Protein Gene Expression Profiling of Yersinia Ruckeri Isolates Grown at Different Temperatures

Abstract

Yersiniosis infection is frequently seen in aquatic products and causes great economic losses. Infection occurs at water temperatures of 15-22 degrees C. In this study, the Yersinia ruckeri strain was developed in the TSA medium and at 1.0 OD density at 600 nm. Then, 100ul of the same concentration of suspension was added to the 25 ml TSA and 25 ml SW medium and incubated for 24 hours at different temperatures (15, 20, 25, 37). Gram staining, catalase, oxidase, and motility tests of developing bacteria were investigated. The API 20E kit was used according to the manufacturer's instructions to determine phenotypic characterization and temperature-related differences. The molecular identification of the agent at different temperatures at the same density was carried out in Real-Time PCR with the primer assay specific for the 16S rRNA gene. Western blot analysis was performed to determine the differences in expression at the protein level at different temperatures. The results of the study revealed that TSA and SW media developed in the media and Gram-negative and catalase oxidase were positive. The motility was lost at 37 degrees C. In the results of phenotypic characterization performed with API 20E, incubation at different temperatures revealed differences in CIT, VP, and GEL tests on the same bacteria. Y. ruckeri strains of the same optical density in the follow-up of the DNA isolation of 16S rRNA gene region-specific primer assays performed Real-Time PCR analysis of all of the bacterial DNA used in the same optic density (OD) because of the very close to the Ct values were found to be positive and give sigmoidal curves give positive results. The results were also confirmed after the analysis whether the samples reacted with the use of standards and automatic threshold values. After the formation of PCR amplicons, melting curve analysis revealed peaks formed by melting curves and it was understood that there was no false positivity. Total protein levels obtained by the Bradford method were determined as 0.1384 at 15 degrees C, 0.1777 at 20 degrees C, 0.1820 at 25 degrees C, and 0.1857 at 37 degrees C. It was found that there was no change in the gene expression profile of the same bacterium developed in TSA and SW media. Given the results of the protein expression obtained from the page ruler prestained protein ladder, significant changes in the level of gene expression are observed under different temperature conditions of the strain. One of the most striking ones is the gene product seen in the 20 and 25 degrees C groups at the level of 110 kDa. While this gene product is not expressed at low and high temperatures, the optimum level of expression is significantly increased. Increased gene products due to temperature increase are seen at approximately 90 kDa, 65 kDa, and 30 kDa. When the temperature-dependent expression levels decreased, it was found to be around 40 kDa and 45 kDa.