Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens. Clostridioides difficile is an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination of in silico predictions and in vitro experiments to demonstrate that C. difficile employs a bipartite origin of replication that shows DnaA-dependent melting at oriC2, located in the dnaA-dnaN intergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region of C. difficile and contributes to our understanding of the initiation of DNA replication in clostridia.

Faithful DNA replication is crucial for viability of cells across all kingdoms. Targeting DNA replication is a viable strategy for inhibition of bacterial pathogens.Clostridioides difficileis an important enteropathogen that causes potentially fatal intestinal inflammation. Knowledge about DNA replication in this organism is limited and no data is available on the very first steps of DNA replication. Here, we use a combination ofin silicopredictions andin vitroexperiments to demonstrate thatC. difficileemploys a bipartite origin of replication that shows DnaA-dependent melting atoriC2, located in thednaA-dnaNintergenic region. Analysis of putative origins of replication in different clostridia suggests that the main features of the origin architecture are conserved. This study is the first to characterize aspects of the origin region ofC. difficileand contributes to our understanding of the initiation of DNA replication in clostridia.

Clostridium difficile is a potentially lethal gut pathogen that causes nosocomial and community-acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA polymerase of Gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E [N2-(3,4-dichlorobenzyl)-7-(2-[1-morpholinyl] ethyl) guanine; MorE-DCBG] is a DNA polymerase inhibitor in preclinical development as a novel therapeutic against C. difficile infection. This synthetic purine shows preferential activity against C. difficile PolC over those of other organisms in vitro and is effective in an animal model of C. difficile infection. In this study, we have determined its efficacy against a large collection of clinical isolates. At concentrations below the MIC, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response of C. difficile to replication inhibition and observed an overrepresentation of upregulated genes near the origin of replication in the presence of PolC inhibitors, but not when cells were subjected to subinhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal and terminus-proximal gene copy number upon exposure to PolC inhibitors. Moreover, we show that certain genes differentially regulated under PolC inhibition are controlled by the originproximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition in C. difficile.

With the impending crisis of antimicrobial resistance, there is an urgent need to develop novel antimicrobials to combat difficult infections and MDR pathogenic microorganisms. DNA replication is essential for cell viability and is therefore an attractive target for antimicrobials. Although several antimicrobials targeting DNA replication proteins have been developed to date, gyrase/topoisomerase inhibitors are the only class widely used in the clinic. Given the numerous essential proteins in the bacterial replisome that may serve as a potential target for inhibitors and the relative paucity of suitable compounds, it is evident that antimicrobials targeting the replisome are underdeveloped so far. In this review, we report on the diversity of antimicrobial compounds targeting DNA replication and highlight some of the challenges in developing new drugs that target this process.

With the impending crisis of antimicrobial resistance, there is an urgent need to develop novel antimicrobials to combat difficult infections and MDR pathogenic microorganisms. DNA replication is essential for cell viability and is therefore an attractive target for antimicrobials. Although several antimicrobials targeting DNA replication proteins have been developed to date, gyrase/topoisomerase inhibitors are the only class widely used in the clinic. Given the numerous essential proteins in the bacterial replisome that may serve as a potential target for inhibitors and the relative paucity of suitable compounds, it is evident that antimicrobials targeting the replisome are underdeveloped so far. In this review, we report on the diversity of antimicrobial compounds targeting DNA replication and highlight some of the challenges in developing new drugs that target this process.

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by a limited understanding of DNA replication in pathogenic micro-organisms. Clostridium difficile is the main cause of health-care associated diarrhea and its DNA replication machinery is virtually uncharacterized. We identified the replicative helicase (CD3657), the helicase loader ATPase (CD3654) and primase (CD1454) of C. difficile and reconstitute helicase and primase activity in vitro . We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. We find that helicase activity is dependent on the presence of primase in vitro . The trinucleotide specificity of primase, which we find to be determined by a single lysine residue, is similar to extreme thermophile Aquifex aeolicus but the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, helicase-primase interactions constitute a crucial aspect of DNA replication initiation in C. difficile and helicase loading and activation in this organism differs critically from that of the Gram-positive model organism Bacillus subtilis .

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro. We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro. The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader–helicase–primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

Background Clostridium difficile strain 630Δ erm is a spontaneous erythromycin sensitive derivative of the reference strain 630 obtained by serial passaging in antibiotic-free media. It is widely used as a defined and tractable C. difficile strain. Though largely similar to the ancestral strain, it demonstrates phenotypic differences that might be the result of underlying genetic changes. Here, we performed a de novo assembly based on single-molecule real-time sequencing and an analysis of major methylation patterns. Results In addition to single nucleotide polymorphisms and various indels, we found that the mobile element CTn 5 is present in the gene encoding the methyltransferase rumA rather than adhesin CD1844 where it is located in the reference strain. Conclusions Together, the genetic features identified in this study may help to explain at least part of the phenotypic differences. The annotated genome sequence of this lab strain, including the first analysis of major methylation patterns, will be a valuable resource for genetic research on C. difficile .