Evgeniya Bystritskaya1,2, Anna Stenkova1,2, Dmitriy Chistuylin1, Nadezhda Chernysheva1,2, Valentina Khomenko1, Stanislav Anastyuk1, Olga Novikova1, Alexander Rakin3, and Marina Isaeva1,2
- G.B. Elyakov Pacific Institute of Bioorganic Chemistry FEB RAS, 690022 Prospect 100-let Vladivostoku 159, Vladivostok, Russia
- The School of Biomedicine, Far Eastern Federal University, 690950 Suchanova str. 8, Vladivostok, Russia
- Max von Pettenkofer Institute for Hygiene and Clinical Microbiology of Ludwig Maximilians-University, 80336 Pettenkofer str. 9a, Munich, Germany
The capability of Yersinia ruckeri to survive in the aquatic systems reflects its adaptation (most importantly through the alteration of membrane permeability) to the unfavorable environments. The nonspecific porins are a key factor contributing to the permeability. Here we studied the influence of the stimuli, such as temperature, osmolarity, and oxygen availability on regulation of Y. ruckeri porins. Using qRT-PCR and SDS-PAGE methods we found that major porins are tightly controlled by temperature. Hyperosmosis did not repress OmpF production. The limitation of oxygen availability led to decreased expression of both major porins and increased transcription of the minor porin OmpY. Regulation of the porin balance in Y. ruckeri, in spite of some similarities, diverges from that system in Escherichia coli. The changes in porin regulation can be adapted in Y. ruckeri in a species-specific manner determined by its aquatic habitats.
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To identify proteins in the sample of a Y. ruckeri bacterial culture, a MALDI-TOF mass spectrometer modified with a CovalX HM1 detection system was used. Prior to analysis, the porins were isolated and purified before being treated with 0.5% sodium sarcosylate solution for 12 hours and then centrifuged. Then, the precipitate was suspended in 30 mmol/L Tris-HCl (Buffer A) which contained 2% SDS and was centrifuged for 1 hour at 25,000 g. In the precipitate, the complex containing peptidoglycan (PG) and OM proteins was treated with DNAse before the PG-associated protein fraction was extracted using Buffer A with 1% SDS and 0.5 mol/L NaCl. The sample was mixed with the matrix (3,5-dimethoxy-4-hydrocinnamic acid in an acetonitrile-0.1% TFA mixture). The sample/matrix mixture was spotted onto a target and dried.
The mass spectrometry was used to identify the molecular masses of porins. The data from the analysis showed the presence of two proteins whose masses corresponded to those of OmpF and OmpC that had been previously calculated.