Purification and properties of nitrate reductase from Mitsuokella multiacidus

Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column. The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands. The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate. Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa. The M. multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor. The maximal activity was found at pH 6.2-7.5 for nitrate reduction. Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective. Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase. The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively. The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).     

Structures of Selenomonas ruminantium phytase in complex with persulfated phytate: DSP phytase fold and mechanism for sequential substrate hydrolysis

Various inositide phosphatases participate in the regulation of inositol polyphosphate signaling molecules. Plant phytases are phosphatases that hydrolyze phytate to less-phosphorylated myo-inositol derivatives and phosphate. The phytase from Selenomonas ruminantium shares no sequence homology with other microbial phytases. Its crystal structure revealed a phytase fold of the dual-specificity phosphatase type. The active site is located near a conserved cysteine-containing (Cys241) P loop. We also solved two other crystal forms in which an inhibitor, myo-inositol hexasulfate, is cocrystallized with the enzyme. In the "standby" and the "inhibited" crystal forms, the inhibitor is bound, respectively, in a pocket slightly away from Cys241 and at the substrate binding site where the phosphate group to be hydrolyzed is held close to the -SH group of Cys241. Our structural and mutagenesis studies allow us to visualize the way in which the P loop-containing phytase attracts and hydrolyzes the substrate (phytate) sequentially.     

Penicillin-Binding Proteins in Selenomonas ruminantium

Diphenyl, o-phenylphenol and thiabendazole were analyzed in citrus fruits. The peel and edible parts were separately homogenized. These fungicides were extracted with dichloromethane from the homogenate, and they were fractionated with Sephadex LH-20 columns. Gas chromatography was used to determine the presence of these fungicides. The fungicides found in edible parts of citrus fruits were confirmed by gas chromatography-mass spectrometry.

Diphenyl, o-phenylphenol and thiabendazole were detected in imported grapefruits, lemons and oranges. Almost all fungicides were found in the peel. The concentrations of the three fungicides in the edible parts were very low. Some samples contained all three fungicides in the edible parts.     

Xylose uptake by the ruminal bacterium Selenomonas ruminantium

Selenomonas ruminantium HD4 does not use the phosphoenolpyruvate phosphotransferase system to transport xylose (S. A. Martin and J. B. Russell, J. Gen. Microbiol. 134:819-827, 1988). Xylose uptake by whole cells of S. ruminantium HD4 was inducible. Uptake was unaffected by monensin or lasalocid, while oxygen, o-phenanthroline, and HgCl2 were potent inhibitors. Menadione, antimycin A, and KCN had little effect on uptake, and acriflavine inhibited uptake by 23%. Sodium fluoride decreased xylose uptake by 10%, while N,N'-dicyclohexylcarbodiimide decreased uptake by 31%. Sodium arsenate was a strong inhibitor (83%), and these results suggest the involvement of a high-energy phosphate compound and possibly a binding protein in xylose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and SF6847 inhibited xylose uptake by 88, 82, and 43%, respectively. The cations Na+ and K+ did not stimulate xylose uptake. The kinetics of xylose uptake were nonlinear, and it appeared that more than one uptake mechanism may be involved or that two proteins (i.e., a binding protein and permease protein) with different affinities for xylose were present. Excess (10 mM) glucose, sucrose, or maltose decreased xylose uptake less than 40%. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, while pH values of 5.0 and 4.0 decreased uptake 28 and 24%, respectively. The phenolic monomers p-coumaric acid and vanillin inhibited growth on xylose and xylose uptake more than ferulic acid did. The predominant end products resulting from the fermentation of xylose were lactate (7.5 mM), acetate (4.4 mM), and propionate (5.1 nM), and the Yxylose was 24.1 g/mol.     

The mechanism of aggregate formation by Selenomonas ruminantium

Summary The mechanism of granule formation by Selenomonas ruminantium was investigated. A basic protein has been isolated from the lysate of S. ruminantium which triggers cluster formation (small aggregates of 20–100 cells) of suspended cells. Evidence is presented that these basic proteins were of ribosomal origin. It is suggested that ribosomes are released into the culture broth by lysis and that the associated basic proteins are subsequently dissociated by high monovalent cation concentrations. It was found that these positively charged basic proteins interact with the negatively charged lipopolysaccharide of the organism to form the clusters. Adding lysate to suspended cells, followed by lowering of the pH from 5.8 to 4.5 also induced clustering. At dilution rates exceeding the maximum growth rate clusters were retained in anaerobic gas-lift reactors and grew into granules (1–3 mm). It is postulated that granules evolve from clusters. Within the clusters, lysis and a low pH are induced due to diffusion limitations. As a consequence dividing cells are entrapped within the clusters, resulting in growth.     

Glucose-induced morphological variation in Selenomonas ruminantium

Abstract Selenomonas ruminantium (strain I10) isolated from the ovine rumen showed considerable morphological variation and lack of motility when cultured in a phosphate-limited chemostat in the presence of high levels of glucose (55.5 mM). Transmission electron microscopy showed that some of these variants were capable of producing daughter cells with a typical selenomonad morphology but lacking flagella.
The reduction of the levels of glucose (27.8 mM) in the media caused the numbers of cells exhibiting variation to decrease, with a corresponding increase in motile cells possessing a typical selenomonad morphology. The removal of trypticase from the media had no effect on the morphology or motility of the cells.
During the initial stages of changeover to reduced glucose levels variants could be found in the chemostat which were flagellate. The flagellae were consistently attached to a concave section of the cells.     

The pectinolytic enzyme of Selenomonas ruminantium

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40 degrees C. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nonagalacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-alpha-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.     

The pectinolytic enzyme of Selenomonas ruminantium

A cell-bound pectinolytic enzyme was isolated from cells of Selenomonas ruminantium and purified about 360-fold. The optimum pH and temperature for enzyme activity was 7.0 and 40°. The enzyme degraded polymeric substrates by hydrolysis of digalacturonic acid units from the non-reducing end; the best substrate was nona-galacturonic acid. Unsaturated trigalacturonate was also degraded, but 30% slower than the saturated analogue. The enzyme was classified as a poly (1,4-aP-D-galactosiduronate) digalacturono-hydrolase; EC 3.2.1.82. Another enzyme, hydrolysing digalacturonic acid to monomers, was also produced in a very small amount by this organism.     

Cytoplasmic reserve polysaccharide of Selenomonas ruminantium.

Selenomonas ruminantium accumulated large quantities of intracellular polysaccharide when grown in simple defined medium in a chemostat, particularly at low dilution rate under NH3 limitation when the carbohydrate content of the cells was greater than 40% of the dry weight. This polysaccharide was used as a source of energy under conditions of energy starvation. Abundant, densely staining cytoplasmic granules were observed by electron microscopy in sections stained by the periodic acid-thiocarbohydrazide-osmium technique. The polysaccharide was extracted in 30% KOH followed by precipitation with 60% ethanol and was found to be a glucose homopolymer. Sepharose 4B gel filtration and iodine-complex spectroscopy showed that the polysaccharide was of the glycogen type with a molecular weight of 5 X 10(5) to greater than 20 X 10(5) and an average chain length of 12 glucose residues.     

Factors affecting lactate and malate utilization by Selenomonas ruminantium.

Lactate utilization by Selenomonas ruminantium is stimulated in the presence of malate. Because little information is available describing lactate-plus-malate utilization by this organism, the objective of this study was to evaluate factors affecting utilization of these two organic acids by two strains of S. ruminantium. When S. ruminantium HD4 and H18 were grown in batch culture on DL-lactate and DL-malate, both strains coutilized both organic acids for the initial 20 to 24 h of incubation and acetate, propionate, and succinate accumulated. However, when malate and succinate concentrations reached 7 mM, malate utilization ceased, and with strain H18, there was a complete cessation of DL-lactate utilization. Malate utilization by both strains was also inhibited in the presence of glucose. S. ruminantium HD4 was unable to grow on 6 mM DL-lactate at extracellular pH 5.5 in continuous culture (dilution rate, 0.05 h-1) and washed out of the culture vessel. Addition of 8 mM DL-malate to the medium prevented washout on 6 mM DL-lactate at pH 5.5 and resulted in succinate accumulation. Addition of malate also increased bacterial protein, acetate, and propionate concentrations in continuous culture. These results suggest that 8 mM DL-malate enhances the ability of strain HD4 to grow on 6 mM DL-lactate at extracellular pH 5.5.     

Localization of lanthanum tracer in oral epithelium using transmission electron microscopy and the electron microprobe

Although water soluble tracers have been used to localize intercellular permeability barriers with the transmission electron microscope, there is the possibility of translocation or loss of the tracer during processing. This study compares the localization of lanthanum tracer in keratinized oral epithelium after routine processing with lanthanum seen after using freeze drying to avoid aqueous fixation and dehydration. The electron probe was used to identify the lanthanum tracer in the tissue and to distinguish it from other electron-dense material. After incubating small biopsies of rat palate mucosa in 1% lanthanum nitrate, specimens were either routinely processed for electron microscopy or quick frozen, dehydrated, fixed in osmium vapour and infiltrated with epoxy resin. Examination in the transmission electron microscope indicated that preservation of the freeze dried tissue did not compare favourably with that of normally processed tissue, but the distribution of the electron-dense tracer in the intercellular spaces and the extent of the penetration through the epithelia was similar in the two types of preparations. Confirmation of the tracer as lanthanum was obtained by wavelength dispersive X-ray analysis with the electron probe. The results indicate that no appreciable shift in the localization of the tracer is introduced by routine aqueous fixation and dehydration for electron microscopic examination.     

Xylose uptake by the ruminal bacterium Selenomonas ruminantium.

Selenomonas ruminantium HD4 does not use the phosphoenolpyruvate phosphotransferase system to transport xylose (S. A. Martin and J. B. Russell, J. Gen. Microbiol. 134:819-827, 1988). Xylose uptake by whole cells of S. ruminantium HD4 was inducible. Uptake was unaffected by monensin or lasalocid, while oxygen, o-phenanthroline, and HgCl2 were potent inhibitors. Menadione, antimycin A, and KCN had little effect on uptake, and acriflavine inhibited uptake by 23%. Sodium fluoride decreased xylose uptake by 10%, while N,N'-dicyclohexylcarbodiimide decreased uptake by 31%. Sodium arsenate was a strong inhibitor (83%), and these results suggest the involvement of a high-energy phosphate compound and possibly a binding protein in xylose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and SF6847 inhibited xylose uptake by 88, 82, and 43%, respectively. The cations Na+ and K+ did not stimulate xylose uptake. The kinetics of xylose uptake were nonlinear, and it appeared that more than one uptake mechanism may be involved or that two proteins (i.e., a binding protein and permease protein) with different affinities for xylose were present. Excess (10 mM) glucose, sucrose, or maltose decreased xylose uptake less than 40%. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, while pH values of 5.0 and 4.0 decreased uptake 28 and 24%, respectively. The phenolic monomers p-coumaric acid and vanillin inhibited growth on xylose and xylose uptake more than ferulic acid did. The predominant end products resulting from the fermentation of xylose were lactate (7.5 mM), acetate (4.4 mM), and propionate (5.1 nM), and the Yxylose was 24.1 g/mol.     

Xylooligosaccharide Utilization by the Ruminal Anaerobic Bacterium Selenomonas ruminantium

Fermentation of xylooligosaccharides by 11 strains of Selenomonas ruminantium was examined. Xylooligosaccharides were prepared by the partial hydrolysis of oat spelt xylan in dilute phosphoric acid (50 mM, 121°C, 15 min) and were added to a complex, yeast extract-Trypticase-containing medium. Strains of S. ruminantium varied considerably in their capacity to ferment xylooligosaccharides. Strains GA192, GA31, H18, and D used arabinose, xylose, and the oligosaccharides xylobiose through xylopentaose, as well as considerable quantities of larger, unidentified oligosaccharides. Other strains of S. ruminantium (HD4, HD1, 20-21a, H6a, W-21, S23, 5-1) were able to use only the simple sugars present in the substrate mixture. The ability of S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. Both enzyme activities were induced by growth on xylooligosaccharides, but no activity was detected in glucose- or arabinose-grown cultures. Xylooligosaccharide-fermenting strains of S. ruminantium exhibited considerable variation in substrate utilization patterns, and the assimilation of individual carbohydrate species also appeared to be regulated. Lactic, acetic, and propionic acids were the major fermentation end products detected. Received: 2 August 1997 / Accepted: 18 September 1997     

Characterization of lateral flagella of Selenomonas ruminantium

Selenomonas ruminantium produces a tuft of flagella near the midpoint of the cell body and swims by rotating the cell body along the cell's long axis. The flagellum is composed of a single kind of flagellin, which is heavily glycosylated. The hook length of S. ruminantium is almost double that of Salmonella.     

Large plasmids in ruminal strains of Selenomonas ruminantium

The plasmid content of six different isolates of Selenomonas ruminantium from the rumen of sheep, cows or goats was examined by electron microscopy. In addition to small plasmids (< 12 kb) studied previously, all six strains contained at least one plasmid larger than 20 kb. Plasmid sizes of 1·4, 2·1, 2·4, 5·0, 6·2, 20·4, 20·8, 22·7, 23·3, 29·3, 30·7, 34·4 and 42·6 kb were estimated from contour length measurements. DNA-DNA hybridization experiments revealed homology among the large plasmids from five strains, while the 20·8 kb plasmid from a sixth isolate showed no apparent relationship with the plasmids of the other strains.     

Purification and properties of Selenomonas ruminantium lysine decarboxylase

Selenomonas ruminantium, a strictly anaerobic, gram-negative bacterium isolated from sheep rumen, contains lysine decarboxylase (Y. Kamio et al., J. Bacteriol. 145:122-128, 1981). This report describes the synthesis, purification, and characterization of the enzyme. Lysine decarboxylase was synthesized in cells grown in chemically defined medium without lysine. The enzyme was purified approximately 1,800-fold to electrophoretic homogeneity. The native enzyme of approximate molecular weight 88,000 consisted of two identical subunits, each with a molecular weight of 44,000. Several properties of the enzyme were determined and compared with those of the lysine decarboxylases from Escherichia coli and Bacterium cadaverisis.     

Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactate by Selenomonas ruminantium

A method is described for increasing the production of H2 from glucose or lactate by Selenomonas ruminantium by sequential transfers in media containing pregrown Methanobacterium ruminantium. The methanogen uses the H2 formed by the selenomonad to reduce CO2 to CH4. Analysis of fermentation products from glucose showed that lactate was the major product formed from glucose by S. ruminantium alone. Several sequential transfers in the presence of the methanogen caused a marked decrease in lactate production, which was accompanied by an increase in acetate. When lactate was the fermentation substrate, S. ruminantium alone produced propionate, acetate, and CO2. Addition to the pregrown methanogen in the sequential transfer procedure caused a significant decrease in the production of propionate and an increase in acetate formed from lactate. These results are interpreted in terms of the influence of H2 utilization by the methanogen on the production of H2 versus lactate or propionate from reduced pyridine nucleotides by S. ruminantium.     

Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactate by Selenomonas ruminantium.

A method is described for increasing the production of H2 from glucose or lactate by Selenomonas ruminantium by sequential transfers in media containing pregrown Methanobacterium ruminantium. The methanogen uses the H2 formed by the selenomonad to reduce CO2 to CH4. Analysis of fermentation products from glucose showed that lactate was the major product formed from glucose by S. ruminantium alone. Several sequential transfers in the presence of the methanogen caused a marked decrease in lactate production, which was accompanied by an increase in acetate. When lactate was the fermentation substrate, S. ruminantium alone produced propionate, acetate, and CO2. Addition to the pregrown methanogen in the sequential transfer procedure caused a significant decrease in the production of propionate and an increase in acetate formed from lactate. These results are interpreted in terms of the influence of H2 utilization by the methanogen on the production of H2 versus lactate or propionate from reduced pyridine nucleotides by S. ruminantium.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.     

Utilization of nucleic acids by Selenomonas ruminantium and other ruminal bacteria.

Species of ruminal bacteria were screened for the ability to grow in media containing RNA or DNA as the energy source. Bacteroides ruminicola D31d and Selenomonas ruminantium HD4, GA192, and D effectively used RNA for growth, but not DNA. B. ruminicola D31d was able grow on nucleosides but not on bases or ribose. The S. ruminantium strains were able to grow when provided with either nucleosides or ribose but not bases. Strains of S. ruminantium, but not B. ruminicola D31d, were also able to use nucleosides as nitrogen sources. These data suggest that RNA fermentation may be a general characteristic of S. ruminantium.