeI fragment of the T. pallidum chromosome which also contains the rrnA and flaA genes. The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T. pallidum cell envelope.     

Identification of Cultivar Differences in Seed Polypeptide Composition of Peanuts (Arachis hypogaea L.) by Two-Dimensional Polyacrylamide Gel Electrophoresis

Seed polypeptides from several cultivars of peanut (Arachis hypogaea L.) have been compared by means of a two-dimensional polyacrylamide gel electrophoresis. Protein was extracted from the defatted peanut meal by homogenizing in 5 millimolar K₂CO₃-9.5 molar urea. After addition of Nonidet P-40 (2%, v/v) and dithiothreitol (0.5%, w/v) the solution was centrifuged at 25,000 g. This procedure led to solubilization of more than 95% of the total protein. The clear supernatant fraction was then subjected to two-dimensional polyacrylamide gel electrophoresis, employing isoelectric focusing in the first dimension and electrophoresis in presence of sodium dodecyl sulfate in the second. After examining several cultivars, it was possible to construct a composite map to include all of the polypeptide species found among all of the cultivars examined. At least 74 major and between 100 and 125 minor components were detectable by Coomassie blue staining. The majority of these had isoelectric points between pH 4.4 and 8.0, and molecular weights between 16,000 and 75,000. Several different cultivars have been compared using this method and it has been shown that considerable variation exists among the major polypeptides present. The method should prove valuable for analyzing different genotypes and selecting varieties with a particular storage protein make-up, as well as for following compositional changes that occur during seed development and germination.     

Autoantibodies to creatine kinase in rabbits infected with Treponema pallidum

Sera from rabbits infected intratesticularly with Treponema pallidum (Nichols) for 30 days were examined for autoantibody reactivity against muscle and testis extracts by Western immunoblotting. Syphilitic sera (30 day) reacted with an autoantigen of 43,000 daltons in muscle extracts. The antigen was shown to be creatine kinase (CK). Studies with the use of an anti-CK ELISA showed that the autoantibody to CK first appeared 3 wk after infection, declined by 7 wk infection, and was absent in rabbits "mock"-infected with heat-killed T. pallidum. CK activity was not detected in sonicated or intact, washed T. pallidum, suggesting that the antibody was not produced in response to treponemal CK.     

Automated, Quantitative Microhemagglutination Assay for Treponema pallidum Antibodies

An automated, quantitative microhemagglutination assay for antibodies to Treponema pallidum was developed by using T. pallidum-sensitized erythrocytes and an automatic serial-dilution instrument. Reactivity was found in sera from 54 rabbits and 6 chimpanzees infected with T. pallidum. Reactivity was also found in sera from animals infected with T. pertenue, T. carateum, and T. cuniculi. No reactivity was found in sera from 75 normal rabbits or from 129 rabbits immunized with cultivatable treponemes or a variety of other bacteria. In approximately 3 min, 13 twofold serial dilutions of each of 8 preabsorbed sera and the addition of sensitized erythrocytes to each dilution were accomplished automatically. The automated assay can serve as a research tool in quantitating antibodies to pathogenic treponemes, and evaluation of its clinical usefulness seems warranted.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens

Lymphoid cells from spleens and lymph nodes of rabbits infected with T. pallidum respond by proliferation to concanavalin A (Con A) and T. pallidum antigens. Spleen cell responsiveness to treponemal antigens appears 6 days after infection, is 100 to 600 fold higher than the response of uninfected control rabbits, and is maintained throughout the 31-day observation period. Specifically responding cells in the inguinal and popliteal lymph nodes of infected animals are demonstrable on day 10, and the magnitude of the response increases throughout the observation period. Specific responsiveness to T. pallidum antigens in vitro is enhanced in purified T cell populations and is abolished by treatment with goat anti-rabbit thymocyte serum and complement. The response of spleen and lymph node cells to Con A is unaffected during syphilitic infection. These results are consistent with a role for T cell-mediated specific immunity to treponemal antigens early after infection and do not support a hypothesis of depressed cellular immunity during syphilitic infection.     

Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide research include continued elucidation of their structural locations and functional activities, identification and characterization of the low-abundance outer membrane proteins, further study of the immunoprotective and immunodiagnostic potential of T. pallidum proteins, and clarification of the roles of treponemal proteins in pathogenesis.     

Molecular and immunochemical analysis of Treponema pallidum

Abstract Molecular analysis of polypeptides and antigens of Treponema pallidum has been used increasingly during the past 5 years in investigation of the immunology, pathogenicity and molecular biology of this organism. Failure to culture the organism has severely limited our knowledge of its constituent polypeptides and antigens, but many profiles of these unknown constituents, revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting techniques have been published. In order to compare meaningfully the results obtained by different groups, we have identified a standard pattern of prominent 'landmark' polypeptides in such gel profiles and where possible have assigned functional identities to them. A preliminary nomenclature for the prominent polypeptides of T. pallidum is proposed. These are: P1, 80 kDa; P2, 60 kDa; P3, 47 kDa, an outer membrane-associated polypeptide; P4, 40 kDa; P5, 37 kDa, the major polypeptide of the axial filament; P6, 34 kDa; and P7, 31.5 kDa.     

Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis.

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.     

Macrophage-mediated suppression of con A-induced IL-2 production in spleen cells from syphilitic rabbits

Blast transformation studies have indicated a diminished T cell response in spleen cell preparations from rabbits infected with Treponema pallidum. IL-2 synthesis by T lymphocytes is required for proliferation of these cells. Thus, Con A-induced IL-2 generation was measured in syphilitic animals infected for 9 to 14 days. IL-2 production in the infected rabbits was only one-half that observed for uninfected rabbits. This marked decrease in IL-2 was not caused by decreased IL-1 secretion by adherent cells from infected animals because similar levels were found in both infected and uninfected splenic cultures. This decrease was also not caused by an increase in infected spleen cell adsorption of IL-2; similar numbers of receptors for this IL were present in Con A-stimulated infected and uninfected splenic preparations. The inhibited IL-2 production in infected spleen cells was reversed upon removal of the adherent cells and also elevated upon addition of indomethacin to the cultures. PGE levels were also elevated in splenic cultures from infected animals. Finally, IL-2 synthesis, when evaluated at various days postinfection, showed that at 4 days, splenic cells generated twice as much IL-2 as uninfected cells. At 9 to 14 days, IL-2 levels were dramatically decreased (50% lower than that observed in uninfected cultures), and suppression of IL-2 by adherent cells was observed as late as 35 days post-infection. We propose that premature down regulation (suppression) of IL-2 secretion is mediated by adherent cells via a cyclo-oxygenase product, most likely PGE. These results may explain why most, but not all, treponemes are cleared during infection, and why the secondary manifestations of the disease occur.     

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.     

Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly.     

S gene product: identification and membrane localization of a lysis control protein.

The product of the bacteriophage S gene has been previously shown to be required for an essential step in triggering host cell lysis. By using two different protein labeling systems, maxicells and UV-irradiated infected cells, we identified the S gene product as an 8,500-molecular-weight polypeptide associated with the cell envelope. The apparent molecular weight is significantly less than the 11,500 predicted from the S gene sequence. We were unable to confirm two previous identifications of S gene products, an acidic 15,000-molecular-weight polypeptide found by two-dimensional gel electrophoresis of infected cells and a 5,500-molecular-weight polypeptide in purified phage particles.