UC Riverside

Mobile genetic elements (MGEs) are important drivers of bacterial evolution, facilitating the exchange of fitness determinant genes such as antibiotic resistance and virulence factors. Although various computational methods exist for identifying potential MGEs, confirming their ability to transfer requires additional experimental approaches. Here, we introduce the transposon (Tn) mutagenesis mobilization method (TMMM) as a means to confirm mobilization without the need for targeted mutations. Using this method, we identified two MGEs, one being a novel CTn labeled PvCTn, present in Phocaeicola vulgatus. Through Tn mutagenesis and subsequent gene deletion, we discovered that a helix-turn-helix motif gene, BVU3433, negatively regulated the conjugation efficiency of PvCTn in vitro. Furthermore, our transcriptomics data revealed that BVU3433 plays a crucial role in the repression concouof PvCTn genes, including genes involved in forming complete conjugation machinery (Type IV Secretion System (T4SS)). Finally, analysis of individual strain genomes and community metagenomes identified the widespread prevalence of PvCTn-like elements with putative BVU3433 homologs among human gut-associated bacteria. Tn mutagenesis detection of MGEs enables in vitro observation of transfer events. Further this method has potential for in vivo identification of additional functional MGEs and characterization of the fitness determinants they encode. For example, there is an increasing threat of antibiotic resistance in clinically relevant bacteria, often exacerbated by MGEs. We specifically investigated CTnDOT, a key contributor to tetracycline resistance in 80% of clinical and community isolates of Bacteroides. In this study, we observed the in vitro mobilization of a novel CTnDOT-like element (PmDOT), from Parabacteroides merdae into Bacteroides thetaiotaomicron. Subsequent genome screens revealed the presence of intact CTnDOT- like elements in ~50% of the 134 human-gut associated Bacteroidota genomes examined. Moreover, through the analysis of human fecal metagenomes, we determined the widespread prevalence of CTnDOT-like elements across diverse global human populations. Our findings confirm CTnDOT-like elements are a diverse and significant group of MGEs within the human gut microbiome.