Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

A network of small RNAs regulates sporulation initiation in Clostridioides difficile.

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

An RNA-centric global view of Clostridioides difficile reveals broad activity of Hfq in a clinically important gram-positive bacterium.

The gram-positive human pathogen Clostridioides difficile has emerged as the leading cause of antibiotic-associated diarrhea. However, little is known about the bacterium's transcriptome architecture and mechanisms of posttranscriptional control. Here, we have applied transcription start site and termination mapping to generate a single-nucleotide-resolution RNA map of C. difficile 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict cis-regulatory RNA elements upstream of multidrug resistance (MDR)-type ATP-binding cassette (ABC) transporters and transcriptional regulators. Despite growing evidence for a role of Hfq in RNA-based gene regulation in C. difficile, the functions of Hfq-based posttranscriptional regulatory networks in gram-positive pathogens remain controversial. Using Hfq immunoprecipitation followed by sequencing of bound RNA species (RIP-seq), we identify a large cohort of transcripts bound by Hfq and show that absence of Hfq affects transcript stabilities and steady-state levels. We demonstrate sRNA expression during intestinal colonization by C. difficile and identify infection-related signals impacting its expression. As a proof of concept, we show that the utilization of the abundant intestinal metabolite ethanolamine is regulated by the Hfq-dependent sRNA CDIF630nc_085. Overall, our study lays the foundation for understanding clostridial riboregulation with implications for the infection process and provides evidence for a global role of Hfq in posttranscriptional regulation in a gram-positive bacterium.

Grad-seq identifies KhpB as a global RNA-binding protein in Clostridioides difficile that regulates toxin production.

Much of our current knowledge about cellular RNA-protein complexes in bacteria is derived from analyses in gram-negative model organisms, with the discovery of RNA-binding proteins (RBPs) generally lagging behind in Gram-positive species. Here, we have applied Grad-seq analysis of native RNA-protein complexes to a major Gram-positive human pathogen, Clostridioides difficile, whose RNA biology remains largely unexplored. Our analysis resolves in-gradient distributions for ∼88% of all annotated transcripts and ∼50% of all proteins, thereby providing a comprehensive resource for the discovery of RNA-protein and protein-protein complexes in C. difficile and related microbes. The sedimentation profiles together with pulldown approaches identify KhpB, previously identified in Streptococcus pneumoniae, as an uncharacterized, pervasive RBP in C. difficile. Global RIP-seq analysis establishes a large suite of mRNA and small RNA targets of KhpB, similar to the scope of the Hfq targetome in C. difficile. The KhpB-bound transcripts include several functionally related mRNAs encoding virulence-associated metabolic pathways and toxin A whose transcript levels are observed to be increased in a khpB deletion strain. Moreover, the production of toxin protein is also increased upon khpB deletion. In summary, this study expands our knowledge of cellular RNA protein interactions in C. difficile and supports the emerging view that KhpB homologues constitute a new class of globally acting RBPs in Gram-positive bacteria.

Growth trajectories in the cave bear and its extant relatives: an examination of ontogenetic patterns in phylogeny.

BACKGROUND: The study of postnatal ontogeny can provide insights into evolution by offering an understanding of how growth trajectories have evolved resulting in adult morphological disparity. The Ursus lineage is a good subject for studying cranial and mandibular shape and size variation in relation to postnatal ontogeny and phylogeny because it is at the same time not diverse but the species exhibit different feeding ecologies. Cranial and mandibular shapes of Ursus arctos (brown bear), U. maritimus (polar bear), U. americanus (American black bear), and the extinct U. spelaeus (cave bear) were examined, using a three-dimensional geometric morphometric approach. Additionally, ontogenetic series of crania and mandibles of U. arctos and U. spelaeus ranging from newborns to senile age were sampled. RESULTS: The distribution of specimens in morphospace allowed to distinguish species and age classes and the ontogenetic trajectories U. arctos and U. spelaeus were found to be more similar than expected by chance. Cranial shape changes during ontogeny are largely size related whereas the evolution of cranial shape disparity in this clade appears to be more influenced by dietary adaptation than by size and phylogeny. The different feeding ecologies are reflected in different cranial and mandibular shapes among species. CONCLUSIONS: The cranial and mandibular shape disparity in the Ursus lineage appears to be more influenced by adaptation to diet than by size or phylogeny. In contrast, the cranial and mandibular shape changes during postnatal ontogeny in U. arctos and U. spelaeus are probably largely size related. The patterns of morphospace occupation of the cranium and the mandible in adults and through ontogeny are different.

Characterisation of an epithelium-like layer of cells in the multicellular Dictyostelium discoideum slug

Ultrarapid freezing (RF) followed by freeze-substitution (FS) provide superior preservation of the Dictyostelium discoideum multicellular slug tissue over conventional methods of chemical fixation at room temperature. The peripheral cells of slugs prepared by RF and FS form a tight layer of flattened cells. This cell layer resembles epithelia of other multicellular organisms in that it has close junctional contact between cells associated with the extracellular matrix (ECM, slime sheath). This is the first report that clearly demonstrates the existence of such peripheral cellular specialisation in this otherwise well-studied model system. Junctional contacts between adjacent cells mean that there is no intercellular space evident between apical membranes of apposing cells, and basally the intermembraneous space between peripheral cells is less than 10 nm. By contrast, the intercellular space between internal cells is approximately 10­25 nm. The shape of the peripheral cells varies with their location around the slug. In the posterior prespore zone, the peripheral cells are squamous and exhibit polarity along their antero-posterior axis. In the anterior prestalk zone, peripheral cells are less flattened, project irregular filipodia between internal cells, and are polarised along their apical-basal axis. Colloidal gold immunocytochemistry with the markers MUD1, MUD50 and MUD62 demonstrated that the peripheral layer is formed of prestalk cells in the anterior region and ventrum, and mostly prespore cells along the dorsum. Thus, the peripheral layer, while having specific cell classes in different regions, is not differentiation-specific. Rather, it appears that the structure of these epithelium-like cells is influenced by interaction with molecules of the ECM (sheath).