Novel Alkaloids from Marine Actinobacteria: Discovery and Characterization

The marine environment is an excellent resource for natural products with therapeutic potential. Its microbial inhabitants, often associated with other marine organisms, are specialized in the synthesis of bioactive secondary metabolites. Similar to their terrestrial counterparts, marine Actinobacteria are a prevalent source of these natural products. Here, we discuss 77 newly discovered alkaloids produced by such marine Actinobacteria between 2017 and mid-2021, as well as the strategies employed in their elucidation. While 12 different classes of alkaloids were unraveled, indoles, diketopiperazines, glutarimides, indolizidines, and pyrroles were most dominant. Discoveries were mainly based on experimental approaches where microbial extracts were analyzed in relation to novel compounds. Although such experimental procedures have proven useful in the past, the methodologies need adaptations to limit the chance of compound rediscovery. On the other hand, genome mining provides a different angle for natural product discovery. While the technology is still relatively young compared to experimental screening, significant improvement has been made in recent years. Together with synthetic biology tools, both genome mining and extract screening provide excellent opportunities for continued drug discovery from marine Actinobacteria.     

Variability in the composition of lupin (Lupinus angustifolius) meals influences their digestible nutrient and energy value when fed to rainbow trout (Oncorhynchus mykiss)

Over a series of seven separate experiments 76 different lupin (Lupinus angustifolius) meals were assessed for their digestible dry matter, protein, amino acid and energy characteristics when fed to rainbow trout. Two reference diets (a common basal diet and a reference lupin meal) were also included in each experiment. Minimal variance in the digestibility parameters of both reference diets was observed among the experiments ensuring that there was a high degree of robustness in the across-experiment evaluations. Using simple and multiple-regression techniques, principal diet and ingredient composition factors affecting diet and ingredient digestibilities and ingredient digestible values were explored within the dataset. Using simple linear regression, it was shown that nitrogen digestibility of the lupin meals was negatively influenced by ingredient lignin content, but positively affected ingredient protein (N × 6.25) content. The energy digestibility of the lupin meals was positively affected by a range of compositional features including protein, sum of amino acids and negatively affected by carbohydrate content. The digestible nutrient and energy content of the meals reflected the combined effects of both ingredient digestibilities and ingredient composition. The digestible nitrogen content of the lupin meals was positively affected by protein (N × 6.25), sum of amino acids and energy content, but was negatively affected by lignin and carbohydrate content. The digestible sum of amino acids was also positively affected by protein, sum of amino acids, but only negatively affected by carbohydrate content, not lignin content. The digestible energy content of the lupin meals was also positively affected by protein, sum of amino acids and their own energy density, but only negatively affected by carbohydrate content. Multiple linear regression modelling supported that together ingredient protein and lignin content were the strongest predictors of digestible protein value, explaining close to 60% of the variability in this parameter. Discrete reassessment of these relationships using non-linear analysis methods provided a stronger interpretation of the ingredient composition effects. In recognition of this, a functional model including terms for ingredient protein and lignin content on the digestible protein value was defined. This study demonstrates that within one raw material type that not only does significant variability in the digestible value of the raw materials exist, but that it is possible to identify compositional features of that raw material that are intrinsically influencing their own digestible value.     

Differences between the ovine tonsils based on an immunohistochemical quantification of the lymphocyte subpopulations

In sheep, the pharyngeal first defence line against oral and inhaled antigens is organized in six tonsils. Since tonsils are regarded as secondary lymphoid tissue and part of the acquired immune system which is subjected to induction through contact with antigens, an evaluation of the different lymphocyte populations in tonsils is useful to determine a tendency of the specific tonsils to more inductive or more effective immunity. By means of immunohistochemistry, different lymphocyte populations were quantified and localized using a panel of eight antibodies, i.e. anti-CD45, anti-CD21, anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-WC1 and anti-Ki67. The CD21+ B lymphocytes were localized within the tonsillar lymphoid follicles. The CD2+/CD3+ T lymphocytes were numerous in the interfollicular regions and were aligned underneath and within the epithelium but were also observed at the CD21+ pole of the lymphoid follicles. Near the lingual and tubal tonsils, and the tonsil of the soft palate, the CD45+ cells around the seromucous glands and in the lamina propria were mainly CD3+ T cells. In all tonsils, the WC1+ gamma delta T cells formed a small lymphocyte population which harboured the lamina propria and the interfollicular region. The relative percentages of the different lymphocyte populations of the large palatine and pharyngeal tonsils, which are macroscopically the most developed, were comparable. In contrast, the lingual tonsil was significantly different from the other tonsils not only by its small size and lack of lymphoid follicles, but also by the lymphocyte populations. Based on the lymphocyte populations, the ovine tonsils can be divided in three groups with the tonsil of the soft palate, the tubal and paraepiglottic tonsil forming an intermediate between the palatine and pharyngeal tonsils as true tonsils on the one side, and the lingual tonsil as a scattered lymphocyte aggregation on the other side.     

Genetic diversity of rhizobia associated with alfalfa in Serbian soils

We have evaluated the genetic diversity and phylogeny of alfalfa rhizobia, originating from different types of soils in Serbia and their ability to establish an effective symbiosis with alfalfa (Medicago sativa L.). A collection of 65 strains isolated from root nodules of alfalfa were characterized by rep-PCR analysis, partial and complete 16S rDNA gene and recA gene sequencing, as well as atpD gene sequencing and DNA–DNA hybridizations. The results of the sequence analyses revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules. Only one strain was identified as Sinorhizobium medicae, two strains as Rhizobium tibeticum and one strain as Rhizobium sp. Despite the fact that the majority of strains were identified as S. meliloti, a high genetic diversity at strain level was detected. Almost all isolates shared the ability to nodulate and fix nitrogen with M. sativa, except 11 of them, which were incapable of fixing nitrogen with this species. About 50% of the isolates showed values of symbiotic effectiveness (SE) above 50%, while 10% of the strains were highly effective with SE values above 70%. Some of the strains which were highly effective in nitrogen fixation at the same time could intensively solubilize phosphates, offering a possibility for multipurpose inoculum development. This was the first genetic study of rhizobia isolated from this region and also the first report of natural presence of R. tibeticum in root nodules of M. sativa.     

Impact of xylanases with different substrate selectivity on gluten-starch separation of wheat flour

The influence on wheat flour gluten-starch separation of a xylanase from Aspergillus aculeatus (XAA) with hydrolysis selectivity toward water extractable arabinoxylan (WE-AX) and that is not inhibited by wheat flour xylanase inhibitors was compared to that of a xylanase from Bacillus subtilis (XBS) with hydrolysis selectivity toward water unextractable arabinoxylan (WU-AX) and that is inhibited by such inhibitors. XAA improved gluten agglomeration through degradation of WE-AX and concomitant reduction in viscosity, which in the laboratory scale batter procedure with a set of vibrating sieves (400, 250, and 125 microm), increased protein recoveries on the 400 microm sieve. In contrast, XBS had a negative effect as it decreased gluten protein recovery on this sieve, probably as a result of the viscosity increase that accompanied WU-AX solubilization. Hence, it was active even if most likely a considerable part of its activity was prevented by xylanase inhibitors. A combination of XAA and XBS at a low dosage yielded a distribution of gluten proteins on the different sieves comparable to that of the control. At a high combined dosage, the gluten agglomeration was better than that with XAA alone, indicating that both WE-AX and WU-AX have a negative impact on gluten agglomeration. Finally, experiments with endoxylanase addition at different moments during the separation process suggest that the status of the arabinoxylan population during dough mixing is far less critical for its impact on gluten agglomeration than that during the batter phase.     

Effect of inoculation with wild type Azospirillum brasilense and A. irakense strains on development and nitrogen uptake of spring wheat and grain maize

Under the controlled conditions of the greenhouse and by varying some biotic and abiotic factors, we tried to identify some of the factors critical to obtain successful Azospirillum inoculation. Spring wheat and grain maize were inoculated with different concentrations of the wild type strains A. brasilense Sp245 and A. irakense KBC1, and grown in a substrate with varying concentrations of organic matter (OM) and N fertiliser. The inoculum concentration was one of the factors that influenced most the outcome of an inoculation experiment on wheat, with lower inoculum concentrations (10⁵-10⁶ cfu plant^-1) stimulating root development and plant dry weight and higher inoculum concentrations (10⁷-10⁸ cfu plant^-1) having no effect or sometimes even inhibiting root development. The effect of inoculation was most pronounced at low to intermediate N fertilisation levels, while the OM content of the substrate had no effect. Inoculation was found to affect early plant and root development, plant and root dry weight, grain yield and the N-uptake efficiency of plants. However, inoculation did not change the N concentration in plants or grains. In addition, a difference in the ability of both strains to stimulate plant growth and N uptake of wheat and maize was observed, with A. brasilense Sp245 having most effect on spring wheat and A. irakense KBC1 being more effective on grain maize. The significance of the obtained results for agriculture is discussed.