Genetic Transformation of Plasmid DNA into Escherichia coli Using High Frequency Electromagnetic Energy.

We present a novel technique of genetic transformation of bacterial cells mediated by high frequency electromagnetic energy (HF EME). Plasmid DNA, pGLO (5.4 kb), was successfully transformed into Escherichia coli JM109 cells after exposure to 18 GHz irradiation at a power density between 5.6 and 30 kW m-2 for 180 s at temperatures ranging from 30 to 40 °C. Transformed bacteria were identified by the expression of green fluorescent protein (GFP) using confocal scanning microscopy (CLSM) and flow cytometry (FC). Approximately 90.7% of HF EME treated viable E. coli cells exhibited uptake of the pGLO plasmid. The interaction of plasmid DNA with bacteria leading to transformation was confirmed by using cryogenic transmission electron microscopy (cryo-TEM). HF EME-induced plasmid DNA transformation was shown to be unique, highly efficient, and cost-effective. HF EME-induced genetic transformation is performed under physiologically friendly conditions in contrast to existing techniques that generate higher temperatures, leading to altered cellular integrity. This technique allows safe delivery of genetic material into bacterial cells, thus providing excellent prospects for applications in microbiome therapeutics and synthetic biology.

Anti-biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins.

The broad-spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio-control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan-rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan-rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD-based peptides PuroA (FPVTWRWWKWWKG-NH2 ) and Pina-M (FSVTWRWWKWWKG-NH2 ) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG-NH2 ) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non-pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina-M at 2 × MIC prevented initial biomass attachment by 85-90% and inhibited >90% of 6-h preformed biofilms of all three organisms. However Hina, with a substitution of Lys-9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan-rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan-rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Unraveling the Effects of Cationic Peptides on Vesicle Structures: Insights into Peptide-Membrane Interactions.

Antimicrobial resistance is a pressing global health issue, with millions of lives at risk by 2050, necessitating the development of alternatives with broad-spectrum activity against pathogenic microbes. Antimicrobial peptides provide a promising solution by combating microbes, modulating immunity, and reducing resistance development through membrane and intracellular targeting. PuroA, a synthetic peptide derived from the tryptophan-rich domain of puroindoline A, exhibits potent antimicrobial activity against various pathogens, while the rationally designed P1 peptide demonstrates enhanced antimicrobial activity with its specific composition. This paper investigates the concentration-dependent effects of these cationic peptides on distinct types of vesicles representing strong-negative bacterial cell membranes (S-vesicles), weak-negative bacterial cell membranes (W-vesicles), and mammalian cell membranes (M-vesicles). To investigate the interactions between the peptides and vesicles, small-angle neutron scattering experiments were conducted. The cationic peptides, PuroA and P1, interact with S-vesicles through electrostatic interactions, leading to distinct effects. PuroA accumulates on the vesicle surface, increasing Rcore and Rtotal, aligning with the carpet model. P1 disrupts the vesicle structure at higher concentrations, consistent with the detergent model. Neither peptide significantly affects W-vesicles, emphasizing the role of charge. In uncharged M-vesicles, both peptides decrease Rcore and Rtotal and increase tshell, indicating peptide insertion and altered bilayer properties. These findings provide valuable insights into peptide-membrane interactions and their impact on vesicle structures. Furthermore, the implications of these findings extend to the potential development of innovative antimicrobial agents and drug delivery systems that specifically target bacterial and mammalian membranes. This research contributes to the advancement of understanding peptide-membrane interactions and lays the foundation for the design of approaches for targeting membranes in various biomedical applications.

Stability of puroindoline peptides and effects on wheat rust.

Peptides modelled on the tryptophan rich domain of puroindolines and the related grain softness protein-1 have a broad range of antibacterial and antifungal activities. With the aims of further investigating the activities of these antimicrobial peptides we studied their activity against wheat rust diseases and environmental stability. PINA-based peptides were found to have high pH and thermal stability in addition to being stable over long periods at room temperature. These properties could make them excellent candidates as preservatives in food. PuroA, Pina-R39G and PuroB peptides adversely affected the morphology of the stripe rust spores (Puccinia striiformis f. sp. tritici), while PuroA and PuroB showed moderate inhibition of their germination. Additionally, GSP-5D reduced the germination of leaf rust spores (P. triticina). PuroA and PuroB sprayed onto stripe rust infected plants effected a moderate reduction in the number of stripe rust uredinia on wheat seedlings, as did PuroB sprayed onto the seedlings and allowed to coat the leaves for 5 day prior to spore infection. The results suggest that the presence of the PIN-based peptides may lower frequency of initial infection foci.

The antimicrobial domains of wheat puroindolines are cell-penetrating peptides with possible intracellular mechanisms of action.

The puroindoline proteins (PINA and PINB) of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD). Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs) made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae) cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity.