Bistable auto-aggregation phenotype in Lactiplantibacillus plantarum emerges after cultivation in in vitro colonic microbiota

Background Auto-aggregation is a desired property for probiotic strains because it is suggested to promote colonization of the human intestine, to prevent pathogen infections and to modulate the colonic mucosa. We recently reported the generation of adapted mutants of Lactiplantibacillus plantarum NZ3400, a derivative of the model strain WCFS1, for colonization under adult colonic conditions of PolyFermS continuous intestinal fermentation models. Here we describe and characterize the emerge of an auto-aggregating phenotype in L. plantarum NZ3400 derivatives recovered from the modelled gut microbiota. Results L. plantarum isolates were recovered from reactor effluent of four different adult microbiota and from spontaneously formed reactor biofilms. Auto-aggregation was observed in L. plantarum recovered from all microbiota and at higher percentage when recovered from biofilm than from effluent. Further, auto-aggregation percentage increased over time of cultivation in the microbiota. Starvation of the gut microbiota by interrupting the inflow of nutritive medium enhanced auto-aggregation, suggesting a link to nutrient availability. Auto-aggregation was lost under standard cultivation conditions for lactobacilli in MRS medium. However, it was reestablished during growth on sucrose and maltose and in a medium that simulates the abiotic gut environment. Remarkably, none of these conditions resulted in an auto-aggregation phenotype in the wild type strain NZ3400 nor other non-aggregating L. plantarum, indicating that auto-aggregation depends on the strain history. Whole genome sequencing analysis did not reveal any mutation responsible for the auto-aggregation phenotype. Transcriptome analysis showed highly significant upregulation of LP_RS05225 (msa) at 4.1–4.4 log2-fold-change and LP_RS05230 (marR) at 4.5–5.4 log2-fold-change in all auto-aggregating strains compared to non-aggregating. These co-expressed genes encode a mannose-specific adhesin protein and transcriptional regulator, respectively. Mapping of the RNA-sequence reads to the promoter region of the msa-marR operon reveled a DNA inversion in this region that is predominant in auto-aggregating but not in non-aggregating strains. This strongly suggests a role of this inversion in the auto-aggregation phenotype. Conclusions L. plantarum NZ3400 adapts to the in vitro colonic environment by developing an auto-aggregation phenotype. Similar aggregation phenotypes may promote gut colonization and efficacy of other probiotics and should be further investigated by using validated continuous models of gut fermentation such as PolyFermS.

Effect of Pseudomonas rhodesiae GRC140 on Cucumis sativus L. seedlings with and without Cadmium

Pseudomonas rhodesiae GRC140 is an endophytic bacterium isolated from the roots of Typha latifolia collected in a Cd-contaminated site. This bacterium has biochemical abilities similar to those exerted by plant growth-promoting rhizobacteria. Moreover, it has been shown that P. rhodesiae GRC140 improves the growth of Arabidopsis thaliana seedlings in the absence and presence of Cd. The aim of this work was to determine the effect of P. rhodesiae GRC140 in Cucumis sativus L. growing in nutritive medium with and without Cd. For this, cucumber seeds were superficially disinfected and exposed to a suspension of P. rhodesiae GRC140. Inoculated seeds were placed in a nutritive medium with and without Cd, then were incubated at 28 oC for eight days. After incubation, seedlings were recovered and determined the length of the primary root, the number of roots per plant, hypocotyl length, and the fresh weight. The results showed that P. rhodesiae GRC140 negatively affects the growth of C. sativus L. seedlings grown in the absence of Cd. On the other hand, in Cd-exposed seedlings, P. rhodesiae GRC140 improves the growth of C. sativus L. These results suggest that P. rhodesiae GRC140 decreases the deleterious effect of Cd in C. sativus L.

Effect of Chemical Composition of Nutritive Medium and Explant Size Over Androgenetic Response in Microspore Culture of Brassica oleracea L.

The dimension of the bud is a key factor for the orientation of microspore culture and the success of obtaining double haploid plants as it is a strong correlation between bud size and the developmental stages of microspores, and it is specific for each plant species and genotype. Our study was focused to determine the correlation between morphological characteristics, namely floral bud size and specific microspore developmental stages in order to determine the proper size, suitable for a successful protocol of obtaining double haploid plants in Brassica oleracea var. italica. Thus, we tested four bud sizes ranging from 2.0 to 4.0 mm measured from the base to the tip of the bud. After the statistical analysis of the results it can be emphasized that the best results were obtained in the case of using as a source of microspores the flower buds with the size between 3.1-3.5 mm. At this dimension, the share of microspores in the uninucleate stage, predominantly in the late uninucleate stage, is 90%, thus ensuring a homogeneous population of microspores in the optimum stage of development. Their evolution is predominantly embryogenic, the percentage of microspores following the gametophytic pathway is reduced, by only 9.12%.

Analysis of the role of kynurenine metabolism in drosophila viability control at the high sugar diet influence

Aim. To analyze viability and life span of drosophila at genetic and pharmacological inhibition of tryptophan-kynurenine metabolism, when cultivating on the standard nutritive medium and at a high sugar diet. Methods. Wild type stock and the stock with vermilion mutation have been used. Viability (number of individuals, mortality at the pupal stage) and median life span of imagoes have been determined. Results. High sugar diet has been found to negatively affect the viability of drosophila, leading to increased mortality at the pupal stage and decrease of males’ life span; wild-type stock is less resistant to the influence of such diet as compared with mutant stock. Berberine (an inhibitor of tryptophan dioxygenase) when added to the high sugar nutritive medium reduces the negative effect of a high sugar diet on life span of the wild-type stock: males’ life span reaches control values and in females life span is even more than in the control. Conclusions. Decrease of kynurenines content in the flies organisms (both at the genetic and pharmacological inhibition of tryptophan-kynurenine metabolism) may attenuate negative influence of the high sugar diet. Keywords: drosophila, viability, life span, kynurenine pathway of tryptophan metabolism, high sugar diet.

IMPACT OF SORBITOL- INDUCED OSMOTIC STRESS ON SOME BIOCHEMICAL TRAITS OF POTATO IN VITRO

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM). The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

STUDY OF THE INFLUENCE OF MEALS OF WHEAT AND OAT GERMS AND WILD ROSE FRUITS ON THE FERMENTING MICROFLORA ACTIVITY OF RYE-WHEAT DOUGH

The aim of the research was to study an influence of meals of wheat germs (WGM) and oat germs (OGM) in amount 10…20 %, and also ones of wild rose fruits (WRFM) in amount 2…6 % of the total mass of flour on the fermenting microflora of rye-wheat dough; and also to establish an influence of the experimental supplements on main microbiological processes in it. It has been established, that adding experimental meals favors the activation of bakery yeast. At introducing WGM, OGM and WRFM, its lifting force grows by 16.0–54.0, 6.0–18.0, 10.0–44.0 % respectively, and zymase and maltase activity – by 16.0–53.3, 6.0–17.7 and 11.1–44.0 % and 18.8–55.0, 6.3 31.3 and 7.5–25.0 % respectively. It has been established, that there also takes place the activity increase of lactate bacteria in rye-wheat dough with adding meals of wheat, oat germs and wild rose fruits. It is possible at the expanse of adding an additional nutritive medium with the supplements. Such action of enriching raw materials on the microflora favors intensification of alcoholic and lactate fermentation that is established by data of acid accumulation and gas formation in rye-wheat dough. The counted indices at introducing WGM, OGM, WRFM increase by 39.0, 27.8, 33.9 % and 18.2, 13.6, 16.7 % respectively.

MICROCLONAL PROPAGATION OF CRATAEGUS MONOGYNA JACQ. IN VITRO

We have researched Crataegus Monogina Jacq. microclonal propagation and in vitro mass regeneration processes. For this experiment we used the asleep buds isolated from maternal plants and in vitro cultivated apical and axillary buds as the effective explants. The high coefficient of micropropagation was provided with Gamborg Medium (B5) nutrient medium added alongside the hormones of cytokinin nature. The best result was achieved in the case of introduction of 2-Isopentenyladenine (2-iP) at concentration of 5-10 mcm and Benzilaminopurine (BAP) at concentration of 10-15 mcm. For stimulation of the mictropropagation process it is reasonable to add a small dose of the auxin hormone 1-Naphtylacetic acid (NAA) together with the cytokinins to the nutritive medium. It has been shown that the growth of micropropagation coefficient in pro rata the kinds and concentration of phytohormones. Regenerated microshoots had a good capacity of rooting. The most effective method for rooting purpose was the introduction of Indole- 3 –butyric acid (IBA) at concentration of 5mcm into 1 /2 B5 nutrient medium. The rooted regenerant plants successfully adopted in vivo.

Fruit Flies & the Gut Microbiome: Redesign-Your-Bacteria Lab Exercise

This lab introduces students to experimental design in an inquiry lab exercise that investigates the gut microbiome, basic microbiology techniques, and the broader topic of bacteriology. Fruit flies are used as a model system to study the impact that foods, food additives, and/or antibiotics have on the gut microbiome. One of the major bacteria in the guts of fruit flies is Lactobacillus, which is easy to grow in the lab. This exercise is done in three consecutive lab sessions. During Lab 1, students prepare a standard nutritive medium that has been mixed with a substance of their choice, add the fruit flies to the medium, and practice serial dilution with a simulation. During Lab 2, students plate mashed fruit flies on MRS medium to look at the change in Lactobacillus levels. During Lab 3, students count and determine the change in the number of Lactobacillus in their tested substance, Gram stain selected colonies, and discuss their results as a class. SALG surveys indicated a significant gain in their understanding of the microbiology concepts introduced in this lab.