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.

Keywords: Clostridium thermocellum; RNA-Seq; cellobiose; cellulosome; ethanol; hydrogen; pretreated yellow poplar (PYP); transcriptomics.

Figure 1

Flowchart for experimental procedures, along with growth curves for cellobiose- and PYP-grown C. thermocellum cells. (A) Flowchart outlining the experimental procedures; RPKM, reads per kilobase of exon model per million mapped reads. (B) OD₆₀₀ for cellobiose-grown cells was measured by spectrophotometry at 600 nm (OD₆₀₀). (C) Cell pellet protein concentration for pretreated yellow poplar (PYP)-grown cell culture. The doubling time for cellobiose- and PYP-grown cells was 3.3 and 7.8 h, respectively. Data are presented as the mean (±SE) of 3 replicates. Arrows indicate the time points sampled for transcriptomic analyses.

Figure 2

Validation and quantification of representative, final RNA-Seq cDNA libraries prior to GA II, using an Agilent 2100 Bioanalyzer and the DNA 1000 Nano Chip Kit (Agilent, Technologies, Santa Clara, CA, USA). The RNA-Seq cDNA libraries were prepared from RNA extracted from C. thermocellum cells grown on cellobiose or PYP. (A) Electrophoresis run of the cDNA libraries by the Bioanalyzer; (B) bioanalyzer trace of cDNA libraries.

Figure 3

RPKM frequency histogram of transcripts from RNA-Seq of pretreated yellow poplar (PYP)- vs. cellobiose-grown C. thermocellum cells. The diagram shows the distribution of the number of genes expressed at different RPKM levels. The total number of protein coding genes aligned with the RNA-Seq was 3081; the percentage value above each bar indicates the genes at specific expression level accounting for the proportion of total number of genes. The ^* mark indicates that significantly different frequencies (i.e., numbers of genes) were observed between the two RNA-Seq data sets from PYP- vs. cellobiose-grown C. thermocellum cells.

Figure 4

COG functional categories of the 1211 up- and 314 down-regulated genes in PYP-grown cells compared with cellobiose-grown cells. Note that since COG annotation classes overlap, the sum of COG annotated genes is larger than the number of total up- and down-regulated genes analyzed.

Figure 5

Correlation between the observed gene abundances in this study and the published protein abundance data of cellulosomal components. (A) Correlation between Log2(RPKM) and emPAI/CipA. (B) Correlation between Log2(RPKM) and NSAF/CipA. The literature sources for data of emPAI/CipA and NSAF/CipA are described in the Results and Discussion section.

Figure 6

Diagram for the primary steps in the conversion of cellulose to fermentation products using C. thermocellum ATCC 27405. SigI-RsgI (sigma factors coupled with membrane-associated RsgI-like anti-sigma factors) are also illustrated in this diagram as they have been proposed in literature for polysaccharide triggered signal transmission in regulation of GH family genes. Detailed nomenclature of enzymes and their associated gene IDs can be found in Supplementary Data Sheet 3, section 3 and Table 4. For enzymes associated with a single gene ID, the gene ID is not included in the figure; instead, its fold change (FC) value is indicated directly beside the enzyme. For enzymes associated with multiple gene IDs, the gene IDs and their FC values are illustrated in sorted lists in the diagram, in order of either their FC values or locus IDs. Text in red and green represents the up-regulated and down-regulated genes, respectively, in PYP- against cellobiose-grown cells; in contrast, text in black represents the genes with no significant transcriptional changes between the two types of cells, i.e., 2.0 > FC value > 0.5. Nomenclature of metabolites: CoA, coenzyme A; ECH, energy conserving hydrogenase; Fd, ferredoxin; Fru, fructose; Glu, glucose; ox, oxidized; P, phosphate; PEP, phosphoenolpyruvate; Pi, inorganic phosphate; PYPO, transcript that was detected in PYP-grown cells only; Pyr, pyruvate; red, reduced.