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.

Mycoplasma non-coding RNA: identification of small RNAs and targets.

BACKGROUND: Bacterial non-coding RNAs act by base-pairing as regulatory elements in crucial biological processes. We performed the identification of trans-encoded small RNAs (sRNA) from the genomes of Mycoplama hyopneumoniae, Mycoplasma flocculare and Mycoplasma hyorhinis, which are Mycoplasma species that have been identified in the porcine respiratory system. RESULTS: A total of 47, 15 and 11 putative sRNAs were predicted in M. hyopneumoniae, M. flocculare and M. hyorhinis, respectively. A comparative genomic analysis revealed the presence of species or lineage specific sRNA candidates. Furthermore, the expression profile of some M. hyopneumoniae sRNAs was determined by a reverse transcription amplification approach, in three different culture conditions. All tested sRNAs were transcribed in at least one condition. A detailed investigation revealed a differential expression profile for two M. hyopneumoniae sRNAs in response to oxidative and heat shock stress conditions, suggesting that their expression is influenced by environmental signals. Moreover, we analyzed sRNA-mRNA hybrids and accessed putative target genes for the novel sRNA candidates. The majority of the sRNAs showed interaction with multiple target genes, some of which could be linked to pathogenesis and cell homeostasis activity. CONCLUSION: This study contributes to our knowledge of Mycoplasma sRNAs and their response to environmental changes. Furthermore, the mRNA target prediction provides a perspective for the characterization and comprehension of the function of the sRNA regulatory mechanisms.