Production, purification and molecular weight determination of the haemolysin of Treponema hyodysenteriae

Kent, Karen; Lemcke, Ruth M.; Lysons, R. J.

doi:10.1099/00222615-27-3-215

Cited by 36 publications

(33 citation statements)

References 10 publications

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“…For the purposes of biological and biochemical characterization, the crude preparations of beta-hemolysin obtained from B. hyo-dysenteriae B204 cells (13) were purified by ultrafiltration concentration followed by two successive separations on native polyacrylamide gels and electroelution. Hemolytic activity of the purified fractions was maintained throughout the purification process ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Phage plates for LE392 (6) Hemolysin preparation and purification. Crude hemolysin was prepared by a modification of a method described previously (13). A 3-liter volume of B. hyodysenteriae culture was centrifuged at 10,000 ϫ g at 4°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

“…Beta-hemolytic activity has been one of the major phenotypic characteristics used to distinguish B. hyodysenteriae from avirulent species of Brachyspira. Numerous methods have been reported for hemolysin production and purification, most involving traditional protein purification techniques (13)(14)(15)23). These studies have been at variance in their reports of the apparent molecular mass of the isolated hemolytic factor, ranging from 19 to 74 kDa.…”

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confidence: 99%

“…None of these three genes, however, have been linked directly by protein sequencing to a B. hyodysenteriae-specific gene product that displays hemolytic activity or to the native beta-hemolysin. Likewise, none of the recombinant products synthesized from the cloned genes have been shown to possess the biochemical properties attributed to the B. hyodysenteriae beta-hemolysin (13,14,22,23), with the possible exception that E. coli harboring tly containing plasmids have hemolytic activity (18,25). Recent studies identifying the cryptic E. coli hemolysin SheA (3) raise the possibility that tlyA, tlyB, and tlyC code for proteins that induce SheA or other cryptic E. coli hemolysins.…”

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confidence: 99%

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Cloning of a Beta-Hemolysin Gene ofBrachyspira(Serpulina)hyodysenteriaeand Its Expression inEscherichia coli

et al. 2001

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Brachyspira (Serpulina) hyodysenteriae induces a mucohemorrhagic diarrheal disease in pigs. The production of a beta-hemolysin has been considered a major virulence attribute of this organism. Previous reports have failed to correlate a specific cloned gene sequence with a purified beta-hemolytic protein sequence. Thus, questions still remain concerning the structural gene sequence of the hemolysin. To answer this question unequivocally, the beta-hemolytic toxin was purified from extracts of log-phase spirochetes, and the N-terminal amino acid sequence was determined (K-D-V-V-A-N-Q-L-N-I-S-D-K) and compared with the translated sequences of previously cloned genes, tlyA to tlyC. The lack of homology between tlyA to tlyC translated sequences and the purified beta-hemolytic toxin sequence resulted in the study that is reported here. A degenerate probe was designed based on the N-terminal amino acid sequence of the purified beta-hemolysin and used to screen a B. hyodysenteriae genomic library. Three overlapping clones were identified, and one was sequenced to reveal an open reading frame coding for a putative 8.93-kDa polypeptide containing the N-terminal sequence of the purified beta-hemolysin. To distinguish this gene from the tlyA to tlyC genes, it has been designated hlyA. A hemolysis-negative Escherichia coli strains containing hlyA was beta-hemolytic on blood agar media. Also, the hemolytic activity of the recombinant protein had identical protease and lipase sensitivities and electrophoretic mobility to those of native B. hyodysenteriae beta-hemolysin. Based on sequence analysis, the translated protein had a pI of 4.3, an ␣-helical structure, and a phosphopantetheine binding motif. Hybridization analysis of genomic DNA indicated that the hlyA gene was present in B. hyodysenteriae and B. intermedia but was not detected in B. innocens, B. pilosicoli, or B. murdochii under high-stringency conditions. The location of hlyA on the chromosomal map was distinct from the locations of tlyA, tlyB, and tlyC.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Cloning of a Beta-Hemolysin Gene ofBrachyspira(Serpulina)hyodysenteriaeand Its Expression inEscherichia coli

et al. 2001

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“…Serpulina hyodysenteriae (S75/ 1 and B78 isolates) was cultured as described by Kent et al (1988). The medium used consisted of Trypticase Soy Broth (TSB) supplemented with 0.2 YO (w/v) yeast extract, 0.25 % (w/v) glucose, 0.05 YO (w/v) L-cysteine hydrochloride, 0.0001 Yo (w/v) resazurin and 800 pg Spectam ml-I (400 pg soluble spectinomycin ml-').…”

Section: -7818 0 1993 Sgm M a Livesley And Othersmentioning

confidence: 99%

Comparison of the fatty acid profiles of Borrelia, Serpulina and Leptospira species

Livesley¹,

Thompson²,

Bailey³

et al. 1993

Journal of General Microbiology

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Fatty acid methyl ester (FAME) derivatives were examined as a means of characterizing Borrelia burgdorferi isolates and distinguishing them from other spirochaetes. Analysis was performed using a gas liquid chromatography column in conjunction with Microbial Identification System (MIS) software. Reproducible FAME profiles were produced which distinguished Borrelia species, Serpulina hyodysenteriae and Leptospira ictevohaemorrhagiae. Furthermore, the FAME profiles of four recognized Borrelia species (including two American isolates of Borrelia burgdorferi, B31 and JDI) were distinct from one another and from the BSK I1 medium in which they were grown. The results confirm previous reports that FAME profiles of bacteria represent a diagnostic phenotypic property and suggest that they may have applications in the chemotaxonomic classification of Borrelia species.

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Brachyspira

Stanton

2015

Bergey's Manual of Systematics of Archaea and Bacteria

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Bra.chy.spi'ra. Gr. adj. brachys short; L. fem. n. spira a coil, spiral; N.L. fem. n. Brachyspira a short spiral, describing a bacterium that resembles a short spiral. Spirochaetes / Spirochaetia / Spirochaetales / Brachyspiraceae / Brachyspira Brachyspira spirochetes are helical shaped bacteria with regular coiling patterns . Cells measure 2–11 µm by 0.2–0.4 µm. Unicellular, but dividing pairs and occasional chains of three or more cells can be observed in growing cultures. Under unfavorable growth conditions, spherical or round bodies are formed. Gram‐stain negative. Obligately anaerobic, aerotolerant . Cell ends may be blunt or pointed. Cells have a typical spirochete cell ultrastructure, consisting of an outer sheath, helical protoplasmic cylinder, and internal flagella in the space between the protoplasmic cylinder and outer sheath. Brachyspire cells have 8–30 flagella per cell depending on the species (flagellar number usually correlates with cell size and species of smaller cells have fewer flagella). Flagella attach subterminally in equal numbers at each cell end, wrap around the protoplasmic cylinder, and their free ends overlap in the middle of the cells. Flexing and creeping motility at 22°C; translational movement in liquids at 37–42°C. Cultured anaerobically on commercially available media (trypticase soy or brain heart infusion broths) containing a carbohydrate growth substrate and supplemented with defibrinated blood or animal (calf) serum. Grows at 36–42°C, optimally at 37–39°C. Population doubling times on glucose in broth cultures are 1–5 h (not reported for Brachyspira aalborgi ). Chemoorganotrophic, using various carbohydrates for growth. Possess NADH oxidase for reducing molecular oxygen. Consume oxygen during growth in culture broth beneath a 1 % oxygen atmosphere. Acetate, butyrate, H 2 , and CO 2 are major endproducts of glucose metabolism. Higher amounts of H 2 than CO 2 are produced . Weakly hemolytic except for Brachyspira hyodysenteriae which exhibits β‐hemolysis (strongly hemolytic). Associated with animal and human hosts. Some species are pathogenic. The genus Brachyspira is distinguished from other spirochete genera based on 16S rRNA gene sequences . Brachyspira species share high 16S rRNA sequence similarity with each other. Species can be differentiated by DNA–DNA relative reassociation and MLEE (multilocus enzyme electrophoresis) analyses. Similar to other spirochete genera, Brachyspira is insensitive to the antibiotic rifampin. DNA G + C content ( mol %): 24.5–27.1 ( T m ). Type species : Brachyspira aalborgi Hovind‐Hougen, Birch‐Andersen, Henrik‐Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1 9 8 3, 896 VP (Effective publication: Hovind‐Hougen, Birch‐Andersen, Henrik‐Nielsen, Orholm, Pedersen, Teglbjaerg and Thaysen 1982, 1135 VP .).

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Production, purification and molecular weight determination of the haemolysin of Treponema hyodysenteriae

Cited by 36 publications

References 10 publications

Cloning of a Beta-Hemolysin Gene ofBrachyspira(Serpulina)hyodysenteriaeand Its Expression inEscherichia coli

Cloning of a Beta-Hemolysin Gene ofBrachyspira(Serpulina)hyodysenteriaeand Its Expression inEscherichia coli

Comparison of the fatty acid profiles of Borrelia, Serpulina and Leptospira species

Brachyspira

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