Ferredoxin from Selenomonas ruminantium

Ferredoxin from the strict rumen anaerobe Selenomonas ruminantium has been purified to homogeneity and characterized with respect to its molecular weight and amino acid composition. The molecular weight of ferredoxin was 9,880. The A₃₈₀/A₂₈₀ absorbance ratio of the pure ferredoxin was 0.54 with a molar extinction coefficient of 31,000 M^-1 cm^-1 at 380 nm. Ferredoxin was reduced by cell-free extracts in the presence of hydrogen gas or pyruvate and acetyl coenzyme A.Abbreviations [Fe_x/S_y] denotes an iron sulfur cluster containing x iron and y sulfur atoms     

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.     

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.     

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.     

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'.     

SruI restriction endonuclease from Selenomonas ruminantium

Abstract Sru I, specific restriction endonuclease, has been characterized from Selenomonas ruminantium isolated from the rumen of fallow deer. Results from the study demonstrate that S. ruminantium 18D possesses a type II restriction endonuclease, which recognizes the sequence 5'-TTT↓AAA-3'. The recognition sequence of Sru I was identified using digestions on pBR322, pBR328, pUC18, M13mp18RF, pACYC184 and λDNA. The cleavage patterns obtained were compared with computer-derived data. Sru I recognises the palindromic hexanucleotide sequence and cleaves DNA after the third T in the sequence, producing blunt ends. The purification and characterization of restriction endonuclease Sru I presented here is the first described for Selenomonas ruminantium spp. and demonstrates that this microorganism pocesses a DNA-cleaving enzyme with the same specificity as Dra I or Aha III.     

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.     

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.     

Monoclonal antibodies against the ruminal bacterium Selenomonas ruminantium.

Monoclonal antibodies were raised against whole cells of two different strains of Selenomonas ruminantium and tested for specificity and sensitivity in immunofluorescence and enzyme-linked immunosorbent assay procedures. Species-specific and strain-specific antibodies were identified, and reactive antigens were demonstrated in solubilized cell wall extracts of S. ruminantium. A monoclonal antibody-based solid-phase immunoassay was established to quantify S. ruminantium in cultures or samples from the rumen, and this had a sensitivity of 0.01 to 0.02% from 10(7) cells. For at least one strain, the extent of antibody reaction varied depending upon the stage of bacterial growth. Antigen characterization by immunoblotting shows that monoclonal antibodies raised against two different strains of S. ruminantium reacted with the same antigen on each strain. For one strain, an additional antigen reacted with both monoclonal antibodies. In the appropriate assay, these monoclonal antibodies may have advantages over gene probes, both in speed and sensitivity, for bacterial quantification studies.     

Cadaverine is covalently linked to peptidoglycan in Selenomonas ruminantium.

Cadaverine was found to exist as a component of cell wall peptidoglycan of Selenomonas ruminantium, a strictly anaerobic bacterium. [14C]cadaverine added to the growth medium was incorporated into the cells, and about 70% of the total radioactivity incorporated was found in the peptidoglycan fraction. When the [14C]cadaverine-labeled peptidoglycan preparation was acid hydrolyzed, all of the 14C counts were recovered as cadaverine. The [14C]cadaverine-labeled peptidoglycan preparation was digested with lysozyme into three small fragments which were radioactive and were positive in ninhydrin reaction. One major spot, a compound of the fragments, was composed of alanine, glutamic acid, diaminopimelic acid, cadaverine, muramic acid, and glucosamine. One of the two amino groups of cadaverine was covalently linked to the peptidoglycan, and the other was free. The chemical composition of the peptidoglycan preparation of this strain was determined to be as follows: L-alanine-D-alanine-D-glutamic acid-meso-diaminopimelic acid-cadaverine-muramic acid-glucosamine (1.0:1.0:1.0:1.0:1.1:0.9:1.0).     

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.     

Regulation of urease and ammonia assimilatory enzymes in Selenomonas ruminantium.

Urease and glutamine synthetase activities in Selenomonas ruminantium strain D were highest in cells grown in ammonia-limited, linear-growth cultures or when certain compounds other than ammonia served as the nitrogen source and limited the growth rate in batch cultures. Glutamate dehydrogenase activity was highest during glucose (energy)-limited growth or when ammonia was not growth limiting. A positive correlation (R = 0.96) between glutamine synthetase and urease activities was observed for a variety of growth conditions, and both enzyme activities were simultaneously repressed when excess ammonia was added to ammonia-limited, linear-growth cultures. The glutamate analog methionine sulfoximine (MSX), inhibited glutamine synthetase activity in vitro, but glutamate dehydrogenase, glutamate synthase, and urease activities were not affected. The addition of MSX (0.1 to 100 mM) to cultures growing with 20 mM ammonia resulted in growth rate inhibition that was dependent upon the concentration of MSX and was overcome by glutamine addition. Urease activity in MSX-inhibited cultures was increased significantly, suggesting that ammonia was not the direct repressor of urease activity. In ammonia-limited, linear-growth cultures, MSX addition resulted in growth inhibition, a decrease in GS activity, and an increase in urease activity. These results are discussed with respect to the importance of glutamine synthetase and glutamate dehydrogenase for ammonia assimilation under different growth conditions and the relationship of these enzymes to urease.     

Outer membrane proteins and cell surface structure of Selenomonas ruminantium.

The protein compositions of the membrane preparations from Selenomonas ruminantium grown in glucose or lactate medium were determined by sodium dodecyl sulfate- and two-dimensional (first, isoelectric focusing; second, sodium dodecyl sulfate) polyacrylamide slab gel electrophoresis. The outer membrane from both glucose- and lactate-grown cells contained two major proteins with apparent molecular weights of 42,000 and 40,000. These proteins existed as peptidoglycan-associated proteins in the outer membrane. The critical temperature at which they were dissociated completely into the monomeric subunits of 42,000 and 40,000 daltons was found to be 85 degrees C. The amount of each protein varied considerably depending upon the cultural conditions. The absence of the lipoprotein of Braun in S. ruminantium was suggested in our preceding paper (Y. Kamio, and H. Takahashi, J. Bacteriol. 141:888--898, 1980), and the possible absence of the protein components corresponding to the Braun lipoprotein in this strain was confirmed by electrophoretic analysis of the outer membrane and the lysozyme-treated peptidoglycan fractions. Examination of the cell surface of S. ruminantium by electron microscopy showed that the outer membrane formed a wrinkled surface with irregular blebs, some of which pinched off forming vesicles of various sizes. Rapid cell lysis occurred with the addition of a low level of lysozyme to the cell suspension. These findings led us to conclude that the physiological and morphological properties of this strain were similar to those of "deep rough" and mlp or lpo mutants of Escherichia coli K-12, respectively.     

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.     

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.     

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.