Abstract
Bacterial plasmids play important roles in the metabolism, pathogenesis and bacterial evolution and are highly versatile biotechnological tools. Stable inheritance of plasmids depends on their autonomous replication and efficient partition to daughter cells at cell division. Active partition systems have not been identified for high-copy number plasmids, and it has been generally believed that they are partitioned randomly at cell division. Nevertheless, direct evidence for the cellular location of replicating and nonreplicating plasmids, and the partition mechanism has been lacking. We used as model pJHCMW1, a plasmid isolated from Klebsiella pneumoniae that includes two β-lactamase and two aminoglycoside resistance genes. Here we report that individual ColE1-type plasmid molecules are mobile and tend to be excluded from the nucleoid, mainly localizing at the cell poles but occasionally moving between poles along the long axis of the cell. As a consequence, at the moment of cell division, most plasmid molecules are located at the poles, resulting in efficient random partition to the daughter cells. Complete replication of individual molecules occurred stochastically and independently in the nucleoid-free space throughout the cell cycle, with a constant probability of initiation per plasmid.
Highlights
Plasmids play an essential role in bacterial metabolism, pathogenesis and evolution by harboring genes coding for diverse factors and facilitating their dissemination.Of particular importance is their role in the dissemination of resistance genes contributing to the current epidemic of resistant infections [1,2]
High copy number plasmids, which are usually smaller than low copy number plasmids, appear not to code for partition systems and their stable inheritance must be assured by the high number of copies of the plasmid molecules
A plasmid derivative containing 48 or 96 copies of tetO inserted into the tnpA gene was introduced into an E. coli strain in which fluorescent Tet repressor was expressed at a low level from a chromosomal tetR gene (Supplementary Figure S1A and B)
Summary
Plasmids play an essential role in bacterial metabolism, pathogenesis and evolution by harboring genes coding for diverse factors and facilitating their dissemination.Of particular importance is their role in the dissemination of resistance genes contributing to the current epidemic of resistant infections [1,2]. Several mechanisms, including regulation of initiation of replication, partition, multimer resolution and post-segregational killing, ensure that plasmids are stably maintained at a constant copy number within the host bacterial cells and are transmitted to the following generations [3,4]. The implementation of fluorescence microscopy in bacteria showed fewer spots per cell than those expected of fluorescently labeled plasmids, leading to the suggestion that hcn plasmids are not randomly located and may be bound or clustered together [3,7,8,9,10] These intriguing results prompted us to further study the segregation and replication of the clinically relevant ColE1-type plasmid pJHCMW1 using quantitative imaging methods. This hcn plasmid was first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis during a nosocomial infection and is responsible for failure of treatment by conferring resistance to several antibiotics [11]
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