Measuring the Plasmid Copy Number of Single E. Coli by Digital PCR: Are Plasmids Partitioned in Clusters during Cell Division?

Abstract

The presence of plasmids is important for the behavior of bacteria, as they often carry antibiotic resistance genes and genes involved in virulence of pathogenic strains. Furthermore plasmids are often responsible for horizontal gene transfer. In order to remain in the bacteria the plasmids have to provide complex regulation mechanisms and often inhere active partitioning during cell division. Fingerprints of the resulting plasmid stability can be found in the plasmid copy number distribution. However, these distributions have been rarely reported, since they have been experimentally difficult to obtain.We performed single cell measurements that allow to piece together the plasmid copy number distribution in a bacterial population. We use fluorescent activated cell sorting (FACS) to isolate single bacteria and subsequently utilize the single molecule resolution of digital PCR to determine the plasmid copy number of single cells.Interestingly, the measurement of the plasmid pZE which has a ColE1 like origin of replication showed a rather broad distribution. An unexpectedly high percentage of the cells had very low plasmid copy numbers, despite the high mean copy number of 120. This characteristic however disappeared for a mutant which additionally contains the active partitioning system parABC. The instability of the pZE copy number is unlikely to be the result of random plasmid partitioning; we explain the findings by clustered partitioning, i.e. clusters of plasmids are partitioned to the daughter cells rather than the individual plasmids. Results are high statistical fluctuations of the plasmid copy number. Other groups showed evidence for clustering of plasmids in E. Coli and also showed, that these clusters get dissolved if the plasmids contain parABC genes. Furthermore, theoretical models which employ clustered partitioning fits our data quantitatively.