Transcriptomic and proteomic changes from medium supplementation and strain evolution in high-yielding Clostridium thermocellum strains.

Clostridium thermocellum is a potentially useful organism for the production of lignocellulosic biofuels because of its ability to directly deconstruct cellulose and convert it into ethanol. Previously engineered C. thermocellum strains have achieved higher yields and titers of ethanol. These strains often initially grow more poorly than the wild type. Adaptive laboratory evolution and medium supplementation have been used to improve growth, but the mechanism(s) by which growth improves remain(s) unclear. Here, we studied (1) wild-type C. thermocellum, (2) the slow-growing and high-ethanol-yielding mutant AG553, and (3) the faster-growing evolved mutant AG601, each grown with and without added formate. We used a combination of transcriptomics and proteomics to understand the physiological impact of the metabolic engineering, evolution, and medium supplementation. Medium supplementation with formate improved growth in both AG553 and AG601. Expression of C1 metabolism genes varied with formate addition, supporting the hypothesis that the primary benefit of added formate is the supply of C1 units for biosynthesis. Expression of stress response genes such as those involved in the sporulation cascade was dramatically over-represented in AG553, even after the addition of formate, suggesting that the source of the stress may be other issues such as redox imbalances. The sporulation response is absent in evolved strain AG601, suggesting that sporulation limits the growth of engineered strain AG553. A better understanding of the stress response and mechanisms of improved growth hold promise for informing rational improvement of C. thermocellum for lignocellulosic biofuel production.

Replicates, Read Numbers, and Other Important Experimental Design Considerations for Microbial RNA-seq Identified Using Bacillus thuringiensis Datasets.

RNA-seq is being used increasingly for gene expression studies and it is revolutionizing the fields of genomics and transcriptomics. However, the field of RNA-seq analysis is still evolving. Therefore, we specifically designed this study to contain large numbers of reads and four biological replicates per condition so we could alter these parameters and assess their impact on differential expression results. Bacillus thuringiensis strains ATCC10792 and CT43 were grown in two Luria broth medium lots on four dates and transcriptomics data were generated using one lane of sequence output from an Illumina HiSeq2000 instrument for each of the 32 samples, which were then analyzed using DESeq2. Genome coverages across samples ranged from 87 to 465X with medium lots and culture dates identified as major variation sources. Significantly differentially expressed genes (5% FDR, two-fold change) were detected for cultures grown using different medium lots and between different dates. The highly differentially expressed iron acquisition and metabolism genes, were a likely consequence of differing amounts of iron in the two media lots. Indeed, in this study RNA-seq was a tool for predictive biology since we hypothesized and confirmed the two LB medium lots had different iron contents (~two-fold difference). This study shows that the noise in data can be controlled and minimized with appropriate experimental design and by having the appropriate number of replicates and reads for the system being studied. We outline parameters for an efficient and cost effective microbial transcriptomics study.