Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii

Abstract

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A spoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the spoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

Model-driven approach for the production of butyrate from CO2/H2by a novel co-culture ofC. autoethanogenumandC. beijerinckii

“…S1). This pH is close to the optimal pH of C. autoethanogenum (40), and acid re-assimilation and ABE production in C. beijerinckii (41,22). Butyrate was the most abundant product with a stoichiometry of 0.6-0.7 mol of butyrate produced and 0.4-0.5 mol acetate consumed per mol of lactate consumed, similar to the stoichiometry reported for C. saccharobutylicum NCP262 in (37).…”

Sporulation in solventogenic and acetogenic clostridia

“…In C. beijerinckii NRRL B598, sporulation initiation was concomitant with an upregulation of the genes encoding a magnesium transporter, and an upregulation of genes encoding potassium, sodium, and iron transporters was detected during stationary phase (Vasylkivska et al 2019). In cultures of the asporogenous C. beijerinckii ΔspoIIE strain, the expression of genes encoding iron transporters were downregulated during stationary phase (Diallo et al 2020b), indicating a potential role of iron in sporulation.…”

“…SpoIIE phosphorylates SpoIIAA, which binds the anti-sigma factor SpoIIAB which then releases σ F in the forespore. In solventogenic clostridia, the role of SpoIIE was studied in C. acetobutylicum and C. beijerinckii through the generation of SpoIIE deficient mutants (Bi et al 2011;Diallo et al 2020b;. Both mutants could no longer sporulate, but phenotypical differences between C. beijerinckii ΔspoIIE and C. acetobutylicum ΔspoIIE were noted.…”

“…Only a few studies on the molecular regulation of sporulation in solventogenic clostridia have been published (Al-Hinai et al 2014;Bi et al 2011;Diallo et al 2020b;Jones et al 2008Jones et al , 2011Ravagnani et al 2000;Steiner et al 2011Steiner et al , 2012Tracy et al 2011). Studies were mainly realized in C. acetobutylicum ATCC 824, and studies with other solventogenic strains show variations in the role of Spo0A and SpoIIE in the regulation of sporulation.…”

“…In both species, the disruption of polyketide clusters decreased sporulation (Table 2). In C. beijerinckii, polyketides might also intervene in the regulation of sporulation; the interruption of sporulation in C. beijerinckii ΔspoIIE affected the expression of the polyketide gene cluster (Diallo et al 2020b). Recently, the role of ranthipeptides, secondary metabolites belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily, was studied in C. beijerinckii and C. ljungdahlii (Chen et al 2020).…”