Isolation and characterization of Selenomonas ruminantium strains capable of 2-deoxyribose utilization.

Microbes from ruminal contents of cattle were selectively enriched by using 2-deoxyribose (2DR) as a substrate for growth. Bacterial isolates growing on 2DR were gram-negative, curved, motile rods. The isolates grew on a broad range of substrates, including deoxyribose, glucose, ribose, mannitol, and lactate as well as ribonucleosides and deoxyribonucleosides. The strains also grew on rhamnose (6-deoxymannose) but not DNA. Organic acids produced from growth on hexoses and pentoses included acetate, propionate, lactate, and succinate. The isolates were identified as Selenomonas ruminantium subsp. lactilytica on the basis of morphology, substrate specificity, and other biochemical characteristics. Several characterized species of ruminal bacteria were also screened for growth on 2DR, with only one strain (S. ruminantium PC-18) found able to grow on 2DR. Ethanol was produced by 2DR when strains were grown on ribose or 2DR.     

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium.

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.     

Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.     

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.     

Isolation of Selenomonas ruminantium from an aquatic ecosystem

A crescentic Gram-negative rod-shaped bacterium motile by a laterally inserted tuft of flagella was isolated from a boggy ditch water habitat. Cells occurred usually singly or in pairs, but sometimes short chains, long helical cells or spheroplasts with flagella still attached were observed. Its metabolism was obligate fermentative. The fermentation of glucose yielded mainly acetate and propionate. It grew with a generation time of 1 h 50 min. The DNA base ratio was found to be 51.6 mol % G+C. The characteristics of this organism indicated that it belongs to the genus Selenomonas closely similar to and by its main characteristics identical with the rumen bacterium Selenomonas ruminantium. The differing characteristics — production of catalase and lower temperature optimum (25°C) — interpretable as the result of adaptation to the specific environmental conditions may justify classification of the isolate into a new subspecies of S. ruminantium named Selenomonas ruminantium subsp. psychrocatalagenes. Additional information on the DNA base composition in strains of Selenomonas ruminantium (GA 192 and HD 1) was obtained.     

Phytase activity of Selenomonas ruminantium: a preliminary characterization

Obligately anaerobic ruminal bacteria have been found to possess phytase activity, in particular, Selenomonas ruminantium . The phytase activity of S. ruminantium JY35 was produced late in growth and required neither phytate for induction nor phosphate limitation for derepression. The activity was completely cell-associated with a significant fraction extractable by a magnesium chloride solution. Zymogram analysis suggested that the activity was the result of a single gene product of a monomeric nature and approximately 46 kDa in size. The phytase had a temperature optimum of 50–55 °C, but activity dropped off sharply at 60 °C. Phytase activity was optimal over the pH range of 4·0–5·5, and dependent on the nature of the buffer used. Activity was inhibited by citric acid buffer and by the addition of 5 mmol l⁻¹ Fe²⁺, Fe³⁺, Cu²⁺, Zn²⁺ and Hg²⁺. The addition of 5 mmol l^–1 Pb²⁺ to the enzyme assay appeared to enhance activity of the enzyme.     

Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes.

The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.     

Isolation and partial characterization of outer and inner membranes from encapsulated Haemophilus influenzae type b.

A method has been developed to separate the cell envelope of encapsulated (type b) Haemophilus influenzae into its outer and inner membrane components with procedures that avoided two problems encountered in fractionation of this envelope: (i) the tendency of the outer and inner membranes to hybridize and (ii) the tendency of the apparently fragile inner membrane to fragment into difficulty sedimentable units. Log phage cells, whose lipids were radioactively labeled, were lysed by passage through a French press. The lysate was applied to a discontinuous sucrose gradient, and envelope-rich material was collected by centrifugation onto a cushion of dense sucrose under carefully controlled conditions. This material was then further fractionated by isopycnic centrifugation in a sucrose gradient to yield four membrane fractions which were partially characterized. On the basis of their radioactivity, buoyant density, ultrastructure, polypeptide composition, and content of phospholipid, protein, lipopolysaccharide, and succinic dehydrogenase, these fractions were identified as follows: fraction 1, outer membrane vesicles with very little inner membrane contamination (less than 4%); fraction 2, outer membrane vesicles containing entrapped inner membrane; fraction 3, a protein-rich fraction of inner membrane; fraction 4, a protein-poor fraction of inner membrane. Fractions 3 and 4 contained about 25% outer membrane contamination.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

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.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

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.     

Two restriction endonucleases in Selenomonas ruminantium subsp. lactilytica

Crude protein extract from a recently isolated ruminal bacterium identified as Selenomonas ruminantium subsp. lactilytica specifically cleaved DNA. This ability was due to the presence of two site-specific restriction endonucleases. Srl I, a Nae I schizomer, recognizes the 5'-GCCGGC-3' sequence. Srl II, a Nsi I schizomer, recognizes 5'-ATGCAT-3'.     

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.     

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.