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# Gene essentiality in the solventogenic Clostridium acetobutylicum DSM 792

Alexandre Delarouzée, Nicolas Lopes Ferreira, Chloé Baum, François Wasels

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Alexandre Delarouzée, Nicolas Lopes Ferreira, Chloé Baum, François Wasels. Gene essentiality in the solventogenic Clostridium acetobutylicum DSM 792. FEMS MICRO, Jul 2025, Milan, Italy. FEMS MICRO Abstract book, 11, pp.334.17P, 2025. [hal-05323908](https://hal-05323908v1)

HAL Id: hal-05323908

https://ifp.hal.science/hal-05323908v1

Submitted on 21 Oct 2025

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Alexandre Delarouzée,<fnref n="1" /> Nicolas Lopes Ferreira<fnref n="1" />, Chloé Baum<fnref n="2" />, & François Wasels<fnref n="1" />

## Context

Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying Acetone-Butanol-Ethanol (ABE) fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived interest in research on this bacterium. The recent development of efficient genetic tools allows us to better understand the physiology of this microorganism, aiming to improve its fermentation capacities.

Knowledge about gene essentiality would guide future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium.

In this work, we applied a transposon insertion site sequencing (TIS) method to generate large mutant libraries containing millions of independent mutants with unique insertion sites, allowing us to identify essential genes.

In total, we identified a core group of 418 critical genes needed for *in vitro* development on rich media containing glucose as a carbon source.

## Methods

* The TIS approach uses transposons for random inactivation mutagenesis. Essential gene inactivation will cause cell death. High-throughput transposon inactivation and sequencing allows the identification of essential gene.

* A 7.4 million transposon mutant library was generated via a *Mariner*-based transposition tool initially dedicated to *C. difficile* (Dembek *et al.*, 2015).

* Genomic DNA was extracted from the surviving mutant pool, and transposon insertion sites were sequenced.

* 136 000 unique insertions were detected throughout the genome using the Bio::TraDIS workflow (Barquist *et al.*, 2016).

A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /><figcaption>**Figure 1:** (A) Transposon insertion sequencing (TIS) strategy. (B) Mapping of transposon junctions on the lower glycolysis locus.</figcaption></figure>

The height of each vertical line, which represents the number of reads at each insertion point, reveals the location of a distinct transposon insertion. Transposon insertions are nearly absent from the CDS of *gapC*, *pgk*, *tpiA*, and *pgm*, indicating that these genes are necessary for *in vitro* growth.

## Results

<figure><img src="image_4.png" /></figure>

Essential changepoint: 0.0692

Ambiguous changepoint: 0.0913

<caption>**Figure 2:** Transposon Insertion Ratio Distribution Plots for Each Gene in the Genome.</caption>

The plot displays a bimodal distribution, where the essential genes are fitted by the black gamma distribution, while the non-essential genes are fitted by the blue gamma distribution. A core group of 418 genes was categorized as essential for growth in rich-medium with glucose as a carbon source.

<figure><img src="image_5.png" /><figcaption>**Figure 3:** Schematic representation of ABE metabolism reactions catalyzed by essential gene products in *C. acetobutylicum*.</figcaption></figure>

Reactions catalyzed by enzymes whose genes have been categorized as essential are shown in red. While acidogenesis-related genes are all essential, solventogenesis-related genes are not required for *in vitro* growth.

A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<table><caption>**Figure 4:** Confirmation of the role of *sigD* in the motility of *C. acetobutylicum*. The *σ* factor *D* encoding gene was significantly highly transposed in the pool of mutants and was inactivated using CRISPR-Cas9 (Wasels *et al.*, 2020). (A) Motility assay on 2YTG agar 0.175% and (B) on 2YTG agar 0.4% petri dishes (C) Maximum growth rates when grown on CGM with 30 mM glucose. Cultures were assayed with five replicates each.</caption><thead><tr><th colspan="2">Maximum Growth rate (h<sup>-1</sup>)</th></tr><tr><th>WT</th><th>ΔsigD</th></tr></thead><tbody><tr><td>0.73 ± 0.03</td><td>0.70 ± 0.08</td></tr></tbody></table>

## Conclusions

* Successful application of the Transposon insertion sequencing strategy to the model solventogenic bacterium *Clostridium acetobutylicum*

* Identification of a core group of 418 essential genes and 48 ambiguous genes

* Future research on this model organism will be guided by the data provided in this work, which will serve as a valuable resource for the community

## References

Delarouzée *et al.* 2024. Gene essentiality in the solventogenic *Clostridium acetobutylicum* DSM 792. Appl Environ Microbiol 0:e00282-24.

Dembek *et al.*, 2015. High-throughput analysis of gene essentiality and sporulation in *Clostridium difficile*. mBio 6:e02383. 4.

Barquist *et al.*, 2016. The TraDIS toolkit. Sequencing and analysis for dense transposon mutant libraries. Bioinformatics 32:1109–1111.

Wasels *et al.*, 2020. A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in *Clostridium acetobutylicum* DSM 792. Appl Environ Microbiol 86.

<footnote n="1">¹ IFP Energies nouvelles, 1 et 4 avenue de Bois-Préau, 92852 Rueil-Malmaison, France</footnote>

<footnote n="2">² Institut Pasteur, Université Paris Cité, Plate-forme Technologique Biomics, F-75015 Paris, France</footnote>