Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq

Abstract

The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism.

Transcriptomic Analysis of Clostridium thermocellum in Cellulolytic Consortium after Artificial Reconstruction to Enhance Ethanol Production

“…The Solexa reads were aligned with the 3173 protein coding genes at the highest ratio of 0.93 in HCt-3. This value was similar to the results from culturing single strain C. thermocellum ATCC 27405 in cellobiose and pretreated yellow poplar, with 95% of the genes in C. thermocellum transcriptionally active and expressed (Wei et al 2014). The quantification of a gene is represented by the RPKM value, as defined in the Experimental section.…”

“…For the phosphorolytic cleavage of cellodextrins, three genes are involved: cellodextrin phosphorylase (Cthe_2989) and cellobiose phosphorylases (Cthe_0275 and Cthe_1221) (Wei et al 2014). The expression level of cellodextrin phosphorylase increased 5-fold, while cellobiose phosphorylase Cthe_1221 increased 3.3-fold and Cthe_0275 remained the same.…”

“…The best local mode alignment was used with no more than two mismatches. The reads that were not mapped unambiguously were excluded from further analysis, as described by Wei et al (2014). The metatranscriptome sequencing data are publicly available in the NCBI Sequence Read Archive under BioProject ID PRJNA280031, and the 16S rRNA gene sequencing data are publicly available under the GenBank accession number SUB883074 Seq1 KR047885.…”

“…RPKM is a widely recognized indicator of gene expression level, especially as a measure of Solexa read density, which indicates the molar concentration of a transcript (Wei et al 2014). The fold-change for an expressed gene was calculated by DEGseq MARS (MA-plot-based method with random sampling model).…”

Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

“…The recA gene was selected as the reference in qRTPCR following previous studies (Stevenson and Weimer, 2005;Wei et al, 2014). Under the optimized fermentation condition (Fig.…”