Bovis JB1 Research Articles

Streptococcus bovis JB1 Protects Against Oxidative Stress Caused by comC Knockout by Increasing Trehalose Secretion

Streptococcus bovis JB1 Protects Against Oxidative Stress Caused by comC Knockout by Increasing Trehalose Secretion

Effects of ruminal lipopolysaccharides on growth and fermentation end products of pure cultured bacteria

Elevated levels of ruminal lipopolysaccharides (LPS) have been linked to ruminal acidosis; however, they result in reduced endotoxicity compared to LPS derived from species like Escherichia coli. Additionally, there is a knowledge gap on the potential effect of LPS derived from ruminal microbiome on ruminal bacteria species whose abundance is associated with ruminal acidosis. The objective of this study was to evaluate the effects of LPS-free anaerobic water (CTRL), E. coli-LPS (E. COLI), ruminal-LPS (RUM), and a 1:1 mixture of E. coli and ruminal-LPS (MIX) on the growth characteristics and fermentation end products of lactate-producing bacteria (Streptococcus bovis JB1, Selenomonas ruminantium HD4) and lactate-utilizing bacterium (Megasphaera elsdenii T81). The growth characteristics were predicted based on the logistic growth model, the ammonia concentration was determined by the phenolic acid/hypochlorite method and organic acids were analyzed with high performance liquid chromatography. Results indicate that, compared to the CTRL, the maximum specific growth rate of S. bovis JB1 decreased by approximately 19% and 23% when RUM and MIX were dosed, respectively. In addition, acetate and lactate concentrations in Se. ruminantium HD4 were reduced by approximately 30% and 18%; respectively, in response to MIX dosing. Compared to CTRL, lactate concentration from S. bovis JB1 was reduced approximately by 31% and 22% in response to RUM and MIX dosing; respectively. In summary, RUM decreased the growth and lactate production of some lactate-producing bacteria, potentially mitigating the development of subacute ruminal acidosis by restricting lactate availability to some lactate-utilizing bacteria that metabolize lactate into VFAs thus further contributing to the development of acidosis. Also, RUM did not affect Megasphaera elsdenii T81 growth.

Characterization of Endolysin LyJH307 with Antimicrobial Activity Against Streptococcus Bovis.

Simple SummaryDevelopment of endolysin is a promising strategy because of having the ability to control problematic bacteria specifically. In this study, we developed and characterized the endolysin having lytic activity against Streptococcus bovis (S. bovis), which is one of the initiators of ruminal acidosis. Based on our findings, endolysin LyJH307 showed potent lytic activity in ruminal pH range and ruminal temperature. In addition, LyJH307 was effective against not only S. bovis isolated from rumen, but also several S. bovis groups. We suggest that LyJH307 may have a lytic effect in the ruminal condition and prevent acute ruminal acidosis by controlling S. bovis specifically.Streptococcus bovis (S. bovis) is one of the critical initiators of acute acidosis in ruminants. Therefore, we aimed to develop and characterize the endolysin LyJH307, which can lyse ruminal S. bovis. We tested the bactericidal activity of recombinant LyJH307 against S. bovis JB1 under a range of pH, temperature, NaCl, and metal ion concentrations. In silico analyses showed that LyJH307 has a modular design with a distinct, enzymatically active domain of the NLPC/P60 superfamily at the N-terminal and a cell wall binding domain of the Zoocin A target recognition domain (Zoocin A_TRD) superfamily at the C-terminal. The lytic activity of LyJH307 against S. bovis JB1 was the highest at pH 5.5, and relatively higher under acidic, than under alkaline conditions. LyJH307 activity was also the highest at 39 °C, but was maintained between 25°C and 55°C. LyJH307 bactericidal action was retained under 0-500 mM NaCl. While the activity of LyJH307 significantly decreased on treatment with ethylenediaminetetraacetic acid (EDTA), it was only restored with supplementation of 10 mM Ca2+. Analyses of antimicrobial spectra showed that LyJH307 lysed Lancefield groups D (S. bovis group and Enterococcus faecalis) and H (S. sanguinis) bacteria. Thus, LyJH307 might help to prevent acute ruminal acidosis.

Lipopolysaccharide Stimulates the Growth of Bacteria That Contribute to Ruminal Acidosis.

Lipopolysaccharide (LPS) has been reported to contribute to a ruminal acidosis of cattle by affecting ruminal bacteria. The goal of this study was to determine how LPS affects the growth of pure cultures of ruminal bacteria, including those that contribute to ruminal acidosis. We found that dosing LPS (200,000 EU) increased the maximum specific growth rates of four ruminal bacterial species (Streptococcus bovis JB1, Succinivibrio dextrinosolvens 24, Lactobacillus ruminis RF1, and Selenomonas ruminantium HD4). Interestingly, all the species ferment sugars and produce lactate, contributing to acidosis. Species that consume lactate or ferment fiber were not affected by LPS. We found that S. bovis JB1 failed to grow in LPS as the carbon source in the media; growth of S. bovis JB1 was increased by LPS when glucose was present. Growth of Megasphaera elsdenii T81, which consumes lactate, was not different between the detoxified (lipid A delipidated) and regular LPS. However, the maximum specific growth rate of S. bovis JB1 was greater in regular LPS than detoxified LPS. Mixed bacteria from a dual-flow continuous culture system were collected to determine changes of metabolic capabilities of bacteria by LPS, and genes associated with LPS biosynthesis were increased by LPS. In summary, LPS was not toxic to bacteria, and lipid A of LPS stimulated the growth of lactate-producing bacteria. Our results indicate that LPS not only is increased during acidosis but also may contribute to ruminal acidosis development by increasing the growth of lactic acid-producing bacteria.IMPORTANCE Gram-negative bacteria contain lipopolysaccharide (LPS) coating their thin peptidoglycan cell wall. The presence of LPS has been suggested to be associated with a metabolic disorder of cattle-ruminal acidosis-through affecting ruminal bacteria. Ruminal acidosis could reduce feed intake and milk production and increase the incidence of diarrhea, milk fat depression, liver abscesses, and laminitis. However, how LPS affects bacteria associated with ruminal acidosis has not been studied. In this study, we investigated how LPS affects the growth of ruminal bacteria by pure cultures, including those that contribute to acidosis, and the functional genes of ruminal bacteria. Thus, this work serves to further our understanding of the roles of LPS in the pathogenesis of ruminal acidosis, as well as providing information that may be useful for the prevention of ruminal acidosis and reducetion of economic losses for farmers.

Heterologous gene expression in the human gut bacteria Eubacterium rectale and Roseburia inulinivorans by means of conjugative plasmids

Commensal butyrate-producing bacteria in the Firmicutes phylum are abundant in the human intestine and are important for maintaining health. However, understanding of the metabolism and host interaction of these bacteria is limited by the lack of genetic modification techniques. Here we establish a protocol enabling the transfer of autonomously-replicating shuttle vectors by conjugative plasmid transfer from an Escherichia coli donor into representatives of an important sub-group of strictly anaerobic human colonic Firmicutes. Five different plasmid shuttle vectors were tested, each carrying a different origin of replication from Gram-positive bacteria. Plasmid pMTL83151 (pCB102 replicon) were successfully transferred into two strains of Eubacterium rectale, while pMTL83151 and pMTL82151 (pBP1 replicon) were transferred into Roseburia inulinivorans A2-194. Plasmids that carried a Streptococcus bovis JB1 glycoside hydrolase family 16 β-(1,3-1,4)-glucanase gene were constructed and conjugated into Roseburia inulinivorans A2-194 and Eubacterium rectale T1-815, resulting in successful heterologous expression of this introduced enzymatic activity in these two strains of butyrate-producing Firmicutes.

Fermentation and chemical composition of guinea grass silage added with wheat meal and Streptococcus bovis

ABSTRACTThe objective of this study was to evaluate the effects of wheat meal alone or combined with Streptococcus bovis on the fermentation and chemical composition of guinea grass silage. A completely randomised design was used in the following treatments: deionised water, untreated; fresh forage plus 100 g kg−1 of wheat meal; S. bovis JB1 plus 100 g kg−1 of wheat meal; and S. bovis HC5 plus 100 g kg−1 of wheat meal, with six replications. There were a higher number of enterobacteria, moulds and yeast populations in the untreated silage (P < .05) compared to treated silages. The association of S. bovis with wheat meal resulted in lower pH and concentration of NH3-N (P < .05) than silages untreated and treated only with wheat meal. The use of both S. bovis JB1 and HC5 combined with 100 g kg−1 wheat meal can also be a good alternative to farmers for improving the silage quality of guinea grass silage.

Effect of biochanin A on corn grain (Zea mays) fermentation by bovine rumen amylolytic bacteria.

The objective was to determine the effect of biochanin A (BCA), an isoflavone produced by red clover (Trifolium pratense L.), on corn fermentation by rumen micro-organisms. When bovine rumen bacterial cell suspensions (n=3) were incubated (24h, 39°C) with ground corn, amylolytic bacteria including group D Gram-positive cocci (GPC; Streptococcus bovis; enterococci) proliferated, cellulolytic bacteria were inhibited, lactate accumulated and pH declined. Addition of BCA (30μgml-1 ) inhibited lactate production, and pH decline. BCA had no effect on total amylolytics, but increased lactobacilli and decreased GPC. The initial rate and total starch disappearance was decreased by BCA addition. BCA with added Strep. bovis HC5 supernatant (containing bacteriocins) inhibited the amylolytic bacteria tested (Strep. bovis JB1; Strep. bovis HC5; Lactobacillus reuteri, Selenemonas ruminatium) to a greater extent than either addition alone. BCA increased cellulolytics and dry matter digestibility of hay with corn starch. These results indicate that BCA mitigates changes associated with corn fermentation by bovine rumen bacteria exvivo. BCA could serve as an effective mitigation strategy for rumen acidosis. Future research is needed to evaluate the effect of BCA on mitigating rumen acidosis invivo.

Chemical composition and nutrient degradability in elephant grass silage inoculated with Streptococcus bovis isolated from the rumen

The objective of the present study was to assess the chemical and bromatological composition and in situ degradability of elephant grass silages inoculated with Streptococcus bovis isolated from cattle rumen. A complete randomized design was used with four treatments and six replications: elephant grass silage, elephant grass silage inoculated with 10(6) CFU/g Streptococcus bovis JB1 strains; elephant grass silage inoculated with 106 CFU/g Streptococcus bovis HC5 strains; elephant grass silage inoculated with 106 CFU/g Enterococcus faecium with six replications each. The pH and ammoniacal nitrogen values were lower (P<0.05) for the silages inoculated with Streptococcus bovis JB1 and HC5, respectively. The silage inoculated with Streptococcus bovis had a higher crude protein content (P<0.05) and there were no differences for the fiber contents in the silage. The (a)soluble fraction degradability, especially in the silages inoculated with Streptococcus bovis JB1 and HC5, had higher values, 30.77 and 29.97%, for dry matter and 31.01 and 36.66% for crude protein, respectively. Inoculation with Streptococcus bovis improved the fermentation profile, protein value and rumen degradability of the nutrients.

Silage fermentation and chemical composition of elephant grass inoculated with rumen strains of Streptococcus bovis

The objective of this study was to evaluate the effects of Streptococcus bovis JB1 and HC5 on the fermentation and chemical composition of elephant grass ensiled in laboratory silos. A complete randomized design was used with four treatments: elephant grass silage, elephant grass silage inoculated with 106CFU/g S. bovis JB1 strains; elephant grass silage inoculated with 106CFU/g S. bovis HC5 strains; elephant grass silage inoculated with 106CFU/g Enterococcus faecium, and six replications. The highest number of enterobacteria, fungi and yeast occurred in the control and elephant grass silages inoculated with E. faecium (P<0.05). The silage inoculated with Streptococcus bovis JB1 and HC5 presented the lowest enterobacteria, fungi and yeast populations and the highest number of lactic acid bacteria, 10.03 and 9.97logCFU/g, respectively. The pH and ammoniac nitrogen values were low (P<0.05) in the treatments with Streptococcus bovis JB1 and HC5, respectively. The observed pH values were 3.83 and 3.96 in the treatments with S. bovis JB1 and HC5, respectively, lower than the observed value of 4.01 in the control treatment. The silages inoculated with S. bovis JB1 and HC5 presented the highest (P<0.05) lactic acid values, 67.8 and 66.2g/kg DM, respectively, and the lowest acetic, butyric and propionic acid values. Inoculation with Streptococcus bovis resulted in a higher (P<0.05) crude protein content in the silages, highest dry matter and crude protein recovery and lowest losses through gases and effluents. Treatments with S. bovis JB1 and HC5 presented values of 93.94 and 92.45 for dry matter recovery. There were no statistical differences (P>0.05) for the other chemical characteristics. Both S. bovis JB1 and HC5 improved the fermentation profile, reduced nutrient losses and increased the nutritional value of the elephant grass silage.

Ruminal degradability of Guinea grass silage inoculated with Streptoccocus bovis isolated from bovine rumen combined or not with com wheat bran

This experiment aimed to evaluate the ruminal degradability in situ of the dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) of Guinea grass silages. The experimental design was completely randomized with six treatments and six repetitions: Guinea grass silage, Guinea grass silage inoculated with 10% wheat bran, Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis JB1; Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis JB1 plus 10% of wheat bran; Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis HC5; Guinea grass silage inoculated with 106 cfu/g of strains of Streptococcus bovis HC5 plus 10% of wheat bran. The silages inoculated with Streptococcus bovis and with added wheat bran resulted in higher values of ruminal degradability of DM, 32.76% and 32.17%, and of crude protein 38.28% and 37.89%. For the effective and potential degradability of DM, CP, NDF and ADF the highest values occurred for silages in these silages as well, in all passage rates. There is a similarity between the responses of Streptococcus bovis JB1 and the HC5, and the wheat bran enhanced the effectiveness of the microbial additive.

Ingestão e digestibilidade aparente em ovinos alimentados com silagens de capim-elefante inoculadas com Streptococcus bovis

Foram avaliadas a ingestão e a digestibilidade aparente de silagens de capim, utilizando-se 20 ovinos adultos, sem raça definida, castrados, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos: silagem de capim-elefante; silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de Enterococcus faecium; silagem de capim-elefante inoculada com 10(6)UFC/g de estirpes de Streptococcus bovis JB1; e silagem de capim-elefante inoculada com 10(6)UFC/g de()estirpes de S. bovis HC5, e cinco repetições por tratamento. O período de avaliação foi de 21 dias, sendo 15 para adaptação e seis para as coletas. Observou-se diferença (P&lt;0,05) entre o consumo de matéria seca, sendo a silagem-controle a que apresentou os valores mais baixos - 1,43%PV ou 42,79g/kg PV0,75/dia -, enquanto a silagem inoculada com S. bovis HC5 apresentou o melhor consumo de matéria seca, 2,3% PV ou 56,10g/kg PV0,75/dia. Menores valores de digestibilidade da matéria seca ocorreram na silagem-controle, 42,8% da MS, seguida pela silagem de capim-elefante inoculada com E. faecium e S. bovis JB1. Para a digestibilidade aparente da fibra em detergente neutro, houve efeito dos inoculantes (P&lt;0,05) com valores acima de cinco pontos percentuais em relação à silagem inoculada, 54,7%.

Growth response of Salmonella enterica Typhimurium in co-culture with ruminal bacterium Streptococcus bovis is influenced by time of inoculation and carbohydrate substrate

Streptococcus bovis (S. bovis) is a normal inhabitant of the gastrointestinal tract of ruminants. It has a high metabolic rate and short generation time. Under conditions of carbohydrate overload, S. bovis can outgrow other ruminal microorganisms and dramatically lower the pH of the rumen. The purpose of this study was to characterize the growth effect on foodborne Salmonella enterica Typhimurium (S. Typhimurium) in the presence of the ruminal S. bovis JB1 isolate under differing incubation conditions. When S. bovis and S. Typhimurium were inoculated simultaneously and co-cultured together in Luria–Bertani broth, growth inhibition of S. Typhimurium was observed. In minimal medium supplemented with different carbon sources, inhibition of growth of S. Typhimurium was greatest in glycerol, followed by maltose, trehalose, and glucose. When the inoculation sequence was staggered in the co-culture with an initial inoculation of S. bovis followed by a subsequent inoculation with S. Typhimurium, growth inhibition of S. Typhimurium was greater for all carbon sources. Based on these studies it appears that carbon substrate and time of inoculation influence S. Typhimurium growth in the presence of actively growing S. bovis.

The ability of non-bacteriocin producing Streptococcus bovis strains to bind and transfer bovicin HC5 to other sensitive bacteria

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5), but S. bovis JB1 does not have antimicrobial activity. Preliminary experiments revealed an anomaly. When S. bovis JB1 cells were washed in stationary phase S. bovis HC5 cell-free culture supernatant, the S. bovis JB1 cells were subsequently able to inhibit hyper-ammonia producing ruminal bacteria ( Clostridium sticklandii, Clostridium aminophilum and Peptostreptococcus anaerobius). Other non-bacteriocin producing S. bovis strains also had the ability to bind and transfer semi-purified bovicin HC5. Bovicin HC5 that was bound to S. bovis JB1 was much more resistant to Pronase E than cell-free bovicin HC5, but it could be inactivated if the incubation period was 24 h. Acidic NaCl treatment (100 mM, pH 2.0) liberates half of the bovicin HC5 from S. bovis HC5, but it did not prevent bovicin HC5 from binding to S. bovis JB1. Acidic NaCl liberated some bovicin HC5 from S. bovis JB1, but the decrease in activity was only 2-fold. Bovicin HC5 is a positively charged peptide, and the ability of S. bovis JB1 to bind bovicin HC5 could be inhibited by either calcium or magnesium (100 mM). Acidic NaCl-treated S. bovis JB1 cells were unable to accumulate potassium, but they were still able to bind bovicin HC5 and prevent potassium accumulation by untreated S. bovis JB1 cells. Based on these results, bovicin HC5 bound to S. bovis JB1 cells still acts as a pore-forming lantibiotic.

Properties and Role of Glyceraldehyde-3-Phosphate Dehydrogenase in the Control of Fermentation Pattern and Growth in a Ruminal Bacterium, Streptococcus bovis

To clarify the control of glycolysis and the fermentation pattern in Streptococcus bovis, the molecular and enzymatic properties of NAD(+)-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined. The GAPDH gene (gapA) was found to cluster with several others, including those that encode phosphoglycerate kinase and translation elongation factor G, however, gapA was transcribed in a monocistronic fashion. Since biochemical properties, such as optimal pH and affinity for glyceraldehyde-3-phosphate (GAP), were not very different between GAPDH- and NADP(+)-specific glyceraldehyde-3-phosphate dehydrogenase (GAPN), the flux from GAP may be greatly influenced by the relative amounts of these two enzymes. Using S. bovis JB1 as a parent, JB1gapA and JB1ldh, which overproduce GAPDH and lactate dehydrogenase (LDH), respectively, were constructed to examine the control of the glycolytic flux and lactate production. There were no significant differences in growth rates and formate-to-lactate ratios among JB1, JB1gapA, and JB1ldh grown on glucose. When grown on lactose, JB1ldh showed a much lower formate-to-lactate ratio than JB1gapA, which showed the highest NADH-to-NAD(+) ratio. However, growth rates did not differ among JB1, JB1gapA, and JB1ldh. These results suggest that GAPDH is not involved in the control of the glycolytic flux and that lactate production is mainly controlled by LDH activity.

The activity and stability of cell-associated activity of bovicin HC5, a bacteriocin from Streptococcus bovis HC5

Streptococcus bovis HC5 cultures released a broad spectrum lantibiotic, bovicin HC5, into the cell-fee culture supernatant after they reached stationary phase, but most of the antibacterial activity remained cell-associated. Cell-associated bovicin HC5 was more resistant to degradation by Pronase E than the cell-free activity. Acidic NaCl (pH 2.0, 100 mM) did not release all of the cell-associated activity, and cells that were sequentially treated with acidic NaCl and Pronase E still had antibacterial activity. Cell-associated activity retained after acidic NaCl treatment was still able to catalyze potassium efflux from S. bovis JB1, a sensitive strain. These results indicate that cell-associated bovicin HC5 is more active and stable than cell-free bovicin HC5.

The Effect of Calcium and Magnesium on the Activity of Bovicin HC5 and Nisin

Some Gram-positive bacteria produce small peptides (bacteriocins) that have antimicrobial activity, but many bacteria can become bacteriocin resistant. Bovicin HC5, a lantibiotic produced by Streptococcus bovis HC5, has the ability to inhibit nisin-resistant bacteria. Because nisin resistance has in many cases been correlated with an alteration of lipoteichoic acids or the polar head groups of membrane phospholipids, we decided to examine the effect of divalent cations on nisin and bovicin HC5 activity. Both bacteriocins catalyzed potassium efflux from S. bovis JB1, a non-bacteriocin-producing strain. The addition of large amounts (100 mM) of calcium or magnesium increased the ability of S. bovis JB1 to bind Congo red (an anionic dye) and counteracted bacteriocin-mediated potassium loss. Calcium was more effective than magnesium in decreasing nisin activity, but the reverse was observed with bovicin HC5. Nisin-resistant S. bovis JB1 cells bound three times as much Congo red as nisin-sensitive cells, and this result is consistent with the idea that changes in cell surface charge can be a mechanism of bacteriocin resistance. The nisin-resistant cells were less susceptible to bovicin HC5, but bovicin HC5 still caused a 50% depletion of intracellular potassium. These results indicate that nisin and bovicin HC5 react differently with the cell surfaces of Gram-positive bacteria.

Assessment of biases associated with profiling simple, model communities using terminal-restriction fragment length polymorphism-based analyses

Community profiles based on terminal-restriction fragment length polymorphism (T-RFLP) analyses of amplified ribosomal RNA genes are used to monitor changes in microbial community structure and are sometimes employed for semi-quantitative estimates of species richness and abundance in environmental samples. To assess the accuracy of T-RFLP community profiles representing the relative abundance of bacteria in a sample, five species of ruminal bacteria were used to construct simple “communities”. Template DNA for PCR amplification was generated either by mixing equal quantities of genomic DNA from pure cultures or by mixing equal numbers of cells prior to DNA extraction. Pairwise mixtures of Fibrobacter succinogenes S85 with Ruminococcus albus 8, Ruminococcus flavefaciens FD-1, Butyrivibrio fibrisolvens 49 and Streptococcus bovis JB1 were created and a 5-member community was constructed. With genomic DNA mixes, relative abundance calculations based on T-RFLP patterns did not reflect input ratios. These discrepancies could not be accounted for by differences in genome size and rRNA operon copy number. In cell mixing experiments, easily lysed cells were overrepresented. To determine if a numerical correction factor could be used to compensate for observed discrepancies, we attempted to quantify biases attributed to DNA extraction and PCR amplification. Biases attributable to these factors led to deviations from expected PCR product ratios by 6% to 38%. We found that interactions were so complex that a suitable factor could not be derived. The unsystematic dependence of T-RFLP peak ratios on variability of DNA extraction and PCR amplification prevents accurate quantification of the relative abundance of microorganisms designed to represent simplified natural populations.

Tolerance mechanisms of streptococci to hydrolysable and condensed tannins

In vitro growth assays demonstrated that Streptococcus bovis JB1 was sensitive to tannic acid (TA), acacia (AT), and calliandra tannins (CT), while Streptococcus gallolyticus 2.2 was tolerant. At low levels of tannin, S. bovis JB1 exhibited some tolerance but this was always less than for S. gallolyticus 2.2. Exopolysaccharide (EPS) has been hypothesised to be produced by S. gallolyticus in response to tannins and may form a protective layer around the cells. The study found that there was a significant increase in cellular polysaccharides and EPS gene expression in response to TA, AT, and CT in S. gallolyticus 2.2 but not S. bovis JB1. To examine the response in vivo, sheep were fed either Acacia angustissima, or Calliandra callothyrsus and the abundance of S. bovis and S. gallolyticus populations was measured using 16S-rRNA based oligonucleotide probes that differentiated these two populations. The S. bovis population declined significantly when acacia was fed at 37% of the diet but recovered when polyethylene glycol (PEG) was added to the ration; PEG has a neutralising effect on tannins. The S. gallolyticus population was more resistant to the presence of tannins in the diet but this effect was diminished when PEG was added to the diet. Similar population dynamics were observed with sheep fed calliandra at 300 g kg −1 diet. In conclusion, it appears that EPS production is correlated with tannin tolerance in vitro. Tannin-resistant S. gallolyticus is more abundant in the rumen in the presence of tannins than is S. bovis indicating that the presence of tannin-resistant organisms may have significant nutritional consequences for the animal.

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

Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. 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. 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. 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.

Molecular Study on Cloned Endoglucanase Gene from Rumen Bacterium

An endoglucanase gene was subcloned from anaerobic rumen bacterium Ruminococcus flavefaciens strain 17. To express endoglucanase gene in Escherichia coli and Streptococcus bovis JB1, an endoglucanase gene fragment was inserted into pVA838-based shuttle vectors. Removal of endoglucanase gene promoter and expression of endoglucanase by promoter of S. bovis JB1 α-amylase gene (pACMCS) was also achieved. Survival of constructs pVACMCI, pTACMC and pACMCS, which carry endoglucanase gene, and stability of endoglucanase gene in S. bovis JB1, were observed. Maximal endoglucanase activities from S. bovis JB1/pVACMCI were 2- to 3-fold higher than from E. coli/pVACMCI. Specific cell activity of E. coli/pACMCS was found to be approximately 2- to -3 fold higher than the both E. coli/pVACMCI and E. coli/pTACMC. Specific cell activity of S. bovis JB1/pACMCS was also found to be approximately 2-fold higher than the both S. bovis/pVACMCI and S. bovis JB1/pTACMC.