Differential expression analysis of a hyperthermophilic archeaon Methanocaldococcus jannaschii upon hydrogen limitation and hydrogen syntrophy with Thermococcus paralvinellae

The purpose of this experiment was to compare the transcriptomes of M. jannaschii using RNA-Seq gene expression analyses to understand the physiology of this organism when it is grown under H2-replete, H2-limited and H2-syntrophy conditions. The RNA-seq reads were mapped to both M. jannaschii and T. paralvinellae genomes using BBSplit from BBMap package. BBSplit is an aligner tool that bins sequencing reads by mapping to them multiple references simultaneously and separates the reads that map to multiple references to a special "ambiguous" file for each of them. For further analyses we removed all ambiguously mapped reads to both genomes and worked with only the reads that unambiguously map to M. jannaschii genome. The mapped reads for M. jannaschii were then aligned to the M. jannaschii genome again and sorted using the STAR aligner version 2.5.1b . Aligned sequence reads were assigned to genomic features and quantified using featureCounts read summarization tool. Genes that were differentially expressed were identified using 'DESeq2' in the Bioconductor software framework in R. The differential gene expression analyses showed that the enzyme responsible for the reduction of methenyl group to a methylene group during carbon fixation switches from a H2-dependent enzyme to a coenzyme F420-dependent enzyme with decreasing H2 availability and into syntrophy. During syntrophy, the genes for energy generation on the membrane decreased in their expression levels. Overall design: Total RNA was extracted from 13 cell pellets from 4 different conditions.1. M. jannaschii was grown in monoculture on high H2 in a chemostat in biological triplicates. 2. M. jannaschii was grown in monoculture on low H2 in a chemostat in biological triplicates. 3. M. jannaschii in co-culture with T. paralvinellae was grown in the bottles with maltose in 3 biological and 2 technical replicates. 4. M. jannaschii in co-culture with T. paralvinellae was grown in the bottles with formate in 4 biological and 1 technical replicates. Technical replicates were pooled for larger RNA yields.