Microbiomes are complex communities of microorganisms that are essential for biochemical processes on Earth and for the health of humans, animals and plants. Many environmental and host-associated microbiomes are dominated by anaerobic microbes, some of which cannot tolerate oxygen. Anaerobic microbial communities have been extensively studied over the last 20 years using molecular techniques, especially next-generation sequencing. However, there is a renewed interest in microbial cultivation because isolates provide the basis for understanding the taxonomic and functional units of biodiversity, elucidating novel biochemical pathways and the mechanisms underlying microbe-microbe and microbe-host interactions and opening new avenues for biotechnological and clinical applications. In this Perspective, we present areas of research and applications that will benefit from advancement in anaerobic microbial cultivation. We highlight key technical and infrastructural hurdles associated with the development and deployment of sophisticated cultivation workflows. Improving the performance of cultivation techniques will set new trends in functional microbiome research in the coming years.

Bacterial noncoding RNAs fulfill a variety of cellular functions as catalysts, as scaffolds in protein complexes or as regulators of gene expression. They often exhibit complex tertiary structures that are a key determinant of their biochemical function. Here, we characterize the structured "raiA motif" RNA from Clostridioides difficile, which is conserved in more than 2,500 bacterial species from the phyla Bacillota and Actinomycetota. We show that its transcript abundance and stability in exponentially growing bacteria rivals that of ribosomal RNAs. Deletion of the "raiA motif" RNA is associated with delayed transition into stationary phase, and changes in stationary phase pathways such as spore formation, hence we rename it ModT (modulator of transition phase). Mechanistically, we show that ModT-mediated changes in cellular cyclic di-GMP levels are linked to the pronounced sporulation defect in the modT mutant. Importantly, we show that expression profiles and isoform patterns of ModT are conserved in Clostridium perfringens and Paeniclostridium sordellii, and that these orthologs can functionally complement ModT in C. difficile. Chemical structure probing of ModT in vivo reveals dynamic refolding and provides initial evidence for a potential association of ModT with proteins. In summary, our findings indicate that ModT fulfills a conserved role in regulating growth transitions in bacteria and provide a crucial step towards delineating its molecular mechanism.

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.

The gram-positive human pathogen 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 5' and 3' untranslated regions, operon structures, and noncoding regulators, including 42 sRNAs. Our results indicate functionality of many conserved riboswitches and predict -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 , 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 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.

Changes in the gut microbiota may underpin many human diseases, but the mechanisms that are responsible for altering microbial communities remain poorly understood. Antibiotic usage elevates the risk of contracting gastroenteritis caused by Salmonella enterica serovars, increases the duration for which patients shed the pathogen in their faeces, and may on occasion produce a bacteriologic and symptomatic relapse. These antibiotic-induced changes in the gut microbiota can be studied in mice, in which the disruption of a balanced microbial community by treatment with the antibiotic streptomycin leads to an expansion of S. enterica serovars in the large bowel. However, the mechanisms by which streptomycin treatment drives an expansion of S. enterica serovars are not fully resolved. Here we show that host-mediated oxidation of galactose and glucose promotes post-antibiotic expansion of S. enterica serovar Typhimurium (S. Typhimurium). By elevating expression of the gene encoding inducible nitric oxide synthase (iNOS) in the caecal mucosa, streptomycin treatment increased post-antibiotic availability of the oxidation products galactarate and glucarate in the murine caecum. S. Typhimurium used galactarate and glucarate within the gut lumen of streptomycin pre-treated mice, and genetic ablation of the respective catabolic pathways reduced S. Typhimurium competitiveness. Our results identify host-mediated oxidation of carbohydrates in the gut as a mechanism for post-antibiotic pathogen expansion.