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.     

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

Aims: The objective of this study was to examine the effect of dilution rates (Ds, varying from 0·05 to 0·42 h^?1) in glucose‐limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. Methods and Results: All glucose‐limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0·10 or 0·20 h^?1. At Ds higher than 0·2 h^?1, fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0·30 and 0·42 h^?1). Glucose was most efficiently utilized by S. ruminantium D at 0·20 h^?1 after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. Conclusions: Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose‐limited continuous culture. Significance and Impact of the Study: Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro‐organism responds to differing growth conditions.     

Alternative strategies of 2-deoxyglucose resistance and low affinity glucose transport in the ruminal bacteria, Streptococcus bovis and Selenomonas ruminantium

Abstract Streptococcus bovis and Selenomonas ruminantium grew in the presence of the glucose analog, 2-deoxyglucose (2-DG), but the cells no longer had high affinity glucose transport. In S. bovis , 2-DG resistance was correlated with a decrease in phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase (PTS) activity. The 2-DG-selected S. bovis cells relied solely upon a low affinity, facilitated diffusion mechanism of glucose transport and a 2-DG-resistant glucokinase (ATP-dependent). The glucokinase activity of S. ruminantium was competitively inhibited by 2-DG, and the 2-DG selected cells continued to use PEP-dependent PTS as a mechanism of glucose transport. In this latter case, the 2-DG selected cells switched from a mannosephosphotransferase (enzyme II) that phosphorylated glucose, mannose, and 2-DG, but not α-methylglucoside to a glucosephosphotransferase (enzyme II) that phosphorylated glucose and α-methylglucoside but not 2-DG or mannose. The glucosephosphotransferase (enzyme II) had a very low affinity for glucose and the transport kinetics were similar to the facilitated diffusion system of S. bovis .     

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.     

Investigation of genetic and morphological variation in the sago palm (Metroxylon sagu; Arecaceae) in Papua New Guinea

BACKGROUND AND AIMS: The genetic and morphological variation in the sago palm (Metroxylon sagu, Arecaceae) in Papua New Guinea (PNG) was investigated. METHODS: Amplified fragment length polymorphism (AFLP) was used to investigate the genetic structure of 76 accessions of M. sagu, collected in seven wild and semi-wild stands in PNG. KEY RESULTS: An analysis of ten quantitative morphological variables revealed that most of these were mutually correlated. Principal component analyses of the same morphological variables showed that neither armature (presence or absence of spines) nor geographical separation was reflected clearly in the quantitative morphological variation. Similarity matrices of genetic, quantitative morphological, geographical and armature data were tested for pair-wise correlations, using Mantel's test. The results only showed a significant correlation between genetic and geographical distances. Visual inspection of principal component analyses plots and a neighbour-joining dendrogram based on genetic distances supported this trend, whereas armature showed no relation with genetic distances. CONCLUSIONS: Geographical distribution defines some weak patterns in the genetic variation, whereas the genetic variation does not reflect any patterns in the morphological variation, including armature. The present study supports the accepted taxonomy of M. sagu, recognizing only one species of M. sagu in PNG.     

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.     

Genetic diversity in Selenmonas ruminantium isolated from the rumen

Abstract Non-denaturing polyacrylamide gel electrophoresis was used to detect genetic variation at loci coding for there intracellular enzymes in the obligately anaerobic rumen bacterium Selenomonas ruminantium . Four mobility variants were detected for lactate dehydrigenase, seven for glucokinase and at least five for NADP-dependent glutamate dehydrogenase among 28 newly isolated, and eitght previously isolated strains from sheep and cattle. No evidence was found for an exclusive association of any particular electrophoretic mobility type with variable metabolic traits such as the ability to utilise lactate, to reduce nitrate or to ferment trehalose, sorbitol, rhamnose or glycerol. The most commonly occurring electromorph type was recovered from more than one animal, while most animals examined were show to harbour more than one electromorph type.     

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'.     

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.     

Evidence to show that an agent that cross-reacts serologically with Cowdria ruminantium in Zimbabwe is transmitted by ticks

The serological diagnosis of heartwater based on reactions to the immunodominant Cowdria ruminantium major antigen protein-1 (MAP-1) is impaired by the detection of false-positive reactions. In this study, the prevalence of false-positive reactions on seven heartwater-free farms in Zimbabwe was determined to be 8-94% by immunoblotting against C. ruminantium antigens. The highest prevalence of false-positives on Spring Valley Farm correlated with the presence of Rhipicephalus evertsi evertsi ticks. The other tick species found on these seven farms were Hyalomma truncatum and Hyalomma marginatum rufipes. Rhipicephalus evertsi evertsi ticks collected from Spring Valley Farm and fed on seronegative sheep caused seroconversion in one of two sheep. This sheep developed a mild febrile reaction and C. ruminantium MAP-1 antigen reactive antibodies 3 weeks after the ticks started feeding. Polymerase chain reactions (PCRs), conducted using C. ruminantium-specific primers on ticks collected from the seven farms and on some of the R. e. evertsi ticks that had caused seroconversion in one sheep, were negative. However, some of these ticks gave positive PCRs with DNA primers which amplify a 350 bp DNA fragment of the 16s rRNA gene from all ehrlichial agents indicating the presence of infection with one or more Ehrlichia species. Although attempts to isolate the cross-reacting agent from the sheep were unsuccessful, this study demonstrates that false-positive reactions with the MAP-1 C. ruminantium antigen are associated with agents transmitted by ticks.     

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).     

Light climate as a factor in the morphological variation of Atherosperma moschatum in a Tasmanian forest

Abstract The growth of saplings of Atherosperma moschatum within a Tasmanian forest during 1987/88 was negatively correlated with canopy closure. The dry weights and specific leaf weights of new leaves were also negatively correlated with canopy closure, but leaf areas showed a maximum at an intermediate canopy closure. Leaf chlorophyll concentration was positively correlated with canopy closure. Internode length was not correlated with canopy closure, but the ratio of leaf dry weight to internode length was negatively correlated with canopy closure. These results indicate that the load of photosynthetically active radiation may be a major determinant of the variation in plant development.     

Pattern of morphological variation and diversity of Cocos nucifera (Arecaceae) in Mexico

The pattern of morphological variation of Cocos nucifera in Mexico was statistically and numerically evaluated. Forty-one populations were analyzed, using 17 morphological fruit characters. Principal components and cluster analyses indicated four main groups of coconut populations that showed high similarity with four different genotypes recently imported into Mexico from areas that could be the origin of Mexican coconut populations. These four genotypes were evaluated with regard to the lethal yellowing disease in Jamaica and showed a differential susceptibility. Therefore it is possible to speculate upon a difference in susceptibility of the Mexican genotypes. The analysis of correlation between morphological and geographical distances showed a high positive correlation that supports: (1) historical evidence that indicates early introductions of coconut from different regions of the world, (2) that on both coasts of Mexico two different patterns of dispersal were involved: continuous and in jumps. Collectively these results suggest that the impact of the lethal yellowing disease on coconut populations will vary depending on the specific area and the origin of its coconuts.     

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.     

Distribution and morphological variation of Streptocephalus torvicornis (Waga, 1842) in Northern Africa

The known range of S. torvicornis is extended to areas of the Western and Central Sahara and Sahel. Morphological variation between populations is important, and cannot be accomodated within the known Iberian and Maghrebian subspecific taxa, which appear untenable. A grouping of populations according to drainage basins appears, however, possible. Populations of the Hoggar, Air, and Tibesti waters, draining towards the Soudanian zone, are more closely related than populations from waters draining towards the Atlantic. Tassili-n-ajjer populations from Central Algeria have individual characteristics, and deserve further study.     

Metabolism of the rumen bacterium Selenomonas ruminantium grown in continuous culture

Selenomonas ruminantium 0078A was grown in a glucose-limited chemostat over a dilution rate range of 0.049-0-137/h. Fermentation products were acetate, propionate, succinate, lactate and C0₂; traces of ethanol were also detected. Succinate accounted for up to 52% of the substrate glucose carbon. When dilution rate was increased without a concomitant increase in glucose supply per unit time there were changes in the fermentation pattern which were not apparent when both dilution rate and glucose supply were simultaneously increased; the molar proportion of acetate increased at the expense of propionate.     

Influence of temperature on morphological variation in populations of Keratella cochlearis (Gosse) in Rybnik Reservoir

Morphological changes in a population of Keratella cochlearis were investigated in a reservoir of changed temperature and high trophy. Four morphological forms were distinguished: Keratella cochlearis f. cochlearis, K. cochlearis var. tecta f. micracantha, K. cochlearis var. tecta f. micracantha, K. cochlearis var. tecta f. typica and K. cochlearis f. macracantha. A correlation between the length of the lorica and the posterior spine, and the temperature of water was observed. These four forms of Keratella cochlearis occurred during the entire period of investigations.