Three orphan histidine kinases inhibit Clostridioides difficile sporulation

The ability of the anaerobic gastrointestinal pathogen, Clostridioides difficile, to survive outside the host relies on the formation of dormant endospores. Spore formation is contingent on the activation of a conserved transcription factor, Spo0A, by phosphorylation. Multiple kinases and phosphatases regulate Spo0A activity in other spore-forming organisms; however, these factors are not well conserved in C. difficile. Previously, we discovered that deletion of a conserved phosphotransfer protein, CD1492, increases sporulation, indicating that CD1492 inhibits C. difficile spore formation. In this study, we investigate the functions of additional conserved orphan phosphotransfer proteins, CD2492, CD1579, and CD1949 which are hypothesized to regulate Spo0A phosphorylation. Disruption of the conserved phosphotransfer protein, CD2492, also increased sporulation frequency, similarly to the CD1492 mutant, and in contrast to a previous study. A CD1492 CD2492 mutant phenocopied the sporulation and gene expression patterns of the single mutants, suggesting that these proteins function in the same genetic pathway to repress sporulation. Deletion of the conserved CD1579 phosphotransfer protein also variably increased sporulation frequency; however, knockdown of CD1949 expression did not influence sporulation. We provide evidence that CD1492, CD2492 and CD1579 function as phosphatases, as mutation of the conserved histidine residue for phosphate transfer abolished CD2492 function, and expression of the CD1492 or CD2492 histidine site-directed mutants or the wild-type CD1579 allele in a parent strain resulted in a dominant negative hypersporulation phenotype. Altogether, at least three phosphotransfer proteins, CD1492, CD2492 and CD1579 (herein, PtpA, PtpB and PtpC) repress C. difficile sporulation initiation by regulating activity of Spo0A.

c-di-GMP inhibits early sporulation in Clostridioides difficile

The formation of dormant spores is essential for the anaerobic pathogen Clostridioides difficile to survive outside of the host gastrointestinal tract. The regulatory pathways and environmental signals that initiate C. difficile spore formation within the host are not well understood. One bacterial second messenger signaling molecule, cyclic diguanylate (c-di-GMP), modulates several physiological processes important for C. difficile pathogenesis and colonization, but the impact of c-di-GMP on sporulation is unknown. In this study, we investigated the contribution of c-di-GMP to C. difficile sporulation. Overexpression of a gene encoding a diguanylate cyclase, dccA, decreased sporulation frequency and early sporulation gene transcription in both the epidemic R20291 and historical 630Δerm strains. Expression of a dccA allele encoding a catalytically inactive DccA that is unable to synthesize c-di-GMP no longer inhibited sporulation, indicating that the accumulation of intracellular c-di-GMP reduces C. difficile sporulation. A null mutation in dccA slightly increased sporulation in R20291 and slightly decreased sporulation in 630Δerm, suggesting that DccA may contribute to the intracellular pool of c-di-GMP in a strain-dependent manner. However, these data were highly variable, underscoring the complex regulation involved in modulating intracellular c-di-GMP concentrations. Finally, overexpression of dccA in known sporulation mutants revealed that c-di-GMP is likely signaling through an unidentified regulatory pathway to control early sporulation events in C. difficile. C-di-GMP-dependent regulation of C. difficile sporulation may represent an unexplored avenue of potential environmental and intracellular signaling that contributes to the complex regulation of sporulation initiation.

Sporulation in solventogenic and acetogenic clostridia

The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

CD25890, a conserved protein that modulates sporulation initiation in Clostridioides difficile

Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50–60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890, which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.

Artificial Sporulation Induction (ASI) by kinA Overexpression Affects the Proteomes and Properties of Bacillus subtilis Spores

To facilitate more accurate spore proteomic analysis, the current study focuses on inducing homogeneous sporulation by overexpressing kinA and assesses the effect of synchronized sporulation initiation on spore resistance, structures, the germination behavior at single-spore level and the proteome. The results indicate that, in our set up, the sporulation by overexpressing kinA can generate a spore yield of 70% within 8 h. The procedure increases spore wet heat resistance and thickness of the spore coat and cortex layers, whilst delaying the time to spore phase-darkening and burst after addition of germinant. The proteome analysis reveals that the upregulated proteins in the kinA induced spores, compared to spores without kinA induction, as well as the ‘wildtype’ spores, are mostly involved in spore formation. The downregulated proteins mostly belong to the categories of coping with stress, carbon and nitrogen metabolism, as well as the regulation of sporulation. Thus, while kinA overexpression enhances synchronicity in sporulation initiation, it also has profound effects on the central equilibrium of spore formation and spore germination, through modulation of the spore molecular composition and stress resistance physiology.

A regulatory protein that represses sporulation in Clostridioides difficile

Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50 to 60% of those bacteria have the ability to produce resilient spores, important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile, and contains several genes of unknown function. We report on the characterization of a signature gene, csiA, which, as we show, is involved in the control of sporulation initiation in C. difficile. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of csiA results in increased sporulation, and increased expression of spo0A per cell. Spo0A also drives transcription of the spoIIA and spoIIG operons, coding for the first forespore-(σF) and mother cell-specific (σE) RNA polymerase sigma factors. Strikingly, deletion of csiA increases expression of the spoIIG operon, but not that of the spoIIA operon. Increased expression of spoIIG results in increased production and proteolytic activation of pro-σE, suggesting that normally, the levels of active σE are limiting for sporulation. While other regulatory proteins affect both sporulation and several processes during the transition phase of growth, including toxin production or motility, deletion of the csiA gene does not alter the expression of the genes coding for the TcdA and TcdB cytotoxins or the genes involved in motility. Thus, our results establish that CsiA acts to modulate sporulation by reducing expression of the spo0A gene.

Multiple and Overlapping Functions of Quorum Sensing Proteins for Cell Specialization in Bacillus Species

ABSTRACT In bacterial populations, quorum sensing (QS) systems participate in the regulation of specialization processes and regulate collective behaviors that mediate interactions and allow survival of the species. In Gram-positive bacteria, QS systems of the RRNPP family (Rgg, Rap, NprR, PlcR, and PrgX) consist of intracellular receptors and their cognate signaling peptides. Two of these receptors, Rap and NprR, have regained attention in Bacillus subtilis and the Bacillus cereus group. Some Rap proteins, such as RapH and Rap60, are multifunctional and/or redundant in function, linking the specialization processes of sporulation and competence, as well as global expression changes in the transition phase in B. subtilis. NprR, an evolutionary intermediate between Rap and RRNPP transcriptional activators, is a bifunctional regulator that modulates sporulation initiation and activates nutrient scavenging genes. In this review, we discuss how these receptors switch between functions and connect distinct signaling pathways. Based on structural evidence, we propose that RapH and Rap60 should be considered moonlighting proteins. Additionally, we analyze an evolutionary and ecological perspective to understand the multifunctionality and functional redundancy of these regulators in both Bacillus spp. and non-Bacillus Firmicutes. Understanding the mechanistic, structural, ecological, and evolutionary basis for the multifunctionality and redundancy of these QS systems is a key step for achieving the development of innovative technologies for health and agriculture.

Quorum sensing in Bacillus subtilis slows down biofilm formation by enabling sporulation bet hedging

1.2ABSTRACTThe ComQXPA quorum sensing (QS) system of Bacillus subtilis, a Gram-positive, industrially relevant, endospore forming bacterium, promotes surfactin production. This lipopeptide increases transcription of several genes involved in biofilm matrix synthesis via the Spo0A-P master regulator. We hypothesized that the inactivation of the QS system will therefore result in decreased rates of floating biofilm formation. We find that this is not the case and that the QS deficient mutant forms pellicles with a faster rate and produces more biofilm matrix components than the wild type. As Spo0A-P is the master regulator of sporulation initiation we hypothesized that the ComQXPA dependent signaling promotes sporulation and consequently slows the growth rate of the wild type strain. Indeed, our results confirm that cells with the inactive QS initiate endospore formation in biofilms later and more synchronously than the wild type, as evidenced by spore frequencies and the PspoIIQ promoter activity. We argue, that the QS system acts as a switch that promotes stochastic sporulation initiation and consequently bet hedging behavior. By committing a subpopulation of cells to sporulation early during growth, wild type population grows slower and produces thinner biofilms but also assures better survival under stressful conditions.1.1IMPORTANCEBacillus subtilis is widely employed model organism to study biofilm formation and sporulation in Gram-positive bacteria. The ComQXPA quorum sensing (QS) system indirectly increases the transcription of genes involved in biofilm matrix formation, which predicts a positive role of this QS in biofilm development Here we show that QS mutants actually form more matrix components per pellicle than the wild type and that their pellicles are thicker and form with a faster rate. We explain this, by showing that cells with an inactive QS exhibit a delay in sporulation entry, which is also more synchronous relative to the wild type. We argue, that the ComQXPA QS system acts as a switch that contributes to the stochastic sporulation initiation and though this path promotes bet hedging behavior. This finding is important in terms of “quorum quenching” strategies aiming to down modulate biofilm development through inhibition of QS signaling and underscores the richness of QS regulated phenotypic outcomes among bacterial species.