Efficiency of the alpha-hairpinin SmAMP-X gene promoter from Stellaria media plant depends on selection of transgenic approach.

The antimicrobial activity of the alpha-HAIRPININ ANTIMICROBIAL PEPTIDE X (SmAMP-X gene, GenBank acc. No. HG423454.1) from Stellaria media plant has been shown in vitro. Here, we isolated the SmAMP-X gene promoter and found two genomic sequences for the promoter (designated pro-SmAMP-X and pro-SmAMP-X-Ψ2) with 83% identity in their core and proximal regions. We found that the abilities of these promoters to express the uidA reporter and the nptII selectable marker differ according to the structural organization of T-DNA in the binary vector used for plant transformation. Analysis of Agrobacterium-infiltrated Nicotiana benthamiana leaves, transgenic Arabidopsis thaliana lines, and transgenic Solanum tuberosum plants revealed that both promoters in the pCambia1381Z and pCambia2301 binary vectors generate 42-100% of the ß-glucuronidase (GUS) activity generated by the CaMV35S promoter. According to 5'-RACE (rapid amplification of cDNA ends) analysis, both plant promoters are influenced by the CaMV35S enhancer used to express selectable markers in the T-DNA region of pCambia1381Z and pCambia2301. The exclusion of CaMV35S enhancer from the T-DNA region significantly reduces the efficiency of pro-SmAMP-X-Ψ2 promoter for GUS production. Both promoters in the pCambia2300 vector without CaMV35S enhancer in the T-DNA region weakly express the nptII selectable marker in different tissues of transgenic N. tabacum plants and enable selection of transgenic cells in media with a high concentration of kanamycin. Overall, promoter sequences must be functionally validated in binary vectors lacking CaMV35S enhancer.

Matrix is everywhere: extracellular DNA is a link between biofilm and mineralization in Bacillus cereus planktonic lifestyle.

To date, the mechanisms of biomineralization induced by bacterial cells in the context of biofilm formation remain the subject of intensive studies. In this study, we analyzed the influence of the medium components on the induction of CaCO3 precipitation by the Bacillus cereus cells and composition of the extracellular matrix (ECM) formed in the submerged culture. While the accumulation of extracellular polysaccharides and amyloids appeared to be independent of the presence of calcium and urea during the growth, the accumulation of extracellular DNA (eDNA), as well as precipitation of calcium carbonate, required the presence of both ingredients in the medium. Removal of eDNA, which was sensitive to treatment by DNase, did not affect other matrix components but resulted in disruption of cell network formation and a sixfold decrease in the precipitate yield. An experiment with a cell-free system confirmed the acceleration of mineral formation after the addition of exogenous salmon sperm DNA. The observed pathway for the formation of CaCO3 minerals in B. cereus planktonic culture included a production of exopolysaccharides and negatively charged eDNA lattice promoting local Ca2+ supersaturation, which, together with an increase in the concentration of carbonate ions due to pH rise, resulted in the formation of an insoluble precipitate of calcium carbonate. Precipitation of amorphous CaCO3 on eDNA matrix was followed by crystal formation via the ACC-vaterite-calcite/aragonite pathway and further formation of larger mineral aggregates in complex with extracellular polymeric substances. Taken together, our data showed that DNA in extracellular matrix is an essential factor for triggering the biomineralization in B. cereus planktonic culture.

Calcifying Bacteria Flexibility in Induction of CaCO3 Mineralization.

Microbially induced CaCO3 precipitation (MICP) is considered as an alternative green technology for cement self-healing and a basis for the development of new biomaterials. However, some issues about the role of bacteria in the induction of biogenic CaCO3 crystal nucleation, growth and aggregation are still debatable. Our aims were to screen for ureolytic calcifying microorganisms and analyze their MICP abilities during their growth in urea-supplemented and urea-deficient media. Nine candidates showed a high level of urease specific activity, and a sharp increase in the urea-containing medium pH resulted in efficient CaCO3 biomineralization. In the urea-deficient medium, all ureolytic bacteria also induced CaCO3 precipitation although at lower pH values. Five strains (B. licheniformis DSMZ 8782, B. cereus 4b, S. epidermidis 4a, M. luteus BS52, M. luteus 6) were found to completely repair micro-cracks in the cement samples. Detailed studies of the most promising strain B. licheniformis DSMZ 8782 revealed a slower rate of the polymorph transformation in the urea-deficient medium than in urea-containing one. We suppose that a ureolytic microorganism retains its ability to induce CaCO3 biomineralization regardless the origin of carbonate ions in a cell environment by switching between mechanisms of urea-degradation and metabolism of calcium organic salts.

Crystal and Supramolecular Structure of Bacterial Cellulose Hydrolyzed by Cellobiohydrolase from Scytalidium Candidum 3C: A Basis for Development of Biodegradable Wound Dressings.

The crystal and supramolecular structure of the bacterial cellulose (BC) has been studied at different stages of cellobiohydrolase hydrolysis using various physical and microscopic methods. Enzymatic hydrolysis significantly affected the crystal and supramolecular structure of native BC, in which the 3D polymer network consisted of nanoribbons with a thickness T ≈ 8 nm and a width W ≈ 50 nm, and with a developed specific surface SBET ≈ 260 m2·g-1. Biodegradation for 24 h led to a ten percent decrease in the mean crystal size Dhkl of BC, to two-fold increase in the sizes of nanoribbons, and in the specific surface area SBET up to ≈ 100 m2·g-1. Atomic force and scanning electron microscopy images showed BC microstructure "loosening"after enzymatic treatment, as well as the formation and accumulation of submicron particles in the cells of the 3D polymer network. Experiments in vitro and in vivo did not reveal cytotoxic effect by the enzyme addition to BC dressings and showed a generally positive influence on the treatment of extensive III-degree burns, significantly accelerating wound healing in rats. Thus, in our opinion, the results obtained can serve as a basis for further development of effective biodegradable dressings for wound healing.

Floating bioplato for purification of waste quarry waters from mineral nitrogen compounds in the Arctic.

A bioplato was organized at Kirovogorskiy pond-settling of OLKON Company (the city of Olenegorsk, in Murmansk region) to reduce the content of nitrogen mineral compounds in water which come into the pond with the quarry waters after blasting operations using nitrogen compounds. The assortment of aboriginal plants was selected, a method of fixing and growing them on the water surface was developed, and observations of their vegetation were carried out. The dynamics of nitrogen compounds was determined in the laboratory and with full-scale tests. The coverage area pond by plants for the effective reduction of mineral nitrogen compounds was calculated. The use of floating bioplato helped to reduce content of ammonium and nitrite to maximum permissible levels or even lower in pond water. Also there was a tendency towards reduction of nitrate concentrations in water. The developmental technology can be used in any climatic zone with a specific assortment of plants-ameliorants.