The solventogenic C . beijerinckii DSM 6423, a microorganism that naturally produces isopropanol and butanol, was previously modified by random mutagenesis. In this work, one of the resulting mutants was characterized. This strain, selected with allyl alcohol and designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 ( sigL ), which encodes the σ 54 sigma factor. Complementation with wild-type sigL fully restored solvent production and sugar assimilation and RT-qPCR analyses revealed its transcriptional control of several genes related to solventogensis, demonstrating the central role of σ 54 in C . beijerinckii DSM 6423. Comparative genomics analysis suggested that this function is conserved at the species level, and this hypothesis was further confirmed through the deletion of sigL in the model strain C . beijerinckii NCIMB 8052.

9Microbial production of butanol and isopropanol, two high value-added chemicals, is 10 naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its 11 ancient discovery, the precise mechanisms controlling alcohol synthesis in this 12 microorganism are poorly understood. In this work, an allyl alcohol tolerant strain 13 obtained by random mutagenesis was characterized. This strain, designated as the AA 14 mutant, shows a dominant production of acids, a severely diminished butanol synthesis 15 capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-16 deficient strain was also found to have a limited consumption of two carbohydrates and 17 to be still able to form spores, highlighting its particular phenotype. Sequencing of the 18 AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which 19 encodes the σ 54 sigma factor. Complementation with the wild-type sigL gene fully 20 restored solvent production and sugar assimilation, demonstrating that σ 54 plays a 21 central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic 22 comparison with other strains further revealed that these functions are probably 23 conserved among the C. beijerinckii strains. The importance of σ 54 in C. beijerinckii was 24 further assessed by the characterization of a sigL deletion mutant of the model strain 25 NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited 26 phenotypic traits similar to the AA strain, and was subsequently complemented with the 27 sigL gene from either the wild type or the AA strains. The results of this experiment 28 confirmed the crucial role of σ 54 in the regulation of both solventogenesis and sugar 29 consumption pathways in C. beijerinckii. 30 3 Importance 31Clostridium beijerinckii shows a significant potential for producing valuable biochemicals 32 and biofuels. One of the major hurdles impeding its widespread usage is its low 33 endogenous production of alcohols, which could be alleviated by metabolic engineering 34 approaches. Despite its former long-time use in the industrial acetone-butanol-ethanol 35 process, the molecular mechanisms controlling solventogenesis in the Clostridium 36 genus still remain elusive, preventing genetic engineering approaches for strain 37 enhancement. In this context, our study provides novel insights into the crucial role of 38 the σ 54 transcriptional factor in solvent synthesis regulation in two C. beijerinckii strains. 39Furthermore, we show that this sigma factor also controls sugar consumption and is 40 therefore a key controller of carbon metabolism in this species. 41 42 80 highly conserved DNA-binding region of the encoded protein. We hypothesize that this 81 mutation results in the loss of nucleic acid binding capacity and thus impedes 82 transcription of the σ 54 regulon. According to in silico analyses, this regulon is apparently 83 conserved in other C. beijerinckii strains. To confirm this hypothesis, sigL (Cbei_0595) 84was deleted in the acet...

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3′-5′ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The ‘prototypical’ Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

In the pathogenic bacterium Helicobacter pylori, post-transcriptional regulation is dominated by the activity of a protein complex, known as the RNA degradosome, composed of the essential ribonuclease RNase J and the DEAD-box RNA helicase RhpA. Here, we describe post-translational modifications of this protein complex that affect its activity. Cell-extracted RNase J is acetylated on multiple residues, one of which, K649, impacts strongly on RNase J oligomerization, which in turn influences recruitment into the degradosome and ribonuclease activity. Corroborating the link between oligomerization and activity, mutations targeting K649 and other residues affect the dimerization and in vitro activity of RNase J. Our crystal structure of RNase J reveals loops that gate access to the active site and rationalizes how oligomerization state influences activity. We show that the acetylated residues of RNase J are important for H. pylori morphology, highlighting that the modifications affect the cellular function of RNase J. We propose acetylation as a regulatory level controlling the activity of RNase J and the H. pylori RNA degradosome.