Absence of YbeY RNase compromises the growth and enhances the virulence plasmid gene expression of Yersinia enterocolitica O:3.

YbeY was recently recognized as an endoribonuclease playing a role in ribosome biosynthesis. In Escherichia coli it functions as a single-strand-specific RNase that processes the 3' end of the 16S rRNA and is crucial for the late-stage 70S ribosome quality control system. Here we report that YbeY is not essential in Yersinia enterocolitica serotype O:3, yet its absence strongly compromised the bacterium. The lack of YbeY resulted in misprocessing of 16S rRNA and a severe decrease of growth rate with complete growth arrest observed at elevated temperatures. Moreover, a ybeY mutation severely disturbed regulation of the Yersinia virulence plasmid (pYV) genes and affected the expression of regulatory small RNA species. Transcription of the pYV genes was upregulated in the ybeY mutant at 22 °C; the same genes were repressed in the wild-type bacterium. Furthermore, ybeY inactivation impaired many virulence-related features, such as resistance to elevated temperature and acid, and hindered utilization of different carbohydrates. In addition, the ybeY mutant strain showed decreased infectivity in a tissue culture infection model, especially at the stage of cell adhesion. Taken together, this study demonstrates the crucial role of YbeY in Y. enterocolitica O:3 physiology and pathogenicity.

Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization.

Onconase, an RNAse extracted from embryos of the Northern leopard frog (Rana pipiens), is in a confirmatory phase IIIb clinical trial for the treatment of unresectable malignant mesothelioma. Because the current purification process for onconase is cumbersome and laborious, the development of more efficient and cost-effective alternative sources is imperative. In this study, we assessed the potential of Pichia pastoris as an expression host for the large-scale production of onconase. Because of its specific N-terminal structure, active onconase with a correct N-terminus could not be secreted by an alpha-mating factor (alpha-MF)-prepro secretion signal, and an alpha-MF-pre secretion signal should be used instead. Onconase accumulated to a high concentration (about 300 and 150 mg L(-1) for glycosylated onconase and aglycosylated mutein, respectively) in high cell density fermentation, and was purified to homogeneity with high yields (56% for glycosylated onconase and 67% for aglycosylated mutein) by a simple purification process consisting of cation exchange chromatography and size exclusion chromatography. In vitro activity assays revealed that glycosylation decreased both the RNAse activity and the cytotoxic activity of onconase. The high expression level and subsequent facile purification process make P. pastoris an efficient and cost-effective host for the large-scale production of onconase.