Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci.

BACKGOUND: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. METHODS: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. RESULTS: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. CONCLUSIONS: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. GENERAL SIGNIFICANCE: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

Dispersion of Echinococcus granulosus eggs from infected dogs under natural conditions in Patagonia, Argentina.

Cystic echinococcosis caused by Echinococcus granulosus is a major zoonosis of public health significance in the Patagonian region of Argentina. This investigation sought to test the hypothesis that the persistence and dispersion of the parasite eggs can be explained by physical and meteorological parameters along with final host infection and behaviour. This observational study was carried out over a five-year period within an enclosure where two dogs harbouring a worm burden ranging from 100 to 1000 mature adult E. granulosus, as well as two uninfected dogs, had previously been kept for six months. Environmental canine faeces, topsoil, pond water, and sediment samples were examined to control for the presence of eggs and coproantigens of the parasite using microscope-based techniques and copro-ELISA plus copro-Western Blot tests. The parasite eggs were detected up to 41 months later in faeces from infected dogs, soil and sediment, and coproantigen tests remained positive for up to 70 months in faeces. Overall, parasite eggs were found within a maximum distance of 115 m from the contaminated dog faeces deposition site. Our findings indicate that under Patagonian environmental conditions, egg persistence and dispersion seem to be related to the worm burden and habits of the infected dog, to prevailing wind direction and to the existence of low bushes as well as natural bodies of water. The present study is the first to provide direct evidence of interaction between bioclimatic conditions and E. granulosus egg dispersion under Patagonian field conditions.

Insights into the mechanisms of desiccation resistance of the Patagonian PAH-degrading strain Sphingobium sp. 22B.

AIM: To analyse the physiological response of Sphingobium sp. 22B to water stress. METHODS AND RESULTS: The strain was grown under excess of carbon source and then subjected to low (60RH) and high (18RH) water stress conditions for 96 h. Quantification of trehalose, glycogen, polyhydroxybutyrate (PHB) and transmission electron microscopy (TEM) was studied. Genes linked with desiccation were searched in Sphingobium sp. 22B and Sphingomonas 'sensu latu' genomes and their transcripts were quantified by real-time PCR. Results showed that, in the absence of water stress, strain 22B accumulated 4·76 ± 1·41% of glycogen, 0·84 ± 1·62% of trehalose and 44·9 ± 6·4% of PHB per cellular dry weight. Glycogen and trehalose were mobilized under water stressed conditions, this mobilization was significantly higher in 60RH in comparison to 18RH. Gene treY was upregulated sixfold in 60RH relative to control condition. TEM and quantification of PHB revealed that PHB was mobilized under 60RH condition accompanied by the downregulation of the phbB gene. TEM images showed an extracellular amorphous matrix in 18RH and 60RH. Major differences were found in the presence of aqpZ and trehalose genes between strain 22B and Sphingomonas genomes. CONCLUSION: Strain 22B showed a carbon conservative metabolism capable of accumulation of three types of endogenous carbon sources. The strain responds to water stress by changing the expression pattern of genes related to desiccation, formation of an extracellular amorphous matrix and mobilization of the carbon sources according to the degree of water stress. Trehalose, glycogen and PHB may have multiple functions in different degrees of desiccation. The robust endowment of molecular responses to desiccation shown in Sphingobium sp. 22B could explain its survival in semi-arid soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the physiology implicated in the toleration of the PAH-degrading strain Sphingobium sp 22B to environmental desiccation may improve the bioaugmentation technologies in semi-arid hydrocarbon-contaminated soils.

A descriptive study of the occurrence and significance of lipids in Taenia hydatigena eggs.

The aim of this work was to investigate the lipid content of Taenia hydatigena eggs and to evaluate the role of lipids in the maintenance of embryo viability. The total lipid content of the egg was 4.5% (w/w). Five classes of neutral lipids were identified: esterified cholesterol, free cholesterol, triacylglycerols, diacylglycerols and free fatty acids. Our results suggest that triacylglycerols play a key role in the maintenance of embryo viability. In addition, we found that T. hydatigena eggs remain metabolically active by mobilisation of stored triacylglycerols. This study contributes to the understanding the survival strategies of a member of the Taeniidae family in the environment outside the host.

Degradation of linear alkylbenzene sulfonate by a bacterial consortium isolated from the aquatic environment of Argentina.

AIMS: To isolate and identify linear alkylbenzene sulfonate (LAS)-degrading bacteria from Río de la Plata and adjacent waters, and to assay their degradation capability as a consortium and as single organisms. METHODS AND RESULTS: A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO(2) release (mineralization) than individual cultures, and degraded 86% of LAS (20 mg l(-1)), whereas pure strains degraded between 21% and 60%. Bacterial desulfonation from LAS was evidenced in the consortium and A. caviae strains. A complete disappearance of LAS (10 mg l(-1)) was accomplished, and LAS levels of 50 and 100 mg l(-1) led to a pronounced decrease in the biodegradation extent and inhibition of culture growth. CONCLUSIONS: A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems.

Evaluación de la actividad antimalárica de algunas plantas utilizadas en la medicina tradicional cubana / Evaluation of the antimalarial activity in some Cuban medicinal plants

The search for new treatments against malaria has a highpriority in the fight to bring this disease under control. The aim of this study was to assess the therapeutic potential of extracts of plants selected on the basis of ethnobotanical information collected from Cuban traditional medicine. Extracts from six plants (Bambusavulgaris, Parthenium hysterophorus, Melaleuca leucadendron, Indigofera suffruticosa, Artemisia absinthium, Simarouba glauca) were tested for their invitro effect against the F32/Tanzania strain ofPlasmodium falciparum. S. glauca, P. hysterophorus, M.leucadendron and A. absinthium exhibited minimum inhibitory concentrations (MIC) in the range from 3.1to 50 g/mL, while B. vulgaris and I. suffruticosa showed negative activity against this strain. The highest in vivo activities against Plasmodium berghei NK65 were shownby A. absinthium, with a 65.9% reduction in parasitemia at a dose of 500 mg/kg, M. leucadendron, with 50%reduction at 250 mg/kg, and S. glauca, with 43.2%reduction at 100 mg/kg. The less toxic extracts wereA. absinthium and M. leucadendron. These results demonstrate the antimalarial properties of some Cuban medicinal plants and pave the way to detailed researchon their active chemical constituents.

Effect of Paecilomyces lilacinus on the viability of oncospheres of Taenia hydatigena.

Paecilomyces lilacinus (Thom) Samson is a saprophytic hyphomycete from the soil with biological activity on helminth eggs. We evaluated the influence in vitro of P. lilacinus on the viability of the oncospheres from Taenia hydatigena, a parasite cestode of dogs and sheep. The eggs were exposed to the fungus strain in sterile distilled water and observed by light microscopy at days 4, 7 and 14 post-inoculation, and the viability was evaluated. The viability found in the exposed P. lilacinus oncospheres was significantly different in all observations. P. lilacinus exercised a negative biological activity on T. hydatigena eggs in vitro.

Triacylglycerols in prokaryotic microorganisms.

Triacylglycerols (TAG) are fatty acid triesters of glycerol; there are diverse types of TAG with different properties depending on their fatty acid composition. The occurrence of TAG as reserve compounds is widespread among eukaryotic organisms such as yeast, fungi, plants and animals, whereas occurrence of TAG in bacteria has only rarely been described. However, accumulation of TAG seems to be widespread among bacteria belonging to the actinomycetes group, such as species of Mycobacterium, Streptomyces, Rhodococcus and Nocardia. Fatty acids in acylglycerols in cells of Rhodococcus opacus PD630 accounted for up to 87% of the cellular dry weight. TAG biosynthesis, justifying an oleaginous status, seems to be restricted mainly to this group of bacteria, but occurs to a minor extent also in a few other bacteria. The compositions and structures of bacterial TAG vary considerably depending on the microorganism and on the carbon source, and unusual acyl moieties, such as phenyldecanoic acid and 4,8,12 trimethyl tridecanoic acid, are also included. The principal function of bacterial TAG seems to be as a reserve compound. Other functions that have been discussed include regulation of cellular membrane fluidity by keeping unusual fatty acids away from membrane phospholipids, or acting as a sink for reducing equivalents. In recent years, basic aspects of the physiology and biochemistry of bacterial TAG accumulation, and the molecular biology of the lipid inclusion bodies have been reported. TAG are used for nutritional, therapeutic and pharmaceutical purposes and serve as a source of oleochemicals.

Biosynthesis of fatty acids and triacylglycerols by 2,6,10,14-tetramethyl pentadecane-grown cells of Nocardia globerula 432.

Nocardia globerula strain 432 was able to synthesize triacylglycerols (TAG) during cultivation on 2,6,10,14-tetramethyl pentadecane (pristane) under nitrogen-limiting conditions. Within these cells, 4,8,12-trimethyl tridecanoic acid was the major fatty acid detected. Fatty acids with an odd number of carbon atoms and minor amounts of even-numbered fatty acids were also observed. Experiments carried out with acrylic acid, an inhibitor of beta-oxidation, suggested that odd-numbered fatty acids such as C15:0, C17:0 and 10-methyl C17:0 were synthesized de novo using propionyl-CoA, the beta-oxidation product, as precursor. Although N. globerula 432 incorporated mainly straight chain fatty acids into TAG, the branched fatty acid 4,8,12-trimethyl tridecanoic acid also appeared, to some extent, in the acylglycerols. The importance of TAG biosynthesis by pristane-grown cells of N. globerula strain 432 is discussed.

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126.

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase.

[Identification of a neutral protease in the intestine of Boophilus microplus with electrophoresis in polyacrylamide gels copolymerized with gelatin]. / Identificación de una proteasa neutra en intestino de Boophilus microplus por electroforesis en geles de poliacrilamida copolimerizados con gelatina.

A neutral activity of Boophilus microplus in the intestine was identified by electrophoresis in polyacrilamide gel copolymerized with gelatin. The maximum of activity was attained at pH 6.0. The highest specific activity at that pH was obtained with casein substrate. The disappearance of this activity was observed in both substrates after the addition of phenylmethylsulphonyl fluoride in the reaction mixtures. It was very interesting to find out an endopeptidase activity with these characteristics in the intestine, in spite of the fact that the digestive activity in ticks is intracellular at very acid pH, which does not occur in other insects.

The research program at AIGLE.

The purpose of this work is to present the research program of a Clinical Center, the AIGLE Foundation in Buenos Aires, Argentina. It describes the potentialities and advantages of research in this context. It also describes the institute, the types of patients treated and its professional staff. The program covers four areas: determining the demand for psychotherapeutic care in the population at large, studying the conditions making participation in community mental health possible, analyzing and follow-up on the outcomes and studying the personal styles of the therapists. Finally, this work discusses the usefulness and applicability of these results for clinical practice.

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630.

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the beta-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized beta-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation.