Presence of double-stranded RNA and virus-like particles in Rhizopus isolates

Fungal isolates belonging to four Rhizopus species were screened for the presence of double-stranded RNA (dsRNA) molecules. Five (two R. stolonifer, two R. microsporus, and one R. oryzae) of the 27 isolates examined harboured such genetic elements. Electrophoresis of the nucleic acids revealed five RNA patterns, with 1-5 discrete dsRNA bands. The molecular sizes corresponding to these bands were 2.2-14.8 kb. Gel electrophoresis of purified virus-like particles (VLPs) indicated only one capsid of similar size in all virus-harbouring strains; when investigated by electron microscopy, they were found to be polyhedral VLPs 40 nm in diameter. In one of the R. microsporus isolates an uncapsidated large dsRNA segment (14.8 kb) was observed. No phenotypic differences were observed between uninfected and virus-harbouring Rhizopus isolates.     

Conjugative transfer of tetracycline resistance in rumen streptococcal strains

In 11% of testedStreptococcus bovis strains a conjugative transfer of tetracycline resistance was observed when mating experiments were carried out on membrane filters. The recipient strain used wasS. bovis BM114 with chromosomal resistance to rifampicin. In addition, in two strains tetracycline resistance was transferred also to recipient strainEnterococcus faecium AL6. The transfer frequencies were in the range of 10⁻⁶ to 10⁻³. The donor strains were screened for the presence of plasmids and one up to four bands of plasmid DNA in all tested strains were revealed. In spite of that isolation of plasmid DNA was successful only in 53/4/114 transconjugants. Transconjugant 32/114 contained amylase activity which was higher than in the donor strain.     

Acrylamide gel analysis of sucrose gradient-derived RNA B fractions transferring delayed sensitivity in vitro

Sucrose-derived RNA fractions transferring specific delayed sensitivity in vitro were extracted from mono-(p-azobenzenearsonate)-N-chloracetyl-l-tyrosine (ARSNAT)- or keyhole limpet hemocyanin (KLH)-sensitized guinea pigs. Fractions having biological activity were assessed by acrylamide gel analysis to enumerate the number of RNA species in active fractions, and to compare and examine the banding patterns of each RNA fraction. Each isolated B fraction of RNA exhibited multiple bands of RNA with molecular weights ranging from 4.0 × 10⁵ to 8.5× 10⁵. Depending on the source and antigen sensitivity of the RNA donor, several differences were observed among the analyzed fractions. These were bands of varying intensity, presence of additional RNA bands, absence of bands in certain positions, and RNA bands migrating in different positions on the gels. Acrylamide gel analysis separation, and resolution of B fractions with specific immunobiological activity now offers an approach for further isolation and resolution of the active species.     

Extrachromosomal ribosomal RNA genes in Tetrahymena: structure and evolution

The macronuclear ribosomal RNA genes from a number of strains within several species of Tetrahymena have been characterized. Restriction enzyme analysis revealed that individual strains all contained entirely homogeneous populations of extrachromosomal palindromic ribosomal DNA, varying in molecular size from 12 × 10⁶ to 14 × 10⁶ in different strains. Considering that the evolutionary distance among some of the species is estimated to be of the order of 10⁶ years, the rDNA from all the species exhibited a strikingly high similarity in the localization of their restriction sites. Nevertheless, differences both inside and outside the gene region were clearly detectable, showing that the rDNA sequences have diverged in all species.Genetic polymorphism with respect to rDNA structure exists in Tetrahymena, but seems to be rare. In only two out of five species examined (T. borealis and T. pigmentosa) interbreeding strains differing in rDNA structure were found. While the differences detected in the T. borealis rDNA were confined to a small size difference located at the non-coding ends of the molecule, several differences were detected in the rDNA from the T. pigmentosa strains. One of the differences was shown to be due to the presence of an intervening sequence within the structural gene for 26 S rRNA in some of the strains. An intervening sequence of similar size located at the same position within the 26 S gene region was found by R-loop mapping in all strains of the species T. thermophila. Restriction enzyme analysis indicates that the rDNA from two other species contains a similar intervening sequence, and we therefore suggest that the size and localization of the intervening sequence is evolutionarily stable. The two intervening sequences examined so far, however, are not identical, as revealed by restriction enzyme mapping.     

Nickel toxicity inhibits ribonuclease and protease activities in rice seedlings: protective effects of proline

Seedlings of two rice cvs Malviya-36 and Pant-12, when grown under increasing levels of nickel (Ni²⁺: 200 and 400 μM) in the nutrient medium, showed increased levels of RNA, soluble proteins and free amino acids, especially proline over a total growth period of 5–20 days. Ribonuclease (RNase) and protease activities decreased in both roots and shoots due to Ni treatment in situ. Under in vitro conditions, a gradual inhibition of RNase activity was observed with increasing concentrations of Ni²⁺ (0–2500 μM) in the assay medium. Artificial desiccation simulated by 40% PEG or the presence of 2.5 mM Ni²⁺ in the reaction medium resulted in about 52–53% loss of RNase activity. Such a loss could be partially restored by 1 M proline in the assay medium. The activity staining of RNase revealed seven and four isoforms of RNase in roots and shoots, respectively. The intensity of most of the bands decreased with increasing levels of Ni²⁺ treatment in situ. The results suggest that Ni toxicity in rice seedlings suppresses the hydrolysis of RNA and proteins by inhibiting the activity of RNase and protease, respectively. Proline appears to act as a protectant of the enzyme RNase against metal- and PEG-induced damages.     

Genetic and fermentation properties of the K2 killer yeast, Saccharomyces cerevisiae T206

Saccharomyces cerevisiae T206 K⁺R⁺, a K₂ killer yeast, was differentiated from other NCYC killer strains of S. cerevisiae on the basis of CHEF-karyotyping and mycoviral RNA separations. Genomic DNA of strain T206 was resolved into 13 chromosome bands, ranging from approximately 0.2 to 2.2 Mb. The resident virus in strain T206 yielded L and M RNA species of approximately 5.1 kb and 2.0 kb, respectively. In micro-scale vinifications, strain T206 showed a lethal effect on a K^-R^- mesophilic wine yeast. Metabolite accumulation and toxin activity were measured over a narrow pH range of 3.2 to 3.5. Contrary to known fermentation trends, the challenged fermentations were neither stuck nor protracted although over 70% of the cell population was killed. Toxin-sensitive cells showed cytosolic efflux.     

Characterization of several LINE-1 elements in Microtus kirgisorum

Several LINE-1s have been isolated and characterized from genomic DNA of the vole, Microtus kirgisorum. Blot hybridization revealed specific restriction patterns of L1 elements in vole genomes. Rehybridization of the genomic blot with a cloned 5′-end fragment revealed two major bands indicating the presence of two different L1 subfamilies. The copy numbers are estimated for different parts of M. kirgisorum L1 elements. Data also demonstrate that most vole L1 elements are truncated at the 5′-end; however, in contrast to mouse, the ORF1 copy number is higher in vole. A difference between the substitution rates of the ORF1 5′-region (approximately 330 nucleotides) and the rest of the L1 coding regions is revealed. Received: 12 January, 1999 / Accepted: 18 March, 1999     

VanA-Type Enterococci from Humans, Animals, and Food: Species Distribution, Population Structure, Tn1546 Typing and Location, and Virulence Determinants

VanA-type human (n = 69), animal (n = 49), and food (n = 36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n = 4) and M49 (n = 13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P > 0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P < 0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.     

Larsenia salina gen. nov., sp. nov., a new member of the family Halomonadaceae based on multilocus sequence analysis

Two Gram-staining-negative, moderately halophilic bacteria, strains M1-18^T and L1-16, were isolated from a saltern located in Huelva (Spain). They were motile, strictly aerobic rods, growing in the presence of 3–25% (w/v) NaCl (optimal growth at 7.5–10% [w/v] NaCl), between pH 4.0 and 9.0 (optimal at pH 6.0–7.0) and at temperatures between 15 and 40 °C (optimal at 37 °C). Phylogenetic analysis based on 16S rRNA gene sequence comparison showed that both strains showed the higher similarity values with Chromohalobacter israelensis ATCC 43985^T (95.2–94.8%) and Chromohalobacter salexigens DSM 3043^T (95.0–94.9%), and similarity values lower than 94.6% with other species of the genera Chromohalobacter, Kushneria, Cobetia or Halomonas. Multilocus sequence analysis (MLSA) based on the partial sequences of atpA, rpoD and secA housekeeping genes indicated that the new isolates formed an independent and monophyletic branch that was related to the peripheral genera of the family Halomonadaceae, Halotalea, Carnimonas and Zymobacter, supporting their placement as a new genus of the Halomonadaceae. The DNA–DNA hybridization between both strains was 82%, whereas the values between strain M1-18^T and the most closely related species of Chromohalobacter and Kushneria were equal or lower to 48%. The major cellular fatty acids were C_18:1ω7c/C_18:1ω6c, C_16:0, and C_16:1ω7c/C_16:1ω6c, a profile that differentiate this new taxon from species of the related genera. We propose the placement of both strains as a novel genus and species, within the family Halomonadaceae, with the name Larsenia salina gen. nov., sp. nov. The type strain is M1-18^T (= CCM 8464 = CECT 8192^T = IBRC-M 10767^T = LMG 27461^T).     

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×10⁶ to 14×10⁶ dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l0⁶ dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.     

Activities of superoxide dismutase (SOD) isoforms during growth of <Emphasis Type="Italic">Scenedesmus</Emphasis> (chlorophyta) species and strains grown in batch-cultures

Growth of Scenedesmus species and strains, grown for 28 days in mineral BBM medium in batch-cultures, displayed sigmoidal kinetics that comprised a lag, exponential and declining growth phases. Total SOD activity in these autotrophically cultured organisms, which oscillated within 0.6 – 1.4 Umg protein⁻¹, was rather species-specific and only to some extent depended on the growth phase. Contrary, three S. obliquus strains: wild type 276-6, mutant with blocked PS I (strain 56.80) and mutant with blocked PS II (strain 57.80), cultured for 7 days on BBM medium supplemented with bacto-tryptone and yeast extract (BBM+) turned out to be time-dependent and to have several times higher total SOD activity than one obtained for Scenedesmus grown autotrophically. Regardless of the media composition, the phase of growth and studied organism, dominant isoforms of total SOD were together determined Fe- and Mn-SOD. Profiles of SOD isoforms, obtained after PAGE analysis of all autotrophically and exponentially growing organisms, revealed that one Mn-SOD and one Cu/Zn-SOD bands located on gels at the same position whereas location of three bands of Fe-SOD depended on the strain. This suggests the presence of two different groups of Fe-SODs in analyzed organisms. Identical SOD profiles found in two S. armatus strains (276-4a and 276-4d) and S. subspicatus correspond well with their taxonomic position. The SOD profile of S. armatus B1-76 distinctly differed from two other S. armatus strains but was identical to S. microspinal B1-76 and S. quadricauda G-15 despite the fact that there were significant growth rate differences between these three species. SODs profiles of S. acutus 437 and S. obliguus 453 were species-specific. In S. obliquus strains cultured on BBM+ medium, there are four SOD bands: one slightly visible band of Mn-SOD, two intensive bands of Fe-SOD and one band of Cu/Zn-SOD. The above finding suggests that antioxidant response of algae kept in batch-cultures differs according to medium composition and the SOD activity mainly restricted to chloroplasts.     

Physical evidence for plasmids in autotrophic,especially hydrogen-oxidizing bacteria

Agarose gel electrophoresis of crude lysates from 23 species of autotrophic bacteria revealed plasmids of various sizes in 12 species. The plasmid pattern varied considerably. While the majority of the plasmid-bearing species harbored one or two plasmids, one species, Alcaligenes latus, exhibited more than six ccc-DNA bands. With one exception the molecular masses of the plasmids were 50×10⁶ or higher. In Achromobacter carboxydus, Alcaligenes latus, Derxia gummosa and three strains of Paracoccus denitrificans large plasmids of molecular masses higher than 300×10⁶ were resolved. The examination of Thiobacillus A2 resulted in the discovery of two plasmids while Pseudomonas oxalaticus was apparently free of resident plasmid DNA. So far these plasmids can only be characterized as cryptic. Future studies may allow to correlate them with specific metabolic activities of their hosts such as the ability to grow on carbon monoxide or thiosulfate, to fix molecular nitrogen and to form soluble NAD-reducing and/or membrane-bound hydrogenases.     

Kinetics of uridine uptake and incorporation into RNA in tobacco pollen culture

The capacity of pollen tubes to utilize exogenous uridine during 8 h of cultivation in shaken suspension in a sugar-mineral medium was examined by continuous and pulse labelling with³H-uridine. The increase of uptake with increasing concentration of the nucleoside indicated a saturable transport system with an approximate K_m of 9.4 × 10⁻⁶ M and 12.5 × 10⁻⁶M as determined in 1-h and 6-h cultures, respectively. Maximal uptake took place at the beginning of germination reaching a rate of about 2 nmol h⁻¹ per 1 mg of dry pollen at 0.1 mM external uridine. The uptake activity decreased with the time of pollen tube growth to less than one third during the 8-h cultivation period. Moreover, the level of radioactivity taken up initially decreased later on during continuous cultivation in the presence of³H-uridine. The uptake took place against a concentration difference and the onset and rate of uridine release depended on its exogenous concentration. The activity of the nucleoside incorporation into RNA increased during the first 4 h of cultivation, decreasing later on. The proportion of RNA radioactivity in continuously labelled pollen tubes grew steadily during 6 h and reached 2.5% with respect to soluble pool at 0.4 μM uridine. The time course of RNA labelling was independent of uridine concentration within the range of 0.4 μM to 40 μM but this concentration rise resulted in an about fiftyfold increase of the total amount of external uridine incorporated.     

Studies in the Mineral and Salt-Catalyzed Formation of RNA Oligomers

Activated mononucleotides oligomerize in the presence of montmorillonite clay to form RNA oligomers. In the present study, effects of salts, temperature and pH on the clay-catalyzed synthesis of RNA oligomers were investigated. This reaction is favored by relatively high concentration of salts, such as 1 M NaCl. It was shown that the presence of divalent cations was not required for this reaction. High concentrations of NH₄ ⁺ and HCO₃ ⁻ and 0.01 M HPO₄ ²⁻ inhibit the reaction. The yields of RNA oligomers decreased as the temperature was raised from 4 ^∘C to 50 ^∘C. A⁵′ ppA was the major product at pH's below 6. The catalytic activity of a variety of minerals and three meteorites were investigated but none of them except galena catalyzed the oligomerization. ATP was generated from ADP but it was due to the presence of HEPES buffer and not due to the minerals. Meteorites catalyzed the hydrolysis of the pyrophosphate bonds of ATP. The results suggest that oligomers of RNA could have formed in pH 7–9 solutions of alkali metal salts in the presence of montmorillonite clay.     

Corynebacterium uberis sp. nov. frequently isolated from bovine mastitis

Several strains belonging to the genus Corynebacterium, but not to any described species of the genus were isolated from bovine mastitic milk samples over the past five years in the diagnostic unit of the University of Bern. Six of these strains (18M0132^T, 17M2518, 18M0913, 19M0083, 20M1046 and 20M1090) that were phenotypically similar were further characterized genotypically. Gram-positive coryneform rods were catalase positive, facultative anaerobe and CAMP-test negative. Whole genome sequencing and subsequent phylogenetic analysis revealed their genome size to be 2.53 Mb and their G + C content to be between 65.4 and 65.5 mol%. Digital DNA-DNA hybridisation (dDDH) showed the highest similarity of only less than 20% with Corynebacterium mastitidis and Corynebacterium frankenforstense, which indicated that the isolates belong to an undescribed Corynebacterium species. This was confirmed by studying the average nucleotide identity (ANI) where the accepted species boundary is around 95% and which ranged between 70.3% and 74.9% with the most closely related species C. mastitidis. We established MALDI-TOF fingerprints of the species, which allows a clear separation from related species and can be used by other laboratories for diagnostic purposes.Based on our analyses we conclude that the selected strains belong to a previously undescribed species and propose the name Corynebacterium uberis sp. nov. The proposed type strain is 18M0132^T (=DSM 111922^T, = CCOS 1972^T).     

Deinococcus aestuarii sp. nov. and Deinococcus aquaedulcis sp. nov., two novel resistant bacteria isolated from pearl river estuary

Two novel species of the genus Deinococcus, designated SYSU M49105^T and SYSU M42101^T, were isolated from freshwater samples of the Pearl River estuary in Guangdong, China. Phylogenetic analysis using 16S rRNA gene sequence indicated that strains SYSU M49105^T and SYSU M42101^T showed the highest sequence similarities to Deinococcus aetherius JCM 11751 ^T (93.6%) and Deinococcus multiflagellatus NBRC 112888 ^T (97.3%), respectively. Cells of both strains were Gram-staining positive, aerobic, coccus-shaped, oxidase-negative and non-motile. The cell wall contained meso-diaminopimelic acid as their diagnostic diamino acid. MK-8 was the predominant respiratory quinone for both strains. The polar lipid profile of SYSU M49105^T contained two unidentified phosphoglycolipids, nine unidentified glycolipids, and five unidentified polar lipids. SYSU M42101^T had one unidentified phosphoglycolipid, nine unidentified glycolipids, one unidentified aminophospholipid and four unidentified polar lipids. The major fatty acids of strains SYSU M49105^T and SYSU M42101^T were summed feature 3 (C_16:1 ω7c and/ or C_16:1 ω6c) and C_16:0. The G?+?C contents of the novel isolates based on genomic DNAs were 69.6% and 67.4%, respectively. On the basis of phenotypic, genotypic and phylogenetic data, strains SYSU M49105^T and SYSU M42101^T should be considered to represent two novel species in the genus Deinococcus, for which the names Deinococcus aestuarii sp. nov. and Deinococcus aquaedulcis sp. nov. were proposed with the type strains SYSU M49105^T (=?KCTC 43258 ^T?=?CGMCC 1.18609 ^T) and SYSU M42101^T (=?KCTC 43257 ^T?=?CGMCC 1.18614 ^T), respectively.

     

Transformation of Mucor circinelloides with Autoreplicative Vectors Containing Homologous and Heterologous ARS Elements and the Dominant Cbx r Carboxine-Resistance Gene

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu⁺ Cbx^r) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx^r(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 μ plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65–120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.     

Stress response in two strains of the aquatic hyphomycete <Emphasis Type="BoldItalic">Heliscus lugdunensis</Emphasis> after exposure to cadmium and copper ions

Biochemical responses to cadmium (Cd²⁺) and copper (Cu²⁺) exposure were compared in two strains of the aquatic hyphomycete (AQH) Heliscus lugdunensis. One strain (H4-2-4) had been isolated from a heavy metal polluted site, the other (H8-2-1) from a moderately polluted habitat. Conidia of the two strains differed in shape and size. Intracellular accumulation of Cd²⁺ and Cu²⁺ was lower in H4-2-4 than in H8-2-1. Both␣strains synthesized significantly more glutathione (GSH), cysteine (Cys) and γ-glutamylcysteine (γ-EC) in the presence of 25 and 50 μM Cd²⁺, but quantities and rates of synthesis were different. In H4-2-4, exposure to 50 μM Cd²⁺ increased GSH levels to 262% of the control; in H8-2-1 it increased to 156%. Mycelia of the two strains were analysed for peroxidase, dehydroascorbate reductase, glutathione reductase and glucose-6-phosphate dehydrogenase. With Cd²⁺ exposure, peroxidase activity increased in both strains. Cu²⁺ stress increased dehydroascorbate reductase activity in H4-2-4 but not in H8-2-1. Dehydroascorbate reductase and glucose-6-phosphate dehydrogenase activities progressively declined in the presence of Cd²⁺, indicating a correlation with Cd²⁺ accumulation in both strains. Cd²⁺ and Cu²⁺ exposure decreased glutathione reductase activity.