Biomedical subjects

James B Russell

Publications and source records attributed to James B Russell.

26 records · Page 2Linked to original sources

PubMed2002

Amino acid deamination by ruminal Megasphaera elsdenii strains.

When ruminal fluid from a cow fed timothy hay was serially diluted (10-fold increments into anaerobic broth containing 15 mg ml(-1) Trypticase), the low dilutions (< or =10(-6)) had optical densities greater than 2.0 and ammonia concentrations greater than 100 m M. The optical densities and ammonia concentrations of the 10(-8) and 10(-9) dilutions were very low, but large cocci were observed in the 10(-8) dilution. The large cocci were isolated and identified by 16S rDNA sequencing as Megasphaera elsdenii. The freshly isolated strain (JL1) grew well on Trypticase, but less than 4% of the amino acid nitrogen in Trypticase was converted to ammonia. Optical density and ammonia production were twice as great if Casamino acids were provided, and similar results were obtained with seven other strains (B159, AW106, YT91, LC1, T81, J1, and YZ70). Specific activities of deamination (based on Casamino acids) of the eight strains ranged from 100 (strain JL1) to 325 (strain B159) nmol mg protein(-1) min(-1). None of the strains could utilize branched-chain amino acids as an energy source for growth, but specific activities of branched-chain amino acid deamination ranged from 15 to 65 nmol mg protein(-1) min(-1). All eight of the M. elsdenii strains grew well in the presence of 5 micro M monensin, and only two of the strains were strongly inhibited by 20 micro M monensin. On the basis of these results, it appears that M. elsdenii is deficient in peptidase activity and can utilize only a few amino acids. Some M. elsdenii strains produced ammonia and branched-chain volatile fatty acids nearly as fast as obligate amino acid-fermenting ruminal bacteria, but the extent of this production was at least fourfold lower. Because all of the strains could tolerate 5 micro M monensin, it is unlikely that this feed additive would significantly inhibit M. elsdenii in vivo.

Amino Acids↗

PubMed2002

Allisonella histaminiformans gen. nov., sp. nov. A novel bacterium that produces histamine, utilizes histidine as its sole energy source, and could play a role in bovine and equine laminitis.

When cattle and horses are fed large amounts of grain, histamine can accumulate in the gastrointestinal tract, and this accumulation can cause an acute inflammation of the hooves (laminitis). When ruminal fluid from dairy cattle fed grain supplements was serially diluted in anaerobic MRS medium containing histidine (50 mM), histamine was detected at dilutions as high as 10(-7). The histidine enrichments were then transferred successively in an anaerobic, carbonate-based medium (50 mM histidine) without glucose. The histamine producing bacteria could not be isolated from the rumens of cattle fed hay; however, histamine producing bacteria could be isolated the feces of cattle fed grain and the cecum of a horse. All of the histamine producing isolates had the same ovoid morphology. The cells stained Gram-negative and were resistant to the ionophore, monensin (25 microM). The doubling time was 110 min, and the yield was 1.5 mg cell protein per mmol histidine. The G+C content was 46.8%. Lysine was the only other amino acid used, but lysine did not allow growth if histidine was absent. Because carbohydrate and organic acid utilization was not detected, it appeared that the isolates used histidine decarboxylation as their sole mechanism of energy derivation. 16s rRNA gene sequencing indicated that the isolates were most closely related to low G+C Gram-positive bacteria (firmicutes), but similarities were < or = 94%. Because the most closely related bacteria (Dialister pneumonsintes, Megasphaera elsdenii and Selenomonas ruminantium) did not produce histamine from histidine, we propose that these histamine producing bacteria be assigned to a new genus, Allisonella, as Allisonella histaminiformans gen. nov., sp. nov. The type strain is MR2 (ATCC BAA610, DSM 15230).

Amino Acids↗

PubMed2002

Bovicin HC5, a bacteriocin from Streptococcus bovis HC5.

Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and alpha-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 2.0) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55%. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.

Amino Acid Sequence↗

PubMed2002

Bacteriocin-like activity of Butyrivibrio fibrisolvens JL5 and its effect on other ruminal bacteria and ammonia production.

When ruminal bacteria from a cow fed hay were serially diluted into an anaerobic medium that had only peptides and amino acids as energy sources, little growth or ammonia production was detected at dilutions greater than 10(-6). The 10(-8) and 10(-9) dilutions contained bacteria that fermented carbohydrates, and some of these bacteria inhibited Clostridium sticklandii SR, an obligate amino acid-fermenting bacterium. Phylogenetic analysis indicated that the most active isolate (JL5) was closely related to Butyrivibrio fibrisolvens B835. Strain JL5 inhibited B. fibrisolvens 49 and a variety of other gram-positive organisms, but it had little effect on most gram-negative ruminal bacteria. Strain JL5 did not produce a bacteriocin-like inhibitory substance (BLIS) until it reached the late log or stationary phase. The JL5 BLIS did not cause the lysis of B. fibrisolvens 49, but the intracellular potassium level, the ATP level, the electrical potential, and the viability decreased rapidly. The JL5 BLIS also caused marked decreases in the viability and cellular potassium level of C. sticklandii SR. The membrane potential and intracellular ATP level also declined. The BLIS was degraded very slowly by pronase E, but it could be precipitated with 60% ammonium sulfate and dialyzed (3,500-Da cutoff). The BLIS could be separated from other peptides by polyacrylamide gel electrophoresis, and C. sticklandii SR overlays indicated that the molecular size of this compound was approximately 3,600 Da. Based on these results, it appeared that the JL5 BLIS was a pore-forming peptide. Because carbohydrate-fermenting ruminal bacteria could inhibit the growth of obligate amino acid-fermenting bacteria, BLIS may play a role in regulating ammonia production in vivo.

Amino Acids↗

PubMed2002

The bacteriocins of ruminal bacteria and their potential as an alternative to antibiotics.

Beef cattle have been fed ionophores and other antibiotics for more than 20 years to decrease ruminal fermentation losses (e.g methane and ammonia) and increase feed efficiency, and these improvements have been explained by an inhibition of gram-positive ruminal bacteria. Ionophores are not used to treat human disease, but there has been an increased perception that antibiotics should not be used as feed additives. Some bacteria produce small peptides (bacteriocins) that inhibit gram-positive bacteria. In vitro experiments indicated that the bacteriocin, nisin, and the ionophore, monensin, had similar effects on ruminal fermentation. However, preliminary results indicated that mixed ruminal bacteria degraded nisin, and the ruminal bacterium, Streptococcus bovis, became highly nisin-resistant. A variety of ruminal bacteria produce bacteriocins, and bacteriocin production has, in some cases, been correlated with changes in ruminal ecology. Some ruminal bacteriocins are as potent as nisin in vitro, and resistance can be circumvented. Based on these results, ruminal bacteriocins may provide an alternative to antibiotics in cattle rations.

Animals↗

PubMed2001

The glucomannokinase of Prevotella bryantii B(1)4 and its potential role in regulating beta-glucanase expression.

Prevotella bryantii B(1)4 has a transport system for glucose and mannose, but beta-glucanase expression is only catabolite-repressed by glucose. P bryantii B(1)4 cell extracts had ATP-dependent gluco- and mannokinase activities, and significant phosphoenolpyruvate- or GTP-dependent hexose phosphorylation was not observed. Mannose inhibited glucose phosphorylation (and vice versa), and activity gels indicated that a single protein was responsible for both activities. Glucose was phosphorylated at a faster rate than was mannose [V(max) 280 nmol hexose (mg protein)(-1) min(-1) versus 60 nmol hexose (mg protein)(-1) min(-1), respectively] and glucose was a better substrate for the kinase (K(m) 0.12 mM versus 1.2 mM, respectively). The purified glucomannokinase (1250-fold) had a molecular mass of 68 kDa, but SDS-PAGE gels indicated that it was a dimer (monomer 34.5 kDa). The N-terminus (25 residues) had an 8 amino acid segment that was homologous to other bacterial glucokinases. The glucomannokinase was competitively inhibited by the nonmetabolizable glucose analogue 2-deoxyglucose (2DG), and cells grown with glucose and 2DG had lower rates of glucose consumption than did cells given only glucose. When the ratio of 2DG to glucose was increased, the glucose consumption rate decreased and the beta-glucanase activity increased. The glucose consumption rate and the glucomannokinase activity of cells treated with 2DG were highly correlated (r(2)=0.98). This result suggested that glucomannokinase activity was either directly or indirectly regulating beta-glucanase expression.

Adenosine Triphosphate↗

PubMed2000

Protonmotive force regulates the membrane conductance of Streptococcus bovis in a non-ohmic fashion.

Because the DCCD (dicyclohexylcarbodiimide)-sensitive, F-ATPase-mediated, futile ATP hydrolysis of non-growing Streptococcus bovis JB1 cells was not affected by sodium or potassium, ATP hydrolysis appeared to be dependent only upon the rate of proton flux across the cell membrane. However, available estimates of bacterial proton conductance were too low to account for the rate of ATP turnover observed in S. bovis. When de-energized cells were subjected to large pH gradients (2.75 units, or -170 mV), internal pH declined at a rate of 0.15 pH units s(-1). Based on an estimated cellular buffering capacity of 200 nmol H+ (mg protein)(-1) per pH unit, H+ flux across the cell membrane (at -170 mV) was 108 mmol (g protein)(-1) h(-1). When potassium-loaded cells were treated with valinomycin in low-potassium buffers, initial K+ efflux generated membrane potentials in close agreement with values predicted by the Nernst equation. These artificial membrane potentials drove H+ uptake, and H+ influx was counterbalanced by a further loss of cellular K+. Flame photometry indicated that the rate of K+ loss was 215 (+/-26) mmol K+ (g protein)(-1) h(-1) at -170 mV, but the potassium-sensitive fluorescent compound CD222 indicated that this rate was only 110 (+/-44) mmol K+ (g protein)(-1) h(-1). As pH gradients or membrane potentials were reduced, the rate of H+ flux declined in a non-ohmic fashion, and all rates were <25 mmol (g protein)(-1) h(-1) at a driving force of -80 mV. Previous estimates of bacterial proton flux were based on low and unphysiological protonmotive forces, and the assumption that H+ influx rate would be ohmic. Rates of H+ influx into S. bovis cells [as high as 9x10(-11) mol H+ (cm membrane)(-2) s(-1)] were similar to rates reported for respiring mitochondria, but were at least 20-fold greater than any rate previously reported in lactic acid bacteria.

Adenosine Triphosphatases↗

PubMed1997

The ability of Escherichia coli O157:H7 to decrease its intracellular pH and resist the toxicity of acetic acid.

Batch cultures of Escherichia coli K-12 grew well in an anaerobic glucose medium at pH 5.9, but even small amounts of acetate (20 mM) inhibited growth and fermentation. E. coli O157:H7 was at least fourfold more resistant to acetate than K-12. Continuous cultures of E. coli K-12 (pH 5.9, dilution rate 0.085 h-1) did not wash out until the sodium acetate concentration in the input medium was 80 mM, whereas E. coli O157:H7 persisted until the sodium acetate concentration was 160 mM. E. coli K-12 cell accumulated as much as 500 mM acetate, but the intracellular acetate concentration of O157:H7 was never greater than 300 mM. Differences in acetate accumulation could be explained by intracellular pH and the transmembrane pH gradient (delta pH). E. coli K-12 maintained a more or less constant delta pH (intracellular pH 6.8), but E. coli O157:H7 let its delta pH decrease from 0.9 to 0.2 units as sodium acetate was added to the medium. Sodium acetate increased the rate of glucose consumption, but there was little evidence to support the idea that acetate was creating a futile cycle of protons. Increases in glucose consumption rate could be explained by increases in D-lactate production and decreases in ATP production. Intracellular acetate was initially lower than the amount predicted by delta pH, but intracellular acetate and delta pH were in equilibrium when the external acetate concentrations were high. Based on these results, the acetate tolerance of O157:H7 can be explained by fundamental differences in metabolism and intracellular pH regulation. By decreasing the intracellular pH and producing large amounts of D-lactate, O157:H7 is able to decrease delta pH and prevent toxic accumulations of intracellular acetate anion.

Acetic Acid↗