Search

Clostridioides difficile causes a wide range of intestinal diseases through the action of two main cytotoxins, TcdA and TcdB. Ingested spores germinate in the intestine establishing a population of cells that produce toxins and spores. The pathogenicity locus, PaLoc, comprises several genes, including those coding for TcdA/B, for the holin-like TcdE protein, and for TcdR, an auto-regulatory RNA polymerase sigma factor essential for tcdA/B and tcdE expression. Here we show that tcdR, tcdA, tcdB and tcdE are expressed in a fraction of the sporulating cells, in either the whole sporangium or in the forespore. The whole sporangium pattern is due to protracted expression initiated in vegetative cells by σD, which primes the TcdR auto-regulatory loop. In contrast, the forespore-specific regulatory proteins σG and SpoVT control TcdR production and tcdA/tcdB and tcdE expression in this cell. We detected TcdA at the spore surface, and we show that wild type and ΔtcdA or ΔtcdB spores but not ΔtcdR or ΔtcdA/ΔtcdB spores are cytopathic against HT29 and Vero cells, indicating that spores may serve as toxin-delivery vehicles. Since the addition of TcdA and TcdB enhance binding of spores to epithelial cells, this effect may occur independently of toxin production by vegetative cells. This study shows that the genes coding for the two main toxins produced by Clostridioides difficile are expressed during sporulation and that the toxins associate with the mature spores, which have a cytopathic effect on human intestinal cell lines. [ABSTRACT FROM AUTHOR]

Sporulation is a finely regulated morphogenetic program important in the ecology and epidemiology of Clostridium botulinum. Exogenous elements disrupting sporulation-associated genes contribute to sporulation regulation and introduce diversity in the generally conserved sporulation programs of endospore formers. We identified a novel prophage-like DNA segment, termed the yin element, inserted within yabG, encoding a sporulation-specific cysteine protease, in an environmental isolate of C. botulinum. Bioinformatic analysis revealed that the genetic structure of the yin element resembles previously reported mobile intervening elements associated with sporulation genes. Within a pure C. botulinum culture, we observed two subpopulations of cells with the yin element either integrated into the yabG locus or excised as a circular DNA molecule. The dynamics between the two observed conformations of the yin element was growth-phase dependent and likely mediated by recombination events. The yin element was not required for sporulation by C. botulinum but triggered an earlier entry into sporulation than in a related isolate lacking this element. So far, the yin element has not been found in any other C. botulinum strains or other endospore-forming species. It remains to be demonstrated what kind of competitive edge it provides for C. botulinum survival and persistence. [ABSTRACT FROM AUTHOR]

Clostridium difficile, a toxin-producing, spore-forming bacillus, is a main cause of nosocomial antimicrobial drug-associated diarrhea in industrialized countries (1). C. difficile infection (CDI) usually develops in previously hospitalized persons with [...]

Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is anaturally competent bacterium that occupies various environments, including plant-associated, soil, and marineniches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43%of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them. [ABSTRACT FROM AUTHOR]

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development. [ABSTRACT FROM AUTHOR]

Engulfment of the forespore by the mother cell is a universal feature of endosporulation. In Bacillus subtilis, the forespore protein SpoIIQ and the mother cell protein SpoIIIAH form a channel, essential for endosporulation, through which the developing spore is nurtured. The two proteins also form a backup system for engulfment. Unlike in B. subtilis, SpoIIQ of Clostridium difficile has intact LytM zinc-binding motifs. We show that spoIIQ or spoIIIAH deletion mutants of C. difficile result in anomalous engulfment, and that disruption of the SpoIIQ LytM domain via a single amino acid substitution (H120S) impairs engulfment differently. SpoIIQ and SpoIIQH120S interact with SpoIIIAH throughout engulfment. SpoIIQ, but not SpoIIQH120S, binds Zn2+, and metal absence alters the SpoIIQ-SpoIIIAH complex in vitro. Possibly, SpoIIQH120S supports normal engulfment in some cells but not a second function of the complex, required following engulfment completion. We show that cells of the spoIIQ or spoIIIAH mutants that complete engulfment are impaired in post-engulfment, forespore and mother cell-specific gene expression, suggesting a channel-like function. Both engulfment and a channel-like function may be ancestral functions of SpoIIQ-SpoIIIAH while the requirement for engulfment was alleviated through the emergence of redundant mechanisms in B. subtilis and related organisms. [ABSTRACT FROM AUTHOR]