Evaluation of growth and gene expression of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in defined medium
Mycoplasmas belong to the Mollicutes class and possess low GC content and lack a cell wall, and also simplified metabolic pathways. Due to its reduced metabolic ability mycoplasmas are fastidious organisms growing with difficult under laboratory conditions. Its complex nutritional requirements render mycoplasmas to depend on external supplies of biosynthetic precursors. Aiming to develop and test defined media that could be used as a tool for Mycoplasma research, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis were cultivated in a complex medium supplemented with serum (Friis broth) and in four different defined media (YUS, YUSm, CMRL and CMRL+, that was developed in the present study). The cell concentration of both Mycoplasma species was assessed, by flow cytometry. Cellular viability was also analyzed in all defined media, indicating the presence of viable mycoplasma cells. All the defined media tested were able to maintain cell concentrations and viability and, amongst them, CMRL+ was the most suitable. For both Mycoplasma species, only the CMRL+ media showed similar cell density when compared to the complex medium. The transcriptional response of M. hyopneumoniae in CMRL+ broth was assessed by RT-qPCR, and the transcriptional profile of 18 genes in three cultures conditions (standard, heat shock and oxidative stress) was analyzed demonstrating gene expression regulation in response to the medium composition and to the culture conditions tested. The medium developed enables the definition of mycoplasmal nutritional requirements and metabolic pathways as well as genetic analysis.
KeywordsMycoplasma Growth measurement Defined medium Gene regulation Flow cytometry
This work was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES. CAPES—Biologia Computacional (Process Number: 23038.010043/2013-02) and Ministério da Ciência, Tecnologia e Inovação/Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCTI/CNPq) Universal (Process Number: 445228/2014-8).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving human participants and/or animals
No human nor animal subjects were involved in this study.
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