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.     

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     

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.     

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.     

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.     

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.     

Characterization of a plasmid from the ruminal bacterium Selenomonas ruminantium

A 4.8-kilobase-pair plasmid was isolated from the ruminal bacterium selenomonas ruminantium HD4 by using a sodium carbonate-EDTA washing buffer to improve cell lysis (R.G. Dean, S.A. Martin, and C. Carver, Lett. Appl. Microbiol. 8:45-48, 1989). This plasmid, designated pSR1, appears to be quite stable. No evidence of plasmid DNA was detected in S. ruminantium D or GA192. All three strains were tested for antibiotic resistance, and all were kanamycin resistant (MIC, 25 to 50 micrograms/ml). Only strain D was tetracycline resistant (MIC, 25 micrograms/ml), and all strains were sensitive to ampicillin (MIC, 1 microgram/ml). pSR1 was cloned into pBR322, and a map of pSR1 was constructed by using HindIII, ClAI, BamHI, and PvuII. Although ClaI, BamHI, ScaI, and EcoRV digested recombined plasmid isolated from Escherichia coli, these restriction endonucleases were not effective in digesting plasmid isolated directly from S. ruminantium HD4.     

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'.     

Purification of a nickel-containing urease from the rumen anaerobe Selenomonas ruminantium

Urease was purified 592-fold to homogeneity from the anaerobic rumen bacterium Selenomonas ruminantium. The urease isolation procedure included a heat step and ion-exchange, hydrophobic, gel filtration, and fast protein liquid chromatography. The purified enzyme exhibited a Km for urea of 2.2 +/- 0.5 mM and a Vmax of 1100 mumol of urea min-1 mg-1. The molecular mass estimated for the native enzyme was 360,000 +/- 50,000 daltons, whereas a subunit value of 70,000 +/- 2,000 daltons was determined. These results are in contrast to the findings of Mahadevan et al. (Mahadevan, S., Sauer, F. D., and Erfle, J. D. (1977) Biochem. J. 163, 495-501) in which isolated rumen urease was reported to be one-third this size (Mr 120,000-130,000) and to catalyze urea hydrolysis at a maximum velocity of only 53 mumol min-1 mg-1. S. ruminantium urease contained 2.1 +/- 0.4 nickel ions/subunit, comparable to the nickel content in jack bean urease (Dixon, N.E., Gazzola, C., Blakeley, R.L., and Zerner, B. (1975) J. Am. Chem. Soc. 97, 4131-4133). Thus, the active site of bacterial urease is very similar to that found in the plant enzymes.     

Characterization of a plasmid from the ruminal bacterium Selenomonas ruminantium.

A 4.8-kilobase-pair plasmid was isolated from the ruminal bacterium selenomonas ruminantium HD4 by using a sodium carbonate-EDTA washing buffer to improve cell lysis (R.G. Dean, S.A. Martin, and C. Carver, Lett. Appl. Microbiol. 8:45-48, 1989). This plasmid, designated pSR1, appears to be quite stable. No evidence of plasmid DNA was detected in S. ruminantium D or GA192. All three strains were tested for antibiotic resistance, and all were kanamycin resistant (MIC, 25 to 50 micrograms/ml). Only strain D was tetracycline resistant (MIC, 25 micrograms/ml), and all strains were sensitive to ampicillin (MIC, 1 microgram/ml). pSR1 was cloned into pBR322, and a map of pSR1 was constructed by using HindIII, ClAI, BamHI, and PvuII. Although ClaI, BamHI, ScaI, and EcoRV digested recombined plasmid isolated from Escherichia coli, these restriction endonucleases were not effective in digesting plasmid isolated directly from S. ruminantium HD4.     

Characterization of Lipid A,A Component of Lipopolysaccharides from Selenomonas ruminantium

Lipid components of a glycolipid, formerly designated as spot A, from the cells of Selenomonas ruminantium were investigated. The basic structure of this material had been previously shown to be β-glucosaminyl-l,6-glucosamine. The major component of O- and N-acyl side chains was β-OH C_13:0 acid when the cells were grown with added valerate. Approximately 85 % of the total amide linked fatty acids was this compound. A considerable amount of C_13:2 acid was also present as a component of O-acyl fatty acids. When the cells were grown in a glucose medium containing caproate, the major fatty acid component of the spot A compound was β-OH myristic and β-OH C_13:0: acids. ¹⁴C-Valerate or ¹⁴C-caproate, supplemented to the glucose medium, was incorporated into O- and N-acyl linked fatty acid moieties of the spot A compound. It was also shown that the spot A compound was the lipid A component of lipopolysaccharides of this organism.     

Sequence Analysis of Small Cryptic Plasmids Isolated from Selenomonas ruminantium S20

Two small cryptic plasmids designated pONE429 and pONE430 were isolated from a rumen bacterium, Selenomonas ruminantium S20. The complete sequence of pONE429 was 2100 bp and contained one open reading frame (ORF) of 201 amino acids. The sequence of pONE430 had 1527 bp and one ORF of 171 amino acids with the similarity of replication protein (Rep protein) of pOM1, pSN2, and pIM13 isolated from Butyrivibrio fibrisolvens, Staphylococcus aureus, and Bacillus subtilis, respectively. In these plasmids, the upstream nucleotide sequence of Rep protein had the conserved nucleotides which could be double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The plasmids of pONE429, pONE430, pJJMI, pJDB21, and pS23 were isolated from S. ruminantium strains and had similar regions that were located within a <450-bp nucleotide. These similar regions may be the location that was recognized by the host strain, S. ruminantium. Received: 10 July 1998 / Accepted: 11 September 1998     

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).     

Characterization of tannin acylhydrolase activity in the ruminal bacterium Selenomonas ruminantium

A strain of the anaerobe Selenomonas ruminantium subsp. ruminantium that is capable of growing on tannic acid or condensed tannin as a sole energy source has been isolated from ruminal contents of feral goats browsing tannin-rich Acacia sp. Growth on tannic acid was accompanied by release of gallic acid into the culture medium but the bacterium was incapable of using gallic acid as a sole energy source. Tannin acylhydrolase (EC 3.1.1.20) activity was measured in crude cell-free extracts of the bacterium. The enzyme has a pH optimum of 7, a temperature optimum of 30-40 degrees C and a molecular size of 59 kDa. In crude extracts, the maximal rate of gallic acid methyl ester hydrolysis was 6.3 micromol min(-1) mg(-1) of protein and the K(m) for gallic acid methyl ester was 1.6 mM. Enzyme activity was displayed in situ in polyacrylamide and isoelectric focusing gels and was demonstrated to increase 17-fold and 36-fold respectively when cells were grown in the presence of gallic acid methyl ester or tannic acid.     

Ammonia assimilation and glutamate formation in the anaerobe Selenomonas ruminantium.

Selenomonas ruminantium was found to possess two pathways for NH4+ assimilation that resulted in net glutamate synthesis. One pathway fixed NH4+ through the action of an NADPH-linked glutamate dehydrogenase (GDH). Maximal GDH activity required KCl (about 0.48 M), but a variety of monovalent salts could replace KCl. Complete substrate saturation of the enzyme by NH4+ did not occur, and apparent Km values of 6.7 and 23 mM were estimated. Also, an NADH-linked GDH activity was observed but was not stimulated by KCl. Cells grown in media containing non-growth-rate-limiting concentrations of NH4+ had the highest levels of GDH activity. The second pathway fixed NH4+ into the amide of glutamine by an ATP-dependent glutamine synthetase (GS). The GS did not display gamma-glutamyl transferase activity, and no evidence for an adenylylation/deadenylylation control mechanism was detected. GS activity was highest in cells grown under nitrogen limitation. Net glutamate synthesis from glutamine was effected by glutamate synthase activity (GOGAT). The GOGAT activity was reductant dependent, and maximal activity occurred with dithionite-reduced methyl viologen as the source of electrons, although NADPH or NADH could partially replace this artificial donor system. Flavin adenine dinucleotide, flavin mononucleotide, or ferredoxin could not replace methyl viologen. GOGAT activity was maximal in cells grown with NH4+ as sole nitrogen source and decreased in media containing Casamino Acids.     

DNA sequence homology in Rhizobium meliloti plasmids

Summary Plasmids were recovered by an alkaline procedure from six symbiotically effective strains of Rhizobium meliloti of diverse geographical origin, reported to harbour only one middle-size large plasmid (ranging from 89 to 143 Megadaltons). Each purified plasmid was digested with eight restriction endonucleases; cleavage patterns were very complex: only KpnI and XbaI gave a limited number of bands. Fingerprints were very different, whatever the restriction enzyme or the geographical origin of the strains. However, Southern DNA-DNA hybridizations revealed that the plasmids showed homologous sequences having a high thermal stability. We gave evidence that some of these sequences are common to all the plasmids of R. meliloti. The biological function of these common sequences is unknown. Hybridization with cloned nitrogen fixation (nif) genes from Klebsiella pneumoniae had demonstrated that nif genes were not located on the middle — size plasmids of R. meliloti studied in this paper.     

Aminoalcohols as probes of the two-subsite active site of beta-D-xylosidase from Selenomonas ruminantium

Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pK(a) 5.0) and catalytic acid (E186, pK(a) 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the catalytically-inactive, dianionic enzyme (D14(-)E186(-)). Enzyme (E) and aminoalcohols (A) form E-A with the affinity progression: triethanolamine>diethanolamine>ethanolamine. E186A mutation raises the K(i)(triethanolamine) 1000-fold. By occupying subsite -1 with aminoalcohols, affinity of monosaccharide inhibitors (I) for subsite +1 is demonstrated. The single access route for ligands into the active site dictates ordered formation of E-A followed by E-A-I. E-A-I forms with the affinity progression: ethanolamine>diethanolamine>triethanolamine. The latter affinity progression is seen in formation of E-A-substrate complexes with substrate 4-nitrophenyl-beta-d-xylopyranoside (4NPX). Inhibition patterns of aminoalcohols versus 4NPX appear competitive, noncompetitive, and uncompetitive depending on the strength of E-A-4NPX. E-A-substrate complexes form weakly with substrate 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA), and inhibition patterns appear competitive. Biphasic inhibition by triethanolamine reveals minor (<0.03%) contamination of E186A preparations (including a His-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols are useful in probing glycoside hydrolases; they cause artifacts when used unwarily as buffer components.     

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.     

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.