In situ detection of horizontal transfer of mobile genetic elements

Abstract

Plasmid transfer was investigated in microbial populations associated with different types of surfaces. The general strategy behind these investigations was to label the transferable plasmid with a gene encoding a fluorescent protein in order to make it a transfer reporter. This was achieved by fusing the reporter gene with a lac promoter expression cassette and combining this with a donor cell-associated lacI repressor cassette. After construction of a range of strains and plasmids with combinations of genes expressing fluorescent proteins from constitutive (cell tagging) or regulated promoters (transfer reporters) it was thus possible to detect transfer events in situ and correlate these with either the location of donor and recipient cells or with the growth activity of the cells. In some cases, expression of unstable Gfp from a growth-controlled promoter, rrnB from Escherichia coli, was used to monitor bacterial growth activity in situ. Differential tagging of mobilizing and mobilizable plasmids with different genes encoding fluorescent proteins with varying emission wavelengths allowed in situ detection of plasmid mobilization and detection of retro-transfer on agar surfaces. The obtained data show that the several different types of fluorescent reporters, which are now available, allow more informative in situ investigations of horizontal gene transfer to be carried out, and by combining these genes with various expression systems it is possible to simultaneously monitor donor/recipient positioning, cellular activity and appearance of transconjugants.

Plasmid transfer in biofilms: a perspective on limitations and opportunities

“…26 In addition, an in silico and experimental model at the individual cell level showed that cell elongation during growth facilitates conjugative plasmid transfer, which may thus be limited for spatially constrained cells in the centre of biofilms. 27,52 This correlation between cell growth and conjugation has been also observed in situ in biofilms and on surface-grown colonies using fluorescent reporters, 25 but the molecular mechanisms remain elusive.…”

“…Several groups observed similar phenomena for other plasmids. 25,29,38,39 In addition, colony growth of a plasmid-bearing population can result in clonal sectors of plasmid-free cells, with no apparent successful reinfection of plasmid-free cells by HGT 37,38,40 (Figure 1c,d). …”

“…23,25,26 Licht et al 27 showed that plasmid invasion was proficient in a biofilm during the initial contact phase, after which no further transfer was detected. In contrast, the efficiency of invasion was initially slower in mixed liquids but constant, and eventually allowed plasmid spread through the entire population.…”

Non-invasive determination of conjugative transfer of plasmids bearing antibiotic-resistance genes in biofilm-bound bacteria: effects of substrate loading and antibiotic selection

“…Destructive samples of biofilms provide gene transfer estimates that are spatially averaged population value that cannot provide direct estimates of local transfer kinetics. However, numerous studies have revealed that the real spatial structure of biofilms is heterogeneous, with numerous pores and channels surrounding dense clusters of biofilm-bound cells (Wuertz et al 2001; Haagensen et al 2002, Molin and Tolker-Nielsen 2003). Only a few studies have been carried out to examine the survival and transfer of plasmid DNA in biofilms (Beaudoin et al 1998; Bathe et al 2004; Maeda et al 2006), and those provide very limited information on the efficiency of transfer on a local scale.…”

Development of Spatial Distribution Patterns by Biofilm Cells

“…strains JH07 and JH08 were constructed by biparental mating between Acinetobacter sp. C6 (CKL01) and SM1921, expressing green fluorescent protein (Gfp), and SM1923, expressing red fluorescent protein (Rfp), respectively, similarly to a previous description (14).…”