Production of recombinant Fibronectin tipe III 9-10 domain in bacterial expression system

Fibronectin (Fn) is a glycoprotein that plays important roles in cell adhesion, growth, differentiation and migration by mediating a wide variety of cellular interactions with the extracellular matrix (ECM). Fn usually exists as a dimer composed of two nearly identical 220~250 kDa subunits where each monomer is composed of homologous repeats of three prototypical domains: type I, type II and type III. Fn interacts with many integrins such as α3β1, α5β1, α8β1, αvβ1, αIIββ3, αvβ3, αvβ5, and αvβ6. In previous studies, the specific integrin-recognition sequences involved in cell adhesion have been identified. The best known of these – Arg-Gly-Asp (RGD) sequence – is located in the central cell-binding domain - FnIII10. It is the most important recognition site that can interact with about half of all known integrins. Another important sequence which acts in synergy with the RGD site is - Pro-His-Ser-Arg-Asn (the ‘synergy site’ PHSRN) found in Fn repeat III9, that promotes specific α5β1 integrin binding. Because of the ECM binding properties Fn is a perfect protein for nanofabrication and integrin-mediated immobilization of cells into synthetic scaffolds. Heterologous expressions of large eucaryotic proteins, such as Fn, in bacterial expression system is complicated. Therefore the aim of this study was to establish a recombinant protein production system for the "cell-binding domain" of Fn protein - including FnIII9-10 fragment and to assess the effect of His-tag position on the recombinant peptide purification efficiency. For this purpose, a sequence of the FnIII9-10 fragment was cloned to pLATE bacterial expression vector using a ligation independent cloning system. This vector includes bacteriophage T7 promoter that ensures high yields of expressed proteins. Two constructs including either amino- or carboxy- terminal 6xHis-tag were developed.[...]Biochemijos katedraGamtos mokslų fakultetasLietuvos agrarinių ir miškų mokslų centro Sodininkystės ir daržininkystės institutasVilniaus universitetasVytauto Didžiojo universiteta

Enduring Effect of Antibiotic Timentin Treatment on Tobacco In Vitro Shoot Growth and Microbiome Diversity

Plant in vitro cultures initiated from surface-sterilized explants often harbor complex microbial communities. Antibiotics are commonly used to decontaminate plant tissue culture or during genetic transformation; however, the effect of antibiotic treatment on the diversity of indigenous microbial populations and the consequences on the performance of tissue culture is not completely understood. Therefore, the aim of this study was to assess the effect of antibiotic treatment on the growth and stress level of tobacco (Nicotiana tabacum L.) shoots in vitro as well as the composition of the plant-associated microbiome. The study revealed that shoot cultivation on a medium supplemented with 250 mg L−1 timentin resulted in 29 ± 4% reduced biomass accumulation and a 1.2–1.6-fold higher level of oxidative stress injury compared to the control samples. Moreover, the growth properties of shoots were only partially restored after transfer to a medium without the antibiotic. Microbiome analysis of the shoot samples using multivariable region-based 16S rRNA gene sequencing revealed a diverse microbial community in the control tobacco shoots, including 59 bacterial families; however, it was largely dominated by Mycobacteriaceae. Antibiotic treatment resulted in a decline in microbial diversity (the number of families was reduced 4.5-fold) and increased domination by the Mycobacteriaceae family. These results imply that the diversity of the plant-associated microbiome might represent a significant factor contributing to the efficient propagation of in vitro tissue culture

Stimulation of Nicotiana tabacum L. In Vitro Shoot Growth by Endophytic Bacillus cereus Group Bacteria

In vitro plant tissue cultures face various unfavorable conditions, such as mechanical damage, osmotic shock, and phytohormone imbalance, which can be detrimental to culture viability, growth efficiency, and genetic stability. Recent studies have revealed a presence of diverse endophytic bacteria, suggesting that engineering of the endophytic microbiome of in vitro plant tissues has the potential to improve their acclimatization and growth. Therefore, the aim of this study was to identify cultivated tobacco (Nicotiana tabacum L.) endophytic bacteria isolates that are capable of promoting the biomass accumulation of in vitro tobacco shoots. Forty-five endophytic bacteria isolates were obtained from greenhouse-grown tobacco plant leaves and were assigned to seven Bacillus spp. and one Pseudomonas sp. based on 16S rRNA or genome sequence data. To evaluate the bacterial effect on in vitro plant growth, tobacco shoots were inoculated with 22 isolates selected from distinct taxonomic groups. Four isolates of Bacillus cereus group species B. toyonensis, B. wiedmannii and B. mycoides promoted shoot growth by 11–21%. Furthermore, a contrasting effect on shoot growth was found among several isolates of the same species, suggesting the presence of strain-specific interaction with the plant host. Comparative analysis of genome assemblies was performed on the two closely related B. toyonensis isolates with contrasting plant growth-modulating properties. This revealed distinct structures of the genomic regions, including a putative enzyme cluster involved in the biosynthesis of linear azol(in)e-containing peptides and polysaccharides. However, the function of these clusters and their significance in plant-promoting activity remains elusive, and the observed contrasting effects on shoot growth are more likely to result from genomic sequence variations leading to differences in metabolic or gene expression activity. The Bacillus spp. isolates with shoot-growth-promoting properties have a potential application in improving the growth of plant tissue cultures in vitro