Saturday, July 19, 2008

Direct Visualization of Plasmid Transfer

Direct evidence of extant in situ plasmid transfer in natural environments has typically been obtained by identifying plasmid-encoded phenotypes following the introduction of donor strains. Many plasmids do not encode any known functions (cryptic plasmids); in many unknown plasmids, functions are not determined or there is no easy method to select plasmid-containing cells. So there is the necessity to use known reporter markers, like antibiotic resistance, lacZ, gusA, luxAB or fluorescent proteins genes to label plasmids prior to study. The main advantage of using luciferase and especially fluorescent proteins is that it is possible to detect the presence of plasmid DNA without plating (non-culturable bacteria) and adding additional substrates to the environment. Using of fluorescent proteins enables direct visualization of plasmid transfer but has also some limitations. First is using epifluorescence microscopy or flow-cytometry-based method to detect fluorescent cells, both of which are technically demanding and require expensive equipment. The second limitation arises from the properties of fluorescent proteins. They need some time - from a few to several hours, to produce the strong, detectable signal. So this make impossible to detect precisely the time of plasmid DNA entering the recipient cell.

In the paper “Direct visualization of horizontal gene transfer” by Ana Babic et al. (2008) authors developed an experimental system that enables them to distinguish the transferred donor DNA from both donor and recipient DNA, and to visualize DNA transfer and recombination by means of fluorescence microscopy in real time, at the level of individual living cells. This tool also allowed them to quantify the ongoing transfer of DNA during conjugation and to acquire time-lapse movies that follow the fate of the newly acquired DNA in individual cells through any number of cell divisions.

This method uses fusion of YFP gene with seqA gene. This translational fusion driven from the native seqA promoter was introduced into E.coli chromosome replacing the wild type seqA allel. SeqA protein has strong affinity for DNA which is hemimethylated by Dam methylase at GATC sequences. Such a hemimethylated DNA usually occurs in the replication forks during replication and SeqA-YFP fusion protein had been found bound to chromosomal DNA previously. When the host strain lacks Dam methylase, the chromosomal DNA is not methylated and SeqA-YFP protein is dispersed in the cytoplasm giving dim background fluorescence.
During conjugation single stranded DNA is transferred from a donor strain to the recipient cells and the second DNA strand is synthesized. When plasmid DNA is methylated by Dam methylase and transferred to a Dam deficient strain, stable hemimethylated duplex is formed. Such a duplex is recognized by SeqA-YFP fusion protein and gives strong fluorescence foci.
Using this technique authors were able to detect presence of transferred plasmid DNA in transconjugants as quickly as 5 minutes after mixing together parental strains. After 30-40 minutes almost all recipient cells in the vicinity of donors showed fluorescent foci. In comparison, the RFP protein expressed from the transferred plasmid from tetracycline promoter showed visible signal 2 hours after transfer. They also showed that in the case of F plasmid direct cell wall contact is not necessary for transfer; this means that single stranded plasmid DNA is transferred from cell to cell thru the sex pili.
To summarize this method allows them to visualize and quantify the DNA of any sequence as it is being transferred from one individual cell to another, and to watch its stable genomic acquisition via genetic recombination (horizontal gene transfer) in real time. This experimental system can be applied to monitor horizontal gene transfer by indefinitely following the fate of DNA acquired in intra- and interspecies crosses.

Ana Babic, Ariel B. Lindner, Marin Vuli, Eric J. Stewart and Miroslav Radman 2008, Direct Visualization of Horizontal Gene Transfer. Science 319, pp. 1533 - 1536.

No comments: