Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2

The aerobic degradation of 3- N -trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment–MS, and GC–MS showed that trimethylamine was the major metabolite, in addition to β-alanine betaine (β-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of β-AB C–N bonds yielded trimethylamine and alkyl chain.     

Isolation of (S)-2,3-dichloro-1-propanol assimilating bacterium,its characterization,and its use in preparation of (R)-2,3-dichloro-1-propanol and (S)-epichlorohydrin

Summary Linear alkylbenzene sulfonate (LAS) is a widely used anionic surfactant. Although approximately 1 million metric tons of LAS are produced annually, relatively little is known about the bacteria or the genetic factors that control LAS degradation in the environment. The objectives of this research were to: i) compare bacterial populations in wastewater and pristine pond systems; ii) determine the frequency of plasmids in bacteria from these sites; and iii) compare the frequency of DNA sequences coding for aromatic catabolism in isolates from these two sites. Plate counts indicated that exposure to wastewater resulted in higher levels of both heterotrophic bacteria and bacteria capable of growing on LAS containing medium (LAS/YEPG). In addition to higher numbers, a higher proportion of heterotrophs from the wastewater system were capable of growth on LAS/YEPG medium. Thus, the high levels of LAS in the wastewater system apparently selected fro organisms that were able to tolerate and/or degrade, it. Mineralization of¹⁴C-ring labelled LAS in any habitat related to the presence of organisms that grew on LAS/YEPG. Although may of these isolates could carry out primary degradation, no isolate, could mineralize¹⁴C-ring LAS in pure culture. A higher incidence of plasmids was found in bacteria from the wastewater pond and among bacteria that grew on LAS containing medium. However, the presence of plasmid, DNA did not necessarily confer the ability to degrade LAS nor was the ability to degrade LAS dependent on the presence of a plasmid. The incidence of selected genotypes for aromatic catabolism was similar among isolates on LAS/YEPG at both sites, suggesting that LAS ring degradation may be present in other populations or encoded by alternative sequences. In conclusion, LAS mineralization is mediated by a consortium and the evidence that initial attack of LAS is plasmid mediated is inconclusive.     

Isolation and characterization of functional insertion sequences of rhizobia

Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.     

Isolation and characterization of cDNA encoding rabbit reticulocyte 2,3-bisphosphoglycerate synthase

We have isolated and sequenced a cDNA clone containing the entire coding region of rabbit reticulocyte 2,3-bisphosphoglycerate (DPG) synthase. The cDNA was verified by translation of the hybridization-selected RNA and by demonstrating identity of the deduced amino acid (aa) sequence to the sequences of CNBr peptides of the purified enzyme. The aa sequence of the enzyme was homologous to the reported sequence of the human enzyme [Haggarty et al., EMBO J. 2 (1983) 1213-1220], especially in the N-terminal half (aa 1-142). Northern blot analysis of rabbit reticulocyte poly(A)+ RNA revealed a single species of mRNA with about 1700 nucleotides.     

Isolation and Characterization of 2,3-Dichloro-1-Propanol-Degrading Rhizobia

2,3-Dichloro-1-propanol is more chemically stable than its isomer, 1,3-dichloro-2-propanol, and is therefore more difficult to degrade. The isolation of bacteria capable of complete mineralization of 2,3-dichloro-1-propanol was successful only from enrichments at high pH. The bacteria thus isolated were found to be members of the α division of the Proteobacteria in the Rhizobium subdivision, most likely Agrobacterium sp. They could utilize both dihaloalcohol substrates and 2-chloropropionic acid. The growth of these strains in the presence of 2,3-dichloro-1-propanol was strongly affected by the pH and buffer strength of the medium. Under certain conditions, a ladder of four active dehalogenase bands could be visualized from this strain in activity gels. The enzyme involved in the complete mineralization of 2,3-dichloro-1-propanol was shown to have a native molecular weight of 114,000 and consisted of four subunits of similar molecular weights.     

Genetic characterization of soybean rhizobia in Paraguay

The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.     

Biosynthesis of cyclic 2,3-diphosphoglycerate. Isolation and characterization of 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus

Starting from 2-phosphoglycerate the biosynthesis of cDPG comprises two steps: (i) the phosphorylation of 2-phosphoglycerate to 2,3-diphosphoglycerate and (ii) the intramolecular cyclization to cyclic 2,3-diphosphoglycerate. The involved enzymes, 2-phosphoglycerate kinase and cyclic 2,3-diphosphoglycerate synthetase, were purified form Methanothermus fervidus. Their molecular and catalytic properties were characterized.     

Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation

Identification of elite nitrogen-fixing rhizobia strains is a continuous and never ending effort, since new legume species can be cultivated in different agro systems or are introduced into new areas. This current study reports on the taxonomic affiliation and symbiotic proficiency of nine strains of Trifolium-nodulating rhizobia isolated from different pasture areas in Spain, as well as three Rhizobium leguminosarum bv. trifolii reference strains, on eleven Trifolium species. Based on 16S rRNA gene sequences the strains belonged to the R. leguminosarum species complex. Additional phylogenetic analyses of the housekeeping genes recA, atpD and rpoB showed the strains were closely related to the species R. leguminosarum, R. laguerreae, R. indicum, R. ruizarguesonis or R. acidisoli. In addition, three strains had no clear affiliation and could represent putative new species, although two of the reference strains were positioned close to R. ruizarguesonis. nodC gene phylogeny allowed the discrimination between strains isolated from annual or perennial Trifolium species and placed all of them in the symbiovar trifolii. Neither geographic origin nor host-plant species could be correlated with the taxonomic affiliation of the strains and a high degree of phenotypic diversity was found among this set of strains. The strong interaction of plant species with the rhizobial strains found for biological nitrogen fixation (BNF) was noteworthy, and allowed the identification of rhizobial strains with a maximum proficiency for certain trefoil species. Several strains showed high BNF potential with a wide range of clover species, which made them valuable strains for inoculant manufacturers and they would be particularly useful for inoculation of seed mixtures in natural or cultivated pastures.     

Isolation and characterization of Arabidopsis thaliana ISU1 gene

We describe the isolation of a cDNA encoding Arabidopsis thaliana ISU1 (AtISU1), which regulates iron homeostasis in the mitochondria. The AtISU1 gene contained an open reading frame that encoded 167 amino acid residues. Northern blot analysis demonstrated that AtISU1 gene was ubiquitously expressed in plant tissues examined. The yeast seo5-1, which harbors a single base-pair deletion in ScISU1, is a suppressor of oxidative damage in sod1-deficient mutant. Based on comparative expression analyses using yeast ISU1 gene (ScISU1) in seo5-1 mutant, we found that AtISU1 acts as a counterpart of ScISU1.     

Isolation and characterization of cytoplasmic cyclin D1 mutants

To elucidate the mechanism governing the subcellular distribution of cyclin D1 protein, we randomly mutagenized human cyclin D1 cDNA and isolated mutants that encode the protein predominantly located in the cytoplasm. Experiments with Leptomycin B suggested a defect in transportation from the cytoplasm to the nucleus rather than enhanced nuclear exportation. Sequencing revealed that the mutations responsible for the cytoplasmic localization of cyclin D1 resided in the vicinity of the cyclin box, which affected interaction with a catalytic partner, Cdk4. We propose that interaction between cyclin D1 and Cdk4 triggers the mechanism controlling the nuclear transportation of this kinase complex.

Structured summary

MINT-7033488:Cdk4 (uniprotkb:P30285) physically interacts (MI:0218) with Cdk1 (uniprotkb:P25322) by anti bait coimmunoprecipitation (MI:0006)MINT-7033511, MINT-7033534:Cdk4 (uniprotkb:P30285) physically interacts (MI:0218) with Cyclin D1 (uniprotkb:P24385) by anti bait coimmunoprecipitation (MI:0006)     

Isolation and characterization of wheat peroxidase isoenzyme B1

Two pure peroxidase isoenzymes B1 and D4 were isolated from the upper parts of 10-day-old wheat seedlings by means of gel and ion-exchange chromatography. Their MWs were 85000 and 24000 respectively. B1 was unstable and under various conditions it was converted to another isoenzyme, electrophoretically identical with D4. B1 contains about 40% of neutral sugars: 17.2% arabinose, 15.3% galactose, 5% glucose and traces of mannose. D4 is free of neutral sugars. None of the isoenzymes contained amino sugars. B1 oxidizes ferulic and p-coumaric acids. This oxidation has two pH optima of 4.4 and 5.4–5.6 and is inhibited by high concentrations of substrates, cyanide and azide. B1 oxidizes IAA in the presence of phenolic cofactor and Mn²⁺ ions. IAA oxidation has two pH optima of 4.5 and 5.6 and is inhibited by high substrate concentration, cyanide and azide, and by a number of indole derivatives. The main products of IAA oxidation are 3-methyleneoxindole and indole-3-methanol. o- and p- diphenols induce a lag period prior to IAA oxidation. Ferulic acid is oxidized during this lag period, probably to a dimer. B1 is able to produce H₂O₂ from oxygen. Mn²⁺ ions, a phenolic cofactor and an electron donor (IAA or NADH) are needed. B1 oxidizes α-keto-γ- methylmercaptobutyric acid to ethylene. D4 has a low peroxidatic activity and is inactive as an IAA oxidase. Thus B1 is probably an active cell wall-bound peroxidase isoenzyme, whereas D4 is its decomposition product.     

Chemotaxonomic characterization of some fast-growing rhizobia nodulating leguminous trees

A total of about 50 strains of rhizobia from two leguminous trees (Acacia andProsopis) were described and compared with 20 reference strains of rhizobia, from other tree and herb legumes on the basis of protein, fatty acid and plasmid profiles, and DNA-DNA hybridization. The rhizobia formed thirteen clusters based on protein profile analysis. These clusters were not in complete agreement with a previously published cluster analysis based on numerical taxonomy of phenotypic characteristics and lipopolysaccharide (LPS) profile analysis (Zhanget al., Int. J. Syst. Bacteriol. 41, 104, 1991; Lindström and Zahran,FEMS Microbiol. Lett. 107, 327, 1993). The fatty acid methyl esters (FAME) of representative strains of rhizobia were analyzed. The rhizobia formed fourteen different clusters based on FAME analysis but the results also conflicted with the phenotypically based methods of analysis. Strains of rhizobia classified in one cluster by any of the above methods of analysis may have shown very different fatty acid profiles. The plasmid profile analysis of the tree rhizobia., on the other hand, was more consistent with the phenotype- and LPS-based numerical analysis. Some strains of the tree rhizobia showed medium or high levels of DNA homology withRhizobium meliloti. The DNA-DNA hybridization correlated well with protein and fatty acid profiles. The described methods provide a significant taxonomic tool for discrimination between rhizobia of leguminous trees. However, further DNA-DNA hybridization studies with other recognized species of rhizobia are needed for proper identification and classification of the diverse rhizobia from leguminous trees.