The safety of ß-carotene from Yarrowia lipolytica.

Crystalline ß-carotene from genetically modified Yarrowia lipolytica is an alternative source of ß-carotene for use as a nutritional supplement. To support the use of ß-carotene from Y. lipolytica as a food ingredient, the genotoxic and subchronic toxicity potential of this compound was determined. Genotoxicity was examined using Salmonella typhimurium and Escherichia coli (Ames test), a chromosomal aberration assay in Chinese Hamster Ovary WBL cells, and the micronucleus test in CD-1 mice. All three assays showed no significant results due to ß-carotene from Y. lipolytica. In a subchronic toxicity study in SD rats, ß-carotene from Y. lipolytica was administered by oral gavage for 13weeks at 0, 125, 250 or 500mg/kg per day. Adverse effects were not observed following clinical, clinical pathology and gross- and histopathological evaluations of dosed rats; thus, the no-observed-adverse effect level (NOAEL) for ß-carotene from Y. lipolytica was 500mg/kg, the highest dose used in the study. In conclusion, ß-carotene derived from Y. lipolytica was shown in genotoxicity models and a standard rat subchronic rat study to have a safety profile similar to that of the current commercial products (synthetic and natural) with no unexpected finding attributable to the alternative source.

Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans.

Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.

Using PATIMDB to create bacterial transposon insertion mutant libraries.

PATIMDB is a software package for facilitating the generation of transposon mutant insertion libraries. The software has two main functions: process tracking and automated sequence analysis. The process tracking function specifically includes recording the status and fates of multiwell plates and samples in various stages of library construction. Automated sequence analysis refers specifically to the pipeline of sequence analysis starting with ABI files from a sequencing facility and ending with insertion location identifications. The protocols in this unit describe installation and use of PATIMDB software.