GATC-specific restriction and modification systems in treponemes

AIMS: To investigate the presence of GATC-specific modification and restriction activities in rumen isolates of Treponema sp. METHODS: The presence of N6-methyladenine within GATC (Dam) sequences was analysed using isoschizomeric restriction endonucleases having different sensitivities to the methylation of the target sequence. A fast screening method was used for testing of site-specific endonuclease activities directly in crude cell extracts. Three out of six rumen isolates of Treponema sp. showed restriction activities. Restriction endonucleases were further purified by Heparin-Sepharose chromatography. Using PCR and specific primers, no sequence homologous to the T. pallidum dam gene was found. CONCLUSIONS: Three rumen treponemal strains were documented to possess MboI isoschizomeric restriction-modification systems. SIGNIFICANCE: This is the first report on restriction activity in rumen treponemes.     

Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

The flagellar hook–basal body (HBB) complex of the Gram-positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram-negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium . The hook protein had an apparent molecular mass of 29 kDa, and its N-terminal sequence was identical to that of B. subtilis FlgG, which was previously reported as a rod protein. The sequence of the reported FlgG protein of B. subtilis is more closely related to that of FlgE (the hook protein) rather than FlgG (the rod protein) of S. typhimurium , in spite of the difference of the apparent molecular masses between the two hook proteins (29 kDa versus 42 kDa). The hook–basal body contained six major proteins (with apparent molecular masses of 82, 59, 35, 32, 29 and 20 kDa) and two minor proteins (23 kDa and 13 kDa), which consistently appeared from preparation to preparation. The N-terminus of each of these proteins was sequenced. Comparison with protein databases revealed the following polypeptide–gene correspondences: 82 kDa, fliF ; 59 kDa, flgK ; 35 kDa, orfF ; 32 kDa, yqhF ; 23 kDa, orf3 of the flaA locus; 20 kDa, flgB and flgC ; 13 kDa, not determined. The band at 20 kDa was a mixture of FlgB and FlgC, as revealed by two-dimensional gel analysis. Characteristic features of B. subtilis HBB are discussed in comparison with those of S. typhimiurium .     

The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.     

Characterization of cytoplasmic fibril structures found in gliding cells of Saprospira sp

The cytoplasmic fibril structures of Saprospira sp. strain SS98-5 grown on a low-nutrient agar medium were purified from cell lysates treated with Triton X-100 and were observed by electron microscopy to be about 7 nm in width and 200-300 nm in length. SDS-PAGE of the fibril structures exhibited a single protein band with a molecular mass of 61 kDa. A Saprospira cytoplasmic fibril protein (SCFP), which is a subunit of the fibril structures, was digested with trypsin to oligopeptides and analyzed for amino acid sequences. A partial nucleotide sequence of the SCFP gene was determined after PCR using primers designated from the amino acid sequences of the oligopeptides. SCFP gene including DNA fragments were detected by Southern hybridization using the PCR product for an SCFP gene as a probe and were cloned to determine whole nucleotide sequences. The SCFP gene indicated relatively higher similarity to conserved hypothetical phage tail sheath proteins. A Western immunoblotting analysis showed that SCFP was significantly expressed in gliding cells as compared with nongliding cells. The above findings with the previously reported results suggest that the cytoplasmic fibril structures are possibly related to the gliding motility of Saprospira sp. strain SS98-5.     

Purification and preliminary characterization of Spiroplasma fibrils.

Fibrils 3.5 nm in diameter were released from the honeybee spiroplasma (BC3) by treatment with detergents and then purified by isopycnic centrifugation. Purified fibrils were flexuous, of indeterminate length, and had an axial repeat of 8.5 nm. The fibrils were associated in pairs, but in 1 M salt formed aggregates with a marked striated appearance. Pronase completely degraded the fibrils, but trypsin had little effect. The fibrils were composed of a single protein of molecular weight 55,000 which represented about 1% of the total cell protein. A protein of molecular weight 26,000 appeared to be associated with the fibrils. The significance of this in relation to membrane attachment and the possible role of fibrils in maintenance of cell shape and in motility are discussed.     

水稻条纹病毒蛋白与核酸的特性

提纯的水稻条纹病毒(云南宜良分离物)在电镜下的形态为多型性,但主要是宽8-10 nm,长80-250的分枝丝状体,有些为直径3 nm或8 nm的开环环状体,有些为13 nm宽,130-190 nm长的丝状体,但其基本结构应是直径3 nm、长度不等的丝状体.经聚丙烯酰胺凝胶电泳分析,vRNA4编码的病害特异蛋白(SP)分子量为19.9 kDa,而vcRNA3编码的外壳蛋白(CP)约为33.6 kDa.在非变性条件下,RSV的4条ssRNAs大小分别为3.0×106(ssRNA1)、1.2×106(ssRNA2)、0.9×106(ssRNA3)和0.8×106 Da(ssRNA4),有时出现一条大小为0.58×106 Da的单链RNA(ssRNA5);而4条dsRNAs的分子量分别为4.9×106(dsRNA1)、2.8×106(dsRNA2)、2.0×106(dsRNA3)和1.7×106 Da(dsRNA4).利用制备电泳分离提纯的外壳蛋白免疫家兔,得到了高特异性的抗血清.A蛋白夹心ELISA检测结果表明,RSV-CP与水稻草状矮化病毒(RGSV)CP抗血清有微弱的反应,但与RSV、RGSV的SP抗血清没有反应,而RSV-CP抗血清与RSV-SP及RGSV的SP、CP都无血清学关系,这个结果表明RGSV与RSV之间在进化上具有一定的亲缘关系.     

Pathological observation on experimental swine dysentery

Experimental swine dysentery caused by 4 cultured strains (S73/2, DJ183, DJ70 and DK762) of Treponema hyodysenteriae was studied pathologically. The distribution and quantity of treponemes were examined on tissue sections stained by the Warthin-Starry method. Of the organs the colon contained the largest number of treponemes and the cecum and rectum the second largest number. Histopathological lesions were restricted to the large intestine. They ranged from mild catarrhal colitis in the mild case to desquamative, hemorrhagic colitis in the severest case. The severity of lesion was closely associated with the quantity of treponemes present. There was no difference in quality of the lesion between any two of the strains used in this study. Electron microscope revealed a large number of free treponemes present in the intestinal lumen and crypts. Treponemes were seen more frequently in the cytoplasm of goblet cells than in that of intestinal epithelial cells. They were also observed in desquamated degenerative epithelia. A small number of them were found in intact epithelia. Morphologically, the treponeme had a granular protoplasmic cylinder at the center which was surrounded by a thin envelope. Between the cylinder and the envelope there were axial fibrils.     

Phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum in virulent, tissue-cultured treponemes

Specific monoclonal antibody and Western blot analysis were used to examine the phenotypic expression of the major 47 kDa surface immunogen of Treponema pallidum among organisms cultivated in vitro. Tissue-cultured treponemes synthesized the 47 kDa immunogen as well as, or better than, organisms cultivated in vivo (rabbit testicles).     

Protein carboxyl methylation in kidney brush-border membranes.

Protein carboxyl methylation activity was detected in the cytosol and in purified brush-border membranes (BBM) from the kidney cortex. The protein carboxyl methyltransferase (PCMT) activity associated with the BBM was specific for endogenous membrane-bound protein substrates, while the cytosolic PCMT methylated exogenous substrates (ovalbumin and gelatin) as well as endogenous proteins. The apparent Km for S-adenosyl-L-methionine with endogenous proteins as substrates were 30 microM and 4 microM for the cytosolic and BBM enzymes, respectively. These activities were sensitive to S-adenosyl-L-homocysteine, a well known competitor of methyltransferase-catalyzed reactions, but were not affected by the presence of chymostatin and E-64, two protein methylesterase inhibitors. The activity of both cytosolic and BBM PCMT was maximal at pH 7.5, while BBM-phospholipid methylation was predominant at pH 10.0. Separation of the = methylated proteins by acidic gel electrophoresis in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride revealed distinct methyl accepting proteins in the cytosol (14, 17, 21, 27, 31, 48, 61 and 168 kDa) and in the BBM (14, 60, 66, 82, and 105 kDa). Most of the labelling was lost following electrophoresis under moderately alkaline conditions, except for a 21 kDa protein in the cytosol and a 23 kDa protein in the BBM fraction. These results suggest the existence of two distinct PCMT in the kidney cortex: a cytosolic enzyme with low selectivity and affinity, methylating endogenous and exogenous protein substrates, and a high-affinity BBM-associated methylating activity.     

Chemotaxis of oral treponemes toward sera and albumin of rabbit

The motility and chemotaxis of human oral spirochetes Treponema denticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580 were examined by a capillary assay method. Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum. The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T. denticola cells swam toward the culture media containing higher concentrations of the rabbit serum. T. denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum. Western blotting analysis demonstrated that these treponemes possessed rabbit albumin-binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa. Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin-binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin-binding polypeptide is responsible for chemotaxis toward rabbit serum.     

Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy

Noncontact mode atomic force microscopy was used to investigate native silk proteins prepared in different ways. Low protein concentrations revealed that single protein molecules exhibit a simple, round shape with apparent diameters of 20-25 nm. Shearing the native protein solutions after extraction from the gland and prior to drying led to a beads-on-a-string assembly at the nanometer scale. Protein concentration had a significant effect on the morphology of the protein assemblies. At higher protein concentrations, shear-induced alignment into nanofibrils was observed, while lower concentrations lead to the formation of much thinner fibrils with a width of about 8 nm.     

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.     

Collagen XI nucleates self-assembly and limits lateral growth of cartilage fibrils

Fibrils of embryonic cartilage are heterotypic alloys formed by collagens II, IX, and XI and have a uniform diameter of approximately 20 nm. The molecular basis of this lateral growth control is poorly understood. Collagen II subjected to fibril formation in vitro produced short and tapered tactoids with strong D-periodic banding. The maximal width of these tactoids varied over a broad range. By contrast, authentic mixtures of collagens II, IX, and XI yielded long and weakly banded fibrils, which, strikingly, had a uniform width of about 20 nm. The same was true for mixtures of collagens II and XI lacking collagen IX as long as the molar excess of collagen II was less than 8-fold. At higher ratios, the proteins assembled into tactoids coexisting with cartilage-like fibrils. Therefore, diameter control is an inherent property of appropriate mixtures of collagens II and XI. Collagen IX is not essential for this feature but strongly increases the efficiency of fibril formation. Therefore, this protein may be an important stabilizing factor of cartilage fibrils.     

Specific interaction of a novel foamy virus Env leader protein with the N-terminal Gag domain

Cryoelectron micrographs of purified human foamy virus (HFV) and feline foamy virus (FFV) particles revealed distinct radial arrangements of Gag proteins. The capsids were surrounded by an internal Gag layer that in turn was surrounded by, and separated from, the viral membrane. The width of this layer was about 8 nm for HFV and 3.8 nm for FFV. This difference in width is assumed to reflect the different sizes of the HFV and FFV MA domains: the HFV MA domain is about 130 residues longer than that of FFV. The distances between the MA layer and the edge of the capsid were identical in different particle classes. In contrast, only particles with a distended envelope displayed an invariant, close spacing between the MA layer and the Env membrane which was absent in the majority of particles. This indicates a specific interaction between MA and Env at an unknown step of morphogenesis. This observation was supported by surface plasmon resonance studies. The purified N-terminal domain of FFV Gag specifically interacted with synthetic peptides and a defined protein domain derived from the N-terminal Env leader protein. The specificity of this interaction was demonstrated by using peptides varying in the conserved Trp residues that are known to be required for HFV budding. The interaction with Gag required residues within the novel virion-associated FFV Env leader protein of about 16.5 kDa.     

Induction of vitellogenesis by estradiol-17beta and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas)

Treatment of juvenile green turtles (Chelonia mydas) with estradiol-17beta resulted in the induction of a 200 kDa plasma protein, consistent with vitellogenin (Vtg). The N-terminal 15 amino acids of the anion exchange purified protein shared sequence homologies with vitellogenins of several vertebrate species. Rabbit antiserum raised against purified Vtg recognized the plasma protein as well as several yolk proteins. Monoclonal antibody (Mab) HL1248, produced by inoculating mice with turtle yolk granules, showed specificity for plasma Vtg as well as a set of yolk proteins 120, 82, 43 and 32 kDa in size. The N-terminal 22 amino acids of the 43 kDa yolk protein was similar to the lipovitellin I subunit of Vtg of several vertebrate species. The peptide mass map of the 82 kDa yolk protein shared enough ions with that of purified plasma Vtg to support the conclusion that this protein was derived from plasma Vtg. Taken together, these results validate the specificity of Mab HL1248 for Vtg. Using purified Vtg concentration standards, competition and antigen capture enzyme-linked immunosorbant assays (ELISAs) were shown to quantitatively detect Vtg in green turtle plasma. Pre-induced plasma of juvenile turtles had Vtg levels of 2-4 micrograms/ml whereas post-estradiol exposure samples had 38-40 mg/ml. The plasma Vtg concentration of a nesting female turtle was 4.6 mg/ml, approximately 20-fold higher than that of a non-nesting adult female. The antigen capture ELISA will be useful in population studies of this endangered species, to detect vitellogenesis in females that will nest in a given year and to detect inappropriate Vtg levels in turtles exposed to xenoestrogens.     

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols).

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.     

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.     

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.     

梅毒螺旋体膜抗原基因在毕赤酵母中的表达和蛋白纯化

以梅毒螺旋体(Treponema pallidumsubsp.pallidum)Nichols菌株基因组DNA为模板,通过PCR扩增梅毒螺旋体47kDa、17kDa和15kDa 3个膜抗原基因,克隆进毕赤酵母表达载体pPICZ B,构建重组表达载体pTP47、pTP17、pTP15,转化酵母菌株GS115,甲醇诱导表达。表达菌体裂解后通过镍离子亲和层析获得3个抗原与6xHis tag的融合蛋白,重组蛋白的获得量分别为His-TP15:4.8mg/L;His-TP 17:6.6mg/L;His-TP47:25mg/L,经SDS-PAGE鉴定纯度都在96%以上,ELISA鉴定均具有很好的抗原性。从而首次在毕赤酵母中表达出梅毒螺旋体膜抗原,为梅毒血清学检测方法开辟了新的抗原制备途径。     

Antigenic and structural analysis of Treponema denticola

Polypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determined by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product of a larger, heat-modifiable polypeptide. Based on the results of SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a 'rough' lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology of the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath and core structure and polypeptide composition characteristic of that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure of antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detailed molecular analysis of the ultrastructure and antigenicity of T. denticola.