Background: There is a worldwide interest for sustainable and environmentally-friendly ways to produce fuels and chemicals from renewable resources. Among them, the production of acetone, butanol and ethanol (ABE) or Isopropanol, Butanol and Ethanol (IBE) by anaerobic fermentation has already a long industrial history. Isopropanol has recently received a specific interest and the best studied natural isopropanol producer is C. beijerinckii DSM 6423 (NRRL B-593). This strain metabolizes sugars into a mix of IBE with only low concentrations of ethanol produced (< 1 g/L). However, despite its relative ancient discovery, few genomic details have been described for this strain. Research efforts including omics and genetic engineering approaches are therefore needed to enable the use of C. beijerinckii as a microbial cell factory for production of isopropanol. Results: The complete genome sequence and a first transcriptome analysis of C. beijerinckii DSM 6423 are described in this manuscript. The combination of MiSeq and de novo PacBio sequencing revealed a 6.38 Mbp chromosome containing 6254 genomic objects. Three Mobile Genetic Elements (MGE) were also detected: a linear double stranded DNA bacteriophage (ϕ6423) and two plasmids (pNF1 and pNF2) highlighting the genomic complexity of this strain. A first RNA-seq transcriptomic study was then performed on 3 independent glucose fermentations. Clustering analysis allowed us to detect some key gene clusters involved in the main life cycle steps (acidogenesis, solvantogenesis and sporulation) and differentially regulated among the fermentation. These putative clusters included some putative metabolic operons comparable to those found in other reference strains such as C. beijerinckii NCIMB 8052 or C. acetobutylicum ATCC 824. Interestingly, only one gene was encoding for an alcohol dehydrogenase converting acetone into isopropanol, suggesting a single genomic event occurred on this strain to produce isopropanol. Conclusions: We present the full genome sequence of Clostridium beijerinckii DSM 6423, providing a complete genetic background of this strain. This offer a great opportunity for the development of dedicated genetic tools currently lacking for this strain. Moreover, a first RNA-seq analysis allow us to better understand the global metabolism of this natural isopropanol producer, opening the door to future targeted engineering approaches. Keywords: Clostridium beijerinckii, DSM6423, Genome, RNA-seq transcriptome, Clustering, IBE fermentation, Isopropanol * Correspondence: firstname.lastname@example.org IFP Energies Nouvelles, 1 et 4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France Full list of author information is available at the end of the article © The Author(s). 2018, corrected publication June 2018. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 2 of 12 Background This study presents the complete genome sequencing The use of petrochemical derived fuels and chemicals of the currently most studied natural isopropanol produ- such as olefins cause severe damages to the environ- cing strain, together with RNA-seq analyses covering its ment and is not sustainable in the long term, due to whole fermentation metabolism for glucose conversion the limited nature of fossil oil. Approaches based on into IBE. Comparative analysis with the model organ- the conversion of renewable biomass into chemicals isms highlights some transcriptomic regulations. of interest may represent a sustainable alternative to replace, at least partially, these petroleum-based che- Methods micals. As an example, isopropanol can be added into Bacterial culture and bioreactor scale fermentations fuel blends  or converted into olefins using chem- Laboratory stocks of C. beijerinckii DSM 6423 spores were ical dehydration steps . Isopropanol is a commer- stored in sterile H Oat − 20 °C. Spores were heat shocked in cial C3 alcohol used as solvent or antifreeze, which boiling water for 1 min, then inoculated at a 2% inoculum can also be used as a precursor of propylene, one of level into 50 mL medium containing per liter: yeast extract, the main platform molecules used in the chemical indus- 5.0 g; KH PO ,0.75g;K HPO ,0.75g;MgSO ·7H O, 0.4 g; 2 4 2 4 4 2 try. Very few microorganisms have been described as nat- MnSO ·H O, 0.01 g; FeSO ·7H O, 0.01 g; NaCl, 1.0 g; 4 2 4 2 ural isopropanol producers and almost none of them has asparagine, 2.0 g; (NH ) SO ,2.0 g;cysteine,0.125 g;and 4 2 4 been thoroughly studied. Solventogenic Clostridia have glucose 12.5 g. The medium was incubated at 37 °C ± 1 °C been mainly studied for their ability to produce a mixture for 24 h in an anaerobic environment under N :CO :H 2 2 2 of acetone, butanol and ethanol (ABE) from a variety of (volume ratio of 85:10:5) atmosphere. Subsequently, the substrates (including C6 and C5 sugars), and a few strains actively growing culture was inoculated at 5% into a fer- are also able to further reduce acetone into Isopropanol, mentation medium (GAPES ) containing per liter: yeast yielding an Isopropanol, Butanol and Ethanol (IBE) extract, 5.0 g; KH PO , 1.0 g; K HPO ,0.76 g; NH Ac, 3. 2 4 2 4 4 mixture. C. acetobutylicum ATCC 824 and C. beijerinckii 0 g; MgSO ·7H O, 1.0 g; FeSO ·7H O, 0.1 g; p-aminoben- 4 2 4 2 NCIMB 8052 are recognized as model organisms for ABE zoic acid, 0.1 g; and glucose, 60 g; in a 500 mL bioreactor production, while C. beijerinckii DSM6423 (formerly system as described previously . Temperature was NRRL B-593) is the best known IBE producer. The gen- controlled at 37 °C ± 1 °C. A stirring at 350 rpm was ome of C. beijerinckii NCIMB 8052 was sequenced a few employed for mixing. Cell density and product concentra- years ago and consists in a 6.0 Mb chromosome , which tion were monitored through the course of fermentation. is 50% larger than that of C. acetobutylicum ATCC 824 . Unlike these strains, the assembled genome of C. beijerinckii DSM 6423 (NRRL B-593) is not available, even Experimental design if it has been sequenced through a Hiseq approach re- In order to get 3 biological replicates, the same proced- cently, together with 29 other Clostridial strains, in order ure was repeated 3 times on 3 different weeks. Each to perform a comparative genomic analysis of saccharoly- week, a fresh preculture was used to inoculate 2 identi- tic strains belonging to the genus of Clostridium . cal bioreactors. Samples were taken over the early expo- Transcriptional analysis is mandatory to gain know- nential, late exponential and stationary phases (samples ledge about gene regulation and thus determine strat- at 3, 6, 8, 11, and 24 h). Following centrifugation of the egies for strain improvement. To date, transcriptomic samples, cell pellets were immediately frozen in liquid analyses of solventogenic Clostridia have focused on nitrogen and supernatants were used for HPLC analysis. ABE strains . Most of these transcriptomic analyses For each timepoint, the RNA samples of the 2 bioreac- were performed using DNA microarray methods and tors were pooled, leading to 1 RNA sample per bioreac- other hybridization techniques exhibiting a relatively tor and timepoint. low dynamic range for the detection of transcriptional levels due to background, saturation and poor sensi- tivity for gene expression. RNA-seq has now supple- Culture growth and fermentation products analysis mented these approaches because of its capacity to Culture growth was measured by following optical density provide a more accurate quantification and a larger at 600 nm (OD600) in the fermentation broth using an dynamic range of expression levels. In addition, it has Ultrospec 2000 (Pharmacia Biotech) spectrophotometer. very low background noise because DNA sequence Metabolites were determined in clear supernatants of reads can be unambiguously mapped to unique samples taken from the fermentation. Sugars, solvents, regions along the genome . The genome-wide tran- and organic acids were determined by HPLC using a gel scriptional dynamics of C. beijerinckii NCIMB 8052 permeation/size exclusion column (Shodex Ionpack KC- over a batch fermentation process was investigated 811) coupled to a refractometer and UV detector as using these technique . described earlier [10, 11]. Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 3 of 12 Genomic DNA extraction and sequencing agarose gel were done before depletion of ribosomal Genomic DNA of C. beijerinckii DSM6423 was purified RNA with MicrobExpress kit (ThermoFischer). Depleted using the GenElute bacterial genomic DNA kit (Sigma- RNA samples were quantified and stored at − 80 °C be- Aldrich, Saint-Louis, USA). Concentration of gDNA was fore being sent for analysis. DNA libraries from RNA determined using Qbit spectrophotometer (Thermo- samples and Hi-seq single read sequencing were per- fisher) and quality checked on 0.5% agar gel. Mi-seq formed by the Imagif platform (I2BC, Orsay, France). analysis was performed by the I2BC plat-form (Saclay, France) using the following protocol. Genomic DNA RNA-seq sequencing data analysis samples was fragmented using a “‘COVARIS S2” sonica- Clean reads of each sample were mapped to the refer- tor, to a mean size of 700 bp. DNA Libraries were con- ence genome of the previously sequenced C. beijerinckii structed from 1 μg of this fragmented DNA, according DSM 6423, with predicted protein encoding genes and to the “Illumina DNA sample prep protocol” (End-repair, rRNA using the TAMARA (Transcriptome Analysis A-tailing, ligation, PCR enrichment), on a Beckman based on MAssive sequencing of RnAs) tool provided SPRI-TE automate and using reagents from the “SPRI- on the MicroScope platform . Table 1 shows statis- works Fragment library system I” kit (Beckman Coulter). tics about the number of total and mapped reads. The final product were gel purified to achieve a library Transcriptomic high throughput sequencing data were mean size of 700 bp. Library quality was assessed on an agi- analyzed using a bioinformatic pipeline implemented in lent Bioanalyzer instrument (Agilent) and sequenced on a the Microscope platform . In a first step, the RNA- Illumina MiSeq instrument, using a Paired-end 2 × 250 bp Seq data quality was assessed by including reads trim- protocol, with a MiSeq Reagent Kit v2 500 cycle (MS-102- ming. In a second step, reads were mapped onto the C. 2003, Illumina), according to the constructor recommenda- beijerinckii DSM6423 genome sequence (previously sub- tions. The run resulted in a 80× coverage of the genome of mitted for sequencing) using the SSAHA2 package  C. beijerinckii and the different reads, spanning 250 nucleo- that combines the SSAHA searching algorithm (sequence tides each, were assembled into DNA fragments called con- information is encoded in a perfect hash function) aiming tigs. De novo sequencing was performed using the PacBio at identifying regions of high similarity, and the cross- RSII platform (GATC, Germany). match sequence alignment program , which aligns Contigs were uploaded to the MicroScope bioinfor- these regions using a banded Smith-Waterman-Gotoh al- matics platform where the putative genes were automat- gorithm . An alignment score equal to at least half of ically annotated through a specific computer workflow the read is required for a hit to be retained. To lower false . MicroScope is a Microbial Genome Annotation & positives discovery rate, the SAMtools (v.0.1.8, ) are Analysis Platform developed by the Genoscope institute then used to extract reliable alignments from SAM for- (Evry, France) . Sequenced strains and RNAseq re- matted files. The number of reads matching each genomic sults can be uploaded and automatically analyzed on this object harbored by the reference genome is subsequently platform. computed with the Bioconductor GenomicFeatures pack- age . If reads matching several genomic objects, the RNA extractions and RNA-seq protocols count number is weighted in order to keep the same total The extraction was performed as previously described number of reads. Finally, the Bioconductor-DESeq pack- . Briefly, cell lysis and RNA isolation was done with age  with default parameters is used to analyze raw the TRIzol Plus RNA purification kit (AMbion). 5 mL counts data and test for differential expression between TRIzol reagent was added to the frozen pellet followed conditions. Moreover, the quality of the RNA-seq data by 1 mL chloroform after cell lysis. After shacking and was strengthened by checking the relative stability of centrifugation, the upper phase was mixed with an equal housekeeping genes such as gyrAand rpoBacross time volume of 70% ethanol before transfer to PureLink RNA points. Spin Cartridges furnished with the kit. Kit instructions allowed to obtain purified total RNA which was further Results processed by cleanup with the RNeasy mini kit (Qiagen). Genome assembly DNA was eliminated with DNAse I treatment (AM1906, Genome sequence of C. beijerinckii DSM6423 was Invitrogen). 20 μL RNA were treated with 2 μL DNAse obtained through PacBio and MiSeq sequencing. The in 50 μL nuclease free water for 30 min at 37 °C before PacBio de novo sequencing yielded 17 unitigs. Unitigs addition of another 1.5 μL DNAse and 30 min extra in- were further combined in four scaffolds when assembled cubation. Treatment was done twice and was followed with the 412 contigs obtained through MiSeq sequen- by RNA cleanup with the RNeasy kit. A full quality as- cing. Junctions between unitigs were confirmed by PCR, sessment of the RNA, quantification using Nanodrop, and the 4 scaffolds were PCR gap-closed, yielding a cu- PCR to check for residual DNA and migration on 1% rated 6,383,364-bp long circular chromosome, with an Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 4 of 12 Table 1 Summary of RNA-Seq sequencing and data analysis results Replicate Time Total read Reliable reads Reads mapped on genomic Number of genes with point (h) number objects (except rRNA) detectable expression A 3 19,158,892 17,799,816 7,024,274 5944 B 3 11,574,716 10,099,244 3,266,941 5829 C 3 15,203,792 12,936,903 4,403,183 5851 A 6 16,007,459 14,775,252 5,341,913 5876 B 6 13,731,477 11,723,511 4,255,219 5884 C 6 13,472,975 11,754,891 3,741,547 5861 A 9 11,050,565 9,196,745 2,854,832 5895 B 9 14,381,148 12,261,616 3,777,589 5906 C 9 11,873,306 10,118,493 3,235,171 5880 A 11 13,663,240 11,531,714 3,327,568 5887 B 11 10,195,910 8,792,217 2,208,510 5789 C 11 19,046,554 16,439,661 4,596,207 5917 A 24 13,578,705 11,371,420 1,153,741 5203 B 24 21,453,820 17,646,890 1,871,469 5606 C 24 9,805,052 8,262,117 1,046,981 5121 average GC content of 29.81% (Fig. 1). Automatic func- predicted (Additional file 1). There is a high probability tional annotation resulted in the prediction of 6052 pro- that this part of the genome was acquired through hori- tein genes and 202 RNA genes on this chromosome. zontal transfer. This NADP(H)-linked, zinc-containing Three Mobile Genetic Elements (MGE) were also iden- secondary alcohol dehydrogenase (sADH) is a tetrameric tified: two natural plasmids named pNF1 and pNF2, and protein having the highest catalytic efficiency on acetone one bacteriophage, named 6423 (Fig. 1). Plasmid pNF1 [22, 23]. Interestingly, This sADH has a significant is 10,278-bp long and contains a gene sharing homology sequence identity with other class I alcohol dehydroge- with both the bacteriocin gene from Clostridium butyri- nases such as the tetrameric enzymes from Entamoeba cum MIYAIRI 588  and the closticin 574 gene from histolytica and bacterial secondary-alcohol dehydrogen- Clostridium tyrobutyricum ADRIAT 932 . The last 83 ase from Thermoanaerobacter brockii which is an excep- aminoacids of the gene product shared 81.9% and 64.6% tionally high degree of identity between enzymes from pairwise identity with the corresponding peptides posses- such distantly related organisms. sing bacteriocin activity from C. butyricum MIYAIRI 588 and C. tyrobutyricum ADRIAT 932, respectively. Plasmid C. beijerinckii cultivation in bioreactor pNF2 is 4282-bp long and carries four predicted ORFs, To further improve genetic knowledge on C. beijerinckii one of them being potentially involved in plasmid replica- DSM6423, a transcriptomic study was performed. A RNA- tion. Phage 6423 have a linear double-stranded DNA Seq approach was chosen in order to have a timelapse study genome of 16,762 bp, with 143 bp inverted terminal re- of the metabolism of the strain throughout the fermentation peats. To our knowledge, this is the first time such a bac- process. Three independent fermentations were carried out teriophage is described in the Clostridium genus. in bioreactors on three different weeks, showing good repro- Identification of Regions of Genomic Plasticity (RGPs) ducibility (Fig. 2). was performed on the DSM6423 chromosome in order Acid re-assimilation and solvent production appeared to identify potentially horizontally transferred genes to start around 6-8 h. This was concomitant with the (HGT) which are gathered in genomic regions. In total, characteristic loss of mobility of the cells and the ap- 47 RGPs were found in the genome of DSM 6423 when pearance of cigar–shape cells as observed under the compared with that of C. beijerinckii NCIMB 8052. One microscope (Additional file 2). of these RGPs contains the specific secondary alcohol The final solvent profile obtained corresponded well to dehydrogenase adh gene whose product allows the con- those described earlier for this strain [24, 25]. Moreover, version of acetone into isopropanol. This gene is located thebiphasicpHis characteristicof the solvantogenicstrains in a 23-kb genomic island having no equivalent in the such as the well-known C. acetobutylicum ATCC824 , NCIMB 8052 strain and harboring 28 coding sequences even if thelatterisabletoleratemoreacidicpHconditions. in which 6 metabolic genes and one transposase were In the presence of sodium acetate in the medium, C. Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 5 of 12 Fig. 1 Circular representation of Clostridium beijerinckii DSM6423 genome. CGView representation  of the circular genome of the DSM6423 strain and its extrachromosomic elements (plasmids: pNF1, pNF2 and double stranded linear phage ɸ6423). Circles display (from the outside): (1) GC percent deviation (GC window - mean GC) in a 1000-bp window. (2) Predicted CDSs transcribed in the clockwise direction. (3) Predicted CDSs transcribed in the counterclockwise direction. Genes displayed in (2) and (3) are color-coded according different categories: red and blue: MaGe validated annotations orange: MicroScope automatic annotation with a reference genome purple: Primary/Automatic annotations. (4) GC skew (G + C/G-C) in a 1000-bp window. (5) rRNA (blue), tRNA (green), misc_RNA (orange), Transposable elements (pink) and pseudogenes (grey) Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 6 of 12 ac bd Fig. 2 Fermentation profile of Clostridium beijerinckii DSM6423 on glucose. C. beijerinckii DSM 6423 was cultivated in bioreactors in GAPES medium. a pH, b biomass followed by OD , c acids and d solvents. Values are the mean and standard deviation of the 6 biological replicates. See Additional file 3 for details on the biological replicates beijerinckii DSM6423 starting its switch to solvantogenesis During our batch fermentation we obtained 3 g/L iso- at a higher pH level (5,2) than C. acetobutilicum ATCC824 propanol and 5.3 g/L butanol after 30 h fermentation (pH 4,2 ). Survase et al. studiedthetime courseof time with 28.8% substrate conversion (Additional file 3). a batch fermentation on glucose and showed that solvent De Vrije et al. reported end concentrations of 10.7 g/L production started at the end of the exponential phase, at total IBE, with 3.2 g/L isopropanol and 6.9 g/L of buta- approximately 6–9 h, which is consistent with our observa- nol after 48 h of fermentation when the strain was tions. Their batch culture showed a maximum concentra- grown on a mix of glucose and xylose . tion of 2.16 g/L (36.8% of total solvents) of isopropanol and For each triplicate, samples were collected for RNA- 3.71 g/L (63.2% of total solvents) of butanol after 24 h fer- Seq analyses at 3 h (acidogenesis phase, exponential mentation with no further significant increase and the max- growth), 6 h (acidogenesis to solventogenesis switch), imum solvent yield was 0.30 g/g glucose consumed with 8 h and 11 h (solventogenesis) and 24 h (late solvento- 33.8% of substrate conversion. Interestingly, the Isopropa- genesis) after inoculation. The 15 resulting RNA samples nol/Butanol ratio was progressively growing along the sol- were sequenced and analyzed using the reconstructed vantogenic phase (Fig. 2a). This may be due to the presence genome of DSM6423. of sodium acetate in the fermentation medium to prevent a severe pH drop linked to acids transport and assimilation RNA seq analysis . This behavior is in contradiction with those observed Highly expressed genes for a batch culture of C. beijerinckii DSM6423 with a con- Determination of the most highly expressed genes was trol of the pH (close to 4.8) using NaOH . In that case, performed using RPKM values during the whole fermen- a constant I/B ratio was maintained during the ten first tation experiment (Additional file 4). About 15% of these hours of solvantogenesis. Interestingly, in the case of C. genes were coding for proteins of unknown functions, acetobutylicum, a significant increase of ABE produc- which represent 34% of the coding sequenced predicted tion but no modification of the A/B ratio was observed in the genome. As expected, the most expressed genes when ammonium acetate is used to control the pH. This after 3 h of fermentation are coding for ribosomal pro- tend to confirm a specific behavior of C. beijerinckii teins, consistently with the protein synthesis activity dur- DSM6423. ing exponential growth phase and cell division. A set of Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 7 of 12 genes involved in cell motility, such as the flagellin main transcriptomic regulations occur between 3 and 11 h, coding protein, was also found to be highly expressed, with only 31 genes being specifically regulated between 11 consistently with the high mobility of the Clostridium and 24 h. Therefore, further clustering analysis focused on cells at this time (Additional file 2). Other highly the 977 genes regulated between 3 and 11 h. expressed genes at 3 h were involved in transcription, translation, protein folding and of course Isopropanol/ Kinetic analysis of transcription regulations Butanol/Ethanol production. Concerning the mobile In order to highlight the transcriptional regulation along genetic elements discovered in C. beijerinckii DSM6423, the fermentation, we calculated the transcription profiles 3 highly expressed genes were located on the bacterio- of each gene, using the 3 h sample as reference. Clustering phage Φ6423 and only one, corresponding to a cell wall was performed using CAST algorithm  with a thresh- binding protein, on the natural plasmid pNF1. old of 0.88 and revealed 8 clusters containing 953 genes. Three clusters (504 genes) correspond to genes up- Differentially expressed genes regulated at 6 h (Fig. 3b), 2 clusters (215 genes) to genes In order to investigate the transcriptional regulations dur- down regulated at 6 h (Fig. 3c) and 3 clusters (234 genes) ing the fermentation, the most differentially expressed to genes up-regulated at 8 h or 11 h (Fig. 3d). geneswereselectedbycomparing theirexpressionata Up-regulated genes at 6 h are divided in 3 different clus- giventime point (6 h, 8h,11h and24h)totheir expres- ters (Additional file 5). A first cluster (6up++) contains 17 sion at the previous time point (3 h, 6 h, 8 h and 11 h re- genes up-regulated at 6 h and 8 h and involved in the spectively) (Fig. 3, a). Thresholds were selected at |log (fold Panthothenate and CoA biosynthetic pathway, oxidative change)| > 1.5 and adjusted p-value< 0.001, which resulted stress or solvantogenesis (butanol production). A second in a list of 1008 significantly differentially expressed genes cluster of 57 genes (6up+), strictly up-regulated at 6 h, (16% of the genome). Interestingly, the majority of these comprises a set of genes related to sporulation, including genes (683 genes) showed a different level of gene expres- the predicted anti-sigma F factor antagonist (SpoIIAA and sion between 3 h and 6 h, which corresponds to the switch SpoIIAB), and a putative operonic structure linked to acet- from acidogenesis to solventogenesis. Most of the genes one production (CIBE_4606 to 4609). (938 genes, 93%) were regulated once during the fermenta- The third cluster (6up) comprises 430 genes that are tion, 69 genes were regulated 2 times, and only 1 gene ap- up-regulated at 6 h and then down-regulated. This clus- peared to be regulated 3 times during the fermentation. ter include genes that are not regulated after 6 h, notably This tends to demonstrate that each time point corre- the anti-sigma factors regulating sporulation, an aceto- sponded to a specific physiological state during the cultiva- lactacte synthase and several genes encoding for en- tion As suggested by the number of regulated genes, the zymes involved in ferrous storage (ferritin). Some genes a b d c Fig. 3 Global transcriptomic analysis of C. beijerinckii DSM6423 fermentation on glucose. a Venn Diagram showing the number of genes regulated in various physiological time points. b to d kinetic expression profiles of various clusters of genes: genes up-regulated at 6 h (B), genes down-regulated at 6 h (c), and genes regulated at 8 h or 11 h (d) Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 8 of 12 of this cluster are directly down-regulated at 8 h, includ- The set of genes up-regulated after 8 h was divided in ing genes involved in solvantogenesis (isopropanol pro- 3 different clusters including a first one (8up, 205 genes) duction), responses to the heat shock effects and suffer mainly comprising genes involved in solvantogenesis. assimilation. Finally, several genes of this cluster are The second cluster (8up+) of genes was strictly up- down-regulated at 11 h including sigma factors related regulated at 8 h and included genes linked to oxidative to the initiation of sporulation and a RNA polymerase stress and the latter phases of sporulation. The last sigma factor 28. The σ28 is a minor sigma factor respon- cluster (11up) corresponded to a set of 22 genes up- sible for the initiation of transcription of a number of regulated at 11 h and mainly linked to sporulation. genes involved in motility and flagellar synthesis . A really small cluster of genes were down regulated at The 235 genes that were down-regulated at 6 h were 8H (8down). Among them, one gene is coding for a mem- divided in two different clusters. The main one (6down) ber of the 2-oxoacid oxidoreductases, a family of enzymes contained a gene cluster involved in the mobility of the that oxidatively decarboxylate different 2-oxoacids to form bacterial cells, in particular motA and motB. This is in their CoA derivatives. Moreover, a putative operonic agreement with the loss of mobility and the change in structure classified in this cluster is potentially involved in cell shape characterizing the onstart of the solventogenic ferrous iron transport (CIBE_5053 to 55). phase (Additional file 2). The second cluster of genes, down-regulated at 6 h then 8 h (6down-) corre- Main transcriptomic regulations sponded to genes coding for ribosomal proteins, Central metabolism oxidativestressresponseproteinsand ahighnumber In order to better understand the transcriptional regulation of putative membrane transporters. This might occurring in C. beijerinckii DSM6423 cells during fermenta- suggest some major changes in the membrane trans- tion, the transcriptomic data allowed us to confirm the role port system for the re-assimiliation of acids linked to of some predicted metabolic genes coming from the auto- solventogenesis. matic annotation. Analysis revealed 22 genes potentially Fig. 4 Main genes and predicted operonic structures involved in the central metabolism of in C. beijerinckii DSM6423. a glycolysis; b acids and solvents production). Number of isozymes, predicted by Microscope tool (Genoscope, Evry, France) are indicated in brackets Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 9 of 12 involved in glycolysis, with multiples candidates (“isozymes”) switch between acidogenic and solventogenic phases appears for some key reactions. A majority of these genes was highly to be less pronounced in the case of C. beijerinckii transcribed throughout the fermentation tending to demon- DSM6423, compared to other solventogenic clostridia, such strate their implication in this central pathway. as C. acetobutylicum ATCC824.Intotal,morethan20genes Transcriptomics analysis of the whole metabolic path- predicted as encoding alcohol dehydrogenases were detected ways also revealed a significant expression level of pre- in the genome of the DSM 6423 strain and most of them dicted operons encoding enzymes involved in the were expressed from the beginning of the fermentation. production of acids and the conversion of acetyl-CoA to butyryl-CoA (Fig. 4). No regulation was observed for Sporulation operonic structures predicted to be involved in acetate It is well established that spo0A is the master regulator for (CIBE_1400 and 1401) or butyrate (CIBE_0223 and sporulation events in Bacillus and in Clostridia [35, 36]. 0224) production. This might be explained by the spe- Phosphorylated Spo0A has been reported to induce the ex- cific medium used in these experiment, containing 3 g/L pression of several targets, including the sol operon and mul- of sodium acetate. Unlike what has been observed in C. tiple sporulation sigma factor genes in C. acetobutylicum acetobutylicum (Schreiber & Dürre, 2015), the gene cod- ATCC 824 . As described previously, the solventogenic ing for the phosphoglycerate mutase (pgm, CIBE_0772) phase started at 6 h and may be linked to the initiation of is predicted to be a part of the operon comprising genes the sporulation phase. This was confirmed by the overex- coding for the glyceraldehyde-3-phosphate dehydrogen- pression at 6 h of a set of 12 genes including the predicted ase (gap), the phospho-glycerate kinase (pgk) and the genes coding for Spo0A (CIBE_2041; Additional file 5), the triosephosphate isomerase (respectively gap, pgk and tpi, sigma factors σF (CIBE_0994) and σG (CIBE_1357) CIBE_0769 to 0771; Fig. 4). but also the anti-anti-sigma factor SpoIIAA and the As observed in other Clostridium strains, genes dedicated anti-sigma factor SpoIIAB (CIBE_0992 and 0993, re- to the production of butyryl-CoA from acetoacetyl-CoA spectively). In C. acetobutylicum ATCC824, a multi- seems to be organized in an operon (CIBE_0339 to 0343, component system involving SpoIIAA and SpoIIAB Fig. 4) and are not regulated in our fermentation conditions. activates Spo0A through phosphorylation, which fur- More interestingly, the 3 genes (CIBE_5186, CIBE _2196 and ther stimulates the expression of sigF . The over- CIBE _1684) apparently involved in the pyruvate decarboxyl- expression at 6 h of the predicted anti-sigma factor ation into acetyl-CoA do not appear to be regulated in the SpoIIAB and the specific regulation of the SpoIVB same way. The first one was highly and constantly tran- predictedgene(repressedat6hthenup-regulatedat scribed throughout the fermentation whereas the two others 8 h; Additional file 5) suggest that the initiation of were slightly transcribed (30× less) and further induced at the sporulation and solventogenesis are simultaneous. 8 h and 11 h. This might suggest a fine-tuned regulation of An increase in the expression of some other the acetyl CoA production along the fermentation. sporulation-related genes, such as spoIVA and spoIVB Another operon containing CIBE_4606 to CIBE_4609 was also detected at 8 h. In Bacillus subtilis, SpoIVB gene, coding for an alcohol dehydrogenase, two CoA trans- protein is the key determinant for inter-compartmental ferases and an acetoacetate decarboxylase, are equivalent to signaling of pro-σK processing [39, 40]. Pro-σK is the in- the ald-ctfA-ctfB-adc operon of C. beijerinckii NCIMB active form of the final transcription factor σK acting in 8052 . Expression of this operon appeared stable the mother cell compartment of the sporulating cell throughout the fermentation. Two genes encoding ctfA and . The σK regulon is then involved in the final stages ctfB analogs (CIBE_3149 and 3150) were then identified of spore maturation including spore coat biosynthesis and appeared to be overexpressed at 8 and 11 h. and release of the spore from the sporangial cell. This is More surprisingly, a high level of transcription was in accordance with the observation that these genes are already observed at 3 h for the predicted operons in- up-regulated at 8 h. volved in butanol (CIBE_2622 to CIBE_2624), acetone (CIBE_4606 to CIBE_4609) or isopropanol (CIBE_3468 The specific regulation of the motility locus to CIBE_3470; Fig. 4) production. Expression of this latter Loss of motility usually comes with the transition from ex- operon was even up-regulated at 6 h. This specificity confirm ponential to stationary growth phases and therefore when the distinct transcriptomic regulation of the solvantogenesis switching from acidogenesis to solventogenesis. Clostridia between C. beijerinckii and C. acetobutylicum strains. In C. cells stop moving and lose their rod shape to gain the acetobutylicum ATCC824, the corresponding genes adhE characteristic cigar shape instead. Expression of motility (CA_P0162), ctfA (CA_P0163) and ctfB (CA_P0164) are genes, such as motA and motB, and of a high number of located on the mega-plasmid pSOL1 and only induced dur- genes coding flagellar proteins was repressed at 6 h, corre- ing solvantogenesis, while adc (CA_P0165) is organized in a sponding to the start of the solventogenic phase. This is in monocistronic operon in the opposite direction [33, 34]. The accordance with previous transcriptomics results observed Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 10 of 12 in Clostridium beijerinckii NCIMB8052 , in Clostridium response using butanol  led to new insights into the acetobutylicum  and more generally in Clostridia and physiological role of several genes or operons involved Bacilli . Most genes in the flagellar/chemotaxis cluster in solvent formation. Concerning C. beijerinckii DSM were down-regulated at the beginning of the solvantogen- 6423, using the MicroScope web-platform to identify the esis, which is related in our strain with the onset of sporula- genes involved in the main metabolic pathways and their tion. Similarly, the flagellar motility coding gene flgC duplications, we were able to detect at least 4 putative is strongly expressed during the early exponential operonic structures related to acidogenesis and solvanto- phase and further down regulated at 6 h, at the onset genesis. One of them, CIBE_4606 to 4609, corresponds of the stationary phase. to the sol operon of C. beijerinckii NCIMB8052 where ald, ctfAB and adc are organized in an operon and co-r- Discussion egulated . The structure of other important gene op- C. beijerinckii is a prominent solvent-producing bacter- erons involved in metabolic pathways for acid and ium with great potential as a microbial cell factory for solvent production in C. beijerinckii NCIMB 8052, in- the biofuel and chemical industries, in particular for iso- cluding pta-ack, ptb-buk, hbd-etfA-etfB-crt operons was propanol production. This strain has already been used also confirmed. for advanced bioprocesses, and increased productivities Most sporulation genes in C. beijerinckii NCIMB8052 were obtained by cell immobilization, in-situ product re- demonstrated similar temporal expression patterns to moval and use of glucose/xylose mixes as substrate . those observed in B. subtilis and C. acetobutylicum. Recently, mutagenesis and genome shuffling have been Sporulation sigma factor genes sigE and sigG exhibited applied to this strain to generate mutants with improved accelerated and stronger expression in C. beijerinckii tolerance to isopropanol . However, a complete gen- NCIMB8052, which is consistent with the more rapid ome characterization of this strain was still lacking. forespore and endspore development in this strain. A Although transcriptional analysis is essential to under- previous study involving insertional inactivation of stand gene functions and regulation and thus elucidate spo0A indicated that Spo0A does control the formation proper strategies for further strain improvement, limited of solvents, spores and granulose in C. beijerinckii information was available for the natural isopropanol NCIMB8052 . A spo0A mutant of C. beijerinckii producer C. beijerinckii DSM 6423. The genome sequen- NCIMB8052 also showed an asporogenous and non- cing of DSM 6423 yielded a 6,4 Mbp chromosome, two septating phenotype . Moreover, in C. acetobutyli- plasmids and one bacteriophage. These mobile genetic cum ATCC 824, spo0H and spo0A are both constitutively elements could be used as a chassis for the development expressed at constant levels throughout the growth cycle of genetic tools dedicated to this strain. . In our strain, production of solvents was detected The genome-wide transcriptional dynamics of C. already at 6 h after inoculation of the fermenters and ini- beijerinckii NCIMB8052 revealed an overexpression of tiation of sporulation was concomitant with the mid to the glycolysis genes throughout the fermentation, es- late solventogenic phase (8 to 11 h). There is a slight pecially during the acidogenesis phase. The expression down-regulation of spo0A at 8 h, similar to what Wang of genes involved in this first part of the metabolism et al. observed with NCIMB8052 , and microscopic was then down-regulated at the beginning of the studies indicated that sporulation was both enhanced well-known metabolic shift from acidogenesis to sol- and accelerated due to spo0A overexpression compared ventogenesis. According to Wang et al. , out of the to parental strains. It is therefore not surprising to see > 20 genes encoding alcohol dehydrogenase in C. sporulation genes, such as spoIVA, highly expressed beijerinckii NCIMB8052, Cbei_1722 and Cbei_2181 starting at 11 h. were highly up-regulated at the onset of solventogen- The expression of motility genes, such as motA and esis, corresponding to their key roles in primary alco- motB, and flagellar protein coding flgC was repressed at hol production. In our fermentation experiments, the 6 h, which corresponds to the onstart of the solvento- expression pattern of glycolysis genes was similar than genic phase in the studied fermentation. In Salmonella in C. beijerinckii NCIMB8052, with high expression enterica, electrostatic interactions between the stator levels throughout the whole fermentation . This protein MotA and the rotor protein FliG are important seems to be specific of C. beijerinckii strainsasa for bacterial flagellar motor rotation . The Salmonella time course transcriptional analysis of C. acetobutyli- flagellar motor, which is embedded within the cell mem- cum ATCC824 showed higher expression during sta- branes, is powered by proton motive force. According to tionary phase for most of the glycolysis genes . the study, five flagellar proteins, MotA, MotB, FliG, FliM, Studies concerning C. acetobutylicum, with genome- and FliN, are involved in motor performance. Therefore wide gene expression analysis of the switch between repression of the motAB genes expression in C. beijerinckii acidogenesis and solventogenesis  or solvent stress might be linked to loss of motility during phase Máté de Gérando et al. BMC Genomics (2018) 19:242 Page 11 of 12 transition. Also, the flagellar motility related gene flgC and Françoise Fayolle-Guichard for their contributions in the design of the fermentation protocol dedicated to the RNA seq analysis and Isabelle is strongly expressed in DSM 6423 during the early Martin-Verstraete (Institut Pasteur, France) for fruitful discussions. exponential phase and down regulated at 6 h at the onset of the stationary phase. Also noticeable is the Availability of data and materials The DSM6423 full genome sequence was deposited on the European overexpression of chaperonins groEL and groES at Nucleotide Archive (ENA) under the accession number PRJEB11626 8h.The groESL genes are involved in stress response (https://www.ebi.ac.uk/ena/data/view/PRJEB11626). of Clostridium acetobutylicum and play a role in The DSM6423 RNA seq data analyzed during this study were deposited on NCBI BioProject Database under the Accession Number GSE100024 solvent tolerance . The toxicity of butanol in particular (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100024). limits its formation in microbial fermentations. Over- expression of groESL, grpE and htpG, significantly im- Authors’ contributions HMG prepared the DSM6423 RNA samples from IBE Fermentation. FB-M and proved butanol tolerance of C. acetobutylicum . EJ performed clustering analysis of the RNA seq data. AB and BC were involved in the IBE fermentation and the extraction of the gDNA and FW performed the physical map of C. beijerinckii DSM6423. NLF and AMLC Conclusions supervised the study and contributed in writing the manuscript. All authors C. beijerinckii DSM6423 is the most well-known natural read and approved the final manuscript. isopropanol producer and harbors the secondary alcohol Ethics approval and consent to participate dehydrogenase gene that was cloned in heterologous Not applicable strains to allow acetone conversion into isopropanol by C. acetobutylicum [10, 49, 50]. Improving the natural pro- Competing interests The authors declare that they have no competing interests. duction of this strain through a targeted approach requires the full sequencing of its genome, together with a Publisher’sNote transcriptomic analyses. Such analyses were carried out in Springer Nature remains neutral with regard to jurisdictional claims in this study and provide useful data to better understand published maps and institutional affiliations. the genetic background and physiology of this strain. Author details Notably, this work described a complete genomic study of Wageningen Food and Biobased Research, Bornse Weilanden 9, 6709WG, a natural IBE producer including a first genome physical Wageningen, The Netherlands. IFP Energies Nouvelles, 1 et 4 avenue de map of a natural IBE producer. This first analysis highlighted Bois-Préau, 92852 Rueil-Malmaison, France. several genetic and metabolic particularities including a Received: 7 September 2017 Accepted: 28 March 2018 specific genomic event occurred on this strain to produce isopropanol. A better understanding of the metabolic path- References ways and various genes involved opens the door for future 1. Li Y, Meng L, Nithyanandan K, Lee TH, Lin Y, Lee C-fF, Liao S. 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