Protein Gene Expression Profiling of Yersinia ruckeri isolates grown at different temperatures


Önalan Ş. , Görgişen G. , Ergöz B.

Fresenius Environmental Bulletin, cilt.29, sa.12, ss.11677-11684, 2020 (SCI Expanded İndekslerine Giren Dergi)

  • Cilt numarası: 29 Konu: 12
  • Basım Tarihi: 2020
  • Dergi Adı: Fresenius Environmental Bulletin
  • Sayfa Sayıları: ss.11677-11684

Özet

Yersiniosis infection is frequently seen in

aquatic products and causes great economic losses.

Infection occurs at water temperatures of 15-22 oC.

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 Gramnegative

and catalase oxidase were positive. The

motility was lost at 37 oC. 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 oC, 0.1777 at 20 oC,

0.1820 at 25 oC, and 0.1857 at 37 oC. 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 oC 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 temperaturedependent

expression levels decreased, it was found

to be around 40 kDa and 45 kDa.