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. 2013 Dec;159(Pt 12):2558–2570. doi: 10.1099/mic.0.069534-0

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052

Walter J Sandoval-Espinola 1, Satya T Makwana 1, Mari S Chinn 2, Michael R Thon 3, M Andrea Azcárate-Peril 4, José M Bruno-Bárcena 1,✉
PMCID: PMC7336276  PMID: 24068240

Abstract

Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l−1 of butanol and 26.3 g l−1 of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g−1, respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.

Introduction

The most recent crisis in rising oil prices has reinvigorated interest in solvent-producing clostridia, well known for their ability to generate acetone/butanol/ethanol (ABE) from plant biomass feedstock. Butanol has attracted historical attention as an energy commodity and solventogenic clostridia have been the traditional organisms selected for its biological production. This taxonomically complex group of organisms, when cultured under the right conditions, possesses a metabolic ability to convert a diverse group of carbohydrates, including pentoses, hexoses and even polysaccharides, into butanol and other subproducts (Mitchell, 1997; Qureshi et al., 2007, 2010; Lee et al., 2008; Jang et al., 2012a). Additionally, a large number of studies on ABE fermentation have been carried out demonstrating that among solventogenic strains, Clostridium beijerinckii exhibits unique cellulolytic and xylanolytic activities as well as desirable properties such as sugar co-fermentation (Lee et al., 1985a, b; Wang & Blaschek, 2011; Heluane et al., 2011). These properties facilitate the exploitation by fermentation of alternative sustainable sugar-based feedstock that also contains vitamins and nitrogenous compounds required for successful ABE fermentations. Therefore, ABE fermentation can be achieved using various agricultural residues, including sorghum juice, cane molasses and other byproducts of the sugar industry (Najafpour & Shan, 2003). Sucrose has been considered an economical and suitable substrate for ABE fermentation (Quratulain et al., 1995; Ni et al., 2012, 2013), and the genes involved in its utilization have been studied and constitute an operon (Reid et al., 1999). Molecular data suggest that sucrose and glucose uptake by C. beijerinckii takes place through a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (Mitchell et al., 1991; Tangney et al., 1998; Reid et al., 1999). However, only a small number of studies have been published evaluating C. beijerinckii utilizing sucrose as the sole carbon and energy source (Wang & Blaschek, 2011).

The history and study of the ABE bioprocess dates back to the early twentieth century and, since then, taxonomic classification of solvent-producing clostridia has been a veritable quagmire that continues to be elucidated (Collins et al., 1994; Johnson et al., 1997; Stackebrandt & Rainey, 1997). Nonetheless, there are physiological characteristics shared by all solventogenic strains that limit the implementation of cost-effective industrial bioprocesses. Problems affecting strain performance include metabolic carbon flow regulation, low butanol tolerance and genetic degeneration. Butanol toxicity has been considered historically to be one of the main limiting factors restricting biological butanol accumulation (Ezeji et al., 2010), whilst genetic degeneration relates to the cell failing to maintain the capacity to produce solvents – a common phenomenon reported in solvent-producing clostridia (Kutzenok & Aschner, 1952; Kashket & Cao, 1993, 1995; Gottschal & Morris, 1981). Historical strategies to overcome poor solvent tolerance and to improve total butanol titres have involved the screening of resistant butanol-producing species after stress-directed evolution (Lin & Blaschek, 1983; Soucaille et al., 1987; Annous & Blaschek, 1991; Quratulain et al., 1995), the isolation of asporogenic, butanol-resistant mutant strains (Jain et al., 1993) and the genetic modification of existing strains (Dai et al., 2012; Mann et al., 2012; Xiao et al., 2011). The stress-directed evolution strategy that emphasized butanol resistance was chosen in 1983 to select a butanol-tolerant mutant of C. beijerinckii NCIMB 8052, initially named SA-1 (Lin & Blaschek, 1983) and later catalogued as C. beijerinckii ATCC 35702. SA-1 has been overlooked the last three decades and only a few studies have evaluated this strain, including the work assessing its α-amylase and glucoamylase activities (Chojecki & Blaschek, 1986). Recently, we evaluated the nutritional requirements of SA-1 and proposed a medium that facilitates its growth under butanol stress, making it possible to obtain reliable continuous butanol production processes when the pH was kept at 6.5 (Heluane et al., 2011). Under these conditions SA-1 also exhibited characteristics of delayed lysis during sporulation similar to a Clostridium acetobutylicum lyt-l mutant – a desirable phenotype that improves butanol accumulation (Van Der Westhuizen et al., 1982). However, the limited amounts of functional data on SA-1 require new physiological evaluations essential for the interpretation of the genotypic differences between SA-1 and its parent strain NCIMB 8052.

Among solventogenic butanol-producing clostridia, there are two reference genomes publicly available: C. acetobutylicum ATCC 824 (Nolling et al., 2001) and C. beijerinckii NCIMB 8052 (GenBank accession number NC_009617). In addition, a metabolic model has been proposed based on the NCIMB 8052 genome sequence (Milne et al., 2011). The availability of these reference genomes facilitates comparative genome analysis and the discovery of new genetic polymorphisms involved potentially in the enhancement of butanol production (Jia et al., 2012; Mann et al., 2012). Thus, choosing closely related strains that show different phenotypic characteristics may facilitate a functional association with genomic polymorphisms.

The present study examined both physiological differences and genetic variations of C. beijerinckii SA-1 (offspring) compared with C. beijerinckii NCIMB 8052 (parent). We compared the solventogenic production capabilities of NCIMB 8052 and SA-1 growing in a carbon- and energy-limiting medium (Heluane et al., 2011), and further identified and confirmed genetic polymorphisms in their genome sequences.

Methods

Strains, media and cultivation methods.

Strain SA-1/ATCC 35702 was deposited by Lin & Blaschek (1983) as an offspring strain derived from C. acetobutylicum ATCC 824. Later, Johnson et al. (1997) identified SA-1 as belonging to the C. beijerinckii species by DNA sequence similarity. After this finding, Blaschek recatalogued their ATCC 824 strain as C. beijerinckii NCIMB 8052 (Formanek et al., 1997). For our study, C. beijerinckii NCIMB 8052 (ATCC 51743) and C. beijerinckii SA-1 (ATCC 35702) were obtained from the American Type Culture Collection. Species identity was verified by PCR amplification and sequencing of the 16S rRNA gene using the prokaryotic 16S rDNA universal primers 515F (5-GCGGATCCTCTAGACTGCAGTGCCA-3) and 1492R (5-GGTTACCTTGTTACGACTT-3). The vegetative cells were routinely maintained at −80 °C in M17 medium (Difco) containing 5 % (v/w) glucose and glycerol (10 %). The organisms were activated without heat shock in M17 containing the sugar or sugar combination to be tested. After activation, cultures were transferred into the same medium used in the reactors. The composition of the medium used for the batch experiments has been reported previously (Heluane et al., 2011). The medium base components were autoclaved without the sugars and trace components, which were always sterilized by filtration and added aseptically to the medium reservoir. Combinations of sugars (sucrose, d-glucose and d-fructose, and d-fructose) were aseptically added into the reactor to reach a 6 % sugar final concentration.

Batch growth experiments were performed in a 2 l BIOSTAT Bplus bioreactor equipped with controllers for pH, temperature and agitation (Sartorius, BBI Systems). The temperature was set at 37 °C, the agitation speed was set at 250 r.p.m. and the pH was kept at 6.5 by the automated addition of 0.5 N KOH or 0.1 M H3PO4 into a working volume of 1400 ml. In order to maintain initial anaerobic conditions after inoculation, a continuous stream of sterile nitrogen gas at 0.1 v/v min–1 was passed through the reactors, usually for between 5 and 10 min. Exact conditions for the fermentations are detailed when the different assays are presented in Results. The purity of the culture was confirmed daily by direct microscopic examination and by plating dilutions on M17 agar plates (Terzaghi & Sandine, 1975).

Preparation of total DNA and genome sequencing.

Genomic DNA was isolated from C. beijerinckii SA-1 cells in exponential growth phase using the modified cetyltrimethylammonium bromide method (Wilson, 1994). DNA quantification was performed by comparison with DNA standards (15, 31, 63, 125, 250 and 500 ng) supplied by the US Department of Energy Joint Genome Institute (DOE JGI). Quantity One software (version 4.5.2) was used to create a calibration curve and gel imaging was performed using a Gel-Doc 2000 (Bio-Rad). Next-generation sequencing was performed on a Genome Analyser IIx (Illumina) by the DOE JGI. Sequences were mapped to the reference genome C. beijerinckii NCIMB 8052, and single nucleotides, small insertions and small deletions were analysed using maq version 0.7.1 (Li et al., 2008). A genome-wide analysis of anomalous read pairs to identify large structural variations (inversions, translocations and large indels) was performed using BreakDancer (Chen et al., 2009). Sequence data were analysed visually using the Integrative Genome Viewer (igv) for confirmation of alignment assembly errors (Robinson et al., 2011).

Nucleotide accession number.

The Whole Genome Shotgun project has been deposited under GenBank sequence read archive accession number SRR191792.

Analytical Methods.

Biomass proliferation in the fermentation tank was monitored using an in-line biomass sensor (Fundalux; Sartorius, BBI Systems) and also by measuring the OD600 on a digital spectrophotometer (SmartSpec Plus; Bio-Rad). Sucrose, d-glucose and d-fructose were quantified by HPLC (Shimadzu) under isocratic conditions at 65 °C and a mobile phase of water at a 0.5 ml min−1 flow rate using a Supelcogel Ca column (300 mm×7.8 mm; Supelco Analytical) coupled to a refractive index detector. The products of the fermentations (butanol, butyric acid, acetone, acetic acid and ethanol) were quantified immediately after sample collection by a gas chromatograph (GC-8A) fitted with a flame ionization detector (Shimadzu). The products were separated in a GC SS Porapak Q 80/100 column (OV) using 200 kPa of nitrogen as the mobile phase with an injection temperature of 220 °C and a column temperature of 140 °C.

Results

Batch cultivation revealed considerable physiological differences between C. beijerinckii NCIMB 8052 and C. beijerinckii SA-1

The literature reported that C. beijerinckii SA-1 was able to tolerate higher concentrations of butanol than NCIMB 8052 (Lin & Blaschek, 1983). Here, we confirmed that these two strains also display fundamental differences with respect to growth, sugar utilization, solvent production and yields (Fig. 1, Table 1). The two strains were grown in pH (6.5)-controlled batch culture. Generation times of 1.5 h were achieved by both strains when growing with sucrose or glucose/fructose as the limiting carbon and energy sources. However, the sugar consumption and the co-fermentation profiles of the two strains differed; whilst NCIMB 8052 was able to co-ferment d-glucose/d-fructose and readily use d-fructose, SA-1 was unable to co-ferment both sugars.

Fig. 1.

Fig. 1.

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Fermentation profile experiments for C. beijerinckii NCIMB 8052 (left) and SA-1/ATCC 35702 (right) at 37 °C, controlled pH (6.5) in a validated medium (Heluane et al., 2011) using as limiting carbon and energy source: (a, b) sucrose (6 %, w/w), (c, d) d-glucose (3 %, w/v) and d-fructose (3 %, w/v), and (e, f) d-fructose (6 %, w/v). Biomass was monitored by two methods: by the in-line biomass sensor Fundalux (arbitrary units) and by optical density.

Table 1.

Kinetic and yield parameters obtained after pH-controlled batch fermentations of C. beijerinckii NCIMB 8052 and SA-1 strains at 37 °C, pH 6.5 in a validated medium (Heluane et al., 2011)

Carbon/energy source Parameter C. beijerinckii
NCIMB 8052 SA-1
Sucrose 6 % (w/v) Biomass (OD600 ml−1) 4.72 5.7
Butanol (g l−1) 8.09±0.27 13.48±0.01
Acetone (g l−1) 6.5±0.13 11.22±0.04
Ethanol (g l−1) 0.49±0.12 0.59±0.01
Total solvents (g l−1) 15.09±0.38 25.30±0.04
Sugar utilized (%) 72.57 100
Specific growth rate µ (h−1) 0.41 0.39
Butanol yield (g g−1) 0.21 0.24
Solvents yield (g g−1) 0.39 0.45
Butanol productivity (g l−1 h−1) 0.13 0.34
d-Glucose 3 %(w/v) and d-fructose 3 %(w/v) Biomass (OD600 ml−1) 9.65 11
Butanol (g l−1) 10.01±0.48 16.05±0.1
Acetone (g l−1) 6.40±0.15 9.92±0.02
Ethanol (g l−1) 0.28±0.03 0.43±0.03
Total solvents (g l−1) 16.85±0.33 26.4±0.1
Sugar utilized (%) 100 100 glucose/67 fructose
Specific growth rate µ (h−1) 0.45 0.46
Butanol yield (g g−1) 0.19 0.3
Solvents yield (g g−1) 0.32 0.49
Butanol productivity (g l−1 h−1) 0.62 1.14
d-Fructose 6 % (w/v) Biomass (OD600 ml−1) 6.82 5.72
Butanol (g l−1) 11.53±0.12 12.6±0.44
Acetone (g l−1) 6.26±0.14 11.33±0.03
Ethanol (g l−1) 0.80±0.007 0.71±0.09
Total solvents (g l−1) 18.59±0.26 24.64±0.53
Sugar utilized (%) 100 100
Specific growth rate µ (h−1) 0.66 0.09
Butanol yield (g g−1) 0.24 0.27
Solvents yield (g g−1) 0.4 0.53
Butanol productivity (g l−1 h−1) 0.54 0.17
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NCIMB 8052 was able to accumulate concentrations of butanol and overall solvents consistent with previously reported maximum levels of butanol >10 g l−1 and solvents >15 g l−1 (Formanek et al., 1997; Wang & Blaschek, 2011). Moreover, while both strains reached comparable biomass values, SA-1 revealed a solventogenic hyper-production phenotype and was able to generate up to 16.1 g butanol l−1 and 26.3 g total ABE l−1, an accumulation improvement of 62.3 % and 63 % compared with NCIMB 8052, respectively. Also, butanol and ABE yield values were significantly higher as compared with NCIMB 8052 under all conditions tested (Table 1).

Sequencing of C. beijerinckii SA-1, comparative assembly to the reference genome C. beijerinckii NCIMB 8052 and validation of the genomic variations

In order to generate a solid basis for understanding the similarities and differences in cell biology and physiology of C. beijerinckii strains, we performed genome sequencing and a comparative analysis. Whole-genome sequencing of strain SA-1 was performed on the Illumina GA IIx platform using one flow cell lane with 36-cycle paired-end chemistry (GenBank sequence read archive accession number SRR191792). This generated 55 995 398 paired-end reads with a mean read length of 98 bp, resulting in a mean sequence depth of 695 reads that corresponds to a 914-fold mean coverage.

The genome sequence evaluated in this study confirms the taxonomic assignment of SA-1 to C. beijerinckii within the clostridial solventogenic group. The ORF identifiers in C. beijerinckii SA-1 also correspond to the same numbers in C. beijerinckii NCIMB 8052. For clarity, we have denoted the C. beijerinckii SA-1 genes as ‘cbs_ORF number’ (Fig. 2a).

Fig. 2.

Fig. 2.

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C. beijerinckii SA-1 genome atlas providing the physical locations of the putative and confirmed polymorphisms. (a) Polymorphisms discovered performing in silico analysis with maq and BreakDancer algorithms. (b) Polymorphisms validated after PCR and Sanger sequencing methods. Image produced using Geneious (http://www.geneious.com). Purple, insertions; navy blue, deletions; black, SNVs; maroon, putative insertions or deletions (indels); gold, putative small indels; green, inversions; cyan, internal translocations. The term ‘Cbs’ is used here to refer specifically to genes that are in the SA-1 strain.

The C. beijerinckii SA-1 genome is 6 000 627 bp in size, does not contain plasmids and is predicted to encode 5243 protein-coding genes. The G+C content of the genome is 29.9 %, which is the same as the parent strain C. beijerinckii NCIMB 8052 (GenBank accession number NC_009617). The SA-1 strain contains 94 tRNA genes; this is the same number of tRNA genes and same genome size as C. beijerinckii NCIMB 8052.

Assembly of the short sequence reads (GenBank sequence read archive accession number SRR191792) and analysis of the results using maq and BreakDancer identified genetic polymorphisms that occurred in the offspring strain. The list of polymorphisms includes eight single nucleotide variants (SNV), one small deletion and one large insertion that is an additional copy of the insertion sequence ISCb1, belonging to the IS4 family (Liyanage et al., 2000) inserted between ORFs cbs_1788 and cbs_1789. To validate all genomic variations, we performed PCR and Sanger sequencing using chromosomal DNA from the strains SA-1 and NCIMB 8052 as templates (Fig. 2b).

SNVs

Overall, most of the SNVs were found within ORFs. Out of the original 12 SNVs identified by maq, eight were confirmed by PCR amplification and Sanger sequencing (Eton Bioscience), whilst four SNVs corresponded to errors that were found in the reference genome of C. beijerinckii NCIMB 8052 (cbei_1046, cbei_R0117, cbei_2885 and cbei_4308). The corrections to these loci are reported in Tables 2 and 3.

Table 2.

Single nucleotide corrections to the reference sequence (GenBank accession number CP000721.1) from C. beijerinckii NCIMB 8052

Table 3 shows physical locations and primer sequences used to verify these polymorphisms. CDS, coding sequence.

ORF number Putative function Position Type Reference sequence Actual sequence
cbei_1046 Band 7 protein CDS 1 255 087 SNV A G
cbei_R0117 23S rRNA CDS 5 747 394 SNV A G
cbei_2885 l-lactate transport protein CDS 3 369 627 Indel –A +A
cbei_4308 Protein glutamate O-methyltransferase CDS 4 962 014 Indel +C –C
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Table 3.

List of point mutations detected and confirmed in the chromosome sequence of C. beijerinckii SA-1/ATCC 35702 using the maq algorithm (Li et al., 2008)

Coverage, variant frequencies and reference frequencies determined through genome assembly using Geneious (www.geneious.com). PCR amplifications were performed using genomic DNA from both NCIMB 8052 (reference strain) and SA-1 as template. COG, Clusters of orthologous groups; CDS, coding sequence. IUPAC naming conventions are used to communicate base and amino acid changes. Primer names are indicated by their position and direction (F, forward; R, reverse). The term ‘cbs’ here is used to emphasize the fact that the mutation occurs in the derivative strain, C. beijerinckii SA-1/ATCC 35702. Term, Termination codon.

ORF number and strand orientation Associated COG CDS size (bp) Mutation position Putative gene function SNV change in CDS and amino acid change in CDS Primers Coverage Variant frequencies (%) Reference frequencies (%)
cbs_0769 + COG1653G 1329 935 449 Solute binding protein C151T Q51Term 934943F 5′-TCGGTTGATACTGATTTATG-3′ 935763R 5′-TTTACCTGGAAAAGTGATAC-3′ 498 100.0
cbs_1854 + S (unknown function) 1737 2 149 286 Peptidase S8/S53 subtilisin kexin sedolisin T698C V323A 2149110F 5′-GTAGTTTGGGAAATCATAAGG-3′ 2149781R 5′-CTGCAACATCTATTCTATCAG-3′ 403 100.0
cbs_1935 + COG2508TQ 1155 2 234 607 PucR transcriptional regulator C139A H47N 2234281F 5′-CATCCGAATCATTTTATAGC-3′ 2234964R 5′-ATGACTGTCTCTCAAATATC-3′ 326 99.4 0.3
cbs_1975 + COG1001F 1707 2 295 776 Adenine deaminase G31T A11S 2295543F 5′-GCCTAAAACAAGCTATATC-3′ 2296459R 5′-CTAAGCATCACATACATAC-3′ 162 99.4
Between cbs_2248 and cbs_2247 – 402 (interval length between coding regions) 2 611 044 Non-coding region between rRNA adenine dimethylase CDS and hypothetical protein CDS G : A 2610380F 5′-AAACAAGATGGCATAAGC-3′ 2611343R 5′-CCTCCAAGATTAATATAGG-3′
cbs_4400 – COG0631T 1839 5 075 403 Serine threonine protein phosphatase-like protein C1766A R589L 5073615F 5′-GCATGATTCTGTCATTTAG-3′ 5074072R 5′-AGCAGGTGATTATGTAAG-3′
cbs_4761 – COG5263R 1218 5 556 196 Cell wall binding repeat-containing protein A101G V34A 5554176F 5′-CCCATTAACGTAATACTG-3′ 5554911R 5′-GTCTATGGCTTATAACAC-3′ 472 99.4
Between cbs_R0121 and cbs_R0122 – 1112 (interval length between coding regions) 5 769 733 Non-coding region between 16S RNA CDS and 5S RNA CDS A : G 5769433F 5′-GTTTCTGACAGCTTACTC-3′ 5769939R 5′-TGTATCCAGGGGTAATTG-3′ 738 57.2 42.5
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cbs_0769 encodes a putative solute binding protein that is interrupted by a nonsense mutation at residue 51, potentially making this protein non-functional. A blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis showed that this protein contains motifs similar to an ABC-type sugar transporter in Clostridium thermocellum. Previous work with C. thermocellum demonstrated that these ABC transport systems are able to transport cellodextrin and laminaribiose into the cell (Nataf et al., 2009). We speculate that the mutation in cbs_0769 may impact SA-1’s ability to transport d-fructose.

In cbs_1854, a valine was substituted for alanine in the amino acid sequence for a putative peptidase S8/S53 subtilisin kexin sedolisin, a member of the serine-carboxyl peptidases (Wlodawer et al., 2003).

ORF cbs_1935 encodes a putative PucR transcriptional regulator. PucR regulators are responsible for regulating purine catabolism (Schultz et al., 2001). In SA-1, the SNV changed a histidine residue, positively charged, for a polar uncharged asparagine residue. This missense mutation at residue 47 (H47N) occurred in a conserved region of the protein and may have an effect on the function of the protein in regulating purine utilization (Xi et al., 2000; Heluane et al., 2011).

cbs_1975 encodes an adenine deaminase, responsible for converting adenosine to inosine in the nucleotide salvage pathway (Kilstrup et al., 2005). In SA-1, a SNV at position 1707 position in the coding sequence of a G→T produced an amino acid change from alanine to serine at position 11 (A11S). This missense mutation does not occur within the conserved protein functional region.

cbs_4400 encodes a serine threonine phosphatase. A SNV at position 1766 position in the coding sequence is a C→A transversion that caused an arginine to leucine substitution at position 589 (R589L) and is outside of a conserved domain of the protein. Multiple cellular processes are regulated by this type of protein, including sugar transport and endospore formation (Obuchowski, 2005).

ORF cbs_4761, encoding a cell wall binding motif, has a mutation at position 5 556 196 (A→G transition; V34A).

PCR and Sanger sequencing also confirmed two mutations occurring in non-coding regions of the genome. The SNV between ORFs cbs_2247 and cbs_2248 is a G→A transition, and the SNV between coding sequences cbs_R0121 (16S RNA) and cbs_R0122 (5S RNA) is an A→G change. In order to determine whether or not these non-coding regions contained regulatory RNAs, an alignment was performed against the Rfam RNA family database located at http://rfam.sanger.ac.uk/ (Griffiths-Jones et al., 2003). No conserved RNA families were found within these non-coding regions.

Small insertions and deletions

Eight small insertions and deletions (1–5 bp) were initially identified using maq and visualized with igv software (Li et al., 2008; Robinson et al., 2011) (Table 4). Seven were found to be false positives, whilst a small deletion of the nucleotide sequence AAATA was confirmed using PCR and Sanger sequencing (primers 5′-TTGCACAAGGAAAACAAG-3′ and 5′-TACAGAATCCCAACTATCAG-3′). At position 1 099 984 in the SA-1 genome, the deletion caused a frame-shift mutation that abolished the stop codon and extended a hypothetical protein (cbs_0917) by three amino acid residues. The detection of false mutations by the maq algorithm has been reported previously (Krawitz et al., 2010; Hamada et al., 2011). With regard to DNA repeats in the sequence, it became apparent that maq and BreakDancer had difficulty in identifying mutations in these regions; this limitation of the alignment software in the in silico analysis has been reported elsewhere and remains to be resolved (Treangen & Salzberg, 2012).

Table 4.

In silico-detected and not corroborated SNVs and small insertions and deletions (indels) from the sequence of C. beijerinckii SA-1/ATCC 35702

Coverage, variant frequencies, and reference frequencies determined through genome assembly using Geneious (http://www.geneious.com). PCR amplifications were performed using genomic DNA from both NCIMB 8052 (reference strain) and SA-1 as template. COG, Clusters of orthologous groups; CDS, coding sequence. Primer positions and directions are indicated by the number followed by F (forward) or R (reverse). NA, Amino acid sequence not available because base change is within a non-coding region.

Type ORF number Associated COG CDS size (bp) Position Gene function Primer Coverage Variant frequency (%) Reference frequency (%)
SNVs cbs_0433 COG0653U 2568 524 879 Sec A 524413F 5′-TTCATCAAGCGATAGAAG-3 525161R 5′-TTGAGCATTCTCAATAGC-3′ 792 34.2 65.0
cbs_2250 COG2205T 981 2 613 440 Histidine kinase 2612973F 5′-GAAAGGGATAATCAACAAAG-3′ 2613589R 5′-CTTTATCCAGTTTCACATAG-3′
cbs_1046 COG0330O 948 1 255 087 Band 7 protein 1254781F 5′-TCAGCTACAACTAATATGAG-3′ 1255488R 5′-GATGCCTTCTTAATTGATTC-3′ 475 99.8
cbs_R0117 NA 2909 5 747 394 23S rRNA 5747160F 5′-GAGCTAATCACGCAAATC-3′ 5748001R 5′-CGCCTCCTAAAATGTAAC-3′
Small indels cbs_2885 COG1620C 1465 3 369 627 l-lactate transport protein CDS 3369256F 5′-CTAGAACTGCTGAAAATG-3′ 3370068R 5′-GTAGCAATTGTTGGAAATG-3′
cbs_4845 S (unknown function) 1173 5 667 579 Mandelate racemase/muconate lactonizing protein CDS 5667420F 5′-TATCATCATATGCTCCTAAG-3′ 5668012R 5′-TAGAGCAGATGGATATACAG-3′
cbs_4963 COG2972T 1761 5 838 970 Integral membrane sensor signal transduction histidine kinase CDS 5838895F 5′-AATAATCCACTTCCCTTTAG-3′ 5839486R 5′-ACAAATAAAGCCTCACTTTC-3′
cbs_5024 COG1506E 1167 5 907 883 α/β-fold family hydrolase CDS 5907769F 5′-CAAGCACGAATGTAACAG-3′5908400R 5′-CATGGTGGAGAAATCTATG-3′
cbs_4308 COG1352T 713 4 962 014 Protein-glutamate O-methyltransferase CDS 4961567F 5′-CTAAGGCTTTCATTATTCC-3′ 4962360R 5′-TGAGCCATATTCATTAGG-3′
Non-coding region NA 392 1 314 429 Located between Cbei_1100 CDS and hypothetical protein CDS 1313971F 5′-GAGTGGTAATATACTAGGTAAG-3′ 1314664R 5′-CTCTAGTTGGTAGTTCATATTC-3′
Non-coding region NA 370 3 369 630 Located between l-lactate transport protein CDS and electron transfer flavoprotein, α subunit-like protein CDS 3369256F 5′-CTAGAACTGCTGAAAATG-3′ 3370068R 5′-GTAGCAATTGTTGGAAATG-3′
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Large insertions, deletions and structural variations

Larger insertions and deletions (20–40 bp) as well as structural variations (e.g. chromosomal translocations and inversions) were analysed using BreakDancer version 1.0 (Chen et al., 2009). The initial in silico analysis produced 10 large putative indels (Table 5). One was found to be an additional copy of the insertion sequence from the IS4 family located between a hypothetical protein (cbs_1788) and a polysaccharide deacetylase (cbs_1789) at position 2 073 281 in the SA-1 genome. Using forward primer 5′-TTGCACCTAATGTGTAATC-3′ and reverse primer 5′-CTATCTCCTCTTTCATTCTATC-3′, this insertion sequence was confirmed by PCR and Sanger sequencing. Previous work has shown that C. beijerinckii NCIMB 8052 contains at least nine copies of an insertion sequence from the IS4 family (Liyanage et al., 2000) (Fig. 3).

Table 5.

In silico-detected and not corroborated large insertions, deletions, and structural variations

PCR amplifications were performed using genomic DNA from both NCIMB 8052 (reference strain) and SA-1 as template. CDS, Coding sequence; COG, clusters of orthologous groups. Primer positions are indicated by the number followed by the direction F (forward) or R (reverse).

Large indels
Location Size (bp) Position Primer
Between hypothetical protein CDS and hypothetical protein CDS 49 3 829 123–3 829 172 3828725F 5′-ATGCAGCCAAATAATGAC-3′
Between hypothetical protein CDS and hypothetical protein CDS 29 3 829 172–3 829 201 3829751R 5′-GAGCTCGATAATTACTAAG-3′
Between hydroxylamine reductase CDS and heavy metal transport detoxification protein CDS 37 1 685 323–1 685 360 1685136F 5′-TATATGCAGCAGAGCTAC-3′ 1685848R 5′-TCTTACCCCTGATGTTTC-3′
Between EmrB/QacA family drug resistance transporter CDS and CBS domain-containing protein CDS 35 1 797 550–1 797 585 1797141F 5′-GCTATGATGCCTATTAGC-3′ 1798121R 5′-CGCCTCTACTAATAACTCC-3′
Between MATE efflux family protein CDS and TPR repeat-containing protein CDS 30 2 039 733–2 039 763 2039609F 5′-TGGCATAAGGATATAATCG-3′ 2040035R 5′-AATGCCTTATCTAGCTTTC-3′
Between GCN5-related N-acetyltransferase CDS and hypothetical protein CDS 26 2 961 163–2 961 189 2960642F 5′-TCGGATTTATCTAGCAAAG-3′ 2961396R 5′-CTTTTATCGCACGAATATG-3′
Between glycosyltransferase family protein CDS and triple helix repeat-containing collagen CDS 26 3 001 786–3 001 812 3001353F 5′-CCAGTATGTAGCTAAGTTTC-3′ 3002219R 5′- CTTCTAAATAGGTCATTAGG-3′
Between thioredoxin reductase CDS and hypothetical protein CDS 28 3 117 231–3 117 259 3117006F 5′-CCAGGATAATTTTCCACTTC-3′ 3002219R 5′-AGACCTAGGCTATAGTTTTG-3′
Between metal-dependent phosphohydrolase CDS and sulfate adenyltransferase large subunit CDS 37 4 819 865–4 819 902 4819453F 5′-GTTAGGCTCATCTAAATAC-3′ 4820145R 5′-GAAAACTCTAGGAGATTTC-3′
Structural variations
ORF number Associated COG/type Size (bp) Position Primer
cbs_4437 COG4974L/prophage inversion 27 5 120 090–5 120 520 5119799F 5′-CACCCTTTCTACAATTAAC-3′ 5120810R 5′-TGCCATTGAGAAATATTGC-3′
cbs_4182 COG1352NT/deletion 483 4 812 971–4 814 517 4812830F 5′-AAGTTATCTGTGGTTCTATC-3′
cbs_4182 COG1352NT/deletion 529 4 814 951–4 814 980 4813942R 5′-ATTGAAGAAATGGGTGTATC-3′
cbs_0060 S (unknown function)/insertion 149 79 389–79 831 79067F 5′-TGCTATATGTAGGGTAAAAG-3′ 80063R 5′-CACGTCATTCAATTTATCC-3′
cbs_0878–cbs_0932 COG1476K/internal translocation (prophage ITX1) 39 411 1 071 995–1 111 510 1071556F 5′-GACCAGAACAAACTATTTAC-3′ 1072303R 5′-TAAGGCTACAGATGATAAAG-3′ 1111245F 5′-AGATGGCACATTGTTTTG-3′ 1111872R 5′-ACTGCTGACCTTTATAGATAG-3′
cbs_1605–cbs_1680 COG1961L/internal translocation (prophage ITX2) 75 644 1 881 845–1 957 557 1881067F 5′-AAAATTGGACGAGGTAATAG-3′ 1958001R 5′-CAGTGAATAGTGCTATATAC-3′ 1957306F 5′-TATGAAAAGGAGCATATCAG-3′ 1883070R 5′-ATGAACTTCAGGTCTATGG-3′
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Fig. 3.

Fig. 3.

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Agarose gel (1 %) showing PCR amplification of the novel insertion sequence within SA-1 (lane 2) compared with the WT NCIMB 8052 (lane 3). Lane 1, 10 kb Logic DNA Marker (Lambda Biotech). Lane 4, PCR with no template added (negative control).

Two overlapping putative indels were also confirmed in a non-coding region between 3 829 123 and 3 829 201, which had AT-rich regions where runs and repeats were common (64 repeats ≥4 bp in length).

Six structural variations were suggested by the in silico analysis; however, through PCR and Sanger sequencing methods we have determined these structural variations were false positives (Table 5). One of these structural variations included a potential inversion at ORF cbs_4437. An attempt to amplify this structural variation with PCR proved to be difficult and upon further analysis it was found that this region contained 93 repeats ≥6 bp in length. In order to determine whether or not these repeats were CRISPR (clustered regularly interspaced short palindromic repeat) elements, a basic local alignment with the SA-1 genome was performed against the CRISPR database available at http://crispr.u-psud.fr/ (Grissa et al., 2007a, b, 2008; Garrett et al., 2011). It was found that there were no previously characterized CRISPR elements present within ORF cbs_4437 or the non-coding region between positions 3 829 123 and 3 829 201.

Discussion

The development and characterization of butanol-producing strains is essential for the continued advancement of renewable energy technology. In this study, we reveal the solventogenic hyper-production phenotype of C. beijerinckii SA-1/ATCC 35702 (a derivative strain of C. beijerinckii NCIMB 8052). Batch cultivations of SA-1 on a previously validated medium (Heluane et al., 2011) compared with NCIMB 8052 showed higher physiological differences in solvent accumulation and sugar utilization than those observed initially in extruded broth (Lin & Blaschek, 1983). SA-1 displays a metabolic carbon flow that is redirected mainly to the production of increased amounts of butanol and acetone. Redirecting the metabolic flow of carbon towards solvents to reach higher final butanol titres and yields has been shown previously by genetically modified C. acetobutylicum strains with disrupted genes responsible for butyric acid production (Harris et al., 2000; Jang et al., 2012b). Moreover, the overall butanol accumulation, ABE yields and solvent productivity of SA-1 are comparable to values previously reported for C. beijerinckii BA101, another butanol hyper-producing offspring of NCIMB 8052 (Formanek et al., 1997; Ezeji et al., 2007a, b; Qureshi & Blaschek, 2000; Lienhardt et al., 2002). BA101 solventogenic carbon flow redirection has been correlated with a 50 % lower PTS system activity, including reduced fructose PTS activity when growing in d-glucose (Lee & Blaschek, 2001; Lee et al., 2005).

The comparative analysis of the genomes of SA-1 and C. beijerinckii NCIMB 8052 enabled us to detect four errors in the reference genome, and a relatively low number of SNVs, indels and chromosomal rearrangements in SA-1, that suggest relative chromosome stability in these important solventogenic Clostridium strains (Kutzenok & Aschner, 1952; Gottschal & Morris, 1981; Kashket & Cao, 1993, 1995). Butanol concentration and tolerance limits are not correlated directly (Harris et al., 2000) and no differences were found to explain previously reported butanol tolerance of SA-1 (Lin & Blaschek, 1983). However, the differences in d-fructose utilization and the SNV found in cbs_0769 could be evidence that this putative solute binding protein may be involved in d-fructose transport. The understanding of sucrose utilization and its regulation has obvious industrial significance as it is possible that d-fructose may repress the uptake of sucrose (Tangney et al., 1998). Additionally, the polymorphism identified in a serine threonine phosphatase (cbs_4400) may be responsible for deregulation of a SA-1 PTS transport system, for delayed sporulation and/or involved in the increased production of butanol (Mitchell et al., 1991).

Once genetic modification tools are more advanced for C. beijerinckii, the insights unveiled here will support future functional and metabolic engineering work. Consequently, genetic modification efforts targeting these polymorphisms will allow us to correlate the proposed gene functions and the phenotypic differences, including metabolic carbon flow redirection and sugar transport in C. beijerinckii.

Acknowledgements

This work was partially supported by the College of Life Sciences at NC State University, the North Carolina Agricultural Research Service and the Biofuels Center of North Carolina. The work conducted by the US DOE JGI was supported by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231.

Footnotes

Abbreviations: ABE, acetone/butanol/ethanol; CRISPR, clustered regularly interspaced short palindromic repeat; DOE JGI, US Department of Energy Joint Genome Institute; PTS, phosphotransferase system; SNV, single nucleotide variation.

The GenBank/EMBL/DDBJ accession number for the sequence read archive of Clostridium beijerinckii ATCC 35702 is SRR191792.

Edited by: S. Kengen

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