Genetic basis of I-complex plasmid stability and conjugation

Abstract

Plasmids are major drivers of increasing antibiotic resistance, necessitating an urgent need to understand their biology. Here we describe a detailed dissection of the molecular components controlling the genetics of I-complex plasmids, a group of antibiotic resistance plasmids found frequently in pathogenic Escherichia coli and other Enterobacteriaceae that cause significant human disease. We show these plasmids cluster into four distinct subgroups, with the prototype IncI1 plasmid R64 subgroup displaying low nucleotide sequence conservation to other I-complex plasmids. Using pMS7163B, an I-complex plasmid distantly related to R64, we performed a high-resolution transposon-based genetic screen and defined genes involved in replication, stability, and conjugative transfer. We identified the replicon and a partitioning system as essential for replication/stability. Genes required for conjugation included the type IV secretion system, relaxosome, and several uncharacterised genes located in the pMS7163B leading transfer region that exhibited an upstream strand-specific transposon insertion bias. The overexpression of these genes severely impacted host cell growth or reduced fitness during mixed competitive growth, demonstrating that their expression must be controlled to avoid deleterious impacts. These genes were present in >80% of all I-complex plasmids and broadly conserved across multiple plasmid incompatibility groups, implicating an important role in plasmid dissemination.

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

“…Typhimurium ST313 L2.2 might have a competitive edge in some situations. Accordingly, we determined bacterial fitness using a mixed-growth competition assay (Wiser and Lenski 2015 , Lian et al 2023 ). The competitive index was calculated in three different growth media using pair-wise combinations of strains D37712 and D23580.…”

Salmonella entericaserovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage

“…Because S. Typhimurium ST313 L2.2 appeared to have displaced S. Typhimurium ST313 L2.0 in Malawi, we speculated that S. Typhimurium ST313 L2.2 might have the competitive edge in some situations. Accordingly, we determined bacterial fitness using a mixed-growth competition assay (Wiser and Lenski, 2015; Lian et al, 2023). The competitive index was calculated in three different growth media using pair-wise combinations of strains D37712 and D23580.…”

Combined functional genomic and metabolomic approaches identify new genes required for growth in human urine by multidrug-resistant Escherichia coli ST131

“…Transposon-directed insertion-site sequencing (TraDIS) and similar transposon-insertion sequencing methods such as TnSeq have been widely used as powerful functional genomics methods to understand the genetic basis of complex phenotypes associated with the ability of UPEC and other bacteria to thrive in infection-related conditions ( 9 , 20 – 25 ). These approaches involve the generation of highly saturated transposon mutant libraries and their subsequent subjection to selection pressure imposed by a condition of interest, followed targeted deep sequencing to determine differences in the frequency of transposon insertions across the genome.…”