The emergence, persistence and spread of antibiotic-resistant human pathogenic bacteria heralds a growing global health crisis. Drug-resistant strains of gram-negative bacteria, such as Pseudomonas aeruginosa, are especially dangerous and the medical and economic burden they impose underscore the critical need for finding new antimicrobials. Recent studies have demonstrated that plant-expressed bacteriocins of the colicins family can be efficient antibacterials against all major enteropathogenic strains of E. coli. We extended our studies of colicin-like bacteriocins to pyocins, which are produced by strains of P. aeruginosa for ecological advantage against other strains of the same species. Using a plant-based transient expression system, we expressed six different pyocins, namely S5, PaeM, L1, L2, L3 and one new pyocin, PaeM4, and purified them to homogeneity. Among these pyocins, PaeM4 demonstrated the broadest spectrum of activity by controlling 53 of 100 tested clinical isolates of P. aeruginosa. The activity of plant-made pyocins was confirmed in the agar drop, liquid culture susceptibility and biofilm assays, and in the Galleria mellonella animal infection model.
Gram-negative bacteria belonging to the genus Klebsiella are important nosocomial pathogens, readily acquiring resistance to all known antibiotics. Bacteriocins, non-antibiotic antibacterial proteins, have been earlier proposed as potential therapeutic agents for control of other Gram-negative species such as Escherichia, Pseudomonas and Salmonella. This study is the first report describing pore-forming and peptidoglycan-degrading bacteriocins klebicins from Klebsiella. We have identified, cloned, expressed in plants and characterized nine pore-forming and peptidoglycan-degrading bacteriocins from different Klebsiella species. We demonstrate that klebicins can be used for broad and efficient control of 101 of the 107 clinical isolates representing five Klebsiella species, including multi-drug resistant pathovars and pathovars resistant to carbapenem antibiotics.
Agrotransfection with viral vectors is an effective solution for the transient production of valuable proteins in plants grown in contained facilities. Transfection methods suitable for field applications are desirable for the production of high-volume products and for the transient molecular reprogramming of plants. The use of genetically modified (GM) Agrobacterium strains for plant transfections faces substantial biosafety issues. The environmental biosafety of GM Agrobacterium strains could be improved by regulating their T-DNA transfer via chemically inducible expression of virE2, one of the essential Agrobacterium virulence genes. In order to identify strong and stringently regulated promoters in Agrobacterium strains, we evaluated isopropyl-β-d-thiogalactoside-inducible promoters Plac, Ptac, PT7/lacO, and PT5/lacOlacO and cumic acid-inducible promoters PlacUV5/CuO, Ptac/CuO, PT5/CuO, and PvirE/CuO. Nicotiana benthamiana plants were transfected with a virE2-deficient A. tumefaciens strain containing transient expression vectors harboring inducible virE2 expression cassettes and containing a marker green fluorescent protein (GFP) gene in their T-DNA region. Evaluation of T-DNA transfer was achieved by counting GFP expression foci on plant leaves. The virE2 expression from cumic acid-induced promoters resulted in 47 to 72% of wild-type T-DNA transfer. Here, we present efficient and tightly regulated promoters for gene expression in A. tumefaciens and a novel approach to address environmental biosafety concerns in agrobiotechnology.
High-value pharmaceutical products are already successfully produced in contained facilities using Agrobacterium-mediated transient transformation of plants. However, transfection methods suitable for open field applications are still desirable as a cheaper alternative. Biosafety concerns related to the use of recombinant agrobacteria in an industrial transfection process include possible transformation or transfection of unintended hosts or spread of the genetically modified agrobacteria in the environment. In this paper, we explored a novel biocontrol approach resulting in greater biosafety of the transient expression process in plants. Our proposed solution involves inducible expression of Agrobacterium tumefaciens toxin PemK and antitoxin PemI that provides for strictly regulated T-DNA transfer from agrobacteria to plants. We also identified several other toxins from putative Agrobacterium toxin-antitoxin modules and demonstrate their potential usefulness in the control of Agrobacterium tumefaciens as a DNA vector.
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