Why microarray analysis of C. beijerinckii8052 transcriptome under furfural stress?
Furfural was chosen as archetypical lignocellulose-derived inhibitory compound for this investigation because it is the most prevalent microbial inhibitor generated during pretreatment and hydrolysis of lignocellulosic biomass to monomeric sugars. However, furfural has been shown previously to enhance solventogenic C. beijerinckii BA101 growth and ABE production when the fermentation medium was supplemented with <3 g/L furfural prior to fermentation [2, 8]. To better understand mechanisms with which furfural affect C. beijerinckii 8052 physiology, the global response of C. beijerinckii 8052 to the challenge of furfural during both acidogenesis and solventogenesis at the mRNA level was profiled using whole genome microarray analysis. The findings from the present study are grouped into different attributes.
Expression of C. beijerinckii8052 redox and cofactor genes in the presence of furfural
After the challenge of furfural during the acidogenic phase some genes expressing redox proteins in C. beijerinckii 8052 increased by up to 16-fold compared with that in the control group (Figure 1A and Additional file 1: Table S4A). Gene ontology (GO) analysis (Additional file 2: Table S2A) shows that three of these redox proteins are involved in antioxidant activity (GO:0016209): thioredoxin reductase (Cbei_2681), redoxin domain-containing protein (Cbei_2680), and glutathione peroxidase (Cbei_0389); the latter two possess oxidoreductase activity acting on peroxide as an acceptor (GO:0016684) and in response to oxidative stress (GO:0006979). Another group of genes that was up-regulated by more than threefold encodes oxidoreductases acting on CH or CH2 groups, with disulfide as an acceptor (GO:0016728) (Additional file 2: Table S2A). According to KEGG enrichment pathway analysis, this group of genes is associated with purine (cbe00230) and pyrimidine (cbe00240) metabolisms (Additional file 3: Table S3), and includes anaerobic ribonucleoside triphosphate reductase (Cbei_0068), adenosylcobalamin-dependent ribonucleoside-triphosphate reductase (Cbei_2522), and ribonucleotide-diphosphate reductase subunits (Cbei_0194 and Cbei_0195). The remaining oxidoreductases (GO:0016491) (Additional file 2: Table S2A) that had higher expression in the furfural treatment culture than in the control culture are aldo/keto reductase (Cbei_3974), short-chain dehydrogenase/reductase (SDR) (Cbei_3904), DSBA oxidoreductase (Cbei_2058), FAD linked oxidase domain-containing protein (Cbei_0312), and alcohol dehydrogenase (Cbei_1464) (Figure 1A and Additional file 1: Table S4A). The transcriptome of C. beijerinckii 8052 after furfural challenge at the solventogenic phase shows some similarities in terms of redox enzymes. All the above genes, except FAD linked oxidase domain-containing protein (Cbei_0312), and alcohol dehydrogenase (Cbei_1464), were also induced by furfural challenge at the solventogenic phase (Figure 1A and Additional file 1: Table S4C).
Besides redox enzymes, components associated with redox reactions were also highly expressed in cultures challenged with furfural at the acidogenic phase. One of the related components affected by furfural treatment is the iron-sulfur cluster. The expression of genes encoding iron-sulfur cluster assembly proteins (Cbei_1848, Cbei_1849, Cbei_1850, Cbei_1851 and Cbei_1852) increased by up to fivefold (Figure 1A and Additional file 1: Table S4A); these genes are classified into the cofactor biosynthetic process (GO:0051188) (Additional file 2: Table S2A). Another group of genes classified into the same group (GO:0051188), as well as into the vitamin biosynthetic process (GO:0009110) (Additional file 2: Table S2A), includes those encoding cobalt ABC transporter ATPase (Cbei_3693), cobalt ABC transporter permease (Cbei_3694) and cobalt transport protein CbiM (Cbei_3695) (Figure 1A and Additional file 1: Table S4A). In addition, differential expression was also observed in furfural-challenged cultures for several members of riboflavin biosynthesis genes (Cbei_1224, Cbei_1225, Cbei_1226, Cbei_1227) (Figure 1A and Additional file 1: Table S4A). This group of genes belongs to the Gene Ontology term riboflavin metabolic process (GO:0006771) (Additional file 2: Table S2A), and if classified by KEGG pathway analysis, these genes are involved in riboflavin metabolism (cbe00740) (Additional file 3: Table S3). However, furfural challenge during solventogenesis affected gene expression differently from that at acidogenesis in terms of redox enzyme cofactors. First, expression of genes that code for iron-sulfur cluster assembly proteins was even higher during solventogenesis (Figure 1A and Additional file 1: Table S4C), and those genes (Cbei_1848, Cbei_1849, Cbei_1850, Cbei_1851 and Cbei_1852) were up-regulated in furfural-challenged cultures by up to 54-fold compared to no more than fivefold during acidogenesis (Figure 1A and Additional file 1: Table S4C). On the other hand, the expression of genes involved in synthesis of other cofactors, including riboflavin and cobalamin, did not show obvious alterations during furfural challenge at solventogenesis (Figure 1A), although these genes were highly induced during furfural challenge at acidogenesis (Figure 1A and Additional file 1: Table S4A).
Expression of membrane transporter genes in C. beijerinckii8052
Gene expression analysis of C. beijerinckii 8052 responding to furfural stress during butanol fermentation was performed to determine not only the effect of furfural on C. beijerinckii 8052 growth and ABE production but also on molecular physiological changes. Furfural in the ABE fermentation medium altered expressions of the membrane transport system, including ATP-binding cassette transporters (ABC-transporter) and phosphotransferase system (PTS), in C. beijerinckii 8052 during both acidogenic and solventogenic phases (Figure 1B). While some ABC-transporter genes such as galactoside ABC transporter (Cbei_3298), multidrug ABC transporter ATPase (Cbei_3299 and Cbei_3300), and cobalt ABC transporter ATPase (Cbei_3693) were expressed up to sevenfold in furfural-challenged C. beijerinckii 8052 at the acidogenic phase (Figure 1B and Additional file 1: Table S4A), expression of these transporter genes was increased by a greater fold during the solventogenic phase (Figure 1B and Additional file 1: Table S4C). According to KEGG pathway analysis, some ABC-transporter-related genes may be classified into what is known as KEGG pathway ABC transporters (cbe02010) (Additional file 3: Table S3), which include transport proteins that catalyze transmembrane movement of different substrates including sulfate, phosphate and branched-chain amino acid. Expression of these genes increased up to twelvefold in furfural-challenged C. beijerinckii 8052 during solventogenesis (Figure 1B and Additional file 1: Table S4C). Specifically, genes involved in sulfate transportation include sulfate ABC transporter ATPase (Cbei_4190), sulfate ABC transporter inner membrane protein (Cbei_4191 and Cbei_4192), and sulfate ABC transporter substrate-binding protein (Cbei_4193); phosphate transporters include phosphate binding protein (Cbei_1127), phosphate ABC transporter permease (Cbei_1128 and Cbei_1129), and phosphate ABC transporter ATPase (Cbei_1130); and genes that code for branched-chain amino acids include extracellular ligand-binding receptor (Cbei_1762, Cbei_1767 and Cbei_5042), inner-membrane translocator (Cbei_1763, Cbei_1764, Cbei_5043, and Cbei_5044), and ABC transporter (Cbei_1765, Cbei_1766, Cbei_5045, Cbei_5046, and Cbei_2145). In addition, genes for cyanate or nitrite transportation that belong to ABC transporters (Cbei_2089 and Cbei_3331) are equally induced in furfural-challenged C. beijerinckii 8052 during solventogenesis (Figure 1B and Additional file 1: Table S4C).
Unlike ABC-transporter genes whose expressions were increased by furfural-challenged C. beijerinckii 8052, another member of the membrane transporter system, phosphotransferase system (PTS), was repressed in furfural-challenged cultures of C. beijerinckii 8052 at both acidogenic and solventogenic phases (Figure 1B). Prominent among the PTS are the PTS system mannose/fructose/sorbose family transporters involved in fructose and mannose metabolism (cbe00051) and amino sugar and nucleotide sugar metabolism (cbe00520) (Additional file 3: Table S3). While mostly genes encoding PTS system mannose/fructose/sorbose family transporter subunits IIA (Cbei_4914), IIB (Cbei_4913), IIC (Cbei_4912 and Cbei_3872), and IID (Cbei_4911 and Cbei_3871) were repressed by up to fourfold when cultures of C. beijerinckii 8052 were challenged with furfural during the acidogenic growth phase (Figure 1B and Additional file 1: Table S4B), a wider spectrum of genes, including PTS system mannose/fructose/sorbose family transporters, was repressed when cultures of C. beijerinckii 8052 were challenged with furfural at the solventogenic growth phase (Figure 1B and Additional file 1: Table S4D). The repressed genes associated with sugar metabolism during the solventogenic growth phase include mannose/fructose/sorbose family transporter subunit IID (Cbei_0958, Cbei_2196, Cbei_3871, Cbei_4557, and Cbei_4911), mannose/fructose/sorbose family IIC subunit (Cbei_3872), sorbose-specific transporter subunit IIC (Cbei_4558), sorbose subfamily transporter subunit IIB (Cbei_4559), mannose-6-phosphate isomerase (Cbei_0996), and glucitol/sorbitol-specific transporter subunit IIC (Cbei_0336) (Figure 1B). Besides the listed genes, N-acetylglucosamine-specific IIBC subunit (Cbei_4532) and glucose subfamily transporter subunit IIA (Cbei_4533) are also involved in amino sugar and nucleotide sugar metabolism (cbe00520) (Additional file 3: Table S3). The repression of these genes may affect the transportation and metabolism of sugars such as those in the glucose family (N-acetyl-D-glucosamine, D-glucosamine and glucosides), the lactose and cellobiose families, the mannose family (mannose and galactosamine), and others such as sorbose, sorbitol, glucitol, and L-ascorbate, many of which are monomeric sugars of lignocellulosic biomass. Furfural challenge of C. beijerinckii 8052 during the solventogenic phase, in addition, inhibited other specific PTS systems, including lactose/cellobiose-specific subunits (Cbei_2663, Cbei_2740, Cbei_4634, Cbei_4639, Cbei_4640, and Cbei_4683), sorbose-specific subunits (Cbei_2907) and subunit IIA-like nitrogen-regulatory protein PtsN (Cbei_2741) (Figure 1B and Additional file 1: Table S4D).
Expression of a two-component signal transduction system, chemotaxis, and cell motility genes in C. beijerinckii8052
As with membrane transporter genes, the expression of genes associated with the two-component signal transduction system (cbe02020) was altered in furfural-challenged C. beijerinckii 8052 at both acidogenic and solventogenic phases (Additional file 3: Table S3). Following furfural challenge of C. beijerinckii 8052 during the acidogenic growth phase, only two genes (Cbei_4019, chemotaxis protein CheA and Cbei_4273, MotA/TolQ/ExbB proton channel) involved in the coding of two-component signal transduction system were repressed by about fourfold (Figure 1C and Additional file 1: Table S4B). When the C. beijerinckii 8052 culture was challenged with furfural at the solventogenic growth phase, more than 40 genes were repressed by up to 18-fold (Figure 1C and Additional file 1: Table S4D). Notably, the two major functional categories of genes belonging to the two-component signal transduction system are bacterial chemotaxis (cbe02030) and flagellar assembly (cbe02040) (Additional file 3: Table S3).
Although chemotaxis is the most widely studied two-component sensory system in bacteria, not much has been reported about the system in relation to furfural stress in solventogenic Clostridium species. When the C. beijerinckii 8052 culture was challenged with furfural at the solventogenic growth phase, many genes associated with the chemotaxis sensory system in C. beijerinckii 8052, such as methyl-accepting chemotaxis sensory transducer (Cbei_0287, Cbei_0804, Cbei_2787, Cbei_3356, Cbei_3671, Cbei_3961, Cbei_4161, Cbei_4821, and Cbei_4828) and genes that code for chemotaxis proteins (CheA Cbei_4307, Cbei_4829, and Cbei_4183; CheB Cbei_4309 and Cbei_4826; CheR Cbei_4827; CheW Cbei_4184 and Cbei_4822; CheY Cbei_4819 and Cbei_4015; and MotA Cbei_4273), were differentially repressed (Figure 1C and Additional file 1: Table S4D). Since chemotaxis directs flagellar motion and controls the swimming pattern of the cell [9], genes encoding flagellar assembly proteins were also differentially repressed by furfural. These flagellar proteins include FliS (Cbei_4292), FliR/FlhB (Cbei_4254), FliH (Cbei_4266), MotA (Cbei_4273), FlgL (Cbei_4297), FliC (Cbei_4274 and Cbei_4289), and FliD (Cbei_4291), as shown in Figure 1C and Additional file 1: Table S4D. Additionally, there are two-component signal transduction systems related to genes encoding proteins that partake in many cellular functions such as quorum sensing and flagella assembly (flagellin domain-containing protein Cbei_4274 and Cbei_4289, and MotA/TolQ/ExbB proton channel Cbei_4273), carbon storage regulation (carbon storage regulator CsrA Cbei_4295), nitrogen assimilation (glutamine synthetase Cbei_0444), and cell cycle progression and development (signal transduction histidine kinase regulating citrate/malate metabolism Cbei_4175, multi-sensor signal transduction histidine kinase Cbei_4430, and histidine kinase internal region Cbei_4458) that were differentially repressed when the C. beijerinckii 8052 culture was challenged with furfural at the solventogenic growth phase (Figure 1C and Additional file 1: Table S4D).
Validation of gene expression data from microarray analysis by Q-RT-PCR
To validate differential gene expressions obtained using microarray analysis, Q-RT-PCR was applied to quantify gene expression levels in biological replicate cultures of C. beijerinckii 8052 using treatment conditions that mimicked microarray treatment but were independent of the cultures used for microarray analysis. Briefly, the C. beijerinckii 8052 culture was challenged with furfural at acidogenic and solventogenic growth phases during which 19 and 23 genes, respectively, were evaluated. The genes were selected randomly within each range of fold change. Differential gene expressions in furfural-challenged C. beijerinckii 8052 determined via microarray analysis and Q-RT-PCR were found to have a high degree of correlation between them at both acidogenic (R = 0.87) and solventogenic phases (R = 0.84) (Figure 2, Additional file 4: Table S1).
Interactive effect of furfural reduction and ABE production
To determine effects of furfural on C. beijerinckii 8052 growth and ABE production at acidogenic and solventogenic phases, a C. beijerinckii 8052 culture grown in P2 medium was challenged with furfural, and changes in cell density, acid production and ABE production were measured relative to cultures grown in P2 medium without furfural. Challenge of C. beijerinckii 8052 with 2 g/L furfural during the acidogenic phase (fermentation time 8 h) when OD600 was between 1.5 and 2.0 resulted in complete depletion of furfural within 4 h, and cell densities of the furfural-challenged C. beijerinckii 8052 and control cultures were nearly indistinguishable (Figure 3A). However, acetone and butanol production by furfural-challenged C. beijerinckii 8052 only increased by 1.1- and 1.2-fold, respectively, during the period, compared to 1.3- and 2.7-fold increases in acetone and butanol production, respectively, by the control culture (Figure 3A-E). The acetic and butyric acid levels measured in both the furfural-challenged C. beijerinckii 8052 and the unchallenged control cultures were reflective of the respective acetone and butanol production profiles (Figure 3). Notably, although ABE production and acid re-assimilation by furfural-challenged C. beijerinckii 8052 were inferior to that of the unchallenged control, the fermentation proceeded rapidly following depletion of furfural, and the maximum concentrations of acetone, butanol, and ethanol produced by the furfural-challenged C. beijerinckii 8052 were higher than that of the control by 28%, 2% and 6%, respectively (Figure 4A-C). Interestingly, acid assimilation was stimulated in the furfural-challenged C. beijerinckii 8052 following furfural depletion in the fermentation medium, and the final concentrations of acetic and butyric acid were lower than that of the control by 22% and 19%, respectively (Figure 4D and E).
While C. beijerinckii 8052 cultures challenged with furfural at the acidogenic phase could tolerate furfural and produce more ABE than the control following the depletion of furfural, challenging C. beijerinckii 8052 culture with furfural during the solventogenic phase (fermentation time 25 h; OD600 5.0-5.5) resulted in shut down of ABE production and rapid accumulation of acetic and butyric acid in the fermentation medium (Figure 5). Unlike the control, C. beijerinckii 8052 grown in P2 medium without furfural underwent a normal fermentation process (Figures 5 and 6). At solventogenesis, furfural reduction was impeded (Figure 5G) when concentrations of acetone, ethanol and butanol were high (3.40 g/L ± 0.27 g/L, 0.22 g/L ± 0.02 g/L, and 5.93 g/L ± 0.12 g/L, respectively) (Figure 5B and C). Although the cell density of C. beijerinckii 8052 in the solventogenic phase culture was four times higher than in the acidogenic phase culture, 3 g/L furfural was reduced by only 80% in 4 h (Figure 5G). To further evaluate the effect of challenging C. beijerinckii 8052 at the solventogenic phase with 3 g/L furfural, the fermentation was allowed to proceed for another 40 h during which no further growth and reduction of furfural were observed (data not shown); and uptake of acetic and butyric acid by C. beijerinckii 8052 did not occur (Figure 6). Similarly, when 2 g/L furfural was used to challenge C. beijerinckii 8052 at the solventogenic phase, the 2 g/L furfural was depleted before 2 h. However, ABE production was shut down (Figure 7A-C) followed by accumulation of acetic and butyric acids (Figure 7D-E) in the fermentation medium, and the culture did not recover following the depletion of furfural.
To independently verify whether the presence of ABE in the fermentation medium was contributing to the toxicity of furfural to C. beijerinckii 8052 and decreasing furfural reduction during the solventogenic phase, the acidogenic phase culture of C. beijerinckii 8052 was supplemented with 2 g/L furfural together with acetone, ethanol and butanol at concentrations that mimic their concentration at the solventogenic phase. Interestingly, this situation reduced the concentration of furfural in the fermentation medium by only 75% after 4 h post-furfural challenge, unlike the control without ABE supplementation, which depleted the furfural in 3 h (Figure 8). However, the presence of furfural in the fermentation medium during the solventogenic growth phase did not have remarkable impact on the expression of ABE production genes in C. beijerinckii 8052 (Additional file 5: Table S5).