A Toxicity Screening Approach to Identify Bacteriophage-Encoded Anti-Microbial Proteins

The rapid emergence of antibiotic resistance among many pathogenic bacteria has created a profound need to discover new alternatives to antibiotics. Bacteriophages, the viruses of microbes, express special proteins to overtake the metabolism of the bacterial host they infect, the best known of which are involved in bacterial lysis. However, the functions of majority of bacteriophage encoded gene products are not known, i.e., they represent the hypothetical proteins of unknown function (HPUFs). In the current study we present a phage genomics-based screening approach to identify phage HPUFs with antibacterial activity with a long-term goal to use them as leads to find unknown targets to develop novel antibacterial compounds. The screening assay is based on the inhibition of bacterial growth when a toxic gene is expression-cloned into a plasmid vector. It utilizes an optimized plating assay producing a significant difference in the number of transformants after ligation of the toxic and non-toxic genes into a cloning vector. The screening assay was first tested and optimized using several known toxic and non-toxic genes. Then, it was applied to screen 94 HPUFs of bacteriophage φR1-RT, and identified four HPUFs that were toxic to Escherichia coli. This optimized assay is in principle useful in the search for bactericidal proteins of any phage, and also opens new possibilities to understanding the strategies bacteriophages use to overtake bacterial hosts.

Genotoxic Profile and Morphological Variation of the Amanita rubescens Complex: Traditional Knowledge for Safe Consumption in Mexico

Wild mushrooms are important to the nutritional health and economic subsistence of rural populations in Mexico, but inaccurate identification of mushrooms has led to reported cases of poisoning. The aim of this study is to establish genotoxic profiles of mushrooms of the putative Amanita rubescens complex and to link those profiles with morphological attributes that suggest a correct identification of mushrooms, in order to prevent poisoning. Several combinations of amplification products (AMA, PHA, POP1, and POP2 genes) were identified in A. rubescens fungi sold in traditional markets; these genes are related to the presence of toxic polypeptides and its enzymatic regulators. The sequences correspond to a previously reported toxic gene family (MSDIM). All samples with the complete toxic gene profile presented reddish to dark-brown sporomes; this is the only attribute that visually distinguishes samples with toxic potential. Our results suggest that the mushrooms sold in traditional Mexican markets do not correspond to the A. rubescens complex. We conclude that morphological variability allows for identification of edible and inedible mushrooms.

The Modulation of Chitosan-DNA Interaction by Concentration and pH in Solution

Chitosan has been widely used to prepare a DNA carrier for highly efficient and non-toxic gene therapy. In the present study, we investigated DNA charge neutralization and compaction by chitosan in solutions of various pH levels by dynamic light scattering (DLS), magnetic tweezers (MT), and atomic force microscopy (AFM). We found that when chitosan concentration is higher than a critical value (0.2 µM), corresponding the ratio of phosphate and NH2 in chitosan k = 1.9 , the electrophoretic mobility of DNA-chitosan complex maintains an almost constant value when pH of solution is less 6.5, the isoelectric point of chitosan. Then it decreases with increasing pH of solution. However, when chitosan concentration is lower than the critical value, the mobility of the complex increases with pH in the range of acidity and reaches the maximum when the pH of the solution approaches the isoelectric point of chitosan. It finally decreases with increasing pH in solutions. The corresponding condensing force of the DNA-chitosan complex measured by single molecular MT changes accordingly with its charge neutralization in the same solution concentration (20 µM) and is consistent with the DLS measurements. This phenomenon might be related to the weakening interaction between DNA and chitosan in low pH solutions, and is verified by measuring the ratio of free chitosan to DNA complex in solutions. We also observed the various morphologies of DNA-chitosan complexes, such as ring, rod, flower, braid, and other structures, under different degrees of deacetylation, molecular weight, solution concentration and pH in solutions by AFM.

A core–shell structured polyplex for efficient and non-toxic gene delivery

We developed a core–shell polyplex with minimal high generation dendrimer to condense DNA and low-molecular-weight linear polyethylenimine coated on the core. The polyplex represented both high transfection efficacy and low toxicity.

Construction of Genome Library for the Toxic Gene of Alexandium minutum

Genome library of toxic Alexandium minutum were constructed. A 1014bp DNA reconstruction fragment was obtained and PCR method testified that this fragment had strong amplification signal in toxic strains while no signal in non-toxic strains. The external sequence of the DNA fragment was analyzed by inverse PCR method, and a 240bp nucleotide sequence which translated into proteins aminophenol was Methionine Aminopeptidase (MAP). The sequence of aminophenol was BLAST in NCBI, and found that it had 97% similarity with Alexandium fundyense. This sequence was exact uniform with the reported sequence in the coding region, but there were three bases mutated in non-coding region behind stop codon. Probably, in the course of growth metabolism of Alexandium minutum, MAP executed different physiological functions and controlled the production of the toxin. In the meantime, map gene induced encoding products of the related poisonous gene, and enabled it has active expressions to starting toxin synthesis.