Project description:Medium chain fatty acids (MCFA) have been shown to inhibit methanogenesis, disrupt the cell envelope, and decrease survival of Methanobrevibacter ruminantium M1 in a dose-, time-, and protonation level dependent way. However, the exact mechanisms behind these observations are still unknown. Although the biochemistry of the metabolic processes of M. ruminantium has been well studied and its genome sequence is now available, little is known about the overall transcriptome regulation of M. ruminantium in response to inhibitors like MCFA. In the present study, we used RNA Sequencing to evaluate the effects of lauric acid (C12) on M. ruminantium. Pure M. ruminantium cell cultures in the mid-exponential growth phase were exposed to C12 in concentrations of 0.4 mg/mL, dissolved in DMSO for 1 h, and the transcriptomic changes compared to DMSO-only treated control samples (final DMSO concentration 0.2 %), were investigated. Gene expression changes upon exposure to C12 were not dramatic in magnitude (log2 fold change mostly below +/- 3) and in gene number (214 genes). However, the observed expression changes affected mostly genes which encoded cell-surface associated proteins (adhesion-like proteins, membrane-associated transporters and hydrogenases), or proteins, which were involved in detoxification or DNA repair processes. The transcriptional response of M. ruminantium M1 to C12 did not specifically inhibit methanogenesis. Instead, the data indicated a non-specific antimicrobial action by lauric acid, which involves destruction of the cell membrane and interferences with cellular energetics in M. ruminantium. To date, there has been no systematic characterization of a ruminal methanogen transcriptome by deep sequencing. Our results give first hints on the molecular inhibitory mechanism of C12 on M. ruminantium.
Project description:Medium chain fatty acids (MCFA) have been shown to inhibit methanogenesis, disrupt the cell envelope, and decrease survival of Methanobrevibacter ruminantium M1 in a dose-, time-, and protonation level dependent way. However, the exact mechanisms behind these observations are still unknown. Although the biochemistry of the metabolic processes of M. ruminantium has been well studied and its genome sequence is now available, little is known about the overall transcriptome regulation of M. ruminantium in response to inhibitors like MCFA. In the present study, we used RNA Sequencing to evaluate the effects of lauric acid (C12) on M. ruminantium. Pure M. ruminantium cell cultures in the mid-exponential growth phase were exposed to C12 in concentrations of 0.4 mg/mL, dissolved in DMSO for 1 h, and the transcriptomic changes compared to DMSO-only treated control samples (final DMSO concentration 0.2 %), were investigated. Gene expression changes upon exposure to C12 were not dramatic in magnitude (log2 fold change mostly below +/- 3) and in gene number (214 genes). However, the observed expression changes affected mostly genes which encoded cell-surface associated proteins (adhesion-like proteins, membrane-associated transporters and hydrogenases), or proteins, which were involved in detoxification or DNA repair processes. The transcriptional response of M. ruminantium M1 to C12 did not specifically inhibit methanogenesis. Instead, the data indicated a non-specific antimicrobial action by lauric acid, which involves destruction of the cell membrane and interferences with cellular energetics in M. ruminantium. To date, there has been no systematic characterization of a ruminal methanogen transcriptome by deep sequencing. Our results give first hints on the molecular inhibitory mechanism of C12 on M. ruminantium. RNA profiles of M. ruminantium treated in vitro (lauric acid disolved in DMSO), non-treated=controls (DMSO supplementation) and non-treated=blanks (no supplementation) were generated by deep sequencing, in triplicates, by using Illumina HiSeq2500
Project description:In order to study the transcriptome of the pathogen, Ehrlichia ruminantium, specific microoarray was designed and validated using genomic DNA of Gardel and Welgevonden strains. Gardel strain was isolated in Guadeloupe and Welgevonden strain in South Africa. DNA from Ehrlichia ruminantium was extracted from cell culture infected with Gardel passage 40 and Welgevonden passage 11, using QIAmp kit (Qiagen). Ehrlichia ruminantium DNA was labeled using BioPrime array CGH labeling system kit (Invitrogen) and Cy3-dCTP (Amersham). Arrays were incubated at 60°C for 20 hours in hybridization chamber. After hybridization, arrays were washed according to the Agilent protocol. Arrays were scanned and the signal intensity of all spots were quantified by Genepix pro 6.0 (Molecular Device Corporation) and data were saved for further analysis.