Isolation and Analysis of Anthocyanin Pathway Genes from Ribes Genus Reveals MYB Gene with Potent Anthocyanin-Inducing Capabilities.

Horticultural crops of the Ribes genus are valued for their anthocyanin-rich fruits, but until now, there were no data about the genes and regulation of their flavonoid pathway. In this study, the coding sequences of flavonoid pathway enzymes and their putative regulators MYB10, bHLH3 and WD40 were isolated, and their expression analyzed in fruits with varying anthocyanin levels from different cultivars of four species belonging to the Ribes genus. Transcription levels of anthocyanin synthesis enzymes and the regulatory gene RrMYB10 correlated with fruit coloration and anthocyanin quantities of different Ribes cultivars. Regulatory genes were tested for the ability to modulate anthocyanin biosynthesis during transient expression in the leaves of two Nicotiana species and to activate Prunus avium promoters of late anthocyanin biosynthesis genes in N. tabacum. Functional tests showed a strong capability of RrMyb10 to induce anthocyanin synthesis in a heterologous system, even without the concurrent expression of any heterologous bHLH, whereas RrbHLH3 enhanced MYB-induced anthocyanin synthesis. Data obtained in this work facilitate further analysis of the anthocyanin synthesis pathway in key Ribes species, and potent anthocyanin inducer RrMyb10 can be used to manipulate anthocyanin expression in heterologous systems.

Broad and Efficient Control of Klebsiella Pathogens by Peptidoglycan-Degrading and Pore-Forming Bacteriocins Klebicins.

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.

Plant-expressed pyocins for control of Pseudomonas aeruginosa.

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.

High-yield production of a functional bacteriophage lysin with antipneumococcal activity using a plant virus-based expression system.

Streptococcus pneumoniae is the causative agent of several serious infectious diseases. It is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are needed. One alternative to antibiotics may be the use of peptidoglycan hydrolases, the bacteriophage lytic enzymes. In this study, we demonstrated high level expression of the S. pneumoniae bacteriophage lysin Pal in Nicotiana benthamiana - TMV (Tobacco Mosaic Virus) transient expression system. The protein was purified to homogeneity and tested for streptococci killing activity in vitro and in vivo. In vitro, Pal was able to lyse three tested S. pneumoniae strains: NCTC12695, NCTC12977 and NCTC11888. The treatment of BALB/c mice with 100 µg, 200 µg and 400 µg of Pal 1h post-challenge with double lethal dose of S. pneumoniae NCTC12695 strain showed a clear dose response and protected from lethal sepsis 30%, 40% and 50% of mice, respectively. The improved mice survival correlated with decreased blood bacterial titers. In conclusion, these results suggest that plant-expressed bacteriophage lysins may have potential use as antimicrobial agents.