We used whole-genome sequencing to study Clostridioides difficile isolates from patients with recurrent or sequential infections in Minnesota, USA. We summ

Combinatorial DNA design and assembly is an efficient and pragmatic way to obtain high-performing metabolic pathway designs quickly. However, implementation may require organism-specific technical barriers to be overcome. Firstly, suitable expression control parts such as promoters and ribosome-binding sites (RBSs) which provide a suitable range of expression levels need to be identified or developed. Secondly, these need to be assembled into pathway-encoding combinatorial libraries of sufficient size, quality and diversity. For organisms with transformation frequencies too low to allow direct transformation of library assembly reactions, such as many Clostridium spp., assembly and amplification is typically carried out using Escherichia coli. However, if constructs are deleterious (or 'burdensome') to E. coli, which is often the case when using Clostridium genetic parts, poor libraries may be obtained. Here we develop a new approach called integration‑coupled activation of promoterless sequences (ICAPS) to overcome this barrier and therefore enable combinatorial assembly in Clostridium. Libraries were designed and assembled as promoterless synthetic operons, preventing expression during DNA assembly, and expression was only activated later, when constructs were integrated into the host genome downstream of a promoter. Variation of expression levels was achieved using a range of context-resistant RBS sequences. This approach was used to produce a Clostridium acetobutylicum library with combinatorial expression variants of an introduced hexanol pathway. This proof of concept provides a generally-applicable approach to implement combinatorial metabolic pathway-encoding libraries in Clostridium spp., circumventing the excessive strength of Clostridium expression control parts in E. coli, and is applicable to other organisms. ### Competing Interest Statement The authors have declared no competing interest. BBSRC, BB/M002454/1, BB/V001396/1

Abstract. Ribosomal RNA (rRNA) methylation is conserved across biology, yet the effect of rRNA methylation on ribosome function is poorly understood. In th

Abstract. Most antibiotics are natural compounds or their derivatives, and bacteria have evolved defensive mechanisms to resist them. Many of these mechani

Here, the authors develop AMPLiT a tool for screening antimicrobial peptides in metagenomic datasets, and apply it to human coprolite metagenomes, finding that Segatella copri, an ancient prevalent hu...

Natural transformation is a widespread mechanism driving genetic exchanges in bacteria. It proceeds by the capture and internalization of exogenous DNA in linear single strands, ultimately integrated in the genome by homologous recombination. It is unknown how the RecA-directed D-loop intermediate of this dedicated recombination pathway is processed. We report that resolution of the transformation D-loop depends on two endonucleases of opposing phylogenetic distribution in bacteria. One is YraN, which has co-evolved and interacts with the ComM helicase, known to extend DNA recombination at the transformation D-loop. The other is CoiA, which is restricted to the Bacillota. CoiA is shown to be a resolvase of the transformation D-loop, extended by the RadA helicase in these species. We demonstrate that both YraN and CoiA act synergistically with their cognate helicases. These findings reveal that bacteria have evolved two helicase/nuclease pairs for the maturation and recombination extension of the transformation D-loop. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-CE12-0004, ANR-10-BLAN-1331, ANR-17-CE13-0031, ANR-22-CE44-0044, ANR-10-LABX-62-IBEID, PIA/ANR-16-CONV-0005 Fondation pour la Recherche Médicale, https://ror.org/04w6kn183, FDT202001010890 European Union's Horizon research and innovation programme. Marie Skłodowska-Curie Postdoctoral Fellowships, 101208987 National Institute of Health, USA, R35GM128674

Proceedings of the National Academy of Sciences (PNAS), a peer reviewed journal of the National Academy of Sciences (NAS) - an authoritative source of high-impact, original research that broadly spans...

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, th...

Metronidazole (MTZ) is a widely used antibiotic for treating anaerobic bacterial infections, including Clostridioides difficile infection. However, the increasing emergence of MTZ resistance among ana...

Peptidoglycan (PG) synthesis is essential to maintain cell integrity during bacterial growth and division. In Bacillus subtilis, PG synthesis involves class A PBPs that act independently and class B P...

Abstract. Infections with epidemic PCR ribotype (RT) 027 strains are characterized by higher mortality and morbidity and have caused outbreaks in North Ame

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the l...

Author summary The environment in which bacteria live puts them under a great deal of stress, forcing them to adapt constantly, either temporarily or permanently. Streptococcus pneumoniae, a pathogeni...

DNA methylation is a widespread phenomenon in bacteria that can regulate gene expression, although the mechanisms underlying this epigenetic regulation are often poorly understood. In Clostridioides d...

Clostridioides difficile, a gram-positive anaerobe, is a leading cause of hospital-acquired diarrhea, responsible for 20,500 deaths from 462,100 infections in 2017 (1, 2). The primary risk factor for ...

When Streptococcus pneumoniae produces low amounts of the second messenger cyclic di-AMP (c-di-AMP), they do not survive in the presence of competence-stimulating peptide (CSP), which is used for tra...

Clostridioides difficile is a pathogenic bacterium and a leading cause of antibiotic-associated diarrhea. Symptoms of the infection arise due to production of large clostridial toxins that disrupt the...