Characterization of certain proteinase isoenzymes produced by benign and virulent strains of Bacteroides nodosus

Three proteinase isoenzymes from one benign strain of Bacteroides nodosus and five proteinase isoenzymes from each of two virulent strains of B. nodosus were purified by horizontal slab polyacrylamide gel electrophoresis. The purified isoenzymes hydrolysed casein, collagen I, collagen III, elastin, alpha-elastin, fibrinogen, gelatin, haemoglobin and alpha-keratin. The pH optima of all the isoenzymes lay between 7.25 and 9.5, the range of 8.75-9.25 being common to all. The isoenzymes were inhibited by phenylmethylsulphonyl fluoride, diphenylcarbamyl chloride, L-(1-tosylamide-2-phenyl)ethyl chloromethyl ketone, EGTA and EDTA, indicating that they were chymotrypsin-like serine proteinases that require a metal ion for stability or activity. EDTA inhibition was not reversed by addition of Ca2+ or Mg2+. Some isoenzymes were activated by Mg2+, Ca2+, Cr3+ and Se4+ and all were inhibited by Fe2+, Co2+, Cu2+, Zn2+, Cd2+ and Hg2+. Isoenzymes from benign strains had a lower temperature stability, losing all activity at 55 degrees C, whereas those from virulent strains lost all activity at 60 degrees C.     

Proteinase isoenzyme patterns of Bacteroides nodosus: distinction between ovine virulent isolates, ovine benign isolates and bovine isolates

Bacteroides nodosus isolates from ovine virulent footrot and ovine benign footrot and bovine isolates of low virulence for sheep were distinguishable from each other by their proteinase isoenzyme patterns after polyacrylamide gel electrophoresis. Variants of low virulence were not always distinguishable from their virulent parent strains. The molecular weights of the isoenzymes ranged from 70000 to 129000. The relationship of isoenzyme patterns to virulence is discussed.     

Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus

The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.     

Multiple proteases from Streptomyces moderatus: I. Isolation and purification of five extracellular proteases

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. Ia and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy-d-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by ϵ-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on ϵ-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.     

Molecular cloning of the fimbrial subunit gene from a benign type B isolate of Bacteroides nodosus

Abstract The fimbrial subunit gene from the benign type B Bacteroides nodosus isolate AC/6 was cloned into the Sph I site of the multicopy vector plasmid pUC19. Five Escherichia coli recombinants that were positive in a colony immunoassay were shown, by Western transfer analysis, to produce an immunologically cross-reacting protein of identical molecular size to fimbrial subunits prepared from B. nodosus AC/6. Restriction endonuclease analysis showed that 4 of the recombinant plasmids carried a 6.7 kb Sph I fragment. Recloning experiments showed that the fimbrial subunit gene was located within a 2.5 kb Eco RI- Sph I fragment and that there was a Pst I site located within the structural gene or its regulatory region. These recombinant clones will prove useful for the construction of a multivalent recombinant vaccine for the control of ovine footrot.     

Characterization and purification of extracellular proteases of Trichomonas vaginalis

Extracellular protease activity was detected in serum-free culture filtrates of Trichomonas vaginalis. The activity was demonstrated by hydrolysis of hide powder azure and possessed the characteristics of cysteine type proteases: inhibition by N-ethyl maleimide, Cu2+, antipain, N-tosyl-L-phenylalanine chloromethyl ketone, N-tosyl-L-lysine chloromethyl ketone, leupeptin, chymostatin, and iodoacetamide, and enhancement by cysteine, EDTA, and dithiothreitol. The activity was optimal at acid pH and the protease was also active on peptide nitroanilides with arginine derivatives. Purification of this activity by ethanol precipitation, ammonium sulfate fractionation, ion exchange chromatography, and gel filtration resulted in the isolation of two proteases estimated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis to have molecular masses of 60 and 30 kilodaltons (kDa), respectively. The larger molecular mass protease broke down during purifications to two subunits of approximately 23 and 43 kDa, as determined by gel electrophoresis. Rabbit sera derived by immunization with the 23-kDa subunit cross-reacted by immunoblot with the 60- and 43-kDa subunits, but not with the 30-kDa protease. These soluble products of T. vaginalis growth could be important pathogenically in establishing T. vaginalis infection in the normally acid (pH less than or equal to 4.5) environment of the vagina.     

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.     

Isoenzyme characterization of proteases and amylases and partial purification of proteases from filamentous fungi causing biodeterioration of industrial paper

Biodeterioration is an undesirable process that can affect cultural heritage and economically important materials. Although several biotic and abiotic conditions can accelerate this process, microorganisms are perhaps its main promoters. Fungi are the most important microbial agents of biodeterioration of industrial paper stored in archives. The high genetic plasticity of these organisms allows them to adapt to different environments, using almost any class of materials as substrate. Fungi produce a wide array of enzymes, including cellulases, amylases, and proteases, which are responsible for their gross biodeterioration activity. Thirty-two morphotypes of filamentous fungi were isolated on different media from industrial paper at an advanced stage of biodeterioration. The isolates showed different degrees of cellulolytic, proteolytic, and amylolytic activities on plate assays. The highest proteolytic and amylolytic activities were selected for isoform characterization, which provided an indication of the biochemical diversity that allowed them to colonize these materials. Eladia sacculum was the morphotype selected for partial purification of basic proteases since it has three basic isoforms, simplifying the purification process. We obtained a protein of 35 kDa with a pI of 8.9.     

Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus

Studies on the potential virulence genes of the ovine footrot pathogen Dichelobacter nodosus have been hindered by the lack of a genetic system for this organism. In an attempt to accomplish the transformation of D. nodosus cells, we constructed a plasmid that contained part of a native D. nodosus plasmid and carried a tetracycline resistance gene that was located between the D. nodosus rrnA promoter and terminator. This plasmid was used to transform several D. nodosus strains to tetracycline resistance. Analysis of two independent transformants from each parental strain showed that in nearly all of these derivatives, the plasmid was not replicating independently, but that the tetracycline resistance gene had inserted by homologous recombination into one of the three rrn operons located on the chromosome. In most of the transformants, double reciprocal crossover events had occurred. These results are highly significant for genetic studies in D. nodosus and for footrot pathogenesis studies, since by using reverse genetics it will now be possible to examine the role of putative D. nodosus-encoded virulence genes in the disease process.     

Separation and partial purification of extracellular amylase and protease from Bacillus caldolyticus

Summary The extracellular amylase and protease from Bacillus caldolyticus can be concentrated by ammonium sulfate precipitation after growth on either solid or in liquid media containing starch, glucose, and brain-heart infusion. Using the Diaflo ultrafiltration system with membranes of various permeability, the enzymes could be separated from each other by extensive flushing with buffer. Best results were obtained with the 50–70% ammonium sulfate fraction as starting material, yielding 72% of the total amylase activity in the low molecular weight fraction (UM-10 fraction: 10000–30000), while 54 and 25% respectively of the protease were retained in the two high molecular weight fractions (50000–100000, and more than 100000). Similar results were obtained with the 20–50% ammonium sulfate fraction, while the fraction of 0–20% saturation contained a low molecular weight protease. The native amylase seems to consist of a number of sub-units, which after extensive flushing accumulate in the fraction with an approximate molecular weight between 10000 and 30000. The enzyme could also be precipitated from cell-free liquid media with ammonium sulfate, followed by separation and purification on ultra-filtration cells. According to the specific activity of the UM-10 fractions a 400-fold purification was obtained compared to the amylase activity of the cell-free medium.Direct concentration and separation from liquid media, omitting ammonium sulfate treatment, was also found to be possible, although prolonged flushing with buffer was necessary to obtain satisfactory separation.During purification from the ammonium sulfate fractions, amylase activity was found to decrease but could be restored by Ca-ions. At 70°C, a final concentration of 0.5 mM CaCl₂, was sufficient for full restoration, while three times that amount was necessary at 80°C. Determination of the K _m-values for Ca at different temperatures resulted in an asymptotically increasing curve at temperatures beyond 75°C. Addition of Ca had a pronounced effect on the stability of the amylase at 80°C but not at 90°C. Protease activity and stability was not affected by Ca-ions.     

Evidence that Bacteroides nodosus belongs in subgroup gamma of the class Proteobacteria, not in the genus Bacteroides: partial sequence analysis of a B. nodosus 16S rRNA gene

The taxonomic status of the anaerobe Bacteroides nodosus has for some time been uncertain. To resolve this uncertainty, the distal portion of a 16S rRNA gene from this important ovine pathogen was cloned, mapped, and sequenced. A comparison of the sequence with the sequences of 16S rRNA molecules from other bacteria indicated that B. nodosus is more closely related to Escherichia coli and other members of the class Proteobacteria than to Bacteroides fragilis or the bacteroides-flavobacterium-cytophaga phylum. The evidence from the comparison of sequence signatures suggests that B. nodosus is not a member of the genus Bacteroides but that it belongs in subgroup gamma of the class Proteobacteria.     

毛栓菌胞外漆酶的纯化及部分性质研究

毛栓菌胞外漆酶经盐析、透析、sephadexG75和G2 5四步纯化 ,粗酶液被纯化了 39 1倍 ,比活力 12 152 ,回收率 4 5 3%。漆酶最适pH值为 4 0 ,最适反应温度为 30℃。K Cu 2 、Zn2 离子可激活漆酶 ,而Ag 、Fe3 离子可抑制漆酶的活性。漆酶的Km值为 1 81× 10 3mol/L。     

Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus

Abstract Ovine footrot is a debilitating and highly infectious disease that is primarily caused by the Gram-negative, anaerobic bacterium Dichelobacter nodosus . The major antigens implicated in virulence are the type IV fimbriae and extracellular proteases. The fimbriae show sequence and structural similarity to other type IV fimbriae, this similarity extends to genes that are involved in fimbrial biogenesis. Several acidic and basic extracellular serine proteases are produced by both virulent and benign isolates of D. nodosus . Subtle functional differences in these proteases appear to be important in virulence. In addition, there are two chromosomal regions that have a genotypic association with virulence. The partially duplicated and rearranged vap regions appear to have arisen from the insertion of a plasmid into a tRNA gene via an integrase-mediated site-specific insertion event. The 27 kb vrl region has several genes often found on bacteriophages and has inserted into an ssrA gene that may have a regulatory role in the cell. The determination of the precise role that each of these genes and gene regions has in virulence awaits the development of methods for the genetic analysis and manipulation of D. nodosus .     

腐蹄病致病因子节瘤拟杆菌蛋白酶在大肠杆菌...

Bacteroides nodosus is the essential causative agent of ovine footrot. It produces extracellular proteases which involved in pathogenesis of footrot. In this paper, we report the subcloning of Bacteroides nodosus protease, its overexpression in E. coli and its N-terminal polypeptide sequence. The subclone library was constructed in E. coli using SphI digested original clone (15 kb) and plasmid PTZ18R and screened using immunological assay. The expression was observed using SDS-PAGE. The subcloned DNA fragment was then cut with Sau3AI, cloned into pKK232-8 vector to perform promoter isolation and analysis. The promoter strength was determined using spectrophotometric assay.     

Ultrastructure of the Bacteroides nodosus cell envelope layers and surface.

The surface structure and cell envelope layers of various virulent Bacteroides nodosus strains were examined by light microscopy and by electron microscopy by using negative staining, thin-section, and freeze-fracture-etch techniques. Three surface structures were described: pili and a diffuse material, both of which emerged from one or both poles of the bacteria (depending on the stage of growth and division), and large rodlike structures (usually 30 to 40 nm in diameter) associated with a small proportion of the bacterial population. No capsule was detected. The cell envelope consisted of four layers: a plasma membrane, a peptidoglycan layer, an outer membrane, and an outermost additional layer. The additional layer was composed of subunits, generally hexagonally packed with center-to-center spacing of 6 to 7 nm. The outer membrane and plasma membrane freeze-fractured through their hydrophobic regions revealing four fracture faces with features similar to those of other gram-negative bacteria. However, some unusual features were seen on the fracture faces of the outer membrane: large raised ring structure (11 to 12 nm in diameter) on cw 3 at the poles of the bacteria; complementary pits or ring-shaped depressions on cw 2; and small raised ring structures (7 to 8 nm in diameter) all over cw 2.     

Morphogenetic expression of Bacteroides nodosus fimbriae in Pseudomonas aeruginosa.

Type 4 fimbriae are found in a range of pathogenic bacteria, including Bacteroides nodosus, Moraxella bovis, Neisseria gonorrhoeae, and Pseudomonas aeruginosa. The structural subunits of these fimbriae all contain a highly conserved hydrophobic amino-terminal sequence preceding a variable hydrophilic carboxy-terminal region. We show here that recombinant P. aeruginosa cells containing the B. nodosus fimbrial subunit gene under the control of a strong promoter (pL, from bacteriophage lambda) produced large amounts of fimbriae that were structurally and antigenically indistinguishable from those produced by B. nodosus. This was demonstrated by fimbrial isolation and purification, electrophoretic and Western transfer analyses, and immunogold labeling and electron microscopy. These results suggest that type 4 fimbriated bacteria use a common mechanism for fimbrial assembly and that the structural subunits are interchangeable, thereby providing a basis for the development of multivalent vaccines.     

Sequence of pilin from Bacteroides nodosus 351 (Serogroup H) and implications for serogroup classification

The nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and Mr 17,150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.