Endophytic and ectophytic potato-associated bacterial communities differ in structure and antagonistic function against plant pathogenic fungi

Differences between endophytic and ectophytic bacterial communities with stress on antagonistic bacteria, were studied by comparing the composition of communities isolated from the rhizosphere, phyllosphere, endorhiza and endosphere of field-grown potato plants using a multiphasic approach. Terminal restriction fragment length polymorphism analysis of 16S rDNA of the bacterial communities revealed discrete microenvironment-specific patterns. To measure the antagonistic potential of potato-associated bacteria, a total of 2648 bacteria were screened by dual testing of antagonism to the soilborne pathogens Verticillium dahliae and Rhizoctonia solani. Composition and diversity of bacterial antagonists were mainly specific for each microenvironment. The rhizosphere and endorhiza were the main reservoirs for antagonistic bacteria and showed the highest similarity in their colonisation by antagonists. The most prominent species of all microenvironments was Pseudomonas putida, and rep-PCR with BOX primers showed that these isolates showed microenvironment-specific DNA fingerprints. P. putida isolates from the rhizosphere and endorhiza gave nearly identical fingerprints confirming the high similarity of bacterial populations. The phlD gene, involved in the production of the antibiotic 2,4-diacetyl-phloroglucinol, was found only among Pseudomonas isolates from the rhizosphere and endorhiza. Evaluation of the bacterial isolates for biocontrol potential based on fungal antagonism and physiological characteristics resulted in the selection of five promising isolates from each microenvironment. The most effective isolate was Serratia plymuthica 3Re4-18 isolated from the endorhiza.     

Kinetic and structural studies of the allosteric conformational changes induced by binding of cAMP to the cAMP receptor protein from Escherichia coli

The cAMP receptor protein, allosterically activated by cAMP, regulates the expression of more than 100 genes in Escherichia coli. CRP is a homodimer of two-domain subunits. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP binding domain to the C-terminal domain of the protein responsible for interaction with specific sequences of DNA. In this study, the stopped-flow and time-resolved fluorescence lifetime measurements were used to observe the kinetics of the distance changes between the N-terminal and C-terminal domain of CRP induced by binding of cAMP to high-affinity binding sites. In these measurements, we used the constructed CRP heterodimer, which possesses a single Trp85 residue localized at the N-terminal domain of one CRP subunit, and fluorescently labeled by 1,5-I-AEDANS Cys178 localized at the C-terminal domain of the same subunit or at the opposite one. The F?rster resonance energy transfer method has been used to study the distance changes, induced by binding of cAMP, between Trp85 (fluorescence donor) and Cys178-AEDANS (fluorescence acceptor) in the CRP structure. The obtained results show that the allosteric transitions of CRP at micromolar cAMP concentrations follow the sequential binding model, in which binding of cAMP to high-affinity sites causes a 4 A movement of the C-terminal domain toward N-terminal domains of the protein, with kinetics faster than 2 ms, and CRP adopts the "closed" conformation. This fast process is followed by the slower reorientation of both CRP subunits.     

Microfluidic chip-based single-cell cloning to accelerate biologic production timelines

Cell line development (CLD) represents a critical, yet time-consuming, step in the biomanufacturing process as significant resources are devoted to the scale-up and screening of several hundreds to thousands of single-cell clones. Typically, transfected pools are fully recovered from selection and characterized for growth, productivity, and product quality to identify the best pools suitable for single-cell cloning (SCC) using limiting dilution or fluorescence-activated cell sorting (FACS). Here we report the application of the Berkeley Lights Beacon Instrument (BLI) in an early SCC process to accelerate the CLD timeline. Transfected pools were single-cell cloned when viabilities reached greater than 85% or during selection when viabilities were less than 30%. Clones isolated from these accelerated processes exhibited comparable growth, productivity, and product quality to those derived from a standard CLD process and fit into an existing manufacturing platform. With these approaches, up to a 30% reduction in the overall CLD timeline was achieved. Furthermore, early process-derived clones demonstrated equivalent long-term stability compared with standard process-derived clones over 50 population doubling levels (PDLs). Taken together, the data supported early SCC on the BLI as an attractive approach to reducing the standard CLD timeline while still identifying clones with acceptable manufacturability.     

Imperatorin–pharmacological meaning and analytical clues: profound investigation

Imperatorin, a furanocoumarin derivative, has many documented pharmacological properties which make it a candidate for possible drug development. In this review, the activity on the central nervous system, the anticancer and antiviral properties and the influence on the cardiovascular system are described. The aim of this review is also to present an overview of the techniques used for the analysis, isolation, and separation of imperatorin from plant material from the practical perspective.