ena-STUDY-IOW-12-10-2017-16:12:00:235-65
Testing the short term and long term effects of fixation on natural marine microbial community metatranscriptomes
Studies of microbial functional dynamics are challenged by the swift turnover of transcripts as the physiological stresses induced by sampling and filtration can alter gene expression profiles. Fortunately distortion of metatranscriptomes can be avoided by fixing seawater in situ with a minimum of 10% volume-to-volume concentration of an alcohol-based fixative. We examined the lower limits of sample volumes and the immediate and long-term preservation of transcripts from a microbial community fixed with 1% and 10% (v/v) stop solution to assess inactivation of biological activity and stability of gene expression levels over time. Stop solution 1% did not inactivate bacterial cells, while 10% inactivated biological activity immediately but caused changes in the quality of RNA. The differences in gene expression observed between the seawater and fixed communities are explained by the assortment of filtration stress on unfixed cells and a latent RNase activity under 10% stop solution before final permanent inactivation. The negligible differential expressions after 12h and for one week concur with a short-lived residual activity and irreversible deactivation by the fixative. Therefore, the use of 10% stop solution for in situ fixation, combined with an autonomous multi-sampler, can improve the collection of time-series and deep-sea investigations of microbial activities.
Studies of microbial functional dynamics are challenged by the swift turnover of transcripts as the physiological stresses induced by sampling and filtration can alter gene expression profiles. Fortunately distortion of metatranscriptomes can be avoided by fixing seawater in situ with a minimum of 10% volume-to-volume concentration of an alcohol-based fixative. We examined the lower limits of sample volumes and the immediate and long-term preservation of transcripts from a microbial community fixed with 1% and 10% (v/v) stop solution to assess inactivation of biological activity and stability of gene expression levels over time. Stop solution 1% did not inactivate bacterial cells, while 10% inactivated biological activity immediately but caused changes in the quality of RNA. The differences in gene expression observed between the seawater and fixed communities are explained by the assortment of filtration stress on unfixed cells and a latent RNase activity under 10% stop solution before final permanent inactivation. The negligible differential expressions after 12h and for one week concur with a short-lived residual activity and irreversible deactivation by the fixative. Therefore, the use of 10% stop solution for in situ fixation, combined with an autonomous multi-sampler, can improve the collection of time-series and deep-sea investigations of microbial activities.