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Applied and Environmental Microbiology, December 2008, p. 7709-7714, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01948-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Transcriptional Analysis of Clostridium beijerinckii NCIMB 8052 and the Hyper-Butanol-Producing Mutant BA101 during the Shift from Acidogenesis to Solventogenesis ^,

Zhen Shi^1* and Hans P. Blaschek^1,2

Institute for Genomic Biology,¹ Department of Food Science and Human Nutrition, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801²

Received 20 August 2008/ Accepted 2 October 2008

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Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone and butanol. The recent availability of genomic sequence information for C. beijerinckii NCIMB 8052 has allowed for an examination of gene expression during the shift from acidogenesis to solventogenesis over the time course of a batch fermentation using a ca. 500-gene set DNA microarray. The microarray was constructed using a collection of genes which are orthologs of members of gene families previously found to be important to the physiology of C. acetobutylicum ATCC 824. Similar to the onset of solventogenesis in C. acetobutylicum 824, the onset of solventogenesis in C. beijerinckii 8052 was concurrent with the initiation of sporulation. However, forespores and endospores developed more rapidly in C. beijerinckii 8052 than in C. acetobutylicum 824, consistent with the accelerated expression of the sigE- and sigG-regulated genes in C. beijerinckii 8052. The comparison of gene expression patterns and morphological changes in C. beijerinckii 8052 and the hyper-butanol-producing C. beijerinckii strain BA101 indicated that BA101 was less efficient in sporulation and phosphotransferase system-mediated sugar transport than 8052 but that it exhibited elevated expression of several primary metabolic genes and chemotaxis/motility genes.

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The gram-positive, spore-forming, anaerobic clostridia constitute a diverse group of species with industrial, agricultural, and medical relevance. Clostridium acetobutylicum and C. beijerinckii are among the prominent solventogenic species capable of acetone and butanol formation via fermentation (8, 15). Butanol is widely used as an industrial chemical. Butanol also exhibits a range of physical properties, including high energy content, water immiscibility, low vapor pressure, and octane-enhancing power, which provide it with potential as a liquid fuel (17). Developing fermentation processes based on the solventogenic clostridia offers the prospect of butanol production from agricultural feedstocks as an alternative to the petrochemical route (8). Much of past efforts has been focused on C. acetobutylicum ATCC 824 (1, 16, 23). However, the functional characterization of genes and metabolic pathways remains to be undertaken. Recently, genome sequencing of C. beijerinckii NCIMB 8052 was completed by the Joint Genome Institute of the Department of Energy. This accomplishment opens up the exciting possibility of investigating the molecular mechanism of solventogenesis on a genome scale. The objective of this study involves using DNA microarray analysis to examine gene expression in relation to physiological changes associated with C. beijerinckii solvent production and cell growth and differentiation. The comparison of gene expression patterns in the C. beijerinckii 8052 parental strain and the C. beijerinckii BA101 hyper-butanol-producing mutant strain (3, 11) will provide insights toward engineering genetically modified C. beijerinckii strains with improved butanol yields, titers, and productivity.

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Bacterial strains, growth conditions, and fermentation experiments.
Spore stocks of the C. beijerinckii NCIMB 8052 parental strain and the C. beijerinckii BA101 mutant (3, 11) were stored in sterile H₂O at 4°C. Batch cultures were grown in complete P2 medium in a 2-liter BioFlo fermentor (New Brunswick Scientific). (Details of the methods are described in File S1 in the supplemental material.)

Fermentation product analysis.
Supernatants from 1-ml aliquots of cultures were analyzed for acetone, butanol, ethanol, acetic acid, and butyric acid by gas chromatography as described previously (11). Cell growth was monitored by measuring absorbance at 600 nm using a UV-visible absorbance spectrophotometer (Biotek Instruments).

Cellular morphology.
Cells from 5-ml culture aliquots collected at 4, 13, and 27 h after the start of the fermentation time course were pelleted by centrifugation at 4°C and 4,000 x g for 10 min. Pellets were suspended in 500 µl of 0.1 M Tris/HCl, pH 7.6, and 10 µl of the suspension was placed onto a coverslip and viewed using a Carl Zeiss microscope equipped with 63x differential inference contrast optics. Cellular morphology, total bacterial counts, clostridial stage counts, and spore counts were determined using AxioVision 4.6 software (Carl Zeiss).

RNA isolation, cDNA probe labeling, and microarray hybridization.
Samples of 10 ml of cultures were harvested at various times and kept on ice immediately after being withdrawn from the fermentor. Cells were collected by centrifugation at 4°C and 4,000 x g for 10 min. Total RNA was extracted using RNeasy minikits according to the protocol of the manufacturer (Qiagen). RNA quality was assessed using a nanochip on a model 2100 bioanalyzer (Agilent Technologies). Concentrations of purified RNA were determined by measuring absorbance at 260 nm using a UV-visible absorbance spectrophotometer (Biotek Instruments). A reference sample from an RNA pool was prepared to generate a probe for the normalization of the microarray hybridization signals. To do so, C. beijerinckii 8052 static flask cultures were grown in 500 ml of complete P2 medium. Cell pellets were collected, and equal quantities of total RNA isolated at various time points were mixed to produce the RNA pool. The DNA microarray contained gene sets which cover the range of functional categories as described in the genome annotation for C. beijerinckii 8052 by the Joint Genome Institute. These genes are orthologs of members of the previously characterized gene families in C. acetobutylicum ATCC 824 (1, 23, 29). The arrays were printed at the Keck Center, University of Illinois, by spotting 70-mer oligonucleotides onto glass slides. cDNA probes were generated using RNA samples obtained at individual time points and the reference RNA pool by following an aminoallyl-labeling procedure, and the probes were used for two-color microarray hybridization (14). The hybridized slides were scanned using an Axon 4000B scanner, and features in the scanned images were extracted using GenePix Pro 6.0 software (Molecular Devices). (Details of the methods are described in Files S2 and S3 in the supplemental material.)

Microarray data analysis.
Data generated from microarray experiments were processed using the TIGR TM4 suite (26). The expression ratios (Cy3/Cy5 ratios) were normalized for all the features on an array by using the TIGR MIDAS program. Lowess intensity-based normalization was applied in most cases. Expression patterns were visualized colorimetrically using TIGR TMEV software.

Real-time quantitative reverse transcription-PCR (Q-RT-PCR).
Total RNA was purified from cell pellets collected from a replicate culture of C. beijerinckii 8052 to prepare cDNA templates by RT. Real-time PCRs were carried out using a Sybr green RT-PCR protocol on a TaqMan ABI 7900T fast real-time PCR machine (Applied Biosystems). (Details of the methods are described in Files S4 and S5 in the supplemental material.)

Microarray data accession number.
DNA microarray data have been deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) under the series accession number GSE12365.

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C. beijerinckii fermentation and morphology.
The C. beijerinckii fermentation exhibited a shift from acidogenesis to solventogenesis. For cells in the early exponential growth phase, the primary fermentation products included acetic and butyric acids (Fig. 1D to E). The initiation of solvent formation was observed during mid-exponential growth, typically at 7 to 8 h after the start of the fermentation time course, and corresponded with a pH minimum (Fig. 1A to C). The acetic acid concentration decreased after the onset of solvent production (Fig. 1E). Butyric acid accumulated in the exponentially growing cells and approached a maximal level as cells entered the stationary phase at 13 h (Fig. 1D). While higher concentrations of butanol and acetone were produced in C. beijerinckii BA101 than in C. beijerinckii 8052, the two strains exhibited similar growth rates and temporal patterns of the shift from the acidogenic to the solventogenic phase (Fig. 1A to C). For C. beijerinckii 8052, elongated vegetative rods were the only form present in an acidogenic culture. Swollen, cigar-shaped clostridial forms began to appear during the onset of solvent formation at 7 h. Forespores were observed shortly afterwards, at 9 h, and were abundant at 13 h. Phase-bright endospores appeared at 20 h and accumulated to high proportions afterwards. While the proportion of vegetative cells of C. beijerinckii 8052 declined from 40% at 13 h to 20% at 27 h, these cells persisted as 50% of the C. beijerinckii BA101 stationary-phase cell population at 13 and 27 h (Fig. 1F). Endospores were found in 20% of the C. beijerinckii 8052 cells but only 10% of the C. beijerinckii BA101 cells at 27 h.

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FIG. 1. Fermentation kinetics and results of morphological assays of the C. beijerinckii 8052 () and C. beijerinckii BA101 () batch cultures. (A) Growth curves with sampling points for microarray analysis indicated by arrows. OD600, optical density at 600 nm. (B, C, D, and E) Concentrations of butanol, acetone, butyric acid, and acetic acid products, respectively. (F) Cell type counts for the distinct morphological stages. WT, wild type.

Expression of primary metabolic genes.
In C. beijerinckii 8052, the expression of the acetate formation genes encoding phosphotransacetylase (pta) and acetate kinase (ack) and the butyrate formation gene encoding butyrate kinase (buk) increased up to twofold during the acidogenic phase and declined with the onset of solvent formation (Fig. 2A). Coordinated changes in pta-ack expression are consistent with the genomic organization of pta and ack as a single operon and the roles of these genes in acetate production. The expression of genes required for butyryl coenzyme A (CoA) formation, including those encoding thiolase (thl), β-hydroxybutyryl-CoA dehydrogenase (hbd), and butyryl-CoA dehydrogenase (bcd), was induced in the late acidogenic phase at 5 h, increased up to threefold with the onset of solvent formation, and remained at a near-maximal level for a few hours (Fig. 2A). Expression decreased as cells entered the stationary phase. The expression of crotonase (crt) did not show significant changes over time. The product of the C. beijerinckii thl gene shows 76% amino acid sequence identity to the product of the C. acetobutylicum thlA gene, and these genes exhibited comparable expression patterns (35). Consistent with previous observations for C. acetobutylicum, bcd expression in C. beijerinckii 8052 was transiently activated, suggesting that the onset of solvent formation is accompanied by high concentrations of butyryl-CoA (5, 12). In C. beijerinckii BA101, the expression of thl, hbd, and crt was similar to that in C. beijerinckii 8052 while the expression of bcd was threefold higher than that in C. beijerinckii 8052 during the stationary phase.

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FIG. 2. Time course profiles of the expression of selected C. beijerinckii 8052 (I) and C. beijerinckii BA101 (II) solventogenesis-related genes (A), sporulation genes (B), sugar transporter and glycolytic genes (C), and chemotaxis and motility genes (D). The onset of solvent formation is indicated by an arrow. Expression levels are represented using a colorimetric scale based on log2 transformation of the normalized expression ratios (sample/reference ratios). The up- and downregulation of expression are indicated in red and green, respectively. WT, wild type.

In C. beijerinckii 8052, the acetone formation genes encoding acetoacetyl-CoA-acetate/butyrate-CoA transferases and β (ctfA and ctfB) and acetoacetate decarboxylase (adc) are organized into a gene cluster (6). However, unlike C. acetobutylicum, which possesses a bifunctional aldehyde-alcohol dehydrogenase gene (adhE1) adjacent to the ctfA-ctfB operon, C. beijerinckii has a NAD-dependent aldehyde dehydrogenase gene (ald) located upstream of and transcribed in the same direction as ctfA-ctfB-adc (30). This ald gene encodes 468 residues, and the product shows 30% sequence identity to the N-terminal aldehyde dehydrogenase domains of the products of adhE1 and adhE2 in C. acetobutylicum ATCC 824 (10, 22). Consistent with their genomic structure, ald, ctfA, ctfB, and adc exhibited coordinated expression (Fig. 2A). Expression was rapidly activated in the late acidogenic phase, increased up to 200-fold shortly after the onset of solvent formation, and was downregulated up to 6-fold as cells entered the stationary phase. Similar expression patterns for the solventogenic genes in C. beijerinckii BA101 were observed. In C. beijerinckii 8052, a putative NAD(P)H-dependent butanol dehydrogenase gene (bdh) was induced in the late acidogenic phase at 5 h and expression increased sixfold during the early solventogenic phase (Fig. 2A). Expression was downregulated as cells entered the stationary phase. This bdh gene exhibits 96 and 99% sequence identity to adhA and adhB of the previously characterized class IV iron-containing primary alcohol dehydrogenase gene family in the solvent-producing C. beijerinckii strain NRRL B592 (7). The bdh gene product also shows 36 and 40% sequence identity to the C-terminal alcohol dehydrogenase domains of the products of adhE1 and adhE2 in C. acetobutylicum ATCC 824 (10, 22). The transient activation of bdh in C. beijerinckii 8052 is in agreement with the previously reported transient increases of bdh transcripts and enzymatic activities associated with active solvent production in batch cultures of C. acetobutylicum ATCC 824 and C. beijerinckii NRRL B592 (7, 32, 34). In C. beijerinckii BA101, bdh expression was upregulated fourfold during the late acidogenic phase and was retained at a high level during the solventogenic phase.

Expression of sporulation genes.
Similar to the corresponding genes in C. acetobutylicum and Bacillus subtilis (9, 16, 24), spo0A and the sigF operon in C. beijerinckii 8052 were among the earliest sporulation genes to be expressed and exhibited similar expression patterns (Fig. 2B). The sigF operon includes the forespore-specific sigma factor gene sigF, the anti-sigF factor gene spoIIAB, and the anti-anti-sigF factor gene spoIIAA, and the transcription of these genes was highly coordinated. The expression of spo0A and the sigF operon was induced during the acidogenic phase and increased 4- and 15-fold during the onset of solvent formation. Expression was rapidly downregulated and decreased to very low levels at 13 h. sigE encodes a mother cell-specific transcription factor necessary for forespore formation (9, 28). Among sigE-regulated genes, spoIIP and spoIIM are required for the engulfment of the forespore by the mother cell, spoIVA is required for spore coat assembly, and spoVR and spoVB are required for spore cortex synthesis and maturation (28). The expression of these genes was upregulated during the onset of solvent formation and increased 10- to 60-fold at 13 h (Fig. 2B). This period which covered the onset of solvent formation to the 13-h time point corresponded to the appearance and expansion of forespores. sigG encodes a forespore-specific transcription factor which regulates the expression of genes important in spore protection and maturation (28, 33). Among them, spoVT encodes an abrB family transcription factor possibly involved in regulating spore coat assembly (28, 33). spoVT expression was activated during the onset of solvent formation and increased 30-fold at 27 h (Fig. 2B). The product of sspA belongs to a family of small, acid-soluble, DNA-binding proteins which protect the spore chromosome from heat, UV, and desiccation (27, 28). The expression of sspA increased as cells entered the stationary phase and was upregulated 80-fold at 27 h (Fig. 2B). The strong expression of the sigG-regulated genes can be correlated with endospore accumulation in the stationary-phase culture after 20 h. In C. beijerinckii BA101, while the overall expression patterns of spo0A, the sigF operon, and the sigE- and sigG-regulated genes were similar to those observed in C. beijerinckii 8052, the maximal levels of induction were lower by two- to eightfold (Fig. 2B).

Expression of sugar transporter and glycolytic genes.
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) consists of a multiprotein complex which plays an important role in sugar uptake in bacteria (4). Extracellular sugar is recognized and transported across the cell membrane by substrate-specific EII proteins and subsequently phosphorylated by the general PTS proteins Hpr and EI on the cytoplasmic side. Specifically, EI mediates the phosphoenolpyruvate-dependent phosphorylation of Hpr. Phosphorylated Hpr then transfers the phosphoryl group to the sugar moiety associated with the EII complex. Phosphorylated sugar is able to enter the glycolytic pathway as glucose-6-phosphate. In the solventogenic clostridia, glucose uptake is essential for generating carbon precursors for solvent synthesis (20). The PTS functions as an important mechanism, while the ATP-dependent glucose uptake serves as an alternate route and requires a permease and glucokinase activity for glucose phosphorylation (20). The levels of glucose-PTS-associated enzymatic activities in C. beijerinckii BA101 were significantly lower than those in C. beijerinckii 8052 (18, 19). These observations appear to agree with the expression of a gene cluster encoding the mannose family PTS (Man-PTS) transporters (Fig. 2C). The Man-PTS transporters are commonly found in gammaproteobacteria and firmicutes, including C. acetobutylicum and C. beijerinckii, and exhibit broad substrate specificity toward glucose, mannose, sorbose, fructose, and a variety of other sugars (4, 37). In the Man-PTS transporter gene cluster, manIIAB encodes a membrane fusion protein of the IIA and IIB subunits involved in sugar phosphorylation while manIIC encodes a sugar-specific permease (4). The expression of manIIAB and manIIC in C. beijerinckii BA101 was 5- to 10-fold lower than that in C. beijerinckii 8052 (Fig. 2C).

The expression of the glycolytic genes necessary for glucose-6-phosphate conversion to pyruvate generally decreased over the time course of the fermentation (Fig. 2C). In C. beijerinckii 8052, glucokinase (glcK) expression did not change significantly. For glucose-6-phosphate isomerase (pgi), 6-phosphofructokinase (pfk), fructose-1,6-bisphosphate aldolase (fba), the gene cluster encoding glyceraldehyde-3-phosphate dehydrogenase (gap), phosphoglycerate kinase (pgk), triosephosphate isomerase (tpi), and phosphoglycerate mutase (pgm), expression was downregulated two- to fourfold during the late acidogenic and early solventogenic phases. The expression of enolase (eno) and pyruvate kinase (pyk) decreased 5- to 10-fold during the acidogenic phase. In C. beijerinckii BA101, similar expression patterns of the glycolytic genes were observed.

Expression of cell motility genes.
Bacterial chemotactic responses and flagellar assembly are mediated by motility-related gene clusters (24, 31). These clusters encode signal transduction protein complexes which typically consist of transmembrane receptors known as the methyl-accepting chemotaxis proteins (MCPs), cheA cytoplasmic histidine kinase for initiating the phosphorelay of the sensory signals, cheW cytoplasmic scaffold protein for clustering the cheA kinase with MCPs, and cheY kinase for phosphorelay from the cheA protein to flagellar proteins (31). Additional components may include cheR methyltransferase for generating highly methylated MCPs, cheB bifunctional protein with a cheY kinase-like receiver domain and a cheR protein-like methyltransferase domain, and cheC and cheD proteins mediating adaptation to chemotactic signals (31). In the clostridia and bacilli, motility-related genes generally exhibit decreased expression in sporulating cells (16, 24). In C. beijerinckii 8052, a che-fli gene cluster containing cheW, cheD, cheB, cheR, cheA, cheC, cheW, fliM, and fliY is homologous to the small flagellar gene cluster including cheABCDRWY, fliDMS, and flgK in C. acetobutylicum ATCC 824 (24). Similar to the downregulation of chemotaxis/motility operons during the onset of sporulation in C. acetobutylicum ATCC 824 (13, 16, 29), a decrease of two- to fourfold in the expression of the che-fli gene cluster in C. beijerinckii 8052 during the late acidogenic and early solventogenic phases was detected (Fig. 2D). However, in C. beijerinckii BA101, the che and fli genes were expressed at relatively stable levels over time, suggesting that mutant cells were more motile in the solventogenic culture than cells of the parental strain.

Real-time Q-RT-PCR.
Q-RT-PCR analysis was applied to quantify the levels of gene expression in a biological replicate culture of C. beijerinckii 8052. Six genes, including spoIVA, spoVR, spoVT, pta, ack, and adc, were analyzed. A high degree of correlation between the results obtained from microarray and Q-RT-PCR (R² = 0.89) was observed (Fig. 3).

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FIG. 3. Q-RT-PCR verification of microarray analysis results for selected genes. Samples representing a replicate of the C. beijerinckii 8052 batch culture were collected at 5, 7, 9, 11, and 13 h. The expression ratios obtained from microarray and Q-RT-PCR analyses of spoIVA (), spoVR (•), spoVT (), pta (), ack (), and adc () are compared.

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DNA microarray profiling revealed for the first time that transcriptional activities are coordinated with physiological changes during the metabolic shift from acidogenesis to solventogenesis in the acetone-butanol fermentation process of C. beijerinckii. Compared with C. beijerinckii 8052, C. beijerinckii BA101 showed increased expression of butyryl-CoA and butanol formation genes, including bcd and bdh, during the solventogenic phase, suggesting differences in the regulation of primary metabolic genes in the two strains. Maximal levels of induction of sporulation genes in C. beijerinckii BA101 were significantly lower than those in C. beijerinckii 8052, consistent with the observed lower level of endospore formation. The spo0A protein is a master regulator of gene expression in the sporulating cells of clostridia and bacilli (2, 9). Phosphorylated spo0A protein has been found to activate a variety of targets, such as the solventogenic operon adhE1-ctfA-ctfB in C. acetobutylicum and adc in C. acetobutylicum and C. beijerinckii, as well as multiple sporulation factor genes including the sigF operon and sigG in C. acetobutylicum and B. subtilis (2, 13, 25, 29, 36). In C. beijerinckii BA101, reduced expression of spo0A may lead to weakened induction of the sigF operon and the sigE and sigG regulons and thus an overall reduction in sporulation. spo0A has also been shown to negatively regulate the expression of chemotaxis/motility operons (21, 24, 29). Possibly, decreased induction of spo0A helps to relieve the inhibition of the che and fli genes during the stationary phase and contributes to the enhanced motility of C. beijerinckii BA101.

Similar to the onset of solventogenesis in C. acetobutylicum ATCC 824, the onset of solvent formation in C. beijerinckii 8052 coincided with the appearance of clostridial forms and high-level expression of spo0A, the sigF operon, and the solventogenic genes ald, ctfA-ctfB, and adc, as well as the downregulation of the chemotaxis/motility genes. However, the expression of spo0A and the sigF operon in C. beijerinckii 8052 quickly declined from maximal levels to very low levels in the following 6 h as cells approached the stationary phase, whereas high levels of expression in C. acetobutylicum ATCC 824 were sustained for 12 to 24 h after the onset of solventogenesis (1, 16). In C. beijerinckii 8052, forespores and endospores were observed 2 and 14 h following the appearance of clostridial forms, while the sigE- and sigG-regulated transcripts rapidly increased within 6 h after the onset of solvent formation and were highly abundant in the early-stationary-phase cells. In C. acetobutylicum ATCC 824, there was a long delay of 12 to 24 h after the onset of solventogenesis before the expression of the sigE- and sigG-regulated genes could be detected and forespores and endospores were observed 18 h after clostridia appeared (1, 13, 16). These results indicate that while the initiation of solvent formation is concurrent with sporulation in both species, forespore and endospore development occurs more rapidly in C. beijerinckii 8052 than in C. acetobutylicum ATCC 824.

 
This work was supported by USDA National Research Initiative award AG 2006-35504-17419. Z.S. is a recipient of a postdoctoral fellowship with the Institute for Genomic Biology at the University of Illinois, Urbana-Champaign.

We thank Mark Band for helpful discussions.

 
* Corresponding author. Mailing address: MC-195, 3409 Institute for Genomic Biology, University of Illinois, 1206 W. Gregory Dr., Urbana, IL 61801. Phone: (217) 333-7786. Fax: (217) 333-0508. E-mail: zshi{at}illinois.edu

Published ahead of print on 10 October 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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Applied and Environmental Microbiology, December 2008, p. 7709-7714, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01948-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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