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Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide‐activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O‐glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco‐profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin‐binding domain that identifies the acceptor. Synopsis: Flagellar subunit glycosylation by specific sialic acids often occurs in bacterial motility systems and is mediated by FlmG glycosyltransferases. Here, the specificity of flagellin glycosylation by pseudaminic acid or its stereoisomer legionaminic acid is shown to depend on the FlmG C‐terminal glycosyltransferase domain with selectivity for the correct sugar. Orthologous pseudaminic or legionaminic acid synthases are functionally interchangeable across eubacterial and archaebacterial kingdomsLegX catalyzes the initial step in the legionaminic acid biosynthesis and is a signature determinant of the pathwayFlagellin glycosylation pathways utilizing either pseudaminic or legionaminic acid are genetically insulated in the related bacteria C. crescentus and B. subvibrioidesC‐terminal glycosyltransferase domain swap dictates sugar specificity in chimeric FlmG flagellin glycosyltransferases [ABSTRACT FROM AUTHOR]

Summary: Clostridia comprise bacteria of environmental, biotechnological and medical interest and many commensals of the gut microbiota. Because of their strictly anaerobic lifestyle, oxygen is a major stress for Clostridia. However, recent data showed that these bacteria can cope with O2 better than expected for obligate anaerobes through their ability to scavenge, detoxify and consume O2. Upon O2 exposure, Clostridia redirect their central metabolism onto pathways less O2‐sensitive and induce the expression of genes encoding enzymes involved in O2‐reduction and in the repair of oxidized damaged molecules. While Faecalibacterium prausnitzii efficiently consumes O2 through a specific extracellular electron shuttling system requiring riboflavin, enzymes such as rubrerythrins and flavodiiron proteins with NAD(P)H‐dependent O2‐ and/or H2O2‐reductase activities are usually encoded in other Clostridia. These two classes of enzymes play indeed a pivotal role in O2 tolerance in Clostridioides difficile and Clostridium acetobutylicum. Two main signalling pathways triggering O2‐induced responses have been described so far in Clostridia. PerR acts as a key regulator of the O2‐ and/or reactive oxygen species–defence machinery while in C. difficile, σB, the sigma factor of the general stress response also plays a crucial role in O2 tolerance by controlling the expression of genes involved in O2 scavenging and repair systems. [ABSTRACT FROM AUTHOR]

Summary: Clostridium difficile is the main cause of antibiotic‐associated diarrhoea. Inside the gut, C. difficile must adapt to the stresses it copes with, by inducing protection, detoxification and repair systems that belong to the general stress response involving σB. Following stresses, σB activation requires a PP2C phosphatase to dephosphorylate the anti‐anti‐sigma factor RsbV that allows its interaction with the anti‐sigma factor RsbW and the release of σB. In this work, we studied the signalling pathway responsible for the activation of σB in C. difficile. Contrary to other firmicutes, the expression of sigB in C. difficile is constitutive and not autoregulated. We confirmed the partner switching mechanism that involved RsbV, RsbW and σB. We also showed that CD2685, renamed RsbZ, and its phosphatase activity are required for RsbV dephosphorylation triggering σB activation. While CD0007 and CD0008, whose genes belong to the sigB operon, are not involved in σB activity, depletion of the essential iron–sulphur flavoprotein, CD2684, whose gene forms an operon with rsbZ, prevents σB activation. Finally, we observed that σB is heterogeneously active in a subpopulation of C. difficile cells from the exponential phase, likely leading to a 'bet‐hedging' strategy allowing a better chance for the cells to survive adverse conditions. [ABSTRACT FROM AUTHOR]

Clostridium difficile is a major cause of diarrhoea associated with antibiotherapy. Exposed to stresses in the gut, C. difficile can survive by inducing protection, detoxification and repair systems. In several firmicutes, most of these systems are controlled by the general stress response involving σB. In this work, we studied the role of σB in the physiopathology of C. difficile. We showed that the survival of the sigB mutant during the stationary phase was reduced. Using a transcriptome analysis, we showed that σB controls the expression of ∼25% of genes including genes involved in sporulation, metabolism, cell surface biogenesis and the management of stresses. By contrast, σB does not control toxin gene expression. In agreement with the up-regulation of sporulation genes, the sporulation efficiency is higher in the sigB mutant than in the wild-type strain. sigB inactivation also led to increased sensitivity to acidification, cationic antimicrobial peptides, nitric oxide and ROS. In addition, we showed for the first time that σB also plays a crucial role in oxygen tolerance in this strict anaerobe. Finally, we demonstrated that the fitness of colonisation by the sigB mutant is greatly affected in a dixenic mouse model of colonisation when compared to the wild-type strain. [ABSTRACT FROM AUTHOR]