Browsing by Author "Sihto, Henna-Maria"

Y. enterocolitica and Y. pseudotuberculosis are the third most common reported zoonoses after Campylobacter and Salmonella. Y. pseudotuberculosis has caused 10 outbreaks in Finland since 1997. The symptoms of yersiniosis include fever, right-sided abdominal pain and diarrhea. Post-infectious complications are common. Refrigeration is the most common modern food preservation method, however, as psychrotrophs, enteropathogenic Yersinia are able to grow at refrigerator temperatures. However, the mechanism behind psychrotrophy is still unknown. The aim of this study was to determine the expression levels of rstB, evgS and the genes coding for the σE- and σS-factors of Y. pseudotuberculosis IP 32953 at 30 °C and after temperature drop to 5 °C. Total RNA had been extracted before and 30 minutes, 3, and 7 hours after the temperature downshift for previously performed DNA microarray gene expression studies. Low temperature had been found to increase the expression levels of these genes in these studies. The gene expression levels were determined using real-time quantitative PCR (RT-qPCR). RNA-samples were reverse transcribed into complementary DNA (cDNA), which was used as a template in the qPCR-reactions. The gene expression levels at different time points were determined by measuring the signal emitted by the fluorescent dye. THE ∆∆Ct-method was used for relative quantification. The 16S rRNA (locus tag YPTB_RNA_102) was used for normalization. The expression levels of all the studied genes were approximately doubled 30 minutes after the cold shock demonstrating the significance of the genes in the immediate cold shock response. The expression levels were at highest 3 hours after the cold shock: 2,4-fold for rstB, 4,4-fold for evgS, 6,6-fold for sigmaE and 5,6-fold for rpoS compared with the expression level before the cold shock. The expression levels of all the four genes were decreased 7 hours after the cold shock illustrating adaptation to the lower temperature. The results confirm the findings of the DNA microarray gene expression experiments done previously in the research group. In the future the knowledge of the genes involved in the cold shock response can be utilized in controlling the growth of Y. pseudotuberculosis in foodstuffs.