Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes

Maize is one of the most important crops worldwide, and in Brazil, the state of Paraná stands as its largest producer. The crop demands high inputs of N fertilizers, therefore all strategies aiming to optimize the grain production with lower inputs are very relevant. Endophytic bacteria have a high potential to increment maize grain yield by means of input via biological nitrogen fixation and/or plant growth promotion, in this last case increasing the absorption of water and nutrients by the plants. In this study, we established a collection of 217 endophytic bacteria, isolated from roots of four lineages and three hybrid genotypes of maize, and isolated in four different N-free culture media. Biochemical―comprising growth in different carbon sources, intrinsic tolerance to antibiotics, and biochemical tests for catalase, nitrate reductase, urease, and growth in N-free media in vitro―and genetic characterization by BOX-PCR revealed great variability among the isolates. Both commercial hybrids and homozygous lineages were broadly colonized by endophytes, and sequencing of the 16S rRNA gene revealed the presence of bacteria belonging to the genera Pantoea, Bacillus, Burkholderia, and Klebsiella. Qualitative differences in endophytic colonization were detected between lineages and hybrid genotypes.     

New structures for goat corrals to control peridomestic populations of Triatoma infestans (Hemiptera: Reduviidae) in the Gran Chaco of Argentina

Goat production is an important economic activity for rural communities in the Gran Chaco of Argentina. Goat corrals are important for the survival of peridomestic populations of Triatoma infestans. This study evaluated the impact of modifying the traditional structure of goat corrals on T. infestans populations and goat productivity in the region of Los Llanos (La Rioja). Thirty-nine experimental corrals were constructed and 57 traditional corrals were used as controls. We evaluated the infestations of the control and experimental corrals for five years following construction of the structures. The results showed that the new structures did not prevent the colonization, although it enhanced the detection of infestation at low densities of T. infestans. No significant difference was found in T. infestans population abundance between control and experimental corrals, probably because of the different detectability in the two types of structures, especially among the small nymphs. Although goat productivity average was higher in experimental than in control corrals, no significant difference was found because of high variability. The new structures can be used as a complement to promote the development of rural communities. Acceptability and adoption of the new corrals by the owners was high, as the enclosures offered better protection for the goats, increased growth of kids and facilitated herd handling.     

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.     

Biochemical and structural characterization of recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh2 gene was heterologously overexpressed in Escherichia coli, and the resulting protein (SaADH2) was purified to homogeneity and both biochemically and structurally characterized. The crystal structure of the SaADH2 NADH-bound form reveals that the enzyme is a tetramer consisting of identical 27,024-Da subunits, each composed of 255 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 80 °C and a 30-min half-inactivation temperature of ～88 °C. It also shows good tolerance to common organic solvents and a strict requirement for NAD(H) as the coenzyme. SaADH2 displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, showing no activity on aldehydes. Interestingly, the enzyme catalyses the asymmetric reduction of benzil to (R)-benzoin with both excellent conversion (98 %) and optical purity (98 %) by way of an efficient in situ NADH-recycling system involving a second thermophilic ADH. The crystal structure of the binary complex SaADH2–NADH, determined at 1.75 Å resolution, reveals details of the active site providing hints on the structural basis of the enzyme enantioselectivity.     

The C-terminal domain of CblD interacts with CblC and influences intracellular cobalamin partitioning

Mutations in cobalamin or B₁₂ trafficking genes needed for cofactor assimilation and targeting lead to inborn errors of cobalamin metabolism. The gene corresponding to one of these loci, cblD, affects both the mitochondrial and cytoplasmic pathways for B₁₂ processing. We have demonstrated that fibroblast cell lines from patients with mutations in CblD, can dealkylate exogenously supplied methylcobalamin (MeCbl), an activity catalyzed by the CblC protein, but show imbalanced intracellular partitioning of the cofactor into the MeCbl and 5′-deoxyadenosylcobalamin (AdoCbl) pools. These results confirm that CblD functions downstream of CblC in the cofactor assimilation pathway and that it plays an important role in controlling the traffic of the cofactor between the competing cytoplasmic and mitochondrial routes for MeCbl and AdoCbl synthesis, respectively. In this study, we report the interaction of CblC with four CblD protein variants with variable N-terminal start sites. We demonstrate that a complex between CblC and CblD can be isolated particularly under conditions that permit dealkylation of alkylcobalamin by CblC or in the presence of the corresponding dealkylated and oxidized product, hydroxocobalamin (HOCbl). A weak CblC·CblD complex is also seen in the presence of cyanocobalamin. Formation of the CblC·CblD complex is observed with all four CblD variants tested suggesting that the N-terminal 115 residues missing in the shortest variant are not essential for this interaction. Furthermore, limited proteolysis of the CblD variants indicates the presence of a stable C-terminal domain spanning residues ∼116–296. Our results are consistent with an adapter function for CblD, which in complex with CblC·HOCbl, or possibly the less oxidized CblC·cob(II)alamin, partitions the cofactor between AdoCbl and MeCbl assimilation pathways.     

A re-evaluation of morphological characters of the invasive ascidian Corella eumyota reveals two different species at the tip of South America and in the South Shetland Islands, Antarctica

The native solitary ascidian Corella eumyota Traustedt, 1882) is commonly found in the Southern Hemisphere in the cold-temperate waters of the Subantarctic and Antarctic regions. Its recent spread into the Northern Hemisphere throughout the NE Atlantic gave the species the status of invasive. Together with its widespread distribution, reports on its wide variability (several distinct morphological characters, genetic discontinuities and also possible misidentifications) cast doubt on the taxonomic status of different populations of this species. This work, based on the observation, quantification and analysis of specific morphological characters in specimens collected at five different localities of South America and Antarctica, strongly indicates that there are two different species: C. eumyota from South America and Corella antarctica Sluiter, 1905) from Antarctica, which has been till now considered a junior synonym of the former. The species clearly differ in the arrangement of the gonadal ducts, the size of the larvae and the shape of the anus, among other characters. Morphological variation displays a defined, discrete grouping supporting a clear differentiation into two species. This result shows the need for careful inspection of specimens to avoid wrong interpretations in a context of changes of marine biota due to biological invasions.     

Ontogenetic diet shift in Commerson’s dolphin (Cephalorhynchus commersonii commersonii) off Tierra del Fuego

The Commerson’s dolphin (Cephalorhynchus c. commersonii, Lacépède, 1804) is the most common endemic dolphin of the coastal waters of Tierra del Fuego incidentally caught in artisanal coastal gill nets. A better understanding of its trophic ecology is essential to facilitate the effective management and conservation of its population. The aim of this study was to analyze possible shifts in diet among age and sex classes by analyzing bone collagen δ¹³C and δ¹⁵N values collected from 220 individuals along the coast of Tierra del Fuego. Additional analysis of potential prey allowed us to use a stable isotope mixing model to quantify resource use. Isotopic comparisons suggest that all age classes share the same foraging areas. We found, however, a significant dietary shift: juveniles consume a higher mean (±SD) proportion (60 ± 7 %) of pelagic Sprattus fuegensis than adults (36 ± 10 %), and the consumption of benthopelagic species such as Illex argentinus and Loligo gahi is higher in adults (33 ± 12 %) than juveniles (20 ± 7 %). This may result from an improvement in foraging skills and expansion of habitats in adulthood. While males move greater distances in search of mates and resources, a lack of isotopic differences between the sexes suggests little evidence for sex-related resource partitioning. A significant decrease in δ¹⁵N values between calves and juveniles indicates a weaning period between 0.5 and 1 year as was shown in previous captive studies on this species. Our results also suggest that artisanal fisheries, despite being a major source of mortality, do not affect the long-term food habits of Commerson’s dolphin since they showed a weak trophic overlap.     

Kappaphycus alvarezii (Gigartinales, Rhodophyta) cultivated in Brazil: is it only one species?

Kappaphycus alvarezii which is endemic to the Indo-Pacific region is the main raw material for kappa carrageenan production. A seedling that was cultivated in Japan (originally from the Philippines) was introduced in a trial, for aquaculture purpose, in 1995, in São Paulo State, southeastern region of Brazil. It was later introduced in Santa Catarina State, in Southern Brazil. In 1998, another seedling that was brought from Venezuela, also originally from the Philippines, was commercially introduced at Ilha Grande Bay and later on at Sepetiba Bay, both in Rio de Janeiro State. The aim of this study was to characterize 21 samples from different farms (Rio de Janeiro and Santa Catarina States) and verify if they are K. alvarezii or other species. Based on the intergenic spacer cox2-3 sequences, phylogenetic relationships were inferred through neighbor joining, maximum likelihood, and Bayesian analyses. The topology of the trees suggests that all samples from the different farms form a monophyletic group of K. alvarezii. Statistical analysis of the cox2-3 marker calculated with median-joining network showed 38 variable positions defining 15 haplotypes for the studied samples of Kappaphycus. The most frequent K. alvarezii haplotype grouped the samples cultivated worldwide with the Brazilian samples. These results are important for better productivity and are environmentally desirable for introduction purposes since the species introduced is known and will help focusing the research on this species. This knowledge can be of assistance to the government in setting up environmental and cultivation protocols to achieve sustainability in macroalgae aquaculture.