High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile

Abstract

(formerly ) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting , and phage c-st, infecting A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a gene. We screened 2,584 genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements. is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect so far. Screening 2,584 genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in to better understand the epidemiology of this clinically important human pathogen.

Integrated mobile genetic elements in Thaumarchaeota

“…The vast majority of known CRSIPR-Cas systems are encoded by cellular organisms and deployed to counter the replication of MGE, but some MGE also carry functional CRISPR-Cas systems. For instance, CRISPR-Cas systems and stand-alone CRISPR arrays have been identified in a number of prophages (Hargreaves et al, 2014;Chénard et al, 2016;Zheng et al, 2016;Garneau et al, 2018) and in the case of a Vibrio-infecting bacteriophage have been shown to target for destruction a pathogenicity island integrated in the host genome (Seed et al, 2013). By contrast, a subgroup of Tn7-like transposons has been hypothesized to employ the encoded CRISPR-Cas system for CRISPR-guided transposition (Peters et al, 2017).…”

Bacteriophages Contribute to Shaping Clostridioides (Clostridium) difficile Species

“…Over 1,300 C. difficile genomes have been fully sequenced and are available in public repositories, but thousands of additional genomes have also been sequenced and are available through collaborative research ( Garneau et al, 2018 ). Although uncommon, as many as 5–6 different prophages were identified in a single C. difficile genome ( Amy et al, 2018 ; Ramírez-Vargas et al, 2018 ).…”

“…However, between 1 and 3 prophages are more frequently observed, in addition to genomic “islands” containing phage-related genes. Recent studies highlighted the prevalence of large phage genomes that reside as extrachromosomal DNA in C. difficile ( Garneau et al, 2018 ; Ramírez-Vargas et al, 2018 ). For example, the large phiCD211/phiCDIF1297T and related phages, with genomes of ≥131-kb, have been detected in 5% of 2,584 C. difficile genomes analyzed, spanning 21 different multi-locus sequence types (MLST) ( Wittmann et al, 2015 ; Garneau et al, 2018 ).…”

“…Recent studies highlighted the prevalence of large phage genomes that reside as extrachromosomal DNA in C. difficile ( Garneau et al, 2018 ; Ramírez-Vargas et al, 2018 ). For example, the large phiCD211/phiCDIF1297T and related phages, with genomes of ≥131-kb, have been detected in 5% of 2,584 C. difficile genomes analyzed, spanning 21 different multi-locus sequence types (MLST) ( Wittmann et al, 2015 ; Garneau et al, 2018 ). Ten other large phage genomes (∼128–135-kb), including phiCD5763, phiCD5774, and phiCD2955, were recently described in C. difficile isolates from around the world and representing seven different MLST sequence types ( Ramírez-Vargas et al, 2018 ).…”

“…However, the presence of partition genes like parA has also been reported in other phages, including phiCD6356 ( Horgan et al, 2010 ), ϕCD38-2 ( Sekulovic et al, 2011 ), and phiSemix9P1 ( Riedel et al, 2017 ), the latter two known to be maintained as extrachromosomal DNAs in lysogenic cells. Large phage genomes such as phiCD211/phiCDIF1296T and phiCD5763 also seem to be frequently found as extrachromosomal DNA, and ParM homologs were identified in some of them ( Garneau et al, 2018 ; Ramírez-Vargas et al, 2018 ). Therefore, in the absence of functional data to assess the inducibility and production of infectious particles from these large “plasmids,” it is hard to conclude on their exact nature.…”