Type I toxin-antitoxin systems contribute to mobile genetic elements maintenance inClostridioides difficileand can be used as a counter-selectable marker for chromosomal manipulation

Abstract

Toxin-antitoxin (TA) systems are widespread on mobile genetic elements as well as in bacterial chromosomes. According to the nature of the antitoxin and its mode of action for toxin inhibition, TA systems are subdivided into different types. The first type I TA modules were recently identified in the human enteropathogen Clostridioides (formerly Clostridium) difficile. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA during normal growth. Here, we report the characterization of five additional type I TA systems present within phiCD630-1 and phiCD630-2 prophage regions of C. difficile 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest. Growth is restored when the antitoxin RNAs, transcribed from the opposite strand, are co-expressed together with the toxin genes. In addition, we show that type I TA modules located within the phiCD630-1 prophage contribute to its stability and mediate phiCD630-1 heritability. Type I TA systems were found to be widespread in genomes of C. difficile phages, further suggesting their functional importance. We have made use of a toxin gene from one of type I TA modules of C. difficile as a counter-selectable marker to generate an efficient mutagenesis tool for this bacterium. This tool enabled us to delete all identified toxin genes within the phiCD630-1 prophage, thus allowing investigation of the role of TA in prophage maintenance. Furthermore, we were able to delete the large 49 kb phiCD630-2 prophage region using this improved procedure.

Identification of RNAs bound by Hfq reveals widespread RNA partners and a sporulation regulator in the human pathogenClostridioides difficile

“…As a positive control of CD1234 protein detection by Western blot, the 3xFLAG-tag sequence was introduced to the C-terminal part of CD1234 gene on pRFP185-derivative plasmid. To introduce the 3xFLAG-tag at the C-terminal part of the coding region of CD1234 gene, directly upstream the stop codon on the C. difficile chromosome, we used an allelic-coupled exchange technique based on a semi-suicidal plasmid vector carrying E. coli cytosine deaminase codA gene as counter-selection marker (35) that was improved by using an inducible toxin expression from type I toxin-antitoxin module instead of codA as counterselection marker (19). We used Gibson assembly to construct a plasmid for further conjugation and homologous recombination in C. difficile.…”

“…Remarkably, significantly enriched peaks mapped to the regions transcribed from both sense and antisense strands forming potential RNA duplexes. Data from our RNAseq and TSS-mapping had revealed the presence of type I TA systems in C. difficile with RNA antitoxin acting as antisense RNA to toxin mRNA (18)(19)(20). We further characterized some of them (18,19) and identified a total of 13 type I TA modules in the reference strain 630 including the previously unannotated TA module with CD630_n00150 antitoxin/CD0440.1 toxin genes (20).…”

“…Data from our RNAseq and TSS-mapping had revealed the presence of type I TA systems in C. difficile with RNA antitoxin acting as antisense RNA to toxin mRNA (18)(19)(20). We further characterized some of them (18,19) and identified a total of 13 type I TA modules in the reference strain 630 including the previously unannotated TA module with CD630_n00150 antitoxin/CD0440.1 toxin genes (20). Our RIP-seq analysis shows that the toxin and antitoxin RNAs of all these systems bind to Hfq, as is the case for CD2517.1 toxin mRNA and RCd8 antitoxin (2,650-fold enrichment) ( Figure 2B) (see Supplementary Figure S5B for additional examples).…”

“…Our RIP-seq data are in complete accordance with all the previously published results from independent studies by various large-scale and targeted approaches on C. difficile ncRNA identification and functional characterization including antisense RNAs within TA systems, CRISPR RNAs, riboswitches and sRNAs. The majority of ncRNAs captured as Hfq ligands have been previously identified by genome-wide approaches including TSS mapping and RNA-seq (15,65) and some of them have been characterized by 5'-RACE, Northern blot, RT-PCR, in vitro interaction analysis and RNA stability assays (15,16,(18)(19)(20)27,65).…”

“…We recently described type I toxin-antitoxin (TA) modules adjacent to CRISPR arrays with the first functional antisense RNAs in this pathogen (18). A total of 13 potential type I TA modules are present in the genome of C. difficile reference strain 630 that could also contribute to its fitness inside the host (19,20). All these studies suggest that the regulatory RNA network controlling C. difficile pathophysiology is in many aspects unique and deserves further special attention (14).…”