Purification and some properties of dextransucrase from Streptococcus bovis 148

The dextransucrase produced by Streptococcus bovis 148 was purified about 20.8-fold to electrophoretic homogeneity (specific activity 246.3 units/mg) from the culture supernatant. Molecular weight of the native enzyme was estimated to be 600 kDa, whereas the molecular weight per subunit was 150 kDa. The dextran synthesized consisted chiefly of α-1,6-glucosidic linkage, containing small numbers of branches that had the α-1,3-glucosidic linkage.     

奶牛瘤胃中牛链球菌的分离鉴定

采用厌氧分离技术从奶牛瘤胃中分离出1株细菌,通过对其形态、培养特性、生理生化特性、16S rRNA基因序列测定与同源性分析等研究,确定分离菌株为牛链球菌（Streptococcus bovis）,为进一步研究其对瘤胃发酵的影响奠定了基础。     

Phenotypic characterization of CO2-requiring strains of Streptococcus bovis from koalas.

We examined phenotypic characteristics of six mannitol-fermenting strains of Streptococcus bovis, including two unusual CO2-requiring strains isolated from koala feces. These strains did not grow in air, but grew in air supplemented with CO2 and under reduced oxygen conditions. All six strains had the same biochemical characteristics, except that the CO2-requiring strains did not produce beta-N-acetylglucosaminidase.     

Phenotypic characterization of CO2-requiring strains of Streptococcus bovis from koalas.

We examined phenotypic characteristics of six mannitol-fermenting strains of Streptococcus bovis, including two unusual CO2-requiring strains isolated from koala feces. These strains did not grow in air, but grew in air supplemented with CO2 and under reduced oxygen conditions. All six strains had the same biochemical characteristics, except that the CO2-requiring strains did not produce beta-N-acetylglucosaminidase.     

Purification and properties of dextransucrase and invertase from Streptococcus mutans

Invertase (beta-d-fructofuranoside fructohydrolase, EC 3.2.1.26) and dextransucrase (alpha-1, 6-glucan: d-fructose 2-glucosyltransferase, EC 2.4.1.5) were purified from the culture fluids of Streptococcus mutans by chromatography on Sepharose 6B and diethylaminoethyl-cellulose followed by treatment with hydroxyapatite. Each of the enzyme preparations gave a single band when analyzed by either polyacrylamide gel electrophoresis or immunodiffusion. The antigenic determinant of invertase was different from that of dextransucrase on immunodiffusion. The pH optima were 5.25 for invertase and 5.75 for dextransucrase, and the K(m) values were 20 mM for invertase and 2.0 mM for dextransucrase. The molecular weights determined by sodium dodecyl sulfate gel electrophoresis were 160,000 for invertase and 170,000 for dextransucrase. The data obtained suggest that the dextransucrase had dextran-synthesizing activity and invertase-like activity.     

Purification and some properties of free and cell-associated dextransucrase from Streptococcus sanguis.

Dextransucrase of Streptococcus sanguis occurred in cell-free and cell-associated forms. Cell-free dextransucrase was purified by four successive chromatographies on Bio-Gel P 60, DEAE-cellulose, and Bio-Gel P 200 from the culture supernatant. The purification of cell-associated dextransucrase was made from the pellet of Streptococcus sanguis culture. Bacterial pellet was extracted with 1 M phosphate buffer (pH 6.0) and chromatographied by using an immunosorbent column. The two enzymes gave single bands in polyacrylamide gel electrophoresis. The molecular weight determined by sodium dodecyl sulfate polyacrylamide gel was about 100 000 daltons for the two forms of dextransucrases. The optimum pH of the cell-free and cell-associated enzymes was around 6 and the temperature optimum was broad for the two enzymes. The KM values for sucrose were respectively 2 mM and 3 mM for cell-free and cell-associated enzymes. When primer dextran was added, the reaction velocity increased but the KM for sucrose remained the same, and the KA for dextran was 200 muM for the two dextransucrases. Trehalose and maltose acted also as glucosyl residue acceptors. Purified enzymes had dextran synthesising activity and invertase-like activity. The same properties of the two forms of enzymes and the positive cross reaction against anti free and anti cell-associated globulins stongly suggest the identity of the two enzymes.     

Deoxyribonuclease activity in Streptococcus bovis

Deoxyribonuclease activity was tested with lambda bacteriophage DNA as a substrate in three Streptococcus bovis strains isolated from the rumen of sheep and cow. Non-specific nuclease activity was detected in the cell extract of Strep, bovis BM 114. Specific endonuclease activity was detected in the cell extract of the strain Strep. bovis II/I, isolated from the rumen of a sheep. A rapid technique is proposed for the detection of endonuclease activity of rumen bacteria.     

The purification and properties of dextransucrase from Streptococcus sanguis ATCC 10558

Dextransucrase has been purified from the culture fluids of S. sanguis 10558 by a combination of hydroxylapatite, ion-exchange, and gel-filtration steps. Two active proteins were isolated with specific activities approaching one order of magnitude higher than other preparations reported. The enzymes have mol. wt. on the order of 100 000 and exhibit pH optima between 5.8 and 6.2. In addition, detailed analysis of one of the enzymes indicates that the enzyme undergoes two ionizations that are important for activity. One pK is at 4.4 and the second at 7.4. The structures of dextrans produced by the two enzymes have been examined by p.m.r. spectroscopy, and a substantial degree of similarity was observed, with only minor differences in the proportion of α-(1→3) and α-(1→6) bonds. No evidence could be obtained that either of the enzymes was capable of catalyzing a rearrangement of α-(1→6) to α-(1→3) bonds.     

Identification of intracellular amylase activity in Streptococcus bovis and Streptococcus salivarius

The ruminal bacterium Streptococcus bovis has been demonstrated to produce an extracellular amylase activity. We previously reported on the cloning of a gene from S. bovis encoding for what was initially believed to be the extracellular amylase. DNA sequence analyses indicated that the amylase produced by the cloned gene did not match the N-terminus amino acid sequence of the purified extracellular amylase and contained no apparent leader sequence for secretion. Analyses of crude extracts demonstrated the presence of an intracellular amylase in S. bovis JB1 that differed in molecular weight (56,000) from that of the extracellular amylase (70,000). The 56,000 molecular weight amylase was identical to the amylase produced by Escherichia coli containing the cloned amylase gene. Low levels of intracellular amylase activity were also detected in other strains of S. bovis and also Streptococcus salivarius. Introduction of the plasmid pVA838 containing the cloned amylase gene into S. bovis and S. sanguis resulted in enhanced intracellular amylase production by both organisms. The amylase gene has been sequenced, and analysis of the deduced amino acid sequence for the amylase indicates a high degree of similarity with secreted amylases from Bacillus species.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Natural genetic transformation in the rumen bacterium Streptococcus bovis JB1

Natural transformation of Streptococcus bovis JB1 was demonstrated after development of competence in normal culture medium. Transformation efficiencies were not significantly increased when heat-inactivated horse serum was added to the medium before growth. This is the first time that a resident rumen bacterial species has been shown to be naturally transformable. Transformation allowed the acquisition of plasmids or integration of sequences into the chromosome. No transformation was observed in the presence of undiluted autoclaved or filter-sterilised ovine rumen fluid or filter-sterilised ovine saliva, suggesting that transformation in the ruminant digestive tract is a rare event, although transformation was observed in the presence of 1% and 0.5% filter-sterilised rumen fluid. The use of natural transformation of S. bovis should facilitate further molecular biological studies on this species.     

Isolation and some properties of extracellular glucan-producing strains of human oral Streptococcus salivarius

A total of eighteen strains of Streptococcus salivarius, which formed rough gelatinous, rough mucoid or smooth mucoid colonies on sucrose agar media, were isolated from the saliva and tongue dorsum of adults. All of the isolates produced glucans as well as fructans from sucrose. The bulk of the glucans was synthesized by the extracellular enzyme fraction and was water insoluble, whereas most of the fructans were synthesized by the cell-associated enzyme fraction and were water soluble. All strains formed microbial deposits on wire and glass surfaces when cultured in sucrose broth, but their sucrose-dependent adhesion was apparently looser than that produced by a cariogenic S. sobrinus strain. The rough gelatinous colony forming strains possessed a greater ability to synthesize water-insoluble glucans and produced heavier deposits with higher cohesion. Preliminary studies showed that the S. salivarius of such characteristic forms of colony were detected primarily in the saliva and tongue dorsum: the smooth mucoid colony formers appeared to predominate in the tongue coat and the rough mucoid and rough gelatinous colony formers were prominent in saliva. Isolation of these S. salivarius from dental plaques was low.     

Production of bacteriocins by Streptococcus bovis strains from Australian ruminants

Aims: To examine the prevalence of bacteriocin production in Streptococcus bovis isolates from Australian ruminants and the feasibility of industrial production of bacteriocin. Methods and Results: Streptococcus bovis strains were tested for production of bacteriocin‐like inhibitory substances (BLIS) by antagonism assay against Lactococcus lactis. BLIS production was associated with source animal location (i.e. proximity of other bacteriocin‐positive source animals) rather than ruminant species/breed or diet. One bacteriocin showing strong inhibitory activity (Sb15) was isolated and examined. Protein sequence, stability and activity spectrum of this bovicin were very similar to bovicin HC5. Production could be increased through serial culturing, and increased productivity could be partially maintained during cold storage of cultures. Conclusions: BLIS production is geographically widely distributed in Eastern Australia, and it appears that the bacteriocin⁺ trait is maintained in animals at the same location. The HC5‐like bacteriocin, originally identified in North America, is also found in Australia. Production of bacteriocin can be increased through serial culturing. Significance and Impact of the Study: The HC5‐like bacteriocins appear to have a broad global distribution. Serial culturing may provide a route towards commercial manufacturing for use in industrial applications, and purified bacteriocin from S. bovis Sb15 could potentially be used to prevent food spoilage or as a feed additive to promote growth in ruminant species.     

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer

AIMS: Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. METHODS AND RESULTS: Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced l-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. CONCLUSIONS: Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.